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Biomedical subjects

D Fischer

Publications and source records attributed to D Fischer.

At least 91 records · Page 5Linked to original sources
PubMed1999

Safety and immunogenicity of an acellular pertussis vaccine in premature infants.

OBJECTIVES: To evaluate the safety and immunogenicity of a two-component acellular pertussis vaccine in preterm infants. STUDY DESIGN: Fifty preterm infants (25-35 weeks of gestation; mean, 30.8 weeks) and 50 term infants as a control group received a two-component acellular pertussis vaccine irrespective of their biological age and actual weight. Adverse reactions were registered by parents on a diary card and reviewed on each visit. Antibodies against pertussis toxoid (PT) and filamentous hemagglutinin (FHA) were determined with an enzyme-linked immunosorbent assay before the first and after the third vaccination. RESULTS: The infants of both groups showed an increase in geometric mean titers (GMT) against PT and FHA after vaccination (3 doses). There was a significant difference of antibody concentration between the preterm and the control group. The GMT for PT antibody of the preterm infants was 64. 16 U/L, and for the term infants it was 98.96 U/L. The GMT for FHA was 50.92 U/L in preterm versus 86.02 U/L in the control group. Efficacy of the immunization (more than a fourfold increase of antibody concentration in each infant) was 93.5% in the preterm group with respect to PT and 82.6% with respect to FHA. The incidence of adverse reactions was low and comparable in both study groups. CONCLUSION: Immunization with an acellular pertussis vaccine is safe for preterm infants. The immune response is significantly lower compared with a control group of term infants, but efficacy is high.

Antibodies, Viral↗
PubMed1999

Modeling three-dimensional protein structures for amino acid sequences of the CASP3 experiment using sequence-derived predictions.

Homology or comparative modeling is aimed at modeling the three-dimensional structure of a target sequence of unknown structure using the framework of an already known fold. Traditionally, homology modeling has been applied to targets with clear sequence similarity to proteins of known structure. Because methods to identify increasingly distant relationships have been developed, homology models can now be built for a wider range of targets. The first challenge in homology modeling is to obtain an initial, accurate, sequence-structure alignment with the most compatible fold. In CASP3, the abilities of fold-recognition methods to fulfill this challenge were evaluated with a number of target sequences of unknown structure. Sequence-structure alignments for 33 of the CASP3 targets using the fold-recognition method SDP were submitted (Fischer and Eisenberg, Protein Sci 1996; 5:947-955). After the three-dimensional structures of the sequences were subsequently released, the quality of the predictions were evaluated. Here I describe three of the predictions for targets with little sequence similarity to proteins of known structure that were judged by the assessors to be of higher quality. For two of these predictions, the sequence-structure alignment corresponded perfectly to the structural alignment (zero average shift), and for the third, the average shift was 0.1. This alignment accuracy entails an ideal starting point for homology modeling.

Amino Acid Sequence↗
PubMed1998

Gender differences in ethanol preference and ingestion in rats. The role of the gonadal steroid environment.

An ethanol oral self administration paradigm showed the existence of gender differences in alcohol preference in rats: whereas males and females initiated alcohol drinking at similar rates, females maintained their preference for ethanol over a longer duration. Neonatal estrogenization of females, which effectively confers a male phenotype on a genetically female brain, resulted in patterns of drinking that were similar to those displayed by intact male rats, indicating that gender differences in alcohol drinking patterns may be, at least partially, accounted for by sexual differentiation of the brain. To test whether gonadal steroids also exert activational effects on ethanol-seeking behavior, we also examined the effects of gonadectomy alone, or in combination with gonadal steroid replacement therapy. Castration did not significantly alter ethanol consumption in males, although treatment of castrated rats with dihydrotestosterone resulted in a significant inhibition of this parameter. As compared with the situation in intact female rats, ethanol ingestion was significantly reduced in ovariectomized female rats receiving estradiol (E2) and in ovariectomized female rats receiving combined E2 and progesterone replacement therapy. However, neither ovariectomy nor progesterone replacement in ovariectomized rats resulted in ethanol drinking patterns that were different compared to those observed in intact female controls. Thus, dihydrotestosterone and E2, respectively, appear to exert modulatory influences on the male and female rats' preference for ethanol, but further investigations are necessary to determine to what extent these effects result from activational actions on the brain.

Alcohol Drinking↗
PubMed1998

Tenascin-C hexabrachion assembly is a sequential two-step process initiated by coiled-coil alpha-helices.

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.

Amino Acid Sequence↗
PubMed1998

Protein phosphatase type-2C isozymes present in vertebrate retinae: purification, characterization, and localization in photoreceptors.

Posttranslational modification of proteins by kinases and phosphatases plays an important role in the regulation of cellular signaling in general and neurochemistry in particular. This also applies to vertebrate photoreceptors where phosphorylation of rhodopsin causes uncoupling from the signal transduction cascade. Functional activity of rhodopsin is restored after substitution of the bleached photopigment 11-cisretinal and by dephosphorylation of the opsin moiety. Phosphatases type-1 and type-2A have been identified in vertebrate retinae. Recently, we have shown by molecular cloning that two isozymes of protein phosphatase type-2C (PP2C, PPM) do exist in retinal tissue. In this report, we have purified PP2Calpha and PP2Cbeta from bovine retinae. Thirty to 40% of PP2C was recovered in the cytosolic fraction. Biochemical properties of native and heterologously expressed recombinant enzymes were similar. Enzymatic activity is strictly dependent on the presence of Mg2+. Addition of Ca2+ ions inhibits Mg2+-sustained activity. Antiserum raised against a C-terminal peptide of PP2Cbeta specifically labeled the outer segments of rod photoreceptor cells. PP2C protein levels were significantly reduced in RCS rats, which develop age-dependent photoreceptor degeneration comparable to the hereditary disease retinitis pigmentosa. Although the retinal substrate(s) remain to be identified, the results suggest that PP2C modulates cellular components of the phototransduction machinery.

Amino Acid Sequence↗
PubMed1998

Detecting distant relatives of mammalian LPS-binding and lipid transport proteins.

In mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three-dimensional structure determined is bactericidal/permeability-increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence-sequence and sequence-structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the "twilight zone," their high sequence-structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.

Acute-Phase Proteins↗
PubMed1998

Norfloxacin primary prophylaxis of bacterial infections in cirrhotic patients with ascites: a double-blind randomized trial.

BACKGROUND/AIMS: Norfloxacin is useful to prevent infections in hospitalized cirrhotic patients with low ascitic fluid protein concentrations. It is also effective in preventing the recurrence of spontaneous bacterial peritonitis. The aim of our study was to determine the efficacy of norfloxacin in the primary prophylaxis of gram-negative bacilli infections in cirrhotic patients with low ascitic fluid protein levels (<15 g/l). METHODS: One hundred and seven patients were randomized to receive norfloxacin (400 mg/day; n=53) or placebo (n=54) for 6 months. The patients had no history of infection since cirrhosis diagnosis and no active infection. RESULTS: The probability of gram-negative infection was significantly lower among patients treated with norfloxacin than among those treated with placebo. Six gram-negative bacilli infections occurred in the placebo group and none in the treatment group. Severe infections (spontaneous bacterial peritonitis, neutrocytic ascites and bacteremia) developed in nine patients in the placebo group (17%) and in one patient in the norfloxacin group (2%; p<0.03). There was no between-group difference in the overall rate of infection or in survival. In ten patients from the norfloxacin group, gram-negative bacilli not present in baseline stool cultures were transiently isolated in follow-up cultures. CONCLUSIONS: These data show that primary prophylaxis with norfloxacin for 6 months is effective in the prevention of infections caused by gram-negative bacilli in cirrhotic patients with low ascitic fluid total protein levels.

Adult↗
PubMed1998

Surface-modified biodegradable albumin nano- and microspheres. II: effect of surface charges on in vitro phagocytosis and biodistribution in rats.

The surface charges on biodegradable albumin nanoparticles were introduced by covalent coupling different primary amines to examine their influence on phagocytosis by macrophages under in vitro conditions. Albumin particles with a zeta potential close to zero showed a reduced phagocytic uptake in comparison with charged particles, especially nanoparticles with a positive zeta potential. The phagocytic uptake in the present study was examined using an established cell culture model based on primary mouse peritoneal macrophages and a human hematopoietic monocytic cell line (U-937) treated with phorbol-12-myristic-13-acetate to induce cell differentiation. The influence of opsonins on in vitro phagocytosis experiments was characterized using carriers pre-treated with human serum. In the presence of human serum the phagocytic activity of U-937 cells was found to be similar to primary mouse macrophages without serum. In contrast to peritoneal macrophages, U-937 cells showed no phagocytic activity in the absence of serum. In particular, only the C3b- complement deposition on the particle surface seems to promote the phagocytic process. The in vivo distribution of albumin carriers in rats was investigated using magnetic resonance imaging (MRI). No differences in blood circulation times and organ accumulation between different nanoparticle preparations with positive, neutral and negative surface charges could be observed in rats, suggesting that the in vivo fate of albumin nanoparticles is significantly influenced by factors not reflected in the in vitro cell culture models.

Albumins↗
PubMed1998

Regulation of RssB-dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator-controlled process.

Sigma(S) (RpoS) is a highly unstable global regulatory protein in Escherichia coli, whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock. As a consequence, these stresses result in the induction of sigma(S)-regulated stress-protective proteins. The two-component-type response regulator, RssB, is essential for the rapid proteolysis of sigma(S) and is probably involved in the transduction of some of these stress signals. Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo. Here, we demonstrate increased in vivo half-lives of sigma(S) and the RpoS742::LacZ hybrid protein (also a substrate for RssB-dependent proteolysis) in acetyl phosphate-free (pta-ackA) deletion mutants, even though no sensor kinase was eliminated. The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB. In vitro, efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp-58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed. Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild-type background. While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB-dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration.

Bacterial Proteins↗
PubMed1998

Inherent glucocorticoid response potential of isolated hypothalamic neuroendocrine neurons.

Within the broader framework of facilitating investigations into the inherent responses of restricted neuronal phenotypes devoid of their in vivo afferents, serum- and steroid-free cultures enriched in corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), and beta-endorphin (beta-END) peptidergic neurons were prepared from the hypothalamic paraventricular (PVN: CRH and AVP) and/or arcuate (ARC: beta-END) nuclei of juvenile male rats. The functional viability of these ARC/PVN cultures was verified by their ability to synthesize and secrete CRH, AVP, and beta-END under basal and depolarizing (veratridine) conditions in vitro. Peptide secretion was shown to be Ca2+ and Na+ dependent in that it was blocked in the presence of verapamil and tetrodotoxin, respectively. Exposure of ARC/PVN cocultures to the glucocorticoid dexamethasone (DEX) resulted in a dose-dependent increase of CRH secretion and an inhibition of AVP and beta-END; the CRH responses deviated strikingly from predictions based on in vivo experiments. Steroid withdrawal or treatment with the glucocorticoid receptor antagonist RU38486 reversed these trends. Opposite effects of DEX on CRH secretion were observed in cultures consisting of PVN cells only. Supported by studies using an opioid receptor agonist (morphine) and antagonist (naloxone), these observations demonstrate that ARC-derived (beta-END) neurons modulate the responses of PVN neurons to DEX.

Animals↗
PubMed1998

The general stress sigma factor sigmaS of Escherichia coli is induced during diauxic shift from glucose to lactose.

The general stress sigma factor sigmaS (RpoS) of Escherichia coli is strongly induced in response to glucose starvation. This increase in the cellular sigmaS level is due to stabilization of sigmaS, which under non-stress conditions is subject to rapid proteolysis. In the present study, it is demonstrated that sigmaS is also induced during the diauxic shift from glucose to lactose, i.e., under conditions of glucose exhaustion in the presence of another, less-preferred carbon source that eventually gets utilized. This sigmaS induction, which is due to stabilization, is transient and precedes the induction of beta-galactosidase. In parallel, sigmaS-dependent genes are transiently activated, as was shown here for osmY. Although sigmaS can mediate transcription of lacZ in vitro, sigmaS does not contribute to the induction of beta-galactosidase during the diauxic lag phase. Rather, the induction of sigmaS and the general stress response during the diauxic shift plays the role of a rapidly activated emergency system, which is shut off again as soon as the cells are able to cope with the stress situation by utilizing a more specific and more economical system.

Bacterial Proteins↗
PubMed1998

Microsatellite enrichment in organisms with large genomes (Allium cepa L.).

To exploit the polymorphism of repeat numbers in short tandem repeat (STR) sequences (microsatellites) as molecular markers, STRs must be isolated and PCR primers must be developed in flanking sequences. In species with large genomes such as Allium cepa L. (onion and shallot), an efficient selection procedure for genomic fragments containing STRs is a crucial step. Here we describe a nonradioactive method for microsatellite isolation based on affinity capture of single-stranded restriction fragments annealed to biotinylated microsatellite oligonucleotides (CA)10, (GAA)8 and (AAC)8 followed by adapter-mediated genomic PCR. Cloning of the products in E. coli and plasmid sequencing revealed more than 60% positive clones. Primers were designed in STR-flanking regions, and one or two bands were amplified in 13 diploid onion and five shallot accessions. Allelism of the bands was confirmed by product sequencing.

Dinucleotide Repeats↗
PubMed1998

Estimated Prevalence of Viral Hepatitis in the General Population of the Municipality of São Paulo, Measured by a Serologic Survey of a Stratified, Randomized and Residence-Based Population.

The present study was done to estimate the prevalence of Hepatitis A (HAV), B (HBV), C (HCV), and E (HEV) infection in the general population residing in the municipality of São Paulo, and to evaluate the level of knowledge related to the various modes of infection transmission by and protection against the different viruses. Blood samples and health questionnaires were collected from 1,059 individuals. The study design used an inductive method of predictive statistical inferences through randomized sampling stratified by Sex, age and residence region. The estimated prevalence rated found were: Hepatitis A = 66.59% (63.75%-69.44% CI); Hepatitis B = 5.94% (4.50%-7.35%); Hepatitis C = 1.42% (0,70%-2.12%); Hepatitis E = 1.68% (0.91%-2.46%). The frequency of hepatitis was similar in males and females. HAV showed an estimated prevalence of 56.16% in the population up to 17 years old, increasing to 65.30% in individuals between 18 and 29 years. The infection reached its peak of 90% in individuals 40 years of age or older. The study showed a greater tendency of dissemination of HBV among the population between 15 and 17 years. This specific age group showed an estimated prevalence of active infection of 1.04% (0.43%-1.65% CI), and also demonstrated an ascending level of acquired immunity with an estimated prevalence of 4.90% (3.60%-6.20% CI). HCV demonstrated an estimated prevalence of 1.42% (0.70%-2.12% CI). This specific infection occurred more frequently among adults 30 years of age or older, with the prevalence reaching a peak of 3.80% among the group aged 50 to 59 years. HEV showed zero prevalence among the age group between 2 and 9 years. This was followed by a slightly ascending rate starting from age 10, with an estimated prevalence of 1.05% (0.94%-3.04% CI) among those 10 to 14 years of age. This infection reached its peak of 3.00% (0.55%-6.74% CI) at the age of 60 years or older. Individuals with lower educational levels had a higher tendency of acquiring HAV and HCV, while there was no statistically significant difference for this parameter related to HBV and HEV. HBV occurred more frequently among inhabitants of the northern region of the city. All other hepatitis forms occurred at similar frequencies among the five regions of the city. Among the population, 1.90% (1.08%-2.72% CI) demonstrated an elevated hepatic enzyme with no serologic evidence indicating the cause was the viruses studied. This observation suggests the presence of other hepatic diseases, possibly including other viral diseases. It was also estimated that 75.12% of the city's population did not know the modes of transmission of hepatitis viruses and 76.70% did not know how to prevent them. This clearly suggests the need for a full-scale education program combined with public health measures regarding prevention of all forms of vial hepatitis.

Journal Article↗
PubMed1998

Does carbohydrate-deficient transferrin predict the severity of alcohol withdrawal syndrome?

Alcohol withdrawal often causes severe complications. However, many addicts deny any abuse. Thus, the diagnosis of alcohol abuse frequently becomes difficult. Laboratory parameters are often used to support the diagnosis of alcohol abuse. Furthermore, laboratory parameters should facilitate the prediction of the severity of alcohol withdrawal syndrome (AWS). The most promising laboratory parameter indicating a recent elevated alcohol consumption is carbohydrate-deficient transferrin (CDT). The aim of this study was to examine whether the measurement of CDT at admission can indicate a higher risk for the development of a complicated AWS. The severity of AWS was assessed by the AWS scale, consisting of two subscales for somatic and mental symptoms. CDT was measured by different methods (radioimmunoassay and HPLC). The radioimmunoassay for CDT (CDTect) yielded the best prediction. Our results showed a weak correlation between CDTect and the severity of AWS. However, there were great gender differences. In men, CDTect had the highest positive predictive value for a severe AWS (86.7%), whereas in women mean corpuscular volume was the best predictor (77.8%). However, the sensitivity of CDTect in men (25.5%), as well as mean corpuscular volume in females (29.2%), was too low for a screening test in a general hospital.

Adult↗
PubMed1997

Assigning folds to the proteins encoded by the genome of Mycoplasma genitalium.

A crucial step in exploiting the information inherent in genome sequences is to assign to each protein sequence its three-dimensional fold and biological function. Here we describe fold assignment for the proteins encoded by the small genome of Mycoplasma genitalium. The assignment was carried out by our computer server (http://www.doe-mbi.ucla.edu/people/frsvr/ frsvr. html), which assigns folds to amino acid sequences by comparing sequence-derived predictions with known structures. Of the total of 468 protein ORFs, 103 (22%) can be assigned a known protein fold with high confidence, as cross-validated with tests on known structures. Of these sequences, 75 (16%) show enough sequence similarity to proteins of known structure that they can also be detected by traditional sequence-sequence comparison methods. That is, the difference of 28 sequences (6%) are assignable by the sequence-structure method of the server but not by current sequence-sequence methods. Of the remaining 78% of sequences in the genome, 18% belong to membrane proteins and the remaining 60% cannot be assigned either because these sequences correspond to no presently known fold or because of insensitivity of the method. At the current rate of determination of new folds by x-ray and NMR methods, extrapolation suggests that folds will be assigned to most soluble proteins in the next decade.

Algorithms↗
PubMed1997

Myocardial contrast echocardiography can be used to quantify intramyocardial blood volume: new insights into structural mechanisms of coronary autoregulation.

BACKGROUND: Changes in intramyocardial blood volume (IBV) mediate autoregulatory adaptations to coronary stenosis. This study investigated whether (1) myocardial contrast echocardiography (MCE) can quantify changes in IBV during coronary stenosis and (2) the relation between coronary resistance- and MCE-derived IBV could yield insight into structural mechanisms of IBV change. METHODS AND RESULTS: A circulating in vitro model with constant flow and varying volume was used to determine whether indicator dilution theory could be applied to MCE. Contrast echo was performed with albumin microbubbles, and time-intensity data were fit to a gamma-variate function. With six different volumes, bubble transit time was linearly related to volume (r=.91). To determine whether changes in IBV could be quantified in vivo, the left anterior descending coronary artery in 12 dogs was instrumented with a flow probe, occluder, and intracoronary pressure catheter, and non-flow-limiting stenoses were created. IBV was derived by use of coronary resistance measurements applied to models that assumed autoregulation to occur via vasodilatation or microvascular recruitment. MCE-IBV was calculated from microbubble transit rates. At constant flow, MCE and resistance IBV increased with stenosis. Although MCE and resistance IBV were linearly related, MCE overestimated IBV derived from the vasodilatation model and underestimated IBV calculated from the recruitment model. CONCLUSIONS: MCE can quantify autoregulatory increases in IBV that maintain resting myocardial perfusion. These data suggest that both microvessel vasodilatation and recruitment are dual mechanisms of IBV change. MCE thus may be a clinically useful technique for the detection and quantification of coronary artery disease at rest.

Animals↗