Simplified methods for construction, assessment and rapid screening of peptide libraries in bacteriophage.

An efficient strategy has been devised for the construction of diverse peptide libraries in bacteriophage vectors. This strategy was used to generate a library of 4 x 10(8) random decapeptide inserts in the pIII protein of bacteriophage fd. A novel method for evaluating the genetic diversity of bacteriophage libraries based on colony hybridization with partially degenerate oligonucleotides has been developed. The decapeptide library was affinity-selected with a previously characterized monoclonal antibody specific for the V3 loop of the gp120 protein of HIV-1. Immunological screening, an efficient technique for the rapid identification of putative binding bacteriophage, is described. Hexapeptide sequences similar to those obtained from affinity selection of a hexapeptide bacteriophage library were obtained from the decapeptide library in all five frames. Immunological screening of 20,000 clones from the two libraries after two rounds of affinity selection rapidly identified antibody-binding sequences; 93% and 86% of the sequences obtained from the hexapeptide and decapeptide libraries, respectively, had IC50 values < or = 10 mM as free peptides.

Effective purging of bone marrow by a combination of immunorosette depletion and complement lysis.

The effectiveness of a simple immunorosette technique for the depletion of common acute lymphatic leukemic (cALL) blasts from autologous bone marrow transplants was studied. Erythrocytes were sensitized with tetramolecular complexes consisting of rat anti-mouse IgG1 monoclonal antibodies (McAbs) that crosslink two different mouse McAbs. One of the McAbs was directed against glycophorin A, and the other was directed against marker glycoproteins of B cells and their precursors (CD9, CD10, CD19, or CD22). Immunorosettes were formed by addition of the sensitized erythrocytes to the cALL+ cells. After density-gradient separation of immunorosettes from mixtures of cALL+/terminal deoxynucleotidyl transferase-positive (TdT+) leukemic blasts and mononuclear bone marrow cells, nearly a 2-log depletion of leukemic cells was measured by flow cytometry. Clonogenic assays with two cALL+B-cell lines (Ros-17 and Nalm-16) were performed to compare the efficacy of complement-mediated cell lysis, immunorosette depletion, and a combination of both procedures. Complement-mediated cytotoxicity with the three McAbs in combination with baby rabbit complement yielded a 1- to 2-log cell kill. Immunorosette depletion resulted in a 3-log reduction of clonogenic units. Sequential application of the two methods (immunorosette depletion with CD19 McAb followed by a complement lysis with CD9 and CD10 McAbs) led to superior results in causing a 4- to 5-log purging effect. These purging procedures did not cause a loss of normal myeloid (granulocyte-macrophage colony-forming units, CFU-GM) or erythroid (erythroid burst-forming units, BFU-e) progenitors from the bone marrow. This study indicates that the combination of the two methods results in a highly efficient purging procedure for the removal of cALL+ cells from autologous bone marrow cells.

Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1).

Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell line (FLG 29.1) during its differentiation induced by phorbol 12-myristate 13-acetate (TPA) was studied using metabolic radiolabeling experiments. The FLG 29.1 cells were metabolically radiolabeled with [3H]glucosamine and [35S]sulfate, and the labeled glycoproteins were analyzed by anion exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiments. One of the major glycoproteins synthesized by the TPA-treated FLG 29.1 cells was sulfated, had an identical electrophoretic mobility to purified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively identified as the BSP. Furthermore, mRNA for BSP was also detected in TPA-treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synthesized by FLG 29.1 cells remained cell-associated, and this is in contrast with those synthesized by osteoblasts, where the protein is rapidly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Golgi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells after permeabilization, while untreated cells were not significantly immunostained. Localization of BSP at the plasma membrane was also demonstrated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expression of various osteoclastic characteristics in FLG 29.1 cells, induction of BSP expression by TPA suggests that the protein may play a role during the differentiation process of osteoclasts or in functions of differentiated osteoclasts.

The cDNA cloning and RNA distribution of bovine osteopontin.

We have isolated and sequenced the bovine cDNA (OPN) counterpart of osteopontin. The cDNA is 1356 nucleotides (nt) in length with an open reading frame of 834 nt, encoding a 278-amino acid (aa) protein. Cell-free transcription and translation of OPN RNA resulted in a major species of approx. 40 kDa in size, in agreement with the predicted size of the deduced aa sequence. Northern analysis of bovine OPN RNA indicated the presence of the message in mineralized, as well as soft tissues. A comparison of the deduced aa sequence among various species indicates both regions of similarity and divergence. One prominent region of dissimilarity in bovine OPN compared to all other species is a 22-aa gap which may represent a loss of a potential Ca(2+)-binding loop. Despite the variability among the species, several regions of conservation are apparent, including a hydrophobic leader sequence, a potential site for Asn-linked glycosylation, a stretch of polyaspartic acid residues, and the cell attachment Arg-Gly-Asp tripeptide. Whether bovine OPN enhances cell attachment is unknown. Furthermore, whether the loss of a potential Ca(2+)-binding loop alters the function of OPN would be interesting to determine.

Structure-activity relationship of 99mTc complexes of phenylenediamine-thiol-thioether ligands (PhAT) to brain uptake in rats.

A novel class of ligands, phenylenediamine-thiol-thioether (PhAT), was synthesized, and their 99mTc complexes were evaluated for potential use as a functional brain imaging agent. The ligands reacted with Na99mTcO4 and SnCl2 to form single, stable, neutral, and lipophilic 99mTc complexes. Several of these complexes showed significant brain uptake and retention in rats. In particular, the S-ethyl, allyl, and propargyl derivatives had high initial brain uptake (0.88, 0.99, and 0.82% dose/g at 5 min, respectively) and good retention (0.71, 0.75, and 0.67% dose/g at 30 min). The structure-activity relationship of alkyl, alkenyl, and alkynyl thioether derivatives is reported.

Discovery of nanomolar ligands for 7-transmembrane G-protein-coupled receptors from a diverse N-(substituted)glycine peptoid library.

Screening a diverse, combinatorial library of ca. 5000 synthetic dimer and trimer N-(substituted)glycine "peptides" yielded novel, high-affinity ligands for 7-transmembrane G-protein-coupled receptors. The peptoid library was efficiently assembled using readily available chemical building blocks. The choice of side chains was biased to resemble known ligands to 7-transmembrane G-protein-coupled receptors. All peptides were screened in solution-phase, competitive radioligand-binding assays. Peptoid trimer CHIR 2279 binds to the alpha 1-adrenergic receptor with a Ki of 5 nM, and trimer CHIR 4531 binds to the mu-opiate receptor with a Ki of 6 nM. This represents the first example of the discovery of high-affinity receptor ligands from a combinatorial library of non-natural chemical entities.

Technetium-99m-N1-(2-mercapto-2-methylpropyl)-N2-(2-propargylthio-2- methylpropyl)-1,2-benzenediamine (T691): preclinical studies of a potential new tracer of regional cerebral perfusion.

We report in vitro and in vivo preclinical studies of a new cerebral blood flow tracer, [99mTc]N1-(2-mercapto-2-methyl-propyl)-N2-(2- propargylthio-2-methylpropyl)-1,2-benzenediamine (T691). The tracer demonstrates excellent in vitro chemical stability and accumulates regionally in the brain in a pattern consistent with that of cerebral blood flow. First-pass cerebral extraction determined with the use of the brain uptake index method in the rat was 0.76. Bolus intracarotid injection in monkeys indicated a cerebral extraction of 68% and prolonged retention of 67% of the initially extracted activity. Autoradiographic studies in rats revealed a pattern characteristic of cerebral blood flow at both 1 and 60 min after systemic injection. Dynamic tomographic imaging following systemic injection in the monkey revealed peak brain activity 1 to 2 min postinjection, without significant decline over 60 min. Chromatographic studies of brain as long as 60 min following systemic injection of [99mTc]T691 showed no evidence of tracer metabolism to account for its retention. Overall, [99mTc]T691 demonstrates promise as a potential new clinical tracer of cerebral perfusion.

25-hydroxycholecalciferol in poultry nutrition.

Vitamin D is a complex of secosteroids that must undergo metabolic alterations to reach optimal biological activity. The parent compounds 1) ergocalciferol (D2) and 2) cholecalciferol (D3) can be synthesized in the leaves of many plants or in the skin of most animals, respectively. Transport of vitamin D steroids after absorption is associated with vitamin D binding proteins (DBP). In general, the relative binding affinities of the vitamin D steroids are: 25-hydroxy vitamin D3 [25-(OH)D3] = 24,25-dihydroxy vitamin D3 [24,25-(OH)2D3] = 25,26-dihydroxy vitamin D3 [25,26-(OH)2D3] > 25-hydroxy vitamin D2 (25-(OH)D2) > 1,25-dihydroxy vitamin D3 [1,25-(OH)2D3] > vitamin D3. The DBP in poultry does not bind D2 forms effectively, and therefore poultry can not use this form of vitamin D adequately. The concentration of 25-(OH)D3 in blood seems to be well correlated with dietary vitamin D intake or exposure to ultraviolet light. The 1 alpha hydroxylase enzyme in the kidney is subject to negative feedback regulation and is critical for formation of the active metabolite 1,25-(OH)2D3. The intracellular vitamin D receptor (VDR) specifically binds 1,25-(OH)2D3 and is necessary for cellular action. Increased levels of two to three orders of magnitude are required for 25-(OH)D3 to compete with 1,25-(OH)2D3 for binding on VDR. Feeding studies with 25-(OH)D3 suggest it has nearly twice the activity of vitamin D3. Hatchability studies have shown that 25-(OH)D3 supports good fertility and hatchability, whereas hens fed only 1,25-(OH)2D3 did not have normal hatchability. Likewise, 1,25-(OH)2D3 seems to reach toxic levels at dietary concentrations only two to three times optimal dietary levels whereas feeding 25-(OH)D3 for extended periods at levels 8 to 10 times requirement seems to have no adverse effects. It seems that 25-(OH)D3 is the most active metabolite of vitamin D3, ultimately capable of supporting both cellular functions and embryonic development in chickens and turkeys when fed as the sole source of vitamin D3.

The intercept method: a novel method for establishing consistency of M-wave recruitment curves.

The stability of the M-wave is an important component of experimental H-reflex methodology. Despite this importance, there is inconsistency in H-reflex literature on the most valid method of M-wave stability analysis. Further, there is currently no specific method for establishing the stability of an M-wave recruitment curve across various trials within an experiment. Therefore, the aim of this study was to investigate the most appropriate method of M-wave stability analysis for use with the recruitment curve methodology. Twenty-five healthy subjects participated in the study. Four M-wave recruitment curve recordings were made in various static positions that imposed stretch on the posterior structures of the back and leg. Four methods of post-data collection M-wave stability analysis were compared. Although on visual inspection, there was clear evidence of marked alterations to the M-wave recruitment curves between trials in some subject's data, analysis of variance of the Ms/p and Mmax found no significant difference. Evaluation of the percent deviation in Mmax found nine subjects with greater than ten percent deviation in their maximum M-wave across the four trials. The intercept method that utilises analysis of the 95% confidence interval of the intercept of the M-wave recruitment curve slope, excluded eight subjects that demonstrated variation. Comparison of the percent deviation and the intercept method revealed that the intercept method was the most appropriate method for M-wave stability analysis in conjunction with the recruitment curve methodology.

Changes in alpha-motoneuron excitability with positions that tension neural tissue.

The slump test assesses the contribution of neural tissue to the referred symptoms associated with spinal pain and musculo-skeletal injuries of the lower limb. The limitation to full range of movement in performing this test has, in the past, been attributed to a mechanical restriction in mobility of neural tissue. Recent literature suggests that the limitation may be caused by protective reflex muscle action. The purpose of this study was to establish whether the slump test was associated with an increase or a decrease in excitability of alpha-motoneurons and, therefore, an alteration in muscle activity at the end of the range of movement of the test. Forty-three normal subjects and eight subjects with abnormal neural tension participated in this study. Changes in alpha-motoneuron excitability in neck flexion, moderate slump, and maximum slump positions were assessed by observing changes in H-reflex recruitment curves. Linear regression analysis on the rising portion of the H-reflex recruitment curve enabled calculation of the dependent variable Hslp for statistical analysis. Normal subjects in the moderate and maximum slump positions demonstrated a significant decrease (p < 0.05) in the slope of the H-reflex recruitment curve. Subjects with abnormal neural tension showed a non-significant increase in slope when in these positions. Subject flexibility had a significant influence on motoneuron excitability in the moderate neural tension position with inflexible subjects demonstrating a significant inhibition of motoneurons. The difference between the flexible or moderately flexible subjects and inflexible subjects was not significant in the maximum neural tension position. These findings have important implications for the rationale for treatment selection and success of treatment outcomes in the clinical setting.

Associations between road traffic accidents and socio-economic deprivation on Scotland's west coast.

Road traffic accidents (RTAs) are declining, but remain a public health concern locally and world-wide. Scottish RTAs killed 316 people and injured over 20,000 in 1996. By 2020, they are predicted to become the world's third-leading cause of sickness and death. Little is know about associations between RTAs and deprivation; it has never been explored on Scotland's West Coast. This study analysed hospital A&E admissions and investigated associations between RTAs and socio-economic status. 1,300 attendance records at a 575-bed NHS Trust Accident & Emergency in North Lanarkshire were reviewed and 1,020 records analysed in conjunction with Health Board socio-economic data. Findings strongly suggest (p = 0.00461) a positive trend between RTA activity and deprivation. Significance held for gender, victim role, purpose of journey and age, except for drivers 60 and over. Given the preventative nature of RTAs and their contribution to morbidity and mortality, further research between RTAs and deprivation is suggested.

Calcium homeostasis in in ovo and ex ovo turkey embryos.

Calcium homeostasis of in ovo (normal) and ex ovo (shell-less) turkey embryos was investigated at 15, 18, and 21 days of incubation. Hypocalcemia and an elevation in circulating 1,25(OH)2D3 in ex ovo embryos were observed by 15 days. Calcium and phosphorus concentrations in femora and tibiae in ex ovo embryos were significantly lower compared to their normal counterparts. These results suggest that shell calcium mobilization is required prior to 15 days of incubation for maintaining serum calcium and supporting bone mineralization. Furthermore, the elevation of 1,25(OH)2D3 is indicative of a functional calcium homeostatic mechanism responding to the absence of the primary calcium source (eggshell) during the second half of turkey embryonic development.

PITX2 AND PITX1 regulate thyrotroph function and response to hypothyroidism.

Pitx2 is a homeodomain transcription factor required in a dose-dependent manner for the development of multiple organs. Pitx2-null homozygotes (Pitx2(-/-)) have severe pituitary hypoplasia, whereas mice with reduced-function alleles (Pitx2(neo/neo)) exhibit modest hypoplasia and reduction in the developing gonadotroph and Pou1f1 lineages. PITX2 is expressed broadly in Rathke's pouch and the fetal pituitary gland. It predominates in adult thyrotrophs and gonadotrophs, although it is not necessary for gonadotroph function. To test the role of PITX2 in thyrotroph function, we developed thyrotroph-specific cre transgenic mice, Tg(Tshb-cre) with a recombineered Tshb bacterial artificial chromosome that ablates floxed genes in differentiated pituitary thyrotrophs. We used the best Tg(Tshb-Cre) strain to generate thyrotroph-specific Pitx2-deficient offspring, Pitx2(flox/-;)Tg(Tshb-cre). Double immunohistochemistry confirmed Pitx2 deletion. Pitx2(flox/-);Tg(Tshb-cre) mice have a modest weight decrease. The thyroid glands are smaller, although circulating T(4) and TSH levels are in the normal range. The pituitary levels of Pitx1 transcripts are significantly increased, suggesting a compensatory mechanism. Hypothyroidism induced by low-iodine diet and oral propylthiouracil revealed a blunted TSH response in Pitx2(flox/-);Tg(Tshb-cre) mice. Pitx1 transcripts increased significantly in control mice with induced hypothyroidism, but they remained unchanged in Pitx2(flox/-);Tg(Tshb-cre) mice, possibly because Pitx1 levels were already maximally elevated in untreated mutants. These results suggest that PITX2 and PITX1 have overlapping roles in thyrotroph function and response to hypothyroidism. The novel cre transgene that we report will be useful for studying the function of other genes in thyrotrophs.

MR imaging of traumatic spinal injuries.

Plain films form the initial evaluation in all cases of spinal trauma. In cases of indeterminate or incomplete plain radiographs, further evaluation should be performed by multiplanar computed tomography (CT) and/or magnetic resonance imaging (MRI). Rapid triage is important to distinguish surgical and nonsurgical cases, as this has implications in terms of relief of cord compression and long-term prognosis. CT is unparalleled in its capacity to demonstrate bony abnormalities. MRI is the modality of choice in the evaluation of soft tissue injuries, in particular where there is a suspicion of ligamentous or intervertebral disc injury and spinal cord injury. MRI has the ability to distinguish between spinal cord edema and hemorrhage, which has important prognostic significance.

Diverse forms of stress result in changes in cellular levels of osteonectin/SPARC without altering mRNA levels in osteoligament cells.

The osteonectin/SPARC gene has been shown to possess motifs for a heat shock element and metal responsiveness. Also, the expression of the protein has been associated with culture stress in endothelial cells. In the present study, osteoligament (OL) cells derived from the patellar ligament were subjected to diverse forms of stress that included (a) exposure to sodium arsenite, (b) heat shock, (c) cadmium ion, and (d) the amino acid analog, AZC. Osteonectin/SPARC levels in OL cells were determined by Western blot analyses, and immunoprecipitation using antiosteonectin antibodies. Expression of osteonectin/SPARC mRNA was determined by Northern analysis using a 1.5 kb EcoRI restriction fragment of bovine osteonectin cDNA. These studies reveal that osteonectin/SPARC is produced following diverse forms of stress, however, the levels are lower than observed in unchallenged OL cells. In all instances, the mRNA levels were comparable to control cells. These studies indicate that expression of osteonectin/SPARC mRNA is tightly controlled in OL cells and that the protein may be regulated at the level of protein translation.

Structure, expression, and regulation of the major noncollagenous matrix proteins of bone.

The noncollagenous proteins (NCPs) that predominate the bone matrix have recently been the focus of intense investigation because of their potential influence on cell attachment, Ca2+ and hydroxyapatite binding, and the mineralization of bone tissue. With the advent of molecular biology, all of the major NCPs of bone have been cloned and their amino acid sequences completely determined. While each of the proteins has distinct structural properties, some proteins appear to be part of gene families. Examples include the small proteoglycans, decorin and biglycan, as well as the gamma carboxyglutamic acid proteins, such as matrix gla protein and osteocalcin (bone gla protein). Some of the NCPs that are clearly not members of any known gene family still share several common characteristics. One such example of this "convergent evolution" is bone sialoprotein and osteopontin. Both are highly posttranslationally modified glycoproteins that share the cell attachment amino acid sequence RGD (arginine-glycine-aspartic acid), which facilitates the attachment of bone cells in vitro, yet they are clearly not related genetically. Using cDNAs and antisera as probes, the precise temporal localization of NCP expression has been determined, and it has been shown that NCPs are produced in skeletal, and in most cases, nonskeletal tissue as well. This observation implies that the functions of the NCPs are not necessarily limited to bone tissue. Many of the promoters for these genes have been isolated and functional domains determined by a combination of chloramphenicol acetyltransferase assay, gel shift, and footprint analyses. The most extensively studied promoter in the NCP category is osteocalcin, whose sensitivity to 1,25-dihydroxycholecalciferol has been delineated in detail. Future studies on the individual and cooperative activities of the NCPs in bone are likely to involve site-directed mutagenesis of cloned DNA and a combination of in vitro and in vivo functional analyses.

Design, construction and application of a fully automated equimolar peptide mixture synthesizer.

A fully automated peptide synthesizer has been constructed that is capable of the synthesis of equimolar peptide mixtures and the simultaneous synthesis of 36 individual peptides. The synthesizer was constructed from a workstation of our own design utilizing a Zymark robot arm. A Macintosh II computer coordinates the movements of the robotic arm, the switching of over 40 solenoid valves and the monitoring of sensors in the workstation. The robot hands are used to deliver solvents from pressurized spigot lines and to pipet amino acid solutions from reservoirs to an array of reaction vessels. Liquid dispensing, reagent mixing and solvent removal are controlled from a multifunction I/O board in the computer. The design features of the synthesizer are presented, as well as the characterization of multiple individual peptides, a simple mixture of 19 components, and a complex mixture of 15,625 components.