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S Watkins

Publications and source records attributed to S Watkins.

At least 19 recordsLinked to original sources
PubMed1995

Structural study of the glycosylated and unglycosylated forms of a genetic variant of human serum albumin (63 Asp-->Asn).

A genetic variant of human serum albumin (alloalbumin) exhibited atypical electrophoretic mobility and chromatographic behavior apparently because of the effect of a point substitution on the molecular conformation. Three forms of albumin were isolated by DEAE HPLC chromatography: normal albumin, and two variant forms V1 and V2. The point substitution (Asp-63-->Asn) generated a canonical tripeptide acceptor sequence for glycosylation with an N-linked oligosaccharide (Asn-Lys-Ser). Neuraminidase digestion followed by electrophoresis showed that the V2 variant form was glycosylated and the V1 form was not. Time-of-flight mass spectrometry yielded a molecular weight of about 2000 for the carbohydrate. Structural analysis of the carbohydrate was done by chromatographic comparison of the pyridylaminated derivatives with standards and was confirmed by proton NMR of the three pronase glycopeptides and of the pyridylaminated oligosaccharide. The oligosaccharide had a complex biantennary structure with two sialic acid residues. In normal albumin Asp-63 is exposed and is adjacent to the first disulfide bond, Cys-62-->Cys-53. The apparent effect on molecular conformation resulting in incomplete glycosylation and atypical electrophoretic mobility suggests that glycosylation may interfere with disulfide bond formation at this site.

Amino Acid Sequence↗
PubMed1995

Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.

Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.

Aging↗
PubMed1995

A genetic variant of albumin (albumin Asola; Tyr140-->Cys) with no free -SH group but with an additional disulfide bridge.

A slow migrating variant of human serum albumin, present in lower amount than the normal protein, has been detected by routine clinical electrophoresis at pH 8.6 in two members of a family living in Asola (Lombardia, Italy). Ion-exchange chromatography of serum samples failed to separate the normal protein from the variant. Analysis of the albumin peak by SDS/PAGE revealed that the variant had a lower apparent molecular mass than its normal counterpart. However, the abnormal band was not detectable when the separation was performed under reducing conditions or when both albumins were carboxymethylated. Isoelectric-focusing analysis of CNBr fragments localized the mutation to fragment CNBr 3 (residues 124-298). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptide revealed that the variant arises from a Tyr140--> Cys substitution. This result was confirmed by DNA sequence analysis, which showed a single transition of TAT-->TGT at nucleotide position 5074. Despite the presence of an additional cysteine residue, several lines of evidence indicated that albumin Asola has no free -SH group; therefore, we propose the formation of a new S-S bond between Cys140 and Cys34, the only free sulphydryl group present in the normal protein. The relatively low level of the variant in serum and its abnormal mobility on cellulose acetate electrophoresis and SDS/PAGE are probably caused by a gross conformational change of the molecule induced by the new S-S bridge.

Amino Acid Sequence↗
PubMed1995

Antitumor activities of subsets of human IL-2-activated natural killer cells in solid tissues.

Human NK cells can be separated into two functionally distinct subpopulations based on the ability to rapidly respond to IL-2 by adherence to solid surfaces. To determine functions of the NK cell subsets in solid tumor tissues, adherent (A) and nonadherent (NA) NK cells were evaluated for their ability to infiltrate multicellular tumor spheroids in vitro, to kill carcinoma (CA) cell targets in these spheroids, and to mediate antitumor activity in vivo. A-NK cells were less cytolytic than NA-NK cells against CA targets in single cell suspensions or in monolayers. However, A-NK cells showed a significantly better ability than NA-NK cells to infiltrate tumor tissues and kill tumor cells in spheroids of human squamous cell CA of the head and neck or breast CA. Perilesional delivery of human A-NK cells and IL-2 resulted in regression of established human squamous cell carcinoma of the head and neck tumors growing subcutaneously in immunosuppressed nude mice. Similarly, in a xenograft model of human gastric CA metastatic to liver of nude mice, a single intrasplenic injection of A-NK cells in combination with i.p. infusions of IL-2 significantly reduced the number of established hepatic metastases (p < 0.007) and prolonged survival of the mice (p < 0.003). In contrast, NA-NK cells were ineffective in either of the in vivo xenograft tumor models. These findings demonstrate that A-NK cells represent a biologically unique and important subset of NK cells that, in contrast to the rest of NK cells, function as effector cells in solid tumor tissues and, consequently, have a great antitumor therapeutic potential.

Adenocarcinoma↗
PubMed1995

Ward sisters' influence on use of care plans.

The ward sister's influence affects nurses' attitudes to care plans. This influence may be positive or negative. There may be other factors that affect nurses' attitudes to care plans, which should be addressed in future research.

Attitude of Health Personnel↗
PubMed1994

Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells.

Bone marrow transplantation is an accepted therapy for hematologic malignancies, aplastic anemia, metabolic disorders, and solid tumors. However, graft-versus-host disease (GVHD) and failure of engraftment have limited the widespread application of this technology to nonmalignant disease states. The use of purified bone marrow stem cells has been suggested as an approach to promote engraftment yet avoid GVHD. Although bone marrow stem cells, purified by cell sorting, engraft and repopulate lethally irradiated genetically identical recipients, they do not engraft in major histocompatibility complex (MHC)-disparate allogeneic recipients. We report for the first time the characterization of a novel cell population of donor bone marrow origin, separate from the hematopoietic stem cell, that facilitates engraftment of purified allogeneic bone marrow stem cells in an MHC-specific fashion without causing GVHD. Although 1,000 purified stem cells (c-kit+/Sca-1+/lineage-) reliably repopulate syngeneic mouse recipients, 10 times that number do not engraft in MHC-disparate allogeneic recipients. The addition of as few as 30,000 facilitating cells (CD8+/CD45R+/TCR-) is sufficient to permit engraftment of purified stem cells in MHC-disparate recipients. The cell surface phenotype of this purified cellular population differs significantly from other characterized lineages of lymphoid or myeloid origin. Based on multiparameter rare-events cell sorting, the facilitating fraction is CD8+, CD3+, CD45R+, Thy 1+, class IIdim/intermediate but alpha beta-TCR- and gamma delta-TCR-. This cellular population comprises approximately 0.4% of the total bone marrow and is separate from the hematopoietic stem cell. The coadministration of purified facilitating cells plus stem cells to optimize engraftment yet avoid GVHD may expand the potential application of bone marrow transplantation to disease states in which the morbidity and mortality associated with conventional BMT cannot be justified.

Animals↗
PubMed1994

Analbuminemia: three cases resulting from different point mutations in the albumin gene.

Analbuminemia is a very rare recessive disorder in which subjects have little or no circulating albumin, although albumin is normally the most abundant plasma protein and has many functions. Analbuminemia is caused by a variety of mutations in the albumin gene and is exhibited only by subjects homozygous for the defect. Previously the mutation had been identified at the molecular level in only two human cases; in one case it resulted from an exon-splicing defect, and in the other case it was caused by a nucleotide insertion that caused a frameshift and premature stop codon. In this investigation we identified the mutations in three unrelated subjects from different countries. In each instance a single-nucleotide mutation produced a stop codon, but the mutations occurred at three different sites: (i) in an Italian male a C-->T transition at nt 2368 in the genomic sequence of albumin, (ii) a C-->T transition at nt 4446 for an American female, and (iii) a G-->A transition at nt 7708 in a Canadian male. The size of the albumin fragment that might have been produced for the three cases varied from 31- to 213-amino acid residues, but no evidence for a circulating albumin fragment was obtained. The paradox is that analbuminemia is extremely rare (frequency < 1 x 10(6)); yet the virtual absence of albumin is tolerable despite its multiple functions.

Adult↗
PubMed1994

The risk of dying in alcohol-related automobile crashes among habitual drunk drivers.

BACKGROUND: Reports suggest that people who drive while intoxicated by alcohol may do so repeatedly. We hypothesized that persons arrested for driving while impaired might be at increased risk for death in an alcohol-related motor vehicle crash. To evaluate this possibility, we studied the deaths of drivers in alcohol-related motor vehicle accidents in North Carolina over a 10-year period. METHODS: We compared drivers who died in motor vehicle crashes from 1980 through 1989 and who had blood alcohol concentrations of at least 20 mg per deciliter (4.3 mmol per liter), referred to as the case drivers, with those who died in crashes but had blood alcohol concentrations below 20 mg per deciliter, referred to as the control drivers. We identified case drivers and control drivers through the state Medical Examiner System. We then searched North Carolina driver-history files for the five years before each death to identify arrests for driving while impaired. RESULTS: We linked a total of 1646 case drivers and 1474 control drivers to their driver-history files. Case drivers were more likely than control drivers to have been arrested for driving while impaired (26 percent vs. 3 percent). After we controlled for potential confounding variables, case drivers 21 to 34 years of age were 4.3 times more likely (95 percent confidence interval, 2.7 to 6.8) than control drivers to have been arrested for driving while impaired; case drivers 35 years of age or older were 11.7 times more likely (95 percent confidence interval, 6.8 to 20.1). The strength of the association appeared to increase with the number of prior arrests. Case drivers were also more likely than the general population of currently licensed drivers to have been arrested. CONCLUSIONS: Arrests for driving while impaired substantially increase the risk of eventual death in an alcohol-related crash. Aggressive intervention in the cases of people arrested for driving while impaired may decrease the likelihood of a future fatal alcohol-related crash.

Accidents, Traffic↗
PubMed1994

Genetic variants of human serum albumin in Italy: point mutants and a carboxyl-terminal variant.

Of the > 50 different genetic variants of human serum albumin (alloalbumins) that have been characterized by amino acid or DNA sequence analysis, almost half have been identified in Italy through a long-term electrophoretic survey of serum. Previously we have reported structural studies of 11 Italian alloalbumins with point mutations, 2 different carboxyl-terminal variants, and 1 case of analbuminemia in an Italian family. This article describes confirmation by DNA sequencing of mutations previously inferred from protein sequencing of 4 of the above alloalbumins; it also reports the mutations identified by protein and DNA sequence analysis of 4 other Italian alloalbumins not previously recorded: albumin Larino, His3-->Tyr; Tradate-2 (protein sequencing only), Lys225-->Gln; Caserta, Lys276-->Asn; and Bazzano, a carboxyl-terminal variant. The first 3 have point mutations that produce a single amino acid substitution, but a nucleotide deletion causes a frameshift and an altered and truncated carboxyl-terminal sequence in albumin Bazzano. In these 4 instances the expression of the alloalbumin is variable, ranging from 10% to 70% of the total albumiN, in contrast to the usual 50% each for the normal and mutant albumin. The distribution of point mutations in the albumin gene is nonrandom; most of the 47 reported point substitutions involve charged amino acid residues on the surface of the molecule that are not concerned with ligand-binding sites.

Amino Acid Sequence↗
PubMed1994

A nucleotide insertion and frameshift cause analbuminemia in an Italian family.

In analbuminemia, a very rare inherited syndrome, subjects produce little or no albumin (1/100th to 1/1000th normal), presumably because of a mutation in the albumin gene; yet, they have only moderate edema and few related symptoms owing to a compensatory increase in other plasma proteins. Because of the virtual absence of albumin the defect must be identified at the DNA level. In this study the mutation causing analbuminemia in an Italian family was investigated by analysis of DNA from a mother and her daughter. The mother was homozygous for the trait and had a serum albumin value of < 0.01 g/dl (about 1/500th normal); the daughter was heterozygous for the trait and had a nearly normal albumin value. Molecular cloning and sequence analysis of DNA from both mother and daughter showed that the mutation is caused by a nucleotide insertion in exon 8; this produces a frameshift leading to a premature stop, seven codons downstream. The methods of heteroduplex hybridization and single-strand conformation polymorphism were used to compare the DNA of the mother and daughter to the DNA of two unrelated analbuminemic individuals (one Italian and one American). This showed that all three analbuminemic individuals had different mutations; these also differed from the mutation in the only human case previously studied at the DNA level, which was a splicing defect affecting the ligation of the exon 6-exon 7 sequences. Thus, analbuminemia may result from a variety of mutations and is genetically heterogeneous.

Amino Acid Sequence↗
PubMed1994

Thermostable variants of bovine beta-lactoglobulin.

The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond. Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121. We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C. Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein. These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method. Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping. In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized. The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.

Amino Acid Sequence↗
PubMed1994

The effectiveness of an intervener model of services for young deaf-blind children.

Project Validation of the Intervener Program (VIP) studied and documented the effectiveness of the Intervener Service Model which provides the services of a paraprofessional (called an intervener) to families of young children who are deaf-blind. The intervener provides auditory, visual, and tactile stimulation for the child and helps the child develop interactive behaviors instead of isolated, defensive, or self-stimulatory behaviors. The intervener also enables the parents to obtain much needed respite. Project VIP obtained abundant quantitative and qualitative data on the effectiveness of the Intervener Service Model. The data strongly support the need for Intervener Services for young children who are deaf-blind and their families.

Blindness↗
PubMed1994

Effect of secretory IgA on transepithelial passage of bacteria across the intact ileum in vitro.

BACKGROUND: Bacterial translocation is a process believed to result in nosocomial infections. Secretory IgA (sIgA) may have a role in the prevention of translocation by its ability to bind and aggregate bacteria, a function termed "immune exclusion." The present study was done to determine the effect of specific binding of sIgA to bacteria on the movement of these organisms across the intact epithelial membrane. STUDY DESIGN: Bacterial translocation across intact intestinal segments of rats were assessed in vitro using the Ussing model. Secretory IgA (0.25 mg per mL) from pooled human colostrum was added to the perfused segments of ileum in the Ussing system. Subsequently, the membranes were exposed to 5 x 10(9) cfu per mL Escherichia coli on their mucosal side. A second experiment tested the effect of human IgG when perfused with E. coli using the same preparation. All experiments had paired matched rats in a control group without immunoglobulin. The ability of sIgA and IgG to bind to E. coli was studied by an in vitro assay, as well as by transmission electron microscopy and immunofluorescence of random IgA/E. coli experiments. Measurements obtained in all experimental and control groups were the incidence and amount of bacterial passage and the potential difference generated by the intestinal segments (an index of viability). RESULTS: There were no differences in potential difference between control and experimental groups in either of the two experiments. Secretory IgA bound E. coli and completely prevented passage of E. coli as compared with rats in the control group. IgG bound E. coli; however, the incidence of passage was equal to that of rats in the control group. However, the presence of IgG resulted in a significantly reduced number of bacteria that passed when compared with controls (p < 0.05). Electron microscopic studies revealed intact surface morphology and immunofluorescence revealed aggregates of IgA and E. coli on the mucosal, but not submucosal, surface of the ileal membranes. CONCLUSIONS: This study provides direct evidence of immune exclusion by sIgA. When bound to bacteria, it prevents passage across a morphologically intact segment of viable intestinal tissue.

Animals↗
PubMed1994

Role of intestinal mucus in transepithelial passage of bacteria across the intact ileum in vitro.

BACKGROUND: Although gastrointestinal mucus is one of a number of putative host defense mechanisms that protect the gut barrier against microbial translocation, little experimental data are available to show its role in this process. The present study sought to determine the role of mucus depletion on the transepithelial passage of bacteria across viable segments of rat ileum mounted in Ussing chambers in vitro. METHODS: Intestinal mucus was depleted in 12 rats after injection with pilocarpine (160 mg/kg intraperitoneally) 45 minutes before intestinal harvest. The mucosal surfaces of the perfused gut segments mounted in the Ussing chamber were exposed to 5 x 10(9) CFU/ml Escherichia coli C-25. Viability was monitored by continuous measurements of the potential difference generated by the membranes. The electrical characteristics were unaltered by pilocarpine pretreatment or exposure to bacteria. RESULTS: Bacterial passage occurred in 100% of pilocarpine membranes as compared with 33.3% in controls (p < 0.05). Pilocarpine-treated membranes resulted in 19.9 +/- 7.5 mg of retrievable mucus as compared with 28.8 +/- 7.2 mg in controls (p < 0.05). Light and transmission electron microscopy revealed an intact epithelial surface in all membranes. There was a marked decrease in mucus on the surface of pilocarpine-treated membranes. CONCLUSIONS: Intestinal mucus secretion is a critical factor in the barrier function of the gut, and its depletion results in a dramatic increase in bacterial passage across the intact rat ileum.

Animals↗
PubMed1993

Protein and DNA sequence analysis of a 'private' genetic variant: albumin Ortonovo (Glu-505-->Lys).

Albumin Ortonovo is a slow moving variant of human serum albumin which has been found only in people coming from the small villages of Ortonovo and Nicola (Liguria, Italy) and reaches polymorphic frequency (> or = 1%) in the poorly admixed population group living in that area. This is the first report of a 'private' variant detected in a Caucasian population. It probably originated as a mutation in a founder individual many generations ago. Isoelectric focusing analysis of CNBr fragments from the purified variant localized the mutation in fragment CNBr VI (residues 447-548). This fragment was isolated on a preparative scale by reversed-phase HPLC and subjected to V8 proteinase digestion. Sequence analysis of the abnormal V8 peptide revealed that the variant arises from a previously unreported substitution at position 505 where glutamic acid has been replaced by lysine. The protein data were confirmed by DNA sequence analysis which indicated a single nucleotide change of GAA-->AAA in the corresponding codon of the structural gene. Since the amino acid substitution found in albumin Ortonovo accords with its electrophoretic mobility on cellulose acetate, residue 505 is probably exposed to the solvent. The clustering of the mutations in the intersubdomain connection linking subdomains IIIA and IIIB (residues 492-511) accords with the fact that this region lies on the molecular surface and is accessible to solvent.

Amino Acid Sequence↗
PubMed1993

Screening procedures used to identify children with hearing loss.

This study focused on the hearing-screening procedures used to identify children who have hearing losses who were later enrolled in SKI*HI parent/infant programs throughout the country. Responses to a questionnaire for 1,404 children indicated that although auditory brainstem response (ABR) technology provided the lowest mean identification age, informal hearing-screening procedures (parental suspicion and referral) were the procedure of identification for 80% of the children. The data suggest that formal screening programs are not presently locating the vast majority of infants who have hearing losses. Recommendations for professional and parental education in the area of hearing-loss screening are provided.

Age Factors↗