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Biomedical subjects

A Stevens

Publications and source records attributed to A Stevens.

At least 163 records · Page 9Linked to original sources
PubMed1993

Acid-induced dissociation of alpha A- and alpha B-crystallin homopolymers.

Homopolymers were constructed from the alpha A and alpha B polypeptides isolated from the lens protein alpha-crystallin. As the pH is lowered from 7.0 to 3.4, these homopolymers dissociate to smaller species with molecular masses ranging from 80 to 250 kDa for the alpha A and around 140 kDa for the alpha B dissociation products. The pKa for this dissociation was 3.8 +/- 0.2 for alpha A and 4.1 +/- 0.1 for alpha B homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alpha B polypeptides, whereas the alpha A dissociation products remained intact. Fractionation of the acid dissociation products from the alpha A homopolymer at pH 2.5 yielded stable species with molecular masses of 220 +/- 30, 160 +/- 20, and 90 +/- 10 kDa. The majority of the population at acid pH consisted of the 160 kDa species. Conformational analysis of these species revealed that most of the secondary structure of the original alpha A homopolymer was retained but that the tertiary structure was perturbed. Fluorescence quenching and energy transfer measurements suggested that the molecule had undergone acid expansion, with the greatest perturbation observed in the smallest particles. The results from this work suggest that alpha A homopolymers are heterogeneous populations of aggregates of a "monomeric" molecule with a molecular mass of 160 kDa. This "monomeric" molecule may be formed from the association of two tetrameric units.

Amino Acids↗
PubMed1993

An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity.

An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomyces cerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as well as activities involving chromosomal DNA pairing and mechanics. Preliminary analysis of an hke1ts mutant reveals a precipitous decline in the translation of mRNA at the nonpermissive temperature. Sporulation of heterozygous HKE1/hke1::URA3 diploids reveals that this gene, unlike the highly related XRN1/KEM1 gene, is essential for cell viability. Overexpression of the homologous gene product, p175, failed to rescue cells lacking a functional p116. In vitro studies demonstrate that p116 is a protein with 5'-->3' exoribonuclease activity, a major activity of the related p175. An immunoreactive RNase activity of 116 kDa is abolished with antiserum against p116. Both the level of this protein and the RNase activity correlate with HKE1 gene dosage. The RNase activity purifies coincidentally with a previously described 116-kDa RNase having 5'-->3' exoribonuclease activity.

Cloning, Molecular↗
PubMed1993

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure.

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.

Base Sequence↗
PubMed1993

A characteristic ganglioside antibody pattern in the CSF of patients with amyotrophic lateral sclerosis.

Paired cerebrospinal fluid and serum samples of patients with amyotrophic lateral sclerosis (n = 35) revealed no consistent abnormalities of CSF cell count, CSF albumin, CSF IgG, CSF IgM, IgG or IgM index, or oligoclonal immunoglobulin band formation in the CSF. Determination of IgG and IgM CSF and serum antibodies to gangliosides GM1, GM2, GM3, AGM1, GD1a, GD1b, and GT1b showed a characteristic pattern which allowed the differentiation of amyotrophic lateral sclerosis from controls and from patients with other neurological disorders including multiple sclerosis. Specifically, patients with the disease had elevated CSF IgM antibodies to all gangliosides except AGM1. The lack of correlation between the CSF findings and corresponding serum antibodies suggests a chronic, compartmental, intrathecal immune response of low activity in amyotrophic lateral sclerosis. Whether this immune response is primary and of pathogenetic significance, or an epiphenomenon of neuronal degeneration, remains to be investigated.

Adult↗
PubMed1992

Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

Sequencing of the XRN1 gene of Saccharomyces cerevisiae, cloned in this laboratory as a gene encoding a 160-kDa 5'-->3' exoribonuclease (XRN1), shows that it is identical to a gene (DST2 or SEP1) encoding a DNA strand transferase and to genes involved in nuclear fusion, KEM1, and plasmid stability, RAR5. To better understand the various phenotypes associated with loss of XRN1 and the enzymatic activities associated with the protein, certain characteristics of our yeast cells lacking an active gene (xrn1) have been examined. Cells are larger (average volume is x 1.5-1.8) and have an increased doubling time (x1.9-2.1). The protein synthesis rate per cell is 80-90% that of wild-type (wt) cells, and the resultant cellular protein levels are higher. The rate of the 25S and 18S rRNA synthesis is approximately 45% that of wt cells and its cellular level is about 90% that of wt cells. Levels of protein bands resolved by one-dimensional PAGE show substantial differences. Synthesis rates observed for the same protein bands, as well as measurements of several specific mRNA levels by Northern analysis, suggest disparities in mRNA levels. Results show two to four times longer half-lives of specific short-lived mRNAs. The variations in levels of protein and RNA species found in the xrn1 cells may be the cause of some of the phenotypes found associated with gene loss.

Exoribonucleases↗
PubMed1992

Tumor cell dissemination triggers an intrathecal immune response in neoplastic meningitis.

The intrathecal immune response in neoplastic meningitis (NM) was studied by quantitation of immune parameters such as immunoglobulin G (IgG); IgM; interleukins (IL) 1, 2, 4, and 6; soluble IL-2 receptors (sIL-2R); interferon gamma (IFNy); tumor necrosis factor-alpha (TNF alpha); and three tumor markers, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and fibronectin (FN), in 47 paired cerebrospinal fluid (CSF) and serum samples from patients with NM from different carcinomas, malignant melanoma, and lymphoma. Elevated IgG and IgM indices, CSF oligoclonal Ig bands, and CSF IL-6 indicated an intrathecal immune activation in most patients with NM. Results for IL-1, IL-2, and IL-4 were always negative. sIL-2R and IFNy were detected occasionally but not associated with specific malignant neoplasms. CSF TNF alpha was detected only in NM from cases of malignant melanoma. None of the immune parameters proved useful for the differentiation of NM from autoimmune or inflammatory conditions. Immune parameters were not correlated with tumor markers CEA, AFP, or FN. Results for AFP were positive only in a case of glioblastoma. CEA was a useful and specific diagnostic parameter in carcinomatous NM. CSF FN levels frequently were elevated but are not specific for NM.

Breast Neoplasms↗
PubMed1992

Ganglioside antibodies: a lack of diagnostic specificity and clinical utility?

Serum IgG and IgM antibodies to gangliosides GM1, GM2, GM3, AGM1, GD1a, GD1b and GT1b were determined in 210 patients with different degenerative and inflammatory disorders including motor neuron diseases, peripheral radiculopathies and neuropathies, multiple sclerosis and neuroborreliosis. No single disorder was associated specifically with ganglioside antibodies. No characteristic patterns of ganglioside antibodies were observed in any disease category. However, 32% of all patients had pathological antibody titres to at least one ganglioside. Four patients had pathological IgG and IgM titres for all gangliosides evaluated. They suffered from systemic lupus erythematosus [2], neuroborreliosis and schizophrenia, respectively. The results of this study indicate that the introduction of ganglioside antibody determination as a differential diagnostic test in clinical neurology is only helpful in a few patients with typical lower motor neuron syndromes.

Biomarkers↗
PubMed1992

Are CSF or serum ganglioside antibodies related to peripheral nerve demyelination in neuroborreliosis, Guillain-Barré syndrome, or chronic inflammatory demyelinating polyradiculoneuropathy?

Cerebrospinal fluid (CSF) and serum IgG and IgM antibodies to seven gangliosides were determined in patients with neuroborreliosis (NB) (n = 20), Guillain-Barré syndrome (GBS) (n = 13), and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) (n = 10). The incidence of elevated antibodies was highest in NB and lowest in CIDP. Correlation between CSF and serum antibodies was only observed for IgG antibodies to GM1, GD1b and GT1b in GBS. The strong IgM antibody reactivity to gangliosides in the CSF of NB patients may be involved in the variety of neurological disorders attributed to Borrelia burgdorferi infection. Since one CIDP and three GBS patients had serologic evidence of prior or concurrent borrelia infection, this infection may belong to the infections that can trigger GBS or CIDP. The lack of specific ganglioside antibody patterns in these four patients suggests that ganglioside antibodies are not the link between Borrelia burgdorferi infection and the demyelination of peripheral nerves in GBS and CIDP.

Adult↗
PubMed1992

Influence of gangliosides on experimental allergic neuritis.

The development of myelin-induced experimental allergic neuritis (EAN) in Lewis rats can be depressed and delayed by adding a ganglioside mixture (GM1, GD1a, GD1b, GT1b) to the immunization compound; however, gangliosides may enhance the induction of adjuvant arthritis. Antibodies against multiple gangliosides are produced in rats after immunization with gangliosides after addition of myelin, but only low titers can be detected in animals immunized with myelin and complete Freund's adjuvant alone. We conclude that this antibody production is not the result of peripheral nerve inflammation but depends rather from external applied gangliosides.

Animals↗
PubMed1992

Localized in vivo 1H spectroscopy of human skeletal muscle: normal and pathologic findings.

To obtain high signal to noise ratio in small volume elements (8 cm3), in vivo 1H NMR spectroscopy of normal and diseased human skeletal muscle was performed using a double spin-echo localization method on a 1.5-T whole body system. High resolved spectra of normal calf muscle show the well known resonances of lipids (methyl, methylene, olefinic, and other fatty acid resonances), creatine/phosphocreatine, choline/carnitine, taurine, and histidine with good intraindividual reproducibility. Pronounced intraindividual differences in the lipid range were found between different upper thigh muscle groups. On pathologic conditions like myopathy, myositis or irradiation damage the spectral lipid content was increased. Three months after local irradiation of the medial vastus muscle (50 Gy), the localized 1H NMR spectrum showed a complete loss of the choline and creatine signals. In a case of M. Behçet with muscular involvement the relative reduction of the choline signal may provide an insight in the pathobiochemistry. The results of our investigations in nine healthy volunteers and three patients are presented in detail including relaxation times of the metabolites.

Adolescent↗
PubMed1992

Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens. Examples of quadrupole ion magnetic relaxation in a crowded protein environment.

Single and multiple quantum nuclear magnetic resonance (NMR) spectroscopic techniques were used to investigate the motional dynamics of sodium and potassium ions in concentrated protein solution, represented in this study by cortical and nuclear bovine lens tissue homogenates. Both ions displayed homogeneous biexponential magnetic relaxation behavior. Furthermore, the NMR relaxation behavior of these ions in lens homogenates was consistent either with a model that assumed the occurrence of two predominant ionic populations, "free" and "bound," in fast exchange with each other or with a model that assumed an asymmetric Gaussian distribution of correlation times. Regardless of the model employed, both ions were found to occur in a predominantly "free" or "unbound" rapidly reorienting state. The fraction of "bound" 23Na+, assuming a discrete two-site model, was approximately 0.006 and 0.017 for cortical and nuclear homogenates, respectively. Corresponding values for 39K+ were 0.003 and 0.007, respectively. Estimated values for the fraction of "bound" 23Na+ or 39K+ obtained from the distribution model (tau C greater than omega L-1) were less than or equal to 0.05 for all cases examined. The correlation times of the "bound" ions, derived using either a two-site or distribution model, yielded values that were at least one order of magnitude smaller than the reorientational motion of the constituent lens proteins. This observation implies that the apparent correlation time for ion binding is dominated by processes other than protein reorientational motion, most likely fast exchange between "free" and "bound" environments. The results of NMR visibility studies were consistent with the above findings, in agreement with other studies performed by non-NMR methods. These studies, in combination with those presented in the literature, suggest that the most likely role for sodium and potassium ions in the lens appears to be the regulation of cell volume by affecting the intralenticular water chemical potential.

Animals↗
PubMed1992

The effects of isolation buffers on the properties of alpha-crystallin.

This work was undertaken in order to resolve some of the controversy in the literature concerning the properties of alpha-crystallins isolated in different laboratories. Bovine lens proteins were extracted and isolated by gel chromatography using 'Hoenders buffer' (0.02 M Tris-HCl, 1 mM EDTA, 80 mM NaCl, pH 7.3), 'Tardieu buffer' (0.04 M phosphate, 1 mM EDTA, 0.2 mM DTT, 0.06 M KCl, pH 6.8) and 'Thomson/Augusteyn' buffer (0.05 M Tris-HCl, 2 mM EDTA, 0.2 mM DTT, pH 8.0). The alpha-crystallin peaks were then divided into 12-16 pools and subjected to detailed physicochemical characterization. Fractionation by HPLC-GPC and quasi-elastic light scattering indicated that the size of the proteins decreased with increasing elution volume and that they were stable for at least 9 months at 20 degrees C. Molecular masses were found to range from over 2 mDa at the front of the peaks to around 600 kDa at the end. The size distributions, for the three buffers, were indistinguishable. No differences could be detected in the polypeptide distributions by SDS-PAGE. The proteins were also identical in their near- and far-UV circular dichroism spectra, accessibility of their sulphydryl groups to DTNB, tryptophan accessibility to quenching by acrylamide and iodide, and immunoreactivity with two monoclonal antibodies with different specificities. It is concluded that identical alpha-crystallins are isolated with the three different buffers and that variations in pH (6.9-8.0), ionic strength (60-150 mM) and cation (K, Na, Tris) during the isolation do not affect the properties of the protein. Claims that differing observations on the properties of alpha-crystallin may be attributed to the buffers used, are untenable.

Animals↗
PubMed1992

Inter- and intra-observer agreement in the assessment of the quality of spontaneous movements in the newborn.

The inter- and intra-observer agreement for the assessment of several aspects of the quality of spontaneous movements in newborn infants was analysed by 4 observers. The agreement rates were corrected for chance by the use of kappa statistics, which revealed kappa values ranging from 0.36 to 0.84. The degree of agreement on various items differed. A moderate-to-good agreement was found for the items "onset of movement," "speed," "speed of arms compared to legs," "amplitude of arms compared to legs," "fluency," "variability in arm patterns" and "global judgement of the movement" (kappa values ranging from 0.51 to 0.84). Less agreement occurred in the items "amplitude," "variability in movement pattern" and "variability in leg patterns" (kappa values ranging from 0.36 to 0.40). Even though it is believed that the qualitative assessment of spontaneous motor behaviour is very subjective, our results suggest that several qualitative aspects of infant motor behaviour can be reliably judged. We concluded that the global judgement of the quality of general movements in full-term infants is a reproducible, and therefore reliable, clinical tool for evaluating the function of the central nervous system in the neonatal period.

Humans↗