Assessing Passeriformes health in South Texas via select venous analytes.

The handheld point of care analyzer is a quick and feasible option to obtain hematology data from individuals. The iSTAT-1® was used to evaluate select venous blood analytes obtained via jugular venipuncture from 238 passerine birds from South Texas. These data were used to assess the health of birds in the area while taking into consideration life history (migratory or sedentary), locale, seasonality, sex, and age. We attributed increased values of pO2 and hematocrit, in addition to hemoglobin and glucose concentrations of migratory birds compared to sedentary birds as the increased need of oxygen carrying capacity and energy for long duration flights. Increased glucose and lower ionized calcium concentrations were observed in migratory birds likely based on breakdown of fat deposits in the body to fuel the muscular endurance of migration. During the hotter months of the year, birds' responses to handling were exhibited by relative respiratory acidosis. When sedentary birds sampled from South Texas were compared to a previous study from Central Texas, venous blood analytes differed by locale but were within the ranges of healthy populations. These findings lead us to conclude that sedentary avian communities can be used as ecosystem bioindicators.

Association of a null allele of SPRN with variant Creutzfeldt-Jakob disease.

BACKGROUND: No susceptibility genes have been identified in human prion disase, apart from the prion protein gene (PRNP). The gene SPRN, encodes Shadoo (Sho, shadow of prion protein) which has protein homology and possible functional links with the prion protein. METHODS: A genetic screen was carried out of the open reading frame of SPRN by direct sequencing in 522 patients with prion disease, including 107 with variant Creutzfeldt-Jakob disease (vCJD), and 861 healthy controls. RESULTS: A common coding variant of SPRN, two further single nucleotide polymorphisms (SNPs) and three rare insertion or deletion variants were found. A single base-pair insertion at codon 46, predicted to cause a frameshift and potentially a novel protein, was found in two patients with vCJD but not in controls (p = 0.01). Two linked SNPs, one in intron 1 and the other a missense variant at codon 7, were associated with risk of sporadic CJD (p = 0.009). CONCLUSION: These data justify the functional genetic characterisation of SPRN and support the involvement of Shadoo in prion pathobiology.

Inherited prion disease with 5-OPRI: phenotype modification by repeat length and codon 129.

BACKGROUND: Human prion diseases have sporadic, acquired and inherited etiologies and show considerable phenotypic heterogeneity. An individual inherited prion disease offers an opportunity to study the determinants of this clinicopathologic heterogeneity among individuals with the same causal mutation. METHODS: We report clinical and pathologic data from three families with different 5-octapeptide repeat insertion (5-OPRI) mutations of the prion protein gene (PRNP), extending the reported phenotypic range of this mutation. RESULTS: The proband of a South African family presented with a rapidly progressive dementia and atypical pathology associated with kuru-like prion protein plaques. The original mutation in this family probably occurred on a PRNP allele encoding a 1-octapeptide repeat deletion polymorphism. This has not been previously reported as a precursor allele in over 30 other OPRI mutation kindreds. An English family with a genetically distinct mutation but identical protein product showed clinical onsets that varied 30 years between father and daughter, an effect that may be explained by their genotypes at PRNP codon 129. A patient from Northern Ireland with a phenotype of sporadic Creutzfeldt-Jakob disease presenting with visual disturbance was unexpectedly found to have a 5-OPRI. CONCLUSIONS: When these cases were combined with the existing world literature, the mean age at onset for patients with 5-octapeptide repeat insertion (5-OPRI) was significantly later than that for patients with 6-OPRI, but both mutations exhibit a similar powerful disease modifying effect of PRNP codon 129.

Early onset familial Alzheimer's disease: Mutation frequency in 31 families.

BACKGROUND: Three causative genes have been identified for autosomal dominant AD. OBJECTIVE: To determine the proportion of patients with early onset AD with a positive family history accounted for by mutations in these genes. METHODS: A mutational analysis of the amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) genes was performed in 31 probands with probable or definite AD from UK families with an age at onset (AAO) <61 years. RESULTS: The mean AAO was 46.9 years (median 45 years; range 33 to 60 years). The majority of patients (23 of 31; 74%) fulfilled recognized criteria for autosomal dominant inheritance. In 17 (55%) probands the authors identified eight novel PSEN1 sequence variants and eight recognized pathogenic mutations. In 4 (13%) probands the authors identified one novel APP sequence variant (H677R) and two recognized mutations. Thus in this series 21 of 31 (68%) probands were associated with a sequence variant in APP or PSEN1. Nine of the 11 (82%) probands with neuropathologically confirmed AD who additionally fulfilled recognized criteria for autosomal dominant inheritance were associated with a sequence variant in APP or PSEN1. The 10 patients in whom the authors were unable to identify a mutation in APP, PSEN1, or PSEN2 were older than the probands with sequence variants (55.4 vs 44.7 years: p = 0.001). CONCLUSIONS: Sequence variants in APP and PSEN1 accounted for the majority of neuropathologically confirmed autosomal dominant early onset AD; no mutations in PSEN2 were detected. There may be a further genetic factor involved in the etiology of autosomal dominant early onset AD.

Oomycete plant pathogens use electric fields to target roots.

Plant roots generate electrical currents and associated electrical fields as a consequence of electrogenic ion transport at the root surface. Here we demonstrate that the attraction of swimming zoospores of oomycete plant pathogens to plant roots is mediated in part by electrotaxis in natural root-generated electric fields. The zones of accumulation of anode- or cathode-seeking zoospores adjacent to intact and wounded root surfaces correlated with their in vitro electrotactic behavior. Manipulation of the root electrical field was reflected in changes in the pattern of zoospore accumulation and imposed focal electrical fields were capable of overriding endogenous signals at the root surface. The overall pattern of zoospore accumulation around roots was not affected by the presence of amino acids at concentrations expected within the rhizosphere, although higher concentrations induced encystment and reduced root targeting. The data suggest that electrical signals can augment or override chemical ones in mediating short-range tactic responses of oomycete zoospores at root surfaces.

Development of a time-resolved immunofluorometric assay for quantitation of mucosal and systemic antibody responses.

We developed a solid phase immunoassay that measured mucosal and systemic antibody responses from mice inoculated with either a staphylococcal enterotoxin B vaccine (SEBv) or noninfectious virus-like particles (VLP) of lentiviral origin. The assay used time-resolved fluorescence (TRF) with affinity-purified goat anti-mouse IgA and IgG conjugated to samarium and europium chelates, respectively. By employing these fluorogenic conjugates with different spectral emissions, IgA and IgG specific for SEB or VLP were readily detected in serum and saliva from mice inoculated intranasally. The TRF assay detected antigen-specific IgA in saliva 10 min after the addition of enhancement solution, while a conventional alkaline phosphatase-based assay for salivary IgA required 18 h after substrate addition. The TRF assay also provided a significantly higher signal-to-noise ratio and exhibited greater sensitivity. TRF assays detected both IgA and IgG in the same well, thereby reducing sample and reagent requirements.

Two-octapeptide repeat deletion of prion protein associated with rapidly progressive dementia.

Insertions of integral numbers of an octapeptide repeat in the prion protein gene are pathogenic mutations associated with inherited prion diseases. Conversely, deletions of a single octapeptide repeat are found as normal polymorphisms in many populations and do not predispose individuals to prion disease. The authors report a two-octapeptide repeat deletion in an elderly woman with a rapidly progressive dementia consistent with Creutzfeldt-Jakob disease. This mutation was absent from more than 3,000 individuals and may be causally related to prion disease and represent a novel disease mechanism.

Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis distinct from other forms of human prion disease: disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and other peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrP(Sc) to investigate the distribution of PrP(Sc) in a range of vCJD tissues. METHODS: We obtained tissues at necropsy from four patients with neuropathologically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrP(Sc) and western blotting using high sensitivity enhanced chemiluminescence. FINDINGS: We could reliably detect PrP(Sc) in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. PrP(Sc) could be detected in tissue homogenates when present at concentrations 10(4)-10(5) fold lower than those reported in brain. Tonsil, spleen, and lymph node were uniformly positive for PrP(Sc) at concentrations in the range of 0.1-15% of those found in brain: the highest concentrations were consistently seen in tonsil. PrP(Sc) was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2.5 and 25%, respectively of those found in brain. Other peripheral tissues studied were negative for PrP(Sc) with the exception of low concentrations in rectum, adrenal gland, and thymus from a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction) were negative for PrP(Sc) at this level of assay sensitivity. INTERPRETATION: We have developed a highly sensitive immunoblot method for detection of PrP(Sc) in vCJD tissues that can be used to provide an upper limit on PrP(Sc) concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should be further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment of the eye, in particular the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for population screening of surgical tissues to assess prevalence of preclinical vCJD infection within the UK and other populations.

Autopsy-confirmed familial early-onset Alzheimer disease caused by the l153V presenilin 1 mutation.

BACKGROUND: Three affected individuals are described from a small English kindred with early-onset autosomal dominant familial Alzheimer disease (FAD) caused by a leucine-to-valine change at codon 153 (L153V) of the presenilin 1 (PSEN1) gene. METHODS: Clinical information on the pedigree was collected directly from family members and from hospital records. Samples of DNA were screened by means of direct sequencing of all coding exons of PSEN1. One patient underwent neuropathological examination. RESULTS: Mean age at onset of symptoms was 35.3 years (95% confidence interval [CI], 34.6-36.0 years); at death, 44.0 years (95% CI, 39.1-48.9 years). Mean duration of illness was 8.3 years (95% CI, 4.7-11.9 years). Myoclonus was a late feature in 1 patient; seizures were not reported in any subjects. Spastic paraparesis and extrapyramidal signs were absent. The neuropsychometric profile of 1 patient showed relatively preserved naming skills in the setting of global cognitive deficits. Results of neuropathological examination demonstrated the signature lesions of Alzheimer disease and the presence of occasional cortical Lewy bodies. CONCLUSIONS: The PSEN1 L153V mutation lies in the main mutation cluster of PSEN1 in the second transmembrane domain. It causes early-onset FAD with clinical features similar to those of other reported FAD pedigrees.

Pediatric toxicologic deaths: a 10-year retrospective study.

A 10-year retrospective study of pediatric toxicologic deaths was performed at the Medical University of South Carolina (Charleston, SC) from January 1989 to December 1998. During this time, 709 pediatric forensic autopsies were performed on children younger than 18 years of age. Eleven deaths were determined to be secondary to toxic exposures (excluding carbon monoxide poisonings secondary to fires). The remaining deaths were reviewed for the presence of alcohol or illicit drugs. The 11 toxicologic deaths were analyzed for age, sex, race, type of toxic exposure, cause and manner of death, location of incident, witness, and, in the younger age group, the primary caregiver at the time of exposure. The deaths had a bimodal age distribution (6 deaths in victims ages 15 to 17 and 5 deaths in victims ages 4 or younger), involving a wide range of toxins. The teenage group was composed of five males and one female, all white. The preschool group had three females and three males, all black. The manner of death ranged from accidental to suicidal to homicidal. In addition, in eight neonatal and fetal deaths, the victims tested positive for maternal cocaine use, and five of these victims tested positive for cocaine or benzoylecgonine. However, the cause of death was not stated to be cocaine in any of these neonatal and fetal cases.

Effect of metabolic acidosis on white-tailed deer antler development.

Metabolic acidosis can result when herbivores consume browse diets high in plant secondary compounds. One mechanism for buffering excess acid is the mobilization of calcium and other alkaline salts from the skeletal system. White-tailed deer (Odocoileus virginianus) and other cervids consuming browse during antler formation may use minerals essential for antler development as buffers, resulting in altered antler characteristics. Our research objectives were to examine the effects of metabolic acidosis on mineral metabolism, acid-base homeostasis, and antler development in white-tailed deer. Fifteen male white-tailed deer were assigned to one of three diets: 2% NH(4)Cl, 3% commercial tannic acid, or a basal ration without additive. Two feeding trials were completed on each deer to determine nutrient use. Urine pH and the percentage of urinary nitrogen excreted as NH+4 varied by diet. No significant diet or trial effects occurred for nitrogen, calcium, phosphorus, magnesium, or sodium use. Urinary calcium excretion varied between diets. No dietary differences were observed for antler characteristics. The NH(4)Cl diet induced metabolic acidosis but did not alter antler development in white-tailed deer. Skeletal mineral reserves and mineral intake appeared sufficient to buffer excess acids and support antler development.

Photoreaction potential of orally administered levofloxacin in healthy subjects.

OBJECTIVE: To evaluate the photoreaction potential of levofloxacin on exposure to solar-simulating radiation. Solar-simulating is ultraviolet (UV) light, defined as UVA in the 320-400 nm range and UVB in the 290-320 nm range. DESIGN: In a single-center, double-blind, randomized study, 30 adults (20 men, 10 women) received oral levofloxacin (500 mg qd x 5 d) or placebo. At baseline photoexposure prior to drug administration, each subject was exposed to UVB light at 0.75, 1.0, and 2.0 times the minimal erythema dose and to UVA light (25 J/cm2). Photoexposure was repeated on day 5, two hours following final drug administration, and response was determined using both a photoreaction rating scale and investigator assessment. RESULTS: Using the photoreaction rating scale, following UVB exposure on day 5, no abnormal photoreactions were observed among levofloxacin recipients. UVA exposure was associated with mild reactions in 20 of 24 levofloxacin-treated and three of six placebo-treated subjects, with no associated symptoms. By investigator assessment, all subjects had a negative reaction to UVB photoexposure, and 10 of 24 levofloxacin-treated and three of six placebo-treated subjects had a photoreaction following UVA photoexposure. Dermal reactions were mild and similar for both treatment groups. No subject experienced an immediate wheal-and-flare reaction. There were no statistically significant differences between treatment groups for any of the comparisons. CONCLUSIONS: Levofloxacin has a low photosensitizing potential when administered to healthy subjects.

Selectively increased growth of fetal hemoglobin-expressing adult erythroid progenitors after brief treatment of early progenitors with transforming growth factor beta.

We have studied the effect of transforming growth factor beta (TGFbeta) on erythropoiesis in cultures from adult peripheral blood, using flow cytometric enumeration of fetal hemoglobin (HbF)-containing cells. TGFbeta caused a dramatic increase in the proportions of cells that accumulated HbF together with adult hemoglobin (HbA) (F+A+ cells). This highly significant (P <.0001) increase in F+ cell proportion was achieved by TGFbeta treatment during the first 4 days of culture and was sustained during further culture expansion in the absence of TGFbeta. The increase in F+ cell proportions did not depend on the cytokine combination (EPO+SCF+IL3, EPO+SCF, EPO+IL3, SCF+IL3) used during the phase of TGFbeta treatment. Increased F+ cell proportions were paralleled by an increased molecular ratio of HbF/ HbF+ HbA, measured by cation exchange high-performance liquid chromatography (HPLC). In addition to the effect on F+ cell proportions, TGFbeta caused a dramatic increase in overall cell division potential. By the time cultures reached terminal growth arrest (12-14 days in controls and 18-26 days after TGFbeta), the overall numbers of F+ cells produced per initially seeded clonogenic cell was approximately 10 times higher in the TGFbeta-treated cultures than in the controls. We propose to investigate whether the TGFbeta-induced increase in relative and absolute numbers of nucleated F+ cells, as demonstrated in vitro, can be translated into increased F+ erythrocytes in vivo, allowing therapeutic application for some beta-hemoglobinopathies. (Blood. 2000;95:2967-2974)

Inherited prion disease with an alanine to valine mutation at codon 117 in the prion protein gene.

A large English family with autosomal dominant segregation of presenile dementia, ataxia and other neuropsychiatric features is described. Diagnoses of demyelinating disease, Alzheimer's disease, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome have been attributed to particular individuals at different times. An Irish family, likely to be part of the same kindred, is also described, in which diagnoses of multiple sclerosis, dementia, corticobasal degeneration and new variant CJD have been considered in affected individuals. Molecular genetic studies have enabled the classification of this disease at the molecular level as one of the group of inherited prion diseases, with the substitution of valine for alanine at codon 117 of the prion protein gene (PRNP). Only three other kindreds have been described world-wide with this mutation and only limited phenotypic information has been reported. Here we describe the phenotypic spectrum of inherited prion disease (PrPA117V). The diversity of phenotypic expression seen in this kindred emphasizes the logic of molecular classification of the inherited prion diseases rather than classification by specific clinicopathological syndrome. Indeed, inherited prion disease should be excluded by PRNP analysis in any individual presenting with atypical presenile dementia or neuropsychiatric features and ataxia, including suspected cases of new variant CJD.

Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora.

Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or beta-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora.

Detection of hemoglobin variants in erythrocytes by flow cytometry.

BACKGROUND: With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedures to monitor the effect of these agents on Hb F levels in individuals will be needed. We developed a rapid procedure that detects fetal hemoglobin in erythrocytes (F cells) using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against Hb F. METHODS: Ten microliters of washed blood was fixed in formaldehyde and glutaraldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cytometry to determine the percentage of F cells. RESULTS: Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by high pressure liquid chromatography (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previously-described method that uses dimethyl 3,3'dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r = 0.96, r2 = 0.93, P<0.001) was observed between the two protocols. CONCLUSIONS: This procedure is easy, reproducible, and gives accurate F cell results. It can be used to measure a wide range of F cell percentages and may also be used to dual-stain Hb F along with other hemoglobin variants and erythrocyte surface antigens.

Pathogenic presenilin 1 mutations (P436S & I143F) in early-onset Alzheimer's disease in the UK. Mutations in brief no. 223. Online.

Familial Alzheimer's disease (AD) is an autosomal dominant disorder characterized by memory impairment and multiple cognitive deficits which occurs in mid to late life. Early onset AD has been associated with mutations in three genes, of which presenilin 1 (PS1) mutations are the most frequent. We sequenced the open reading frame from genomic DNA of a series of 21 early onset AD (AD3) UK families in which there were at least two affected individuals in two or more generations with a diagnosis of probable or definite AD. We found PS1 mutations in six of these families with no sequence variation in the remaining 15. The six families contained between them five different mutations of which two, I143F and P436S, have not been found elsewhere. I143F shows incomplete penetration within the affected family. P436S is the most carboxy-terminal presenilin 1 mutation reported to date.

Quantification of axonal damage in traumatic brain injury: affinity purification and characterization of cerebrospinal fluid tau proteins.

Diffuse axonal injury is a primary feature of head trauma and is one of the most frequent causes of mortality and morbidity. Diffuse axonal injury is microscopic in nature and difficult or impossible to detect with imaging techniques. The objective of the present study was to determine whether axonal injury in head trauma patients could be quantified by measuring levels of CSF tau proteins. Tau proteins are structural microtubule binding proteins primarily localized in the axonal compartment of neurons. Monoclonal antibodies recognizing the form of tau found in the CSF of head trauma patients were developed by differential CSF hybridoma screening using CSF from head trauma and control patients. Clones positive for head trauma CSF tau proteins were used to characterize this form of tau and for ELISA development. Using the developed ELISA, CSF tau levels were elevated >1,000-fold in head trauma patients (mean, 1,519 ng/ml of CSF) when compared with patients with multiple sclerosis (mean, 0.014 ng/ml of CSF; p < 0.001), normal pressure hydrocephalus (nondetectable CSF tau), neurologic controls (mean, 0.031 ng/ml of CSF; p < 0.001), or nonneurologic controls (nondetectable CSF tau; p < 0.001). In head trauma, a relationship between clinical improvement and decreased CSF tau levels was observed. These data suggest that CSF tau levels may prove a clinically useful assay for quantifying the axonal injury associated with head trauma and monitoring efficacy of neuroprotective agents. Affinity purification of CSF tau from head trauma patients indicated a uniform cleavage of approximately 18 kDa from all six tau isoforms, reducing their apparent molecular sizes to 30-50 kDa. These cleaved forms of CSF tau consisted of the interior portion of the tau sequence, including the microtubule binding domain, as judged by cyanogen bromide digestion. Consistent with these data, CSF cleaved tau bound taxol-polymerized microtubules, indicating a functionally intact microtubule binding domain. Furthermore, epitope mapping studies suggested that CSF cleaved tau proteins consist of the interior portion of the tau sequence with cleavage at both N and C terminals.