Search PubMed⌕ Search

Biomedical subjects

J Klein

Publications and source records attributed to J Klein.

At least 199 records · Page 11Linked to original sources
PubMed1999

Formation of N-methylnicotinamide in the brain from a dihydropyridine-type prodrug: effect on brain choline.

The enhancement of brain choline levels is a possible therapeutic option in neurodegenerative diseases; however, brain choline levels are held within narrow limits by homeostatic mechanisms including the rapid clearance of excess choline from the brain. The present study tests whether N-methylnicotinamide (NMN), an inhibitor of the outward transport of choline from the brain, can elevate brain choline levels in vivo. As NMN does not cross the blood-brain barrier, we synthesized and administered the brain-permeable prodrug, 1,4-dihydro-N-methyl-nicotinamide (DNMN), and tested its effect on the levels of NMN and choline in brain extracellular fluid, using the microdialysis procedure. Administration of DNMN (1 mmol/kg s.c.) caused a 4- and 9-fold increase in plasma and liver NMN levels, respectively, as determined by HPLC. Concomitantly, the brain tissue levels of NMN were increased by a factor of twenty. In brain extracellular fluid, the injection of DNMN (1-3 mmol/kg s.c.) elevated NMN levels by 3- to 10-fold to maximum levels of >10 microM. In spite of these enhanced NMN levels, the choline concentrations in the brain extracellular fluid and in the cerebrospinal fluid (4.7 microM) remained unchanged or were even slightly decreased. Microsomal incubations of DNMN indicated that cytochrome P-450 3A isoforms may be involved in NMN formation in the liver, but not in the brain. We conclude that DNMN, a brain-permeable prodrug of NMN, is efficiently oxidized to NMN in the brain, but a 10-fold increase in extracellular NMN levels is not sufficient to reduce the clearance of choline from the brain.

Animals↗
PubMed1999

Validation of assays for use with combination vaccines.

A general methodology is presented for the validation of assays used for testing combination vaccines. The presentation is detailed and technical as our intention is to address challenges that we have encountered in the design and statistical analysis of assay validation studies. There are several noteworthy features which render the approach particularly useful in practice. It employs a statistical experimental design approach to the investigation of assay ruggedness with respect to manufacturing variability; it makes use of the assay variability results to determine the level of test-run replication necessary to achieve precision compatible with the product specifications; and, it provides a generic approach to assay validation. With combination vaccines, as with other pharmaceuticals, the analytical methods for release and stability must be validated early in the development programme Several things, though, distinguish this task with combination vaccines: (1) assays are typically pre-existing and often have been validated for use with an established sample matrix, e.g. a monovalent formulation; (2) sample matrices are complex and therefore more subject to manufacturing variability and more likely to cause assay interferences; and (3) the analytical workload is considerable due to the number of antigens. The methodology presented here was developed jointly by Merck Research Laboratories (West Point, PA) and Pasteur Mérieux Connaught, Inc. (Swiftwater, PA). Many of the issues discussed here have application outside of combination vaccines and are common features of all assay validations.

Biological Assay↗
PubMed1999

The dentin matrix protein 1 gene of prototherian and metatherian mammals.

Mineralization of tooth dentin (the deposition of hydroxyapatite crystals in and around collagen type I fibers of the extracellular matrix) requires the involvement of several genes, among them the gene coding for the dentin matrix protein 1, DMP1. We determined the exon-intron organization of the cattle DMP1 gene and used this information to amplify by the polymerase chain reaction homologous gene fragments from the genomic DNA of two species of metatherian (marsupial) mammals and one prototherian (monotreme) species. The translated proto- and metatherian protein sequences are highly divergent from the eutherian sequences but retain the general characteristics of the DMP1 (high acidity, serine-richness, multiple glycosylation sites, and the presence of the RGD cell attachment tripeptide). They therefore appear to be functional even though, evolutionarily, teeth are in a regression phase in prototherians. It is possible, therefore, that DMP1 is also involved in other functions besides dentinogenesis. The DMP1 gene appears to evolve rapidly and apparently tolerates non-frame-shifting insertions/deletions throughout the coding sequence.

Amino Acid Sequence↗
PubMed1999

Evolution of Mhc-DRB introns: implications for the origin of primates.

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders-Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera.

Animals↗
PubMed1999

Bilobalide, a constituent of Ginkgo biloba, inhibits NMDA-induced phospholipase A2 activation and phospholipid breakdown in rat hippocampus.

In rat hippocampal slices superfused with magnesium-free buffer, glutamate (1 mM) caused the release of large amounts of choline due to phospholipid breakdown. This phenomenon was mimicked by N-methyl-D-aspartate (NMDA) in a calcium-sensitive manner and was blocked by NMDA receptor antagonists such as MK-801 and 7-chlorokynurenate. The NMDA-induced release of choline was not caused by activation of phospholipase D but was mediated by phospholipase A2 (PLA2) activation as the release of choline was accompanied by the formation of lyso-phosphatidylcholine (lyso-PC) and glycerophospho-choline (GPCh) and was blocked by 5-[2-(2-carboxyethyl)-4-dodecanoyl-3,5-dimethylpyrrol-1-yl]pentano ic acid, a PLA2 inhibitor. Bilobalide, a constituent of Ginkgo biloba, inhibited the NMDA-induced efflux of choline with an IC50 value of 2.3 microM and also prevented the formation of lyso-PC and GPCh. NMDA also caused a release of choline in vivo when infused into the hippocampus of freely moving rats by retrograde dialysis. Again, the effect was completely inhibited by bilobalide which was administered systemically (20 mg/kg i.p.). Interestingly, convulsions which were observed in the NMDA-treated rats were almost totally suppressed by bilobalide. We conclude that release of choline is a sensitive marker for NMDA-induced phospholipase A2 activation and phospholipid breakdown. Bilobalide inhibited the glutamatergic excitotoxic membrane breakdown both in vitro and in vivo, an effect which may be beneficial in the treatment of brain hypoxia and/or neuronal hyperactivity.

Animals↗
PubMed1999

Bioassays for risk assessment of coal conversion products.

Traditional as well as biotechnological processing of coal leads to complex mixtures of products. Besides chemical and physical characterization, which provides the information for product application, there is a need for bioassays to monitor properties that are probably toxic, mutagenic or cancerogenic. Investigations carried out focused on the selection, adaptation and validation of bioassays for the sensitive estimation of toxic effects. Organisms like bacteria, Daphnia magna and Scenedesmus subspicatus, representing different complexities in the biosphere, were selected as test systems for ecotoxicological and mutagenicity studies. The results obtained indicate that bioassays are, in principle, suitable tools for characterization and evaluation of coal-derived substances and bioconversion products. Using coal products, coal-relevant model compounds and bioconversion products, data for risk assessment are presented.

Animals↗
PubMed1999

Mapping and genomic characterization of the gene encoding diacylglycerol kinase gamma (DAGK3): assessment of its role in dominant optic atrophy (OPA1).

The family of diacylglycerol kinases (DAGKs) is known to play an important role in signal transduction linked to phospholipid turnover. In the fruitfly Drosophila melanogaster, a human DAGK ortholog, DGK2, was shown to underlie the phenotype of the visual mutant retinal degeneration A (rdgA). Previously, the gene encoding a novel member of the human DAGK family, termed DAGK3, was cloned and demonstrated to be abundantly expressed in the human retina. Based on these findings we reasoned that DAGK3 might be an excellent candidate gene for a human eye disease. In the present study, we report the genomic organization of the human DAGK3 gene, which spans over 30 kb of genomic DNA interrupted by 23 introns. In addition, we have mapped the gene locus by fluorescence in situ hybridization to 3q27-28, overlapping the chromosomal region known to contain the gene underlying dominant optic atrophy (OPA1), the most common form of hereditary atrophy of the optic nerve. Mutational analysis of the entire coding region of DAGK3 in 19 unrelated German OPA1 patients has not revealed any disease-causing mutations, therefore excluding DAGK3 as a major cause underlying OPA1.

Adolescent↗
PubMed1999

Cyclophosphamide and paclitaxel as initial or salvage regimen for the mobilization of peripheral blood progenitor cells.

Peripheral blood progenitor cells are now commonly used for hematologic reconstitution after myelosuppressive chemotherapy for hematologic and solid malignancies. The purpose of this study was to evaluate the activity of paclitaxel 170 mg/m2 and cyclophosphamide 2 g/m2 (CP) with filgrastim (human G-CSF) for mobilization of PBPCs as the first or second maneuver after failure with filgrastim alone. Sixty-four patients with stage II-IV breast cancer received (CP) followed by filgrastim (10 microg/kg/day). In 35 (55%) this was the first maneuver while it was for salvage in 29 (45%) patients. The median number of aphereses was two (range, 1-7). In 83% of the patients apheresis was initiated on days 10-11 following chemotherapy. The median numbers of CD34+ cells/kg, CD34+ cells/apheresis/kg and total nucleated cells/kg collected were 8.7 x 10(6) (2.11-73.5), 3.97 x 10(6) (0.3-36.75) and 164.15 x 10(8) (9-660), respectively. All the patients yielded at least 2 x 10(6) CD34+ cells/kg. CP mobilization salvaged the 29 patients who failed mobilization with filgrastim alone. When used as first-line mobilization the yield of CD34+ cells x 10(6)/kg was higher than in the salvage group (16.93 vs 3.94, P < 0.001). Patients receiving CP as salvage reached the target of 5 x 10(6) CD34+ cells/kg in only 45% (13/29) of cases vs 94.3% as first maneuver. CP followed by filgrastim is a safe and effective regimen for the mobilization of PBPCs in patients with breast cancer and shows significant activity in patients who failed to mobilize with filgrastim, suggesting a higher mobilization potential.

Adult↗
PubMed1999

cDNA sequence coding for the alpha'-chain of the third complement component in the African lungfish.

cDNA clones coding for almost the entire C3 alpha-chain of the African lungfish (Protopterus aethiopicus), a representative of the Sarcopterygii (lobe-finned fishes), were sequenced and characterized. From the sequence it is deduced that the lungfish C3 molecule is probably a disulphide-bonded alpha:beta dimer similar to that of the C3 components of other jawed vertebrates. The deduced sequence contains conserved sites presumably recognized by proteolytic enzymes (e.g. factor I) involved in the activation and inactivation of the component. It also contains the conserved thioester region and the putative site for binding properdin. However, the site for the interaction with complement receptor 2 and factor H are poorly conserved. Either complement receptor 2 and factor H are not present in the lungfish or they bind to different residues at the same or a different site than mammalian complement receptor 2 and factor H. The C3 alpha-chain sequences faithfully reflect the phylogenetic relationships among vertebrate classes and can therefore be used to help to resolve the long-standing controversy concerning the origin of the tetrapods.

Amino Acid Sequence↗
PubMed1999

Contralateral trigeminal nerve lesion reduces polyneuronal muscle innervation after facial nerve repair in rats.

Functional recovery after facial nerve surgery is poor. Axotomized motoneurons (hyperexcitable upon intracellular current injections, but unable to discharge upon afferent stimulation) outgrow supernumerary branches which are misrouted towards improper muscles. We hypothesized that alterations in the trigeminal input to axotomized electrophysiologically silent facial motoneurons might improve specificity of reinnervation. To test this we compared, in the rat, behavioural, electrophysiological, and morphological parameters after transection and suture of the buccal facial nerve (buccal-buccal anastomosis, BBA) with those after BBA plus excision of the ipsi- or contralateral infraorbital nerve (ION). After BBA, the mystacial vibrissae dropped and remained motionless until 18-21 days post operation (days PO). After BBA plus ipsilateral ION excision, there was no recovery of vibrissae whisking at all. Following BBA plus contralateral ION excision, full restoration of whisking occurred at 7-10 days PO. Electromyography of whiskerpad muscles showed normal waveform and amplitude was also most rapidly restored after BBA plus contralateral ION excision. Neuron counts after retrograde tracing showed that the intact buccal nerve contained axons of the superior (91%) and inferior (9%) buccolabial nerves. After BBA, the superior nerve comprised 56%, the inferior 21%, and 23% of the motoneurons projected within both nerves. After BBA plus ipsilateral ION excision, misdirection worsened and values changed to 48, 39 and 13%, respectively. After BBA plus contralateral ION excision, portions improved to 69, 23 and 8%. We conclude that, by reducing the redundant axon branching, lesion of contralateral ION provides the best conditions for recovery of vibrissae rhythmical whisking after reconstructive surgery on the facial nerve.

Anastomosis, Surgical↗
PubMed1999

Ethanol inhibits astroglial cell proliferation by disruption of phospholipase D-mediated signaling.

The activation of phospholipase D (PLD) is a common response to mitogenic stimuli in various cell types. As PLD-mediated signaling is known to be disrupted in the presence of ethanol, we tested whether PLD is involved in the ethanol-induced inhibition of cell proliferation in rat cortical primary astrocytes. Readdition of fetal calf serum (FCS) to serum-deprived astroglial cultures caused a rapid, threefold increase of PLD activity and a strong mitogenic response; both effects were dependent on tyrosine kinases but not on protein kinase C. Ethanol (0.1-2%) suppressed the FCS-induced, PLD-mediated formation of phosphatidic acid (PA) as well as astroglial cell proliferation in a concentration-dependent manner. Moreover, exogenous bacterial PLD increased astroglial proliferation in an ethanol-sensitive manner, whereas exogenous PA or lysophosphatidic acid was less effective. Formation of PA and astroglial proliferation were strongly inhibited by 1-butanol (0.1-1%), a substrate of PLD, but were unaffected by t-butanol, a non-substrate; 2-butanol had intermediate effects. Platelet-derived growth factor and endothelin-1 mimicked the mitogenic effect of FCS; their effects were also inhibited by the butanols in the potency order 1-butanol > 2-butanol > tert-butanol. Our results, in particular, the differential effects of 1-, 2-, and tert-butanol with respect to PA formation and astroglial proliferation, strongly suggest that the antiproliferative effects of ethanol in glial cells are due to the disruption of the PLD signaling pathway. This mechanism may also contribute to the inhibition of astroglial growth and brain development observed in alcoholic embryopathy.

1-Butanol↗
PubMed1999

Origin of gene overlap: the case of TCP1 and ACAT2.

The human acetyl-CoA acetyltransferase 2 gene, ACAT2, codes for a thiolase, an enzyme involved in lipid metabolism. The human T-complex protein 1 gene, TCP1, encodes a molecular chaperone of the chaperonin family. The two genes overlap by their 3'-untranslated regions, their coding sequences being located on opposite DNA strands in a tail-to-tail orientation. To find out how the overlap might have arisen in evolution, the homologous genes of the zebrafish, the African toad, caiman, platypus, opossum, and wallaby were identified. In each species, standard or long polymerase chain reactions were used to determine whether the ACAT2 and TCP1 homologs are closely linked and, if so, whether they overlap. The results reveal that the overlap apparently arose during the transition from therapsid reptiles to mammals and has been retained for >200 million years. Part of the overlapping untranslated region shows remarkable sequence conservation. The overlap presumably arose during the chromosomal rearrangement that brought the two unrelated and previously separated genes together. One or both of the transposed genes found by chance signals that are necessary for the processing of their transcripts to be present on the noncoding strand of the partner gene.

Acetyl-CoA C-Acetyltransferase↗
PubMed1999

Fatty acid ethyl esters: a novel biologic marker for heavy in utero ethanol exposure: a case report.

The authors report testing the meconium of a newborn for the presence of FAEE. Meconium from a newborn of a woman who acknowledged drinking beer throughout pregnancy was tested. The authors also tested the meconiums of 3 newborns whose mothers did not drink at all while pregnant. The FAEE were extracted from the meconium samples using solid phase extraction (SPE), and were identified and quantitated by gas chromatography with flame ionization detection (FID). For assignment of retention times and determination of individual concentrations, authentic mixtures of FAEE were injected. The total FAEE concentration in the meconium of the alcohol-exposed infant was 13126 ng/g compared to a mean of 410 ng/g in the control meconiums. Also, in this case, palmitic, linoleic, and stearic ethyl esters were found in the alcohol-exposed infant's meconium while they were not found in the unexposed infant's meconium. In a parallel experiment, the authors spiked increasing amounts of ethyl alcohol (0-40mM) into the meconium from a newborn that was not exposed to ethanol in utero. The spiked samples were incubated for 4 hours at 37 degrees C and subsequently assayed for the presence of ethyl linoleate. In these experiments, they document for the first time that FAEE is produced in meconium. If confirmed by large studies, FAEE may become the first neonatal biologic marker for babies at risk for alcohol-related birth defects.

Alcohol Drinking↗
PubMed1999

Multivariate predictive models for group A beta-hemolytic streptococcal pharyngitis in children.

OBJECTIVES: To create predictive models for the clinical diagnosis of group A beta-hemolytic streptococcal (GABHS) pharyngitis in children. METHODS: Patients aged 6 months to 18 years presenting to a pediatric ED with suspected GABHS pharyngitis were prospectively enrolled in the study. Clinicians recorded pertinent clinical information using a standardized form and obtained a throat swab to culture GABHS using a reference standard method. Twelve demographic and clinical features of patients with positive throat cultures were compared with the features of patients with negative throat cultures. Significantly different features were entered in a stepwise logistic regression analysis to create predictive models for the diagnosis. RESULTS: Eighty-five patients (29%) were culture-positive and 212 (71%) were culture-negative for GABHS. Respective mean ages were 6.2 years and 6.1 years in the two groups. Univariate chi-square analysis of the 12 features identified six variables that were significantly associated with GABHS. All significant features were initially included in a stepwise logistic regression analysis. In model I, four independent variables were identified: moderate to severe presentation of tonsillar swelling, moderate to severe tenderness and enlargement of cervical lymph nodes, the presence of scarlatiniform rash, and the absence of moderate to severe coryza, yielding a 95% probability for GABHS. Excluding the rare scarlatiniform rash, the remaining variables were used in the second regression analysis. In model II, three independent variables were identified: moderate to severe tonsillar swelling, moderate to severe tenderness and enlargement of cervical lymph nodes, and absence of moderate to severe coryza, yielding a probability of 65% for the diagnosis. A probability of <15% was observed in the absence of scarlatiniform rash, the absence of moderate to severe tenderness and enlargement of cervical lymph nodes, and the presence of moderate to severe coryza. CONCLUSIONS: In children with moderate to severe presentation of tonsillar swelling, tenderness and enlargement of cervical lymph nodes, and the absence of coryza, the probability of a positive throat culture is >65%. Conversely, in the absence of a moderate to severe presentation of tonsillar swelling, enlargement of cervical nodes, and the presence of coryza, the probability of a positive throat culture is <15%. If prospectively validated, these models could be integrated into a consistent treat, test, and no treatment/no testing approach to the clinical management of childhood pharyngitis.

Adolescent↗