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Biomedical subjects

S Anderson

Publications and source records attributed to S Anderson.

At least 199 records · Page 11Linked to original sources
PubMed1998

Tubulointerstitial disease in aging: evidence for underlying peritubular capillary damage, a potential role for renal ischemia.

Aging is associated with a progressive decline in renal function and the development of glomerulosclerosis and interstitial fibrosis. Although many studies have addressed the cellular mechanisms of age-related glomerulosclerosis, less is known about the tubulointerstitial fibrosis. In this study, aging (24 mo) rats develop tubulointerstitial fibrosis characterized by tubular injury and focal tubular cell proliferation, myofibroblast activation, macrophage infiltration with increased immunostaining for the adhesive proteins osteopontin and intercellular adhesion molecule-1, and collagen IV deposition. Aging rats demonstrated immunostaining for endothelial nitric oxide synthase (eNOSIII) in renal tubular epithelial cells and infiltrating mononuclear cells in areas of tubulointerstitial injury, with a relative loss of staining of the peritubular capillaries compared with young rats. The aging rats also displayed focal loss of peritubular capillaries (as noted by focally decreased RECA-1 and OX-2 staining) in areas of tubulointerstitial injury. The areas of fibrosis and hypocellularity were associated with increased apoptosis of tubular and interstitial cells compared with young (3 mo) rats (25.4 +/- 5.3 versus 3.5 +/- 2.5 TUNEL-positive cells/0.25 mm2 in old versus young rats, P = 0.0001). It is concluded that tubulointerstitial fibrosis in aging is an active process associated with interstitial inflammation and fibroblast activation. The progressive loss of cells in areas of fibrosis may be due to accelerated apoptosis. Furthermore, the tubulointerstitial injury may be the consequence of ischemia secondary to peritubular capillary injury and altered eNOS expression.

Actins↗
PubMed1998

Structural-functional bioinformatics: knowledge-based NMR interpretation.

This paper describes a knowledge-based approach to a problem of structural-functional bioinformatics, specifically the determination of protein structure through the automated analysis of NMR data. Highly successful results in carrying out sequence-specific assignments of residues from multidimensional NMR datasets has led us to automation of NOE dataset interpretation and a design for integrating these results with other protein structure and function analysis programs.

Computational Biology↗
PubMed1998

Juvenile rheumatoid arthritis in a Canadian First Nations (aboriginal) population: onset subtypes and HLA associations.

OBJECTIVE: To determine onset subtypes and HLA associations of juvenile rheumatoid arthritis (JRA) in a First Nations (aboriginal) population; to determine whether population frequencies of HLA antigens may explain the distribution of subtypes of JRA in this population. METHODS: All patients were children from Manitoba and Northwestern Ontario seen in a single pediatric rheumatology clinic between 1975 and 1996. Patients were identified from a clinic registry. Controls were adults of Algonkian Cree and Ojibway heritage. Class I and II major histocompatibility (HLA) typing was performed for First Nations patients and controls. RESULTS: There were a total of 74 First Nations patients with JRA. The relative frequency of rheumatoid factor (RF) positive polyarticular JRA was higher and that of pauciarticular JRA was lower in First Nations compared with Caucasian patients (42 versus 3% and 22 versus 58%, respectively; p = 0.00000). HLA-DRB1*04 (63%), 08 (43%), and 1402 (25%) were the most common DRB1 antigens among controls. The main subtypes of DRB1*04 were 0404 (33% of controls) and 0407 (23%). HLA typing was performed for 39 First Nations patients; 27 were Cree or Ojibway, 4 were from other tribes, and 8 were part First Nations. Among Cree and Ojibway, 59% of controls and 63% of patients with RF positive polyarticular JRA (n = 16) had HLA-DRB1 antigens bearing the rheumatoid arthritis (RA) shared epitope (OR 1.16, 95% CI: 0.38, 3.48). The OR for polyarticular RF positive JRA in those with DRB1*0802 and 0901 were 0.15, 95% CI: 0.02; and 1.24 and 5.83, 95% CI: 1.58, 28.38, respectively. CONCLUSION: There was a high frequency of the RA shared epitope represented by both HLA-DRB1*0404 and 1402 in this Algonkian population. This high frequency may explain the high frequency of RF positive polyarticular JRA. DRB1*0802 may be protective, whereas DRB1*0901 may increase the risk for this subtype of JRA.

Adult↗
PubMed1998

Beyond the dichotomy: linking HIV prevention with care.

In the past decade, the global strategy against AIDS has focused primarily upon prevention. Regardless of the effectiveness of prevention efforts being made today and advances in treatments, the numbers of persons infected globally continues to grow at an alarming rate, especially in developing countries. With numbers of infections increasing, and the trend to more people learning their HIV status earlier, demands for care will mount dramatically into the next century. This paper examines the virtually unexplored role care can play in prevention and its potential to have a mitigating effect on the pandemic. Critical issues addressed include (i) the relationship between care, HIV and productivity; (ii) the role of both care and prevention in promoting acceptance of HIV/AIDS as a community problem; (iii) the role of care in decreasing the vulnerability to HIV in specific populations such as women and children; (iv) the role of care in sustaining behavior change over time for infected persons; and (v) the synergy between improved treatments and prevention. Future areas of research are proposed examining these prevention and care issues that move beyond the traditional dichotomy.

AIDS-Related Opportunistic Infections↗
PubMed1997

Nested genetic bit analysis (N-GBA) for mutation detection in the p53 tumor suppressor gene.

There is a growing and significant demand for reliable, simple and sensitive methods for repeated scanning of a given gene or gene fragment for detection and characterization of mutations. Solid-phase sequencing by single base primer extension of nested GBATM primers on miniaturized DNA arrays can be used to effectively scan targeted sequences for missense, insertion and deletion mutations. This paper describes the use of N-GBA arrays designed to scan the sequence of a 33 base region of exon 8 of the p53 gene (codons 272-282) encompassing a hot spot for mutations associated with the development of cancer. Synthetic DNA templates containing various missense, insertion and deletion mutations, as well as DNA prepared from pancreatic and biliary tumor cells, were genotyped using the exon 8 arrays.

Adenocarcinoma↗
PubMed1997

v-Ras and v-Raf block differentiation of transformable C3H10T1/2-derived preadipocytes at lower levels than required for neoplastic transformation.

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiative insulin signaling pathway, we introduced a leu61-activated ras gene into a Ras-transformable, C3H10T1/2-derived preadipocytic cell line. The results demonstrate that rasleu61 expression in this line blocks differentiation and that this block appears at lower levels than required for full neoplastic transformation. In addition, to examine whether the inability of Rasleu61 to induce differentiation by replacing the insulin signal could be attributed to its transforming effect in this system, we examined the effect of Rasleu61 at levels below the baseline, by expressing rasleu61 in a series of preadipocytes which were rendered deficient in endogenous c-Ras activity. The results show that even very low Rasleu61 levels, insufficient to restore the growth rate of these cells to normal, blocked rather than enhanced differentiation, indicating that rasleu61 expression alone is not sufficient to promote adipocytic differentiation in this system, even in the absence of neoplastic transformation. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrored the v-Ras effects upon adipocytic differentiation and transformation.

Adipocytes↗
PubMed1997

MARCKS regulates membrane ruffling and cell spreading.

The dynamic rearrangement of the actin cytoskeleton is fundamental to most biological processes including embryogenesis, morphogenesis, cell movement, wound healing and metastasis [1]. Membrane ruffling and reversible cell-substratum interactions underlie actin-driven cell movement. Protein kinase C (PKC) stimulates membrane ruffling and adhesion [2], but the mechanism by which this occurs is unknown. Myristoylated alaninerich C kinase substrate (MARCKS) is a PKC substrate that cycles on and off membranes by a mechanism termed the myristoyl-electrostatic switch [3-6]. While at the membrane, MARCKS binds to and sequesters acidic phospholipids including phosphatidyl-inositol-4,5-bisphosphate (PIP2) [7]. MARCKS also binds and cross-links filamentous actin, an activity which is regulated by PKC-dependent phosphorylation and calcium-calmodulin [3]. In this report, we demonstrate that expression, in fibroblasts, of MARCKS containing a mutation which abrogates the myristoyl-electrostatic switch prevents cell spreading. The MARCKS mutant arrests the cell during an early stage of spreading, characterized by profuse membrane blebbing, and prevents the formation of membrane ruffles and lamellae usually found at the leading edge of spreading cells. This defect in the regulation of the actin cytoskeleton is accompanied by a decrease in cell-substratum adhesion. Our results provide direct evidence that MARCKS and PKC regulate actin-dependent membrane ruffling and cell adhesion, perhaps via a PIP2-dependent mechanism.

Actins↗
PubMed1997

"Designing out" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor.

We have used a combination of spectroscopic and calorimetric techniques to assess the thermodynamic and extrathermodynamic consequences of paired amino acid substitutions at positions 30 and 51 in bovine pancreatic trypsin inhibitor (BPTI). Correctly folded, wild type BPTI contains a disulfide at the 30-51 positions, with the nonbackbone atoms of this cystine being relatively solvent inaccessible. Mutants missing this buried 30-51 disulfide adopt a conformation very similar to that of the native state of wild type BPTI (Eigenbrot et al., 1990, 1992), although they are severely destabilized relative to the wild type molecule (Hurle et al., 1990). We have conducted a systematic effort to find the energetically most favorable substitution for this buried 30-51 disulfide in BPTI. To this end, we have studied and characterized the thermally induced and guanidine hydrochloride-induced denaturation transitions for a family of mutants in which the amino acid residue(s) at positions 30 and/or 51 have been systematically altered. Specifically, we studied the unfolding transitions of the following series of residue 30/residue 51 paired substitution mutants: C30A/C51A, C30V/C51A, C30G/C51A, C30S/C51A, C30T/C51A, C30A/C51S, C30S/C51S, and C30G/C51M. For this series of mutants, comparisons between the relative stabilization free energies, derived from analysis of the denaturation profiles, allow us to reach the following conclusions: (a) side chains containing polar moieties (Ser and Thr) are destabilizing, with this effect being position dependent (i.e., a serine substitution is more destabilizing at position 51 than at position 30); (b) the destabilizing effects of two serine substitutions are approximately additive, suggesting that side chain-side chain hydrogen bonds between the two serine hydroxyl groups probably are weak or nonexistent; (c) the thermodynamic impact of a Gly30 substitution is consistent with a glycine-induced increase in the configurational entropy of the unfolded state; (d) the C30G/C51M mutant is highly destabilized relative to the C30A/C51A mutant despite the fact that, based on considerations of hydrophobicity and steric fit, substitution of a buried disulfide by Gly30 and Met51 would be expected to be optimal. Methionine may be particularly ill-suited as a buried disulfide substitute due to the large loss of side chain conformational entropy it undergoes during the transition from the unfolded to the native state. In the aggregate, our data provide insight into the residue-, position-, and context-dependent consequences on protein stability of "designing out" the buried 30-51 disulfide bond in the BPTI molecule. These data also suggest that a previously unrecognized component of disulfide bridge stabilization of proteins is the relatively minor penalty in side chain conformational entropy incurred by cystine residues during folding due to their severely restricted rotation even in the unfolded state.

Amino Acid Sequence↗
PubMed1997

Correlation between disulfide reduction and conformational unfolding in bovine pancreatic trypsin inhibitor.

The native-like two-disulfide intermediate of bovine pancreatic trypsin inhibitor (BPTI), with the disulfide between Cys14 and Cys38 reduced, plays a particularly important role in the disulfide-coupled folding pathway of BPTI because of its participation in the rate-determining step of the reaction [Creighton & Goldenberg (1984) J. Mol. Biol. 179, 497-526; Weissman & Kim (1991) Science 253, 1386-1393]. In order to study directly the relationship between conformational stability and reductive unfolding kinetics, and to gain insight concerning the rate-limiting transition state in the thiol/disulfide-mediated folding/unfolding reaction of BPTI, BPTI variants based on a native-like two-disulfide analog of this intermediate, BPTI(Ala14)Ala38, were examined. The amino acid replacements introduced into BPTI(Ala14)Ala38 rendered it thermodynamically less stable. The kinetic stability, with respect to reduction by dithiothreitol, of the disulfides in these BPTI(Ala14)Ala38 variants was also decreased by the substitutions. The stabilization free energy (deltaG), obtained from chemical denaturation measurements, and the activation energy of the conformational transition (deltaG(++)conf), from the reductive unfolding reaction for this series of variants, were highly correlated. The observed correlation implies a direct coupling of disulfide reduction to conformational stability in this set of protein variants. It also strongly suggests that the transition state in the rate-limiting step of the reductive unfolding reaction involves a highly unfolded conformation of the protein. These data are consistent with a conformation-coupled redox folding pathway for BPTI(Ala14)Ala38 involving two parallel paths with unfolded (30-51) and unfolded (5-55) as the reactive species. Furthermore, the results provide a theoretical explanation for the observed 1000-fold diminution in the rate of 5-55 disulfide bond formation, relative to that of 14-38 bond formation, from the one-disulfide (30-51) intermediate in the wild-type BPTI refolding reaction. The data fit a general paradigm for protein disulfide formation during protein folding whereby native-like structure in folding intermediates accelerates formation of solvent-exposed disulfides but inhibits formation of core disulfides. This model predicts that a "rearrangement" mechanism (i.e., with non-native disulfides involved in the rate-limiting step) to form buried disulfides at a late stage in the folding reaction may be a common feature of redox folding pathways for surface disulfide-containing proteins of high stability.

Animals↗
PubMed1997

Novel transcribed sequences neighbouring a translocation breakpoint associated with schizophrenia.

A 1.3Mb chromosome 11-specific yeast artificial chromosome (YAC) that spans a t(1;11) translocation breakpoint associated with major psychosis has been used to enrich cDNAs that are encoded within it and expressed in the human foetal brain. Database analysis of the selected fragments led to the identification of 54 clones matching alpha-tubulin, 4 fragments matching two anonymous human expressed sequence tags (ESTs) and 8 fragments giving no database matches. The clones matching alpha-tubulin led to the identification of a novel alpha-tubulin locus located approximately 250 kb proximal to the translocation breakpoint. Extensive sequence and expression analysis of this locus suggests that this is a processed pseudogene, although a long open reading frame is maintained and the possibility that an abnormally acting protein may be expressed in a highly tissue or developmental specific manner cannot be discounted. The novel cDNA fragments map up to 700 kb proximal to the translocation breakpoint and are associated with potential CpG islands. Reverse transcriptase polymerase chain reaction (RT-PCR) expression analysis and high resolution genomic mapping suggest that they may comprise up to three novel genes. No major disruption of the identified fragments could be detected in the genomic DNA of translocation carriers. The psychosis associated with this translocation may therefore be due to position effects on the transcription of these genes or an involvement of translocated chromosome 1 sequences.

Chromosomes, Artificial, Yeast↗
PubMed1997

Binding of amino acid side-chains to S1 cavities of serine proteinases.

The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (+/-2) degrees C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1, cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin A alpha (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 10(6) to 10(8). The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu- and Asp- are strongly unfavorable. The Pearson pro duct moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (-0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.

Amino Acid Sequence↗
PubMed1997

Alterations of the p16 gene in head and neck cancer: frequency and association with p53, PRAD-1 and HPV.

Alterations, especially homozygous deletions, of the putative tumor suppressor gene, p16 (p16INK4A, MTS1, CDKN2) have been found in tumor cell lines from a variety of neoplasms. Recent studies have reported frequent p16 gene deletions in cell lines from squamous cell carcinomas of the head and neck (SCCHN), although the prevalence of alterations was variable in primary tumors. This study determined the prevalence of point mutations and deletions of the p16 gene in 33 SCCHN. In addition, the association of p16 gene alterations and abnormalities of p53, PRAD-1 (cyclin D1), and the presence of human papillomavirus (HPV) was examined. We found an overall prevalence of p16 alterations of 36% (nine deletions, three single base substitutions, including one polymorphism). Seven tumors (of 29, 24%) had an alteration of p16 and p53; five (of 33, 15%) had alterations of p16 and PRAD-1; three (of 29, 10%) had alterations of all three genes. In addition, of the five tumors with human papillomavirus detected, only one also had a p16 gene alteration. The results indicate a potentially important role for the p16 gene in head and neck tumorigenesis. In addition, the presence of tumors with multiple somatic gene alterations suggest a possible interaction in the dysregulation of the cell cycle.

Adult↗