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Biomedical subjects

S G Welch

Publications and source records attributed to S G Welch.

At least 19 recordsLinked to original sources
PubMed2001

Distribution of human kappa locus IGKV2-29 and IGKV2D-29 alleles in Swedish Caucasians and Hong Kong Chinese.

Polymorphism in the IGKV2-29 gene was shown to decrease the recombination frequency in B cells and to be important for immune responses to Haemophilus influenzae type b polysaccharide. By using the combination of PCR and restriction enzyme mapping, the distribution of IGKV2D-29 and IGKV2-29 gene alleles was estimated in two geographically and ethnically different groups. We found that V2D-29*01 homozygous individuals were most common in Swedish Caucasians (82%), but less common in the Chinese population of Hong Kong (28%). The homozygous V2D-29*02 genotype was found in 19% Chinese, but only in one Caucasian (1%). The frequency of the heterozygous V2D-29*01/V2D-29*02 genotype was also higher in the Chinese population (46%) compared with the Caucasians (7%). V2-29*01 homozygosity was more frequent among Caucasians (85%) than among Chinese (19%). In contrast, homozygous V2-29*02 individuals were over-represented in the Chinese population (18%), whereas only one was found among Caucasians (1%). Heterozygous V2-29*01/V2-29*02 individuals were also more common in the Chinese (63%) than the Caucasian (15%) population. Most Caucasians had the combination of V2D-29*01/V2D-29*01+V2-29*01/V2-29*01 (74%), while the most common genotype for Chinese was V2D-29*01/V2D-29*02+ V2-29*01/V2-29*02 (41%). Analysis of the association of V2D-29*02 and V2-29*02 alleles demonstrated a high degree of linkage, as for V2D-29*01 with V2-29*01. These data show a significant difference in the distribution of IGKV2D-29 and IGKV2-29 alleles among Swedish Caucasians and Hong Kong Chinese. This may help to explain differences in the occurrence of H. influenzae type b infection in the two populations. Evaluated methods for IGKV2D-29 and IGKV2-29 allele detection can be used for the screening allele polymorphisms in other particular patient groups.

Alleles↗
PubMed1998

Cloning and thermostability of TaqI endonuclease isoschizomers from Thermus species SM32 and Thermus filiformis Tok6A1.

Two TaqI endonuclease (hereafter referred to as TaqI) isoschizomer genes, tsp32IR from Thermus species SM32 of Azores and tfiTok6A1I from T. filiformis Tok6A1 of New Zealand, were cloned in Escherichia coli. The overexpressed enzymes were partly purified and their thermostability was determined. In the medium-salt buffer, Tsp32IR, TfiTok6A1I and one previously cloned TaqI isoschizomer (TthHB8I) were more thermostable than TaqI. Tsp32IR remained partly active up to 90 degreesC in the low-salt buffer. Six amino acid residues that are identical in the three high thermostability isoschizomers (Tsp32IR, TfiTok6A1I and TthHB8I) but differ in TaqI might provide added rigidity for thermostabilization. These include four proline residues located in or near loop regions, and one alanine and one arginine located at helix regions in the predicted TaqI endonuclease secondary structure. The possible role of these residues in thermostabilization was evaluated by mutagenizing the TaqI enzyme. Mutants generated at these six positions were less thermostable than wild-type TaqI. The results suggest that the surrounding sequence or structural context might be as important as the mutation itself.

Amino Acid Sequence↗
PubMed1998

BstB7SI (R decreases CCGGY), a thermostable isoschizomer of Cfr10I, from a strain of Bacillus stearothermophilus isolated from oil-contaminated soil in Kuwait.

Isolates of thermophilic bacteria from desert soil in Kuwait, heavily contaminated with crude oil, have been screened for the presence of restriction endonuclease activity. One of the isolates (B7S), identified as Bacillus stearothermophilus, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda bacteriophage DNA at 65 degrees C. A type II restriction endonuclease (BstB7SI) has been partially purified from this isolate. BstB7SI recognises the six-base sequence RCCGGY (R = A or G; Y = T or C) and hydrolyses the phosphodiester bond in both strands of the DNA substrate between the first and second bases of the recognition sequence 5'-R decreases CCGGY-3'producing four-base 5' overhangs. BstB7SI is therefore an isoschizomer of the mesophilic prototype restriction endonuclease Cfr10I. BstB7SI has a pH optimum of 9.7, requires 10 mM MgCl2 and 75 mM NaCl for maximum activity, and retains full enzyme activity when incubated for 5 min at temperatures up to 70 degrees C.

Base Sequence↗
PubMed1998

Progesterone 6-hydroxylation is catalysed by cytochrome P-450 in the moderate thermophile Bacillus thermoglucosidasius strain 12060.

The moderate thermophile, Bacillus thermoglucosidasius, transforms progesterone into four metabolites. These are 6alpha- and 6beta-hydroxyprogesterone, androstenedione and testosterone. This is the first report of bacterial 6alpha-hydroxylation of steroids. The identity of the progesterone metabolites shows that there are three major types of transforming activity in this organism; C-17-C-20 lyase that cleaves the pregnane side chain of the substrate, C-17 oxidoreductase that interconverts the metabolites androstenedione and testosterone, and 6-hydroxylation. 6-hydroxylation activity was purified virtually to homogeneity and was shown to be catalysed by a cytochrome P-450 monooxygenase enzyme. This is the first report of a thermostable cytochrome P-450.

Bacillus↗
PubMed1996

Tsp49I (ACGT/), a thermostable neoschizomer of the Type II restriction endonuclease MaeII (A/CGT), discovered in isolates of the genus Thermus from the Azores, Iceland and New Zealand.

One hundred and forty eight isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (SM49) from the island of Sao Miguel, in the Azores, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Tsp49I) has been partially purified from this isolate and the recognition and cleavage site determined. Tsp49I recognizes the four base sequence ACGT, which is the same as the recognition sequence of the mesophilic Type II restriction endonuclease MaeII. However, unlike MaeII, which cleaves DNA between the first and second bass of the recognition sequence (A/CGT), Tsp49I hydrolyses the phosphodiester bond in both strands of the substrate after the last base of the recognition sequence 5'-ACGT/-3', producing four base 3'-OH overhangs (sticky ends). The enzyme has a pH optimum of 9.0, requires 2 mM MgCl2 for maximum activity and retains full enzyme activity following incubation for 10 min at temperatures up to 8O degrees C. Two further examples of the same restriction endonuclease specificity as Tsp491 were detected in Thermus isolates from Iceland (TspIDSI) and New Zealand (TspWAM8AI). The three MaeII neoschizomers, Tsp49I, TspIDSI and TspWAM8AI, exhibit similar pH optima, heat stabilities and MgCl2 requirements, but differ in their requirements for NaCl and KCl.

Bacteriophage lambda↗
PubMed1996

Thermus oshimai sp. nov., isolated from hot springs in Portugal, Iceland, and the Azores, and comment on the concept of a limited geographical distribution of Thermus species.

We examined aerobic, thermophilic, gram-negative bacteria that were isolated from hot springs in Portugal and were identified as Thermus strains and placed in phenetic groups on the basis of their phenotypic characteristics. We determined the composition of the peptidoglycan, identified the respiratory quinones, and determined the mean base composition of the DNA, and the levels of DNA-DNA homology were determined by both the filter hybridization and reassociation rate methods. Thermus aquaticus, Thermus brockianus, and Thermus filiformis were not detected in this collection of organisms, although three Thermus thermophilus strains were identified. We propose that the isolates that belonged to phenetic clusters E and F are members of a new species, Thermus oshimai; the type strain of T. oshimai is strain SPS17.

Azores↗
PubMed1995

Two different isoschizomers of the type-II restriction endonuclease Taq I (T/CGA) within the same Thermus isolate: Tsp32 I, an enzyme with similar heat stability properties to the prototype enzyme Taq I, and Tsp32 II, a hyperthermostable isoschizomer of Taq I.

We have recently screened 112 separate isolates of the genus Thermus, collected from neutral and alkaline hot water springs on four continents, for the presence of the Type-II restriction endonuclease Taq I (T/CGA). One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recognition and cleavage site as Taq I. Initial studies revealed that the partially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Taq I, being able to withstand temperatures at least 10 degrees C higher than Taq I, before showing evidence of heat inactivation. Subsequently it became clear that the partially purified extract from strain 32 contains two separate enzymes, both of which are isoschizomers of Taq I. One of the enzymes, Tsp32 I, has similar thermal stability characteristics to Taq I, whereas the second Taq I isoschizomer, Tsp32 II, found in the same Thermus isolate as Tsp32 I, is considerably more thermostable than Taq I, retaining full enzyme activity up to a temperature of 85 degrees C. Tsp32 I and Tsp32 II were further distinguished by virtue of their different requirements for magnesium ions.

Base Sequence↗
PubMed1995

Taq52 I, a novel and thermostable type II restriction endonuclease from the genus Thermus, recognising the pentanucleotide sequence GC(A or T)GC and cleaving DNA between the first and second bases of the recognition sequence: G decreased or reduced C(A or T)GC.

127 isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (YS52) from Yellowstone National Park, USA, showed a high level of restriction endonuclease activity when a cell free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Taq52 I) has been partially purified from this isolate and the recognition and cleavage site determined. Taq52 I has a novel interrupted palindromic tetranucleotide recognition sequence GCWGC, where W can be either adenine (A) or thymine (T). It hydrolyses the phosphodiester bond in both strands of the substrate between the first and second bases of the recognition sequence: 5'G decreased or reduced CWGC3', producing three-base 5'-OH overhangs (sticky ends). The enzyme has a pH optimum of 7.0, requires 8 mM MgCl2 for maximum activity and is thermally stable, retaining full enzyme activity following incubation at 79 degrees C for 10 min. Taq52 I not only represents a new addition to the Type II restriction endonucleases, but also increases the small list of thermostable restriction endonucleases.

Bacteriophage lambda↗
PubMed1995

Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC).

Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3'-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3'-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.

Base Sequence↗
PubMed1995

DNA relatedness of Thermus strains, description of Thermus brockianus sp. nov., and proposal to reestablish Thermus thermophilus (Oshima and Imahori)

Aerobic, thermophilic, gram-negative bacteria obtained from Yellowstone National Park that were placed in the genus Thermus on the basis of phenotypic data were examined by chemotaxonomic techniques to determine their peptidoglycan compositions, their respiratory quinones, their mean DNA base compositions, and their levels of DNA-DNA homology as determined by both the filter hybridization and reassociation rate methods. These isolates from hot springs included Thermus aquaticus strains and strains of a new genospecies. We propose the name Thermus brockianus for this new genospecies; strain YS38 is the type strain of this taxon. A collection of 10 strains, including the type strain of "Thermus thermophilus", which were isolated from widely separated geothermal sites, exhibited high levels of DNA-DNA homology with each other and had similar physiological properties. Therefore, we propose that the species Thermus thermophilus (Oshima and Imahori) should be reestablished, with strain HB8 as the type strain.

Base Composition↗
PubMed1986

Carrier-mediated efflux of ketone bodies in isolated rat hepatocytes.

The rate of efflux of ketone bodies has been studied in isolated hepatocytes prepared from starved rats and preloaded with D-3-[14C]hydroxybutyrate. Efflux of ketone bodies was temperature-dependent, saturable and inhibited by alpha-cyano-3-hydroxycinnamate and phloretin. The rate of efflux was also reduced by 6 mmol/l lactate and pyruvate added to the external medium. Under conditions of simulated metabolic acidosis in the hepatocyte suspension medium, ketone body efflux rate was reduced. The experimental data suggest that hepatic plasma membrane ketone body transit is carrier-mediated.

3-Hydroxybutyric Acid↗
PubMed1986

Inhibition of lactate removal by ketone bodies in rat liver. Evidence for a quantitatively important role of the plasma membrane lactate transporter in lactate metabolism.

We studied the effect of DL-3-hydroxybutyrate and acetoacetate on lactate transport into isolated hepatocytes and on lactate removal in the isolated perfused rat liver. Ketone bodies inhibited lactate transport into isolated hepatocytes (maximum, 35% at concentrations of 10-20 mM). Lactate removal and glucose production by perfused livers were examined before and after the introduction of a constant infusion of hydroxybutyrate, acetoacetate, or appropriate control into the portal venous limb. Lactate removal was significantly inhibited within 10 s of the appearance of increasing concentrations of ketone bodies in the effluent. Corresponding decreases in glucose production were observed. The dependence of inhibition on D-3-hydroxybutyrate concentration was documented in isolated perfused livers (maximum inhibition of lactate removal, 58% at 14 mM). This phenomenon could be a factor in the development of lactic acidosis accompanying ketoacidosis, and indicates that plasma membrane lactate transport may determine the rate of hepatic lactate removal.

3-Hydroxybutyric Acid↗
PubMed1984

L(+)-Lactate binding to preparations of rat hepatocyte plasma membranes.

Incubation of rat hepatocyte plasma membranes with L-[14C]lactate resulted in the labeling of protein(s) of apparent molecular weight 40,000 when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The binding was saturable, irreversible, and inhibited by pyruvate, 2-oxoglutarate, and alpha-cyano-3-hydroxycinnamate, but not by D-lactate. It was markedly enhanced by L-alanine, but not D-alanine or beta-alanine. The binding protein(s) could be solubilized in cholic acid giving a single peak on gel filtration corresponding to a molecular weight of 26,000 and an isoelectric point of 5.1. This peak, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ran in a position corresponding to an apparent molecular weight of 40,000. When membranes were treated with Triton X-100, lactate binding was retained by the Triton-insoluble fraction. The binding of L-[14C]lactate increased with incubation time, due apparently to the appearance of new binding sites and not to sequestration into vesicles. As many of the characteristics of lactate binding to rat hepatocyte plasma membranes were found to be similar to those of lactate entry into isolated hepatocytes, we speculate that the lactate-binding protein could represent part or whole of a plasma-membrane lactate transporter. Lactate-binding proteins of the same molecular weight were identified in the plasma membranes from rat erythrocytes, cardiac muscle, skeletal muscle, lung, and brain.

Animals↗
PubMed1983

A collaborative study of the measurement of glycosylated haemoglobin by several methods in seven laboratories in the United Kingdom.

A collaborative study compared methods for measuring glycosylated haemoglobin in seven laboratories in the United Kingdom. No satisfactory standard for general use was found. Satisfactory internal quality control systems were in use for each assay which allowed the maintenance of a normal range in each participating laboratory. No satisfactory quality control system suitable for general use could be identified. Costs and convenience of the assays are reported. The technical problems with each type of assay and precautions for their use were identified, such as the need for standardization in incubation times, the practicability of automation of colorimetric assays, and the precision of pH needed for buffers in column separation methods. The relevance of the technical problems to interpretation of measurements is also considered. It is concluded that laboratories measuring glycosylated haemoglobin should maintain a normal range, use 'in-house' quality controls to monitor assay performance and keep clinical colleagues informed of the findings and of any changes in methodology that might affect the interpretation of results.

Autoanalysis↗