Mebendazole in simultaneous combination with dexamethasone-(C21-phosphoramidate)-[anti-EGFR] generated utilizing a novel synthesis regimen: dual anti-neoplastic cytotoxicity against pulmonary adenocarcinoma (A549).

PURPOSE: The short and long-term resolution of neoplastic conditions with conventional low molecular weight chemotherapeutics is frequently restricted by limitations associated dose-dependent toxic sequelae. Penetration into neoplastic cells occurs non-selectively where their intracellular concentration following simple passive diffusion from the extracellular fluid compartment becomes essentially equivalent to levels found in normal healthy cell populations residing within tissues and organ systems. Selective "targeted" delivery of conventional low molecular weight chemotherapeutics represents one molecular strategy that can both increase potency and reduce dose-dependent toxic sequela. A second strategy is the identification of synergistic or additive combinations of chemotherapeutics and pharmaceutical agents, in addition to the discovery of re-purposed pharmaceutical agents that possess anti-cancer properties. DISCUSSION: Mebendazole evoked anti-neoplastic cytotoxicity as both a single entity, and contributed to the potency of the covalent immunoglucocorticoid, dexamethasone-(C21-phosphoramidate)-[anti-EGFR] when applied in a dual-combination challenge against populations of pulmonary adenocarcinoma (A549). In this capacity mebendazole demonstrated a role as a candidate re-purposed pharmaceutical that possessing potential as a [-i-] substitute alternative for conventional tubulin inhibitors in scenarios of idiosyncratic reactions, therapeutic resistance, or anticipated toxic sequelae; [-ii-] a new monotherapy; or [-iii-] a component in the design of new multi-therapeutic protocols.

Genetic structure and ecological niche space of lentil's closest wild relative, Lens orientalis (Boiss.) Schmalh.

Crops arose from wild ancestors and to understand their domestication it is essential to compare the cultivated species with their crop wild relatives. These represent an important source of further crop improvement, in particular in relation to climate change. Although there are about 58,000 Lens accessions held in genebanks, only 1% are wild. We examined the geographic distribution and genetic diversity of the lentil's immediate progenitor L. orientalis. We used Genotyping by Sequencing (GBS) to identify and characterize differentiation among accessions held at germplasm collections. We then determined whether genetically distinct clusters of accessions had been collected from climatically distinct locations. Of the 195 genotyped accessions, 124 were genuine L. orientalis with four identified genetic groups. Although an environmental distance matrix was significantly correlated with geographic distance in a Mantel test, the four identified genetic clusters were not found to occupy significantly different environmental space. Maxent modelling gave a distinct predicted distribution pattern centred in the Fertile Crescent, with intermediate probabilities of occurrence in parts of Turkey, Greece, Cyprus, Morocco, and the south of the Iberian Peninsula with NW Africa. Future projections did not show any dramatic alterations in the distribution according to the climate change scenarios tested. We have found considerable diversity in L. orientalis, some of which track climatic variability. The results of the study showed the genetic diversity of wild lentil and indicate the importance of ongoing collections and in situ conservation for our future capacity to harness the genetic variation of the lentil progenitor.

A hypervariable intron of the STAYGREEN locus provides excellent discrimination among Pisum fulvum accessions and reveals evidence for a relatively recent hybridization event with Pisum sativum.

An analysis of 82 non-synonymous Pisum fulvum accessions for sequence variation in a fragment of the STAYGREEN (SGR) locus revealed 57 alleles, most of which differed in indel structure. Eight additional P. fulvum accessions, each supposedly synonymous with a different accession of the initial group, were also analyzed. In every case the paired synonymous accessions possessed the same SGR sequence but varied slightly for a 6-trait morphological phenotype, indicating that SGR sequence is a much more reliable indicator of accession identity than is a morphological characterization. SGR sequence analysis confirmed our previous finding that P. fulvum accessions separate into two allele groups. This division was not supported by results of previous studies that were based on sequences distributed across the entire genome, suggesting that the division may have been produced by selection at a nearby locus and that the SGR phylogeny may not be good indicator of overall relationships within the species. One P. fulvum accession, PI 595941 (=JI1796), displayed an SGR sequence outside the variation typical of the species. Instead, its allele resembled alleles limited to a set of Pisum sativum landraces from the Middle East, suggesting hybridization between ancestors of PI 595941 and some primitive form of domesticated P. sativum. With one exception from the extreme northwest corner of Israel, P. fulvum accessions collected north of latitude 35.5° N were fixed for alleles from group A. These northern accessions also displayed greatly reduced SGR sequence diversity compared to group A accessions collected from other regions, suggesting that the northern populations may represent recent extensions of the range of the species. Group B accessions were distributed from Lake Tiberias south and were generally sympatric with the southern group A accessions. Although group B accessions occupied a smaller area than group A, the SGR sequence diversity in this group (28 alleles in 33 accessions) exceeded that for group A.

Synthesis of a covalent gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic and its cytotoxic anti-neoplastic activity against chemotherapeutic-resistant SKBr-3 mammary carcinoma.

UNLABELLED: Gemcitabine is a potent chemotherapeutic that exerts cytotoxic activity against several leukemias and a wide spectrum of carcinomas. A brief plasma half-life in part due to rapid deamination and chemotherapeutic-resistance frequently limit the utility of gemcitabine in clinical oncology. Selective 'targeted' delivery of gemcitabine represents a potential molecular strategy for simultaneously prolonging its plasma half-life and minimizing exposure of innocent tissues and organ systems. MATERIALS AND METHODS: Gemcitabine was combined in molar excess with N-[p-maleimidophenyl]-isocyanate (PMPI) so that the isocyanate moiety of PMPI which exclusively reacts with hydroxyl groups preferentially created a carbamate covalent bond at the terminal C(5)-methylhydroxy group of gemcitabine. Monoclonal immunoglobulin with binding-avidity specifically for HER2/neu was thiolated with 2-iminothiolane at the terminal ε-amine group of lysine amino acid residues. The gemcitabine-(carbamate)-PMPI intermediate with a maleimide moiety that exclusively reacts with reduced sulfhydryl groups was then combined with thiolated anti-HER2/neu monoclonal immunoglobulin. Western-blot analysis was utilized to delineate the molecular weight profile for gemcitabine-(carbamate)-[anti-HER2/neu] while cell binding characteristics were determined by cell-ELISA utilizing SKBr-3 mammary carcinoma which highly over-expresses HER2/neu receptors. Cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] between the gemcitabine-equivalent concentrations of 10(-12) and 10(-6)M was determined utilizing vitality staining analysis of chemotherapeutic-resistant SKBr-3 mammary carcinoma. RESULTS: Gemcitabine-(carbamate)-[anti-HER2/neu] was synthesized at a molar incorporation index of 1:1.1 (110%) and had a molecular weight of 150kDa that was indistinguishable from reference control immunoglobulin fractions. Cell-ELISA detected progressive increases in SKBr-3 mammary carcinoma associated immunoglobulin with corresponding increases in covalent gemcitabine immunochemotherapeutic concentrations. The in vitro cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] was approximately 20% and 32% at 10(-7) and 10(-6)M (gemcitabine-equivalent concentrations) after a 182-h incubation period. DISCUSSION: The investigations describes for the first time a methodology for synthesizing a gemcitabine anti-HER2/neu immunochemotherapeutic by creating a covalent bond structure between the C(5)-methylhydroxy group of gemcitabine and thiolated lysine amino acid residues of monoclonal antibody or other biologically active protein fractions. Gemcitabine-(carbamate)-[anti-HER2/neu] possessed binding-avidity at HER2/neu receptors highly over-expressed by chemotherapeutic-resistant SKBr-3 mammary carcinoma. Alternatively, gemcitabine can be covalently linked at its C(5)-methylhydroxy group to monoclonal immunoglobulin fractions that possess binding-avidity for other receptors and membrane complexes uniquely highly over-expressed by a variety of neoplastic cell types. Compared to chemotherapeutic-resistant SKBr-3 mammary carcinoma, gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic is anticipated to exert higher levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic carcinoma, small-cell lung carcinoma, neuroblastoma, glioblastoma, oral squamous cell carcinoma, cervical epithelioid carcinoma, or leukemia/lymphoid neoplastic cell types based on their reportedly greater sensitivity to gemcitabine and gemcitabine covalent conjugates.

Birth of live triplets in a mare.

An 11-year-old American Buckskin mare gave birth to live triplets unattended at approximately 300 days gestation. All foals were small and dysmature, requiring intensive care. The smallest foal died 4 days after admission, the second was subjected to euthanasia 24 days after admission due to poor healing of a third metatarsal fracture. The remaining foal survived to discharge and was considered small but otherwise normal at age one year.

First Report of Bean yellow mosaic virus from Diseased Lupinus luteus in Eastern Washington.

Lupine accessions from the Cool Season Food Legume Seed Collection are grown for seed regenerations in Pullman, WA by the Agricultural Research Service, Western Regional Plant Introduction Station. Selected seed was germinated in the greenhouse and assayed by indirect ELISA using antiserum for potyvirus group detection (Agdia, Inc., Elkhart, IN). Healthy transplants were grown for seed collection on outside plots. In July of 2005, more than 90% of 307 Lupinus luteus L. transplants developed severe yellowing, necrosis, and stunting with an estimated 5% plant death. Plants were heavily infested with aphids and leaf sap was serologically positive for potyvirus. Partially purified virus preparations from infected plants contained filamentous particles and a 35-kDa protein that reacted with universal potyvirus antiserum on western blots. Reverse transcription (RT)-PCR using potyvirus universal primers (2) and cDNA derived from virion RNA generated a ~1.7-kbp product that was cloned and sequenced. The sequenced portion of the genomic RNA contained 1,610 nucleotides (nt) on its 3'-terminus (GenBank Accession No. EU144223) that included a partial nuclear inclusion protein, NIb, (1 to 637 nt) with the conserved amino acid (aa) replicase motif GDD (131 to 139 nt), the coat protein (CP) gene of 821 nt (638 to 1,459 nt), and a 171-nt untranslated region (1,460 to 1,630 nt) attached to a poly(A)tail. The CP sequence contained a NAG motif instead of the DAG motif commonly associated with aphid transmission. Searches in the NCBI GenBank database revealed that the CP aa and nt sequences contained conserved domains with isolates of Bean yellow mosaic virus (BYMV). A pairwise alignment (ClustalX) (4) of the CP aa from 20 BYMV isolates with the BYMV-Pullman isolate revealed identities from 96% (BYMV-S, U47033) to 88% (BYMV-MI [X81124)] -MI-NAT [AF434661]). This meets the species demarcation criteria of more than ~80% identity for inclusion with BYMV (1). Virion mechanical inoculations resulted in local lesions on Chenopodium amaranticolor Coste et Reyn and C. quinoa Willd., necrotic blotches on Phaseolus vulgaris L., and yellow spots and systemic movement in L. succulentus Douglas ex. K. Koch, L. texensis 'Bluebonnet', and L. texensis 'Maroon'; BYMV was confirmed by western blots and ELISA. The experimental inoculations represent the first documented report of BYMV in the annual L. succulentus and biennial L. texensis species. Since BYMV is seedborne and transmitted by many aphid species (3), it is possible that several lupine transplants escaped potyvirus detection, and secondary transmission of BYMV to plants occurred by aphids. During the 1950s, BYMV was confirmed in several annual lupines grown as crops in the southeastern United States (3). To our knowledge, this is the first report of BYMV occurring naturally in a lupine species in Washington. BYMV is a destructive virus to lupine species worldwide and has a wide host range in Fabaceae. This research directly contributes toward the maintenance of virus-free lupine seed for distribution to scientists focusing on lupine research. References: (1) P. H. Berger et al. Family Potyviridae. Page 819 in: Virus Taxonomy: Eighth Report of the ICTV. C. M. Fauquet et al. eds., 2005. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) R. A. C. Jones and G. D. Mclean, Ann. Appl. Biol. 114:609, 1989. (4) J. D. Thompson et al. Nucleic Acids Res. 24:4878, 1997.

Intrastriatal dopamine D1 antagonism dampens neural plasticity in response to motor cortex lesion.

Motor cortex lesions in rats partially denervate the striatum, producing behavioral deficits and inducing reactive neuroplasticity. Plastic responses include changes in growth-associated protein marker expression and anatomical restructuring. Corticostriatal plasticity is dependent on dopamine at the striatal target, where D1 receptor signaling reinforces behaviorally relevant neural activity. To determine whether striatal dopamine D1 receptor signaling is important for the growth-associated protein responses and behavioral recovery that follow unilateral motor cortex aspiration, the dopamine D1 receptor antagonist SCH23390 was intrastriatally infused in cortically lesioned animals. After a cortical aspiration lesion in Long Evans rats, the growth-associated proteins SCG10 and GAP-43 were upregulated in the cortex contralateral to the lesion at 30 days post-lesion. However, continuous unilateral intrastriatal infusion of SCH23390 prevented this aspiration-induced upregulation. Furthermore, lesioned rats demonstrated spontaneous sensorimotor improvement, in terms of limb-use symmetry, about 1 month post-lesion. This improvement was prevented with chronic intrastriatal SCH23390 infusion. The D1 receptor influence may be important to normalize corticostriatal activity (and observable behavior), either in a long-term manner or temporarily until other more permanent means of synaptic regulation, such as sprouting or synaptogenesis, may be implemented.

Biochemical mechanisms that interact with membrane-associated IL-1 RII (60-kDa decoy) receptors in populations of adherent macrophages and vascular endothelium.

The aim of this investigation was to identify the potential biochemical mechanisms that alter the integrity of membrane-associated IL-1 RII (decoy) receptor complexes expressed by populations of adherent macrophages and vascular endothelium. The initial research strategy utilized to achieve this objective involved delineating the ability of macrophage activation or exposure of macrophages and vascular endothelium to a spectrum of enzyme proteases to influence the expression of membrane-associated IL-1 RII (decoy) or generate soluble fragments of this receptor complex. Results from these investigations revealed that stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-1 RII (decoy) and release of soluble receptor fragments. Exposure of macrophages and vascular endothelium to the reference proteases discovered the ability of cathepsin-D to biochemically deplete membrane-associated IL-1 RII (decoy) in addition to generating soluble fragments of this receptor complex. Complementary investigations isolated a carboxyl/aspartate protease from activated macrophages utilizing pepstatin-A affinity chromatography. Exposure of vascular endothelium to pepstatin-A binding proteins resulted in a detectable depletion of membrane-associated IL-1 RII (decoy) and generation of soluble receptor fragments. Evaluation of pepstatin-A binding proteins by SDS-PAGE identified a primary protein fraction with a molecular mass of 47-52 kDa that closely correlates with the known molecular size of leukocyte cathepsin-D fractions. Macrophage pepstatin-A binding protein fractions evaluated by nondenaturing haemoglobin-substrate PAGE (Hb-PAGE) analysis detected a lucent proteolytic band at 47-52 kDa. Macrophage pepstatin-A binding proteins also hydrolyzed a synthetic enzyme-specific substrate that selectively recognizes cathepsin-D biochemical activity. In conclusion, the leukocyte carboxyl/aspartate protease cathepsin-D can biochemically alter the integrity and generate soluble fragments of membrane-associated IL-1 RII (60-kDa decoy) receptor complexes expressed by macrophages and vascular endothelium.

Consistent Quantitative Trait Loci in Pea for Partial Resistance to Aphanomyces euteiches Isolates from the United States and France.

ABSTRACT Development of pea cultivars resistant to Aphanomyces root rot, the most destructive root disease of pea worldwide, is a major disease management objective. In a previous study of a mapping population of 127 recombinant inbred lines (RILs) derived from the cross 'Puget' (susceptible) x '90-2079' (partially resistant), we identified seven genomic regions, including a major quantitative trait locus (QTL), Aph1, associated with partial resistance to Aphanomyces root rot in U.S. fields (21). The objective of the present study was to evaluate, in the same mapping population, the specificity versus consistency of Aphanomyces resistance QTL under two screening conditions (greenhouse and field, by comparison with the previous study) and with two isolates of Aphanomyces euteiches originating from the United States and France. The 127 RILs were evaluated in the greenhouse for resistance to pure culture isolates SP7 (United States) and Ae106 (France). Using the genetic map previously described, a total of 10 QTL were identified for resistance in greenhouse conditions to the two isolates. Among these were Aph1, Aph2, and Aph3, previously detected for partial field resistance in the United States. Aph1 and Aph3 were detected with both isolates and Aph2 with only the French isolate. Seven additional QTL were specifically detected with one of the two isolates and were not identified for partial field resistance in the United States. The consistency of the detected resistance QTL over two screening environments and isolates is discussed with regard to pathogen variability, and disease assessment and QTL detection methods. This study suggests the usefulness of three consistent QTL, Aph1, Aph2, and Aph3, for marker-assisted selection.

Biochemical alteration of membrane-associated IL-6 RI (80-kDa) in adherent macrophages and vascular endothelium.

The potential biochemical mechanisms that mediate the 'shedding' of soluble IL-6 RI (80-kDa) receptor fragments in populations of adherent macrophages were explored. Stimulated macrophages displayed proportional increases in both the expression of membrane-associated IL-6 RI (80-kDa) and the release of soluble receptor fragments. The use of protease inhibitors implicated thiol/cysteine and carboxyl/aspartate proteases in this process. Cathepsin-D depleted membrane-associated IL-6 RI (80-kDa) complexes and generated soluble receptor fragments. A carboxyl/aspartate protease from activated macrophages isolated utilizing pepstatin-A affinity chromatography, was also found to affect membrane-associated IL-6 RI (80-kDa) complexes and generate soluble receptor fragments. Most likely, this fraction corresponded to cathepsin-D based upon its origin, pepstatin-A binding avidity, Hb-PAGE zymography, and hydrolysis of an enzyme-specific substrate. We conclude that cathepsin-D can generate soluble fragments of IL-6 RI (80-kDa) expressed by both macrophages and vascular endothelium.

Fludarabine- (C2-methylhydroxyphosphoramide)- [anti-IGF-1R]: Synthesis and Selectively "Targeted"Anti-Neoplastic Cytotoxicity against Pulmonary Adenocarcinoma (A549).

INTRODUCTION: Many if not most conventional small molecular weight chemotherapeutics are highly potent against many forms of neoplastic disease. Unfortunately, majority of an administered dose unintentionally diffuses passively into normal tissues and healthy organ systems following intravenous administration. One strategy for both increasing potency and reducing dose-limited sequela is the selective "targeted" delivery of conventional chemotherapeutic agents. MATERIALS AND METHODS: The fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] was synthesized by initially reacting fludarabine with a carbodiimide to form a fludarabine carbodiimide phosphate ester intermediate that was subsequently reacted with imidazole to create an amine-reactive fludarabine- (C2-phosphorylimidazolide) intermediate. Monoclonal anti-IGF-1R immunoglobulin was combined with the amine-reactive fludarabine- (C2-phosphorylimidazolide) intermediate resulting in the synthesis of covalent fludarabine-(C2-methylhydroxyphosphoramide)- [anti-IGF-1R] immunochemotherapeutic. Residual fludarabine and un-reacted reagents were removed by serial microfiltration (MWCO 10,000) and monitored by analytical-scale HP-TLC. Retained IGF-1R binding-avidity of fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] was established by cell-ELISA using pulmonary adenocarcinoma cell (A549) which over-expresses IGF-1R and EGFR. Anti-neoplastic cytotoxic potency of fludarabine-(C2-methylhydroxyphosphoramide)-[anti- IGF-1R] was determined against pulmonary adenocarcinoma (A549) using an MTT-based vitality stain methodology. RESULTS: The fludarabine molar-incorporation-index for fludarabine- (C2-methylhydroxyphosphoramide)-[anti-IGF-R1] was 3.67:1 while non-covalently bound fludarabine was not detected by analytical scale HP-TLC following serial micro-filtration. Size-separation fludarabine-(C2-methylhydroxyphosphoramide)-[anti- IGF-1R] by SDS-PAGE with chemo luminescent autoradiography detected only a single 150-kDa band. Cell-ELISA of fludarabine- (C2-methylhydroxyphosphoramide)-[anti-IGF-1R] measuring total immunoglobulin bound to exterior surface membranes of pulmonary adenocarcinoma (A549) increased with elevations in immunoglobulin-equivalent concentrations of the covalent fludarabine immunochemotherapeutic. Between the fludarabine-equivalent concentrations of 10-10 M and 10-5 M both fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] and fludarabine had ex-vivo anti-neoplastic cytotoxic potency levels that increased rapidly between the fludarabine-equivalent concentrations of 10-6 M and 10-5 M where cancer cell death percentages increased from 24.4% to a maximum of 94.7% respectively. CONCLUSION: The molecular design and organic chemistry reaction schemes were developed for synthesizing fludarabine-(C2- methylhydroxyphosphoramide)-[anti-IGF-1R] which possessed both properties of selective "targeted" delivery and anti-neoplastic cytotoxic potency equivalent to fludarabine chemotherapeutic.

Simultaneous Dual Selective Targeted Delivery of Two Covalent Gemcitabine Immunochemotherapeutics and Complementary Anti-Neoplastic Potency of [Se]-Methylselenocysteine.

The anti-metabolite chemotherapeutic, gemcitabine is relatively effective for a spectrum of neoplastic conditions that include various forms of leukemia and adenocarcinoma/carcinoma. Rapid systemic deamination of gemcitabine accounts for a brief plasma half-life but its sustained administration is often curtailed by sequelae and chemotherapeutic-resistance. A molecular strategy that diminishes these limitations is the molecular design and synthetic production of covalent gemcitabine immunochemotherapeutics that possess properties of selective "targeted" delivery. The simultaneous dual selective "targeted" delivery of gemcitabine at two separate sites on the external surface membrane of a single cancer cell types represents a therapeutic approach that can increase cytosol chemotherapeutic deposition; prolong chemotherapeutic plasma half-life (reduces administration frequency); minimize innocent exposure of normal tissues and healthy organ systems; and ultimately enhance more rapid and thorough resolution of neoplastic cell populations. MATERIALS AND METHODS: A light-reactive gemcitabine intermediate synthesized utilizing succinimidyl 4,4-azipentanoate was covalently bound to anti-EGFR or anti-HER2/neu IgG by exposure to UV light (354-nm) resulting in the synthesis of covalent immunochemotherapeutics, gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu]. Cytotoxic anti-neoplastic potency of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] between gemcitabine-equivalent concentrations of 10-12 M and 10-6 M was determined utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKRr-3). The organoselenium compound, [Se]-methylselenocysteine was evaluated to determine if it complemented the anti-neoplastic potency of the covalent gemcitabine immunochemotherapeutics. RESULTS: Gemcitabine-(C4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] and the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] all had anti-neoplastic cytotoxic potency against mammary adenocarcinoma. Gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] produced progressive increases in anti-neoplastic cytotoxicity that were greatest between gemcitabine-equivalent concentrations of 10-9 M and 10-6 M. Dual simultaneous combinations of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] produced levels of anti-neoplastic cytotoxicity intermediate between each of the individual covalent gemcitabine immunochemotherapeutics. Total anti-neoplastic cytotoxicity of the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) was substantially higher when formulated with [Se]-methylsele-nocysteine.

Temporal sequence of plaque formation in the cerebral cortex of non-demented individuals.

One of the hallmarks of Alzheimer's disease is the presence of argyrophilic plaques (arg-P) accompanying dementia and other forms of cognitive alterations. In the present investigation 195 non-demented, cognitively normal patients were grouped according to the presence or absence of critical coronary artery disease (cCAD), defined as a 75% or greater stenosis of one of the epicardial arteries. None of the subjects had significant cerebral vascular disease. The parahippocampal gyrus (PHG) and frontal pole were analyzed for the presence of arg-P, A4 deposition, ALZ-50 immunoreactive (IR) neurons and neuropil threads (NT). Individuals with cCAD have a significantly greater incidence of plaques than non-heart disease (non-HD) subjects. Every cCAD subject had ALZ-50 IR neurons in the PHG and a greater incidence of NT as compared to the non-HD subjects. Every subject with plaques also had IR neurons and NT in the PHG. Based on the presumption that early neurodegeneration labeled by ALZ-50 antibody and amyloid deposition are in some way linked, then the sequence of plaque formation is initiated by the presence of ALZ-50 IR neurons followed in order by NT, A4 deposition and diffuse form arg-P.

Neurochemical and histopathologic alterations characteristic of Pick's disease in a non-demented individual.

In the course of investigating a large number of non-demented subjects, a 68 year old female dying of coronary artery disease was found to have Pick bodies in her grossly normal brain. Although only mild subcortical gliosis and no neuron loss were observed. Pick bodies were found throughout the brain and occasional balloon cells were noted. Pick bodies and numerous neurons were also ALZ-50 and Tau-1 immunoreactive. Retrospective studies indicated a lack of overt intellectual decline or depression in this individual. Frontal, temporal and occipital poles, amygdala, hypothalamus and nucleus basalis of Meynert (nbM) were analyzed for ChAT, AChE and MAO-A and -B enzymatic activities and for the binding of 5HT and imipramine. Cholinergic decreases were found only in subcortical structures. Serotonin binding decreases were widespread, excluding the nbM. Altered MAO-B activity was regionally variable, and no differences in MAO-A activity or imipramine binding were observed. Few differences in neurochemical alterations were observed in the current non-demented subject with abundant Pick bodies compared to previous studies of demented Pick's patients. This case strongly suggests that chemical dysfunction and neuropathological features of Pick's disease occur in advance of overt clinical manifestations of the disorder.

Alterations in membrane-associated CD14 expression and the simultaneous liberation of soluble CD14 fragment in adherent macrophages mediated by a leukocyte carboxyl/aspartate protease.

Investigations sought to discover the biochemical mechanisms in macrophages that mediate the 'shedding' of soluble CD14 fragment. Stimulated macrophages display both increased liberation of soluble CD14 fragment and decreases in residual membrane-associated CD14 complexes following exposure to activating agents (fMLP/A23187). Application of 'class-specific' protease inhibitors revealed that a thiol/cysteine was involved in the biochemical production of soluble CD14 fractions and that a metalloprotease enzymatically degraded soluble CD14 fragment. Exposure of macrophages to individual proteases revealed that both cathepsin-D and elastase promoted variable depletion of membrane-associated CD14 complexes. Additionally, cathepsin-D, and to a lesser extent elastase, generated soluble CD14 fragment. Related studies isolated a carboxyl/aspartate protease from activated macrophages using pepstatin-A affinity chromatography. The physical and functional properties of macrophage pepstatin-A binding protein fractions closely corresponded with the known characteristics of cathepsin-D with respect to: (i) cellular origin; (ii) binding-avidity of carboxyl/aspartate proteases for pepstatin-A; (iii) non-specific proteolysis of haemoglobin detected by Hb-PAGE zymography; and (iv) hydrolysis of a synthetic cathepsin-D-specific peptide substrate. Interpretation of these findings collectively implies that activated leukocytes can biochemically alter membrane-associated CD14 complex expression and promote the liberation of soluble CD14 fragment in both activated and non-activated cell populations.

Breast and cervical cancer screening interventions: an assessment of the literature.

An extensive body of intervention research to promote breast and cervical cancer screening has accumulated over the last three decades, but its coverage and comprehensiveness have not been assessed. We evaluated published reports of these interventions and propose a framework of critical elements for authors and researchers to use when contributing to this literature. We identified all articles describing breast and cervical cancer screening interventions published between January 1960 and May 1997 in the United States and abstracted specified critical elements in the broad areas of: (a) needs assessment; (b) intervention study design; and (c) analysis methods and study outcomes from each article using a template developed for that purpose. Fifty-eight studies met our criteria for inclusion. Thirty-eight focused exclusively on breast cancer screening, 7 promoted cervical cancer screening, and 13 were designed to promote screening for both cancers. The amount of detail reported varied among the 58 studies. All studies reported the outcome measures used to assess the effectiveness of the intervention, yet only 40% of the studies reported the investigators' original hypotheses or research questions. Needs assessment data were reported in 84% of the studies. Data sources ranged from national surveys to local intervention baseline surveys. Population characteristics reported also varied, with most studies reporting age and race of the study population (78 and 71%, respectively), and fewer studies reporting income and education (53 and 38%, respectively). As the field of behavioral intervention research progressed, we found that more recent studies included and reported many of the parameters we had identified as critical. If this trend continues, it will enhance the reproducibility of studies, enable comparisons between interventions, and provide a reference point for measuring progress in this area. To facilitate this trend toward uniform reporting, we propose an evaluative framework of critical elements for authors to use when developing and reporting their research. The comprehensive assessment of literature that this article provides should be useful background to investigators planning and reporting cancer control interventions, to funding agencies choosing and guiding quality research, and to publishers to help them enhance the quality and utility of their publications.

Identification and isolation of two rat serum proteins with A-esterase activity toward paraoxon and chlorpyrifos-oxon.

The active metabolites (oxons) of phosphorothionate insecticides can be detoxified via A-esterase hydrolysis. Two enzymes with A-esterase activity have been isolated from rat serum. Whole serum was applied to anion exchange gel (DEAE Sepharose Fast Flow) and incubated (1 hr). Tris-HCl buffer (0.05 M; pH 7.7, at 5 degrees) containing 0.25 M NaCl was added to the slurry and incubated. The decant, containing low A-esterase activity but a high protein concentration, was discarded. Further displacement of A-esterase from DEAE gel was achieved with 1.0 M NaCl in 0.05 M Tris-HCl buffer (Ph 7.7 at 5 degrees). Following desalting and concentration, further separation was achieved by gel filtration (Sephacryl S-100 HR) and two sequential preparative scale isoelectric focusings. Final fractions contained two proteins of high molecular mass (one about 200 kDa and one between 137 and 200 kDa). The apparent range of isoelectric points for the two enzymes was 4.5 to 5.6. Following native-PAGE analysis, activity stains with beta-naphthyl acetate and Fast Garnet GBC in the presence of paraoxon (10-5 M) verified that A-esterase activity was associated with both proteins. Spectropho-tometric assay detected A-esterase activity toward paraoxon, chlorpyrifos-oxon, and phenyl acetate in the final preparation.

Characterisation of Haemonchus contortus-derived cell populations propagated in vitro in a tissue culture environment and their potential to induce protective immunity in sheep.

Cell populations derived from viable Haemonchus contortus L(3) larvae were propagated in vitro in a tissue culture environment for a prolonged period (>48 months). Microscopic evaluation of H. contortus-derived cell populations revealed gross morphological characteristics highly analogous to those described for cell types originating from species of plant nematodes propagated in vitro in a tissue culture environment for a briefer period of time (<6 months). The characterisation of extracts harvested from tissue culture populations of H. contortus-derived cells by SDS-PAGE analysis detected molecular fractions of approximately 29, 45, 55, and 200-kDa that closely correlated with reports for preparations obtained from intact/viable H. contortus larvae. Complementary investigations detected the dual biochemical expression of phosphohydrolase and aminopeptidase-M activities based on the hydrolysis of the synthetic enzyme-specific substrates, para-nitrophenylphosphate and leucine-para-nitroanaline, respectively. The identification of phosphohydrolase and aminopeptidase-M-like biochemical activity in fractions harvested from H. contortus-derived cell populations and propagated in vitro in tissue culture served as evidence validating their parasitic-origin. Further validation of H. contortus-derived cell populations propagated in tissue culture entailed the formulation of Triton X-100 extracts containing potential immunoprotective antigens with SEAM adjuvant and its administration by intramuscular injection (100 microg total protein) to healthy sheep (n=8) on day 0 (left rear-limb) and day +14 (right rear-limb). Animals on day 28 subsequently received a single oral challenge of 10,000 infective L(3)-stage H. contortus larvae. Applying ELISA methodologies, increases in antigen-specific IgM and IgG were detected in ovine serum samples. Interpretation of experimental findings revealed that sheep with the greatest antigen-specific humoral immune responses (IgG titre 1/3125) also demonstrated a degree of reduced abomasal H. contortuslarvae burdens (60% reduction). Polyclonal antibody from immunoprotected sheep was subsequently found to recognise both the: (i), digestive tract; and (ii), antigen extracts associated with intact/viable H. contortus larvae. These experimental findings reveal the potential feasibility of propagating parasite-derived cell populations in an in vitro tissue culture environment in a manner that retains their ability to express immunoprotective antigenic fractions.

A mechanism of TNFR type II (75 kDa) "shedding" in macrophages.

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.

Increased incidence of neurofibrillary tangles (NFT) in non-demented individuals with hypertension.

The incidence rates and numerical densities of argryophilic neurofibrillary tangles (NFT) and senile plaques (SP) were determined in non-demented individuals and subjects with Alzheimer's disease (AD). The non-AD subjects were grouped according to cardiac status; those individuals with critical coronary artery disease (cCAD), those hypertensive individuals without cCAD (HyperT), and those without heart disease (non-HD). The incidence and densities of SP and NFT were significantly greater in AD than any of the non-demented groups. The prevalence of SP was increased in both HyperT and cCAD compared to non-HD controls, while NFT occurrence was accentuated in non-demented HyperT subjects only. The densities of SP and NFT in HyperT were elevated compared to cCAD or both cCAD and non-HD controls; NFT densities were similar in cCAD and non-HD. NFT density increased with increasing age in only the non-HD and cCAD groups, suggesting a possible relationship between disease process and NFT formation in the AD and HyperT populations.