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Biomedical subjects

K C Smith

Publications and source records attributed to K C Smith.

At least 91 records · Page 5Linked to original sources
PubMed1994

Distribution of equid herpesvirus-1 (EHV-1) in respiratory tract associated lymphoid tissue: implications for cellular immunity.

Twelve adult ponies and 2 conventional foals were exposed intranasally to EHV-1, strain Ab4 (TCID50 10(-6.6) and samples of respiratory tract associated lymphoid tissues were recovered between 12 h and 13 days after infection. Infectious virus was detected in tissue homogenates using susceptible cell monolayers and expression of viral antigens was monitored using indirect immunoperoxidase histochemistry on paraffin sections. The results showed both infectious EHV-1 and viral antigens in respiratory tract associated lymph nodes 12 h after exposure. Infected leucocytes were identified morphologically as lymphocytes, monocytes, macrophages and plasma cells. The rapid intracellular localisation of EHV-1 in lymph nodes implies that cell mediated immunity is an important aspect of the equine response to this virus.

Animals↗
PubMed1993

Replication of equid herpesvirus-1 in the vaginal tunics of colts following local inoculation.

Equid herpesvirus-1 (EHV-1; Ab4 isolate) was inoculated unilaterally into the cavum vaginale of four pony colts under general anaesthesia. The animals were monitored daily for evidence of scrotal or testicular swelling and euthanased electively on days 3, 4, 6 and 12 after infection. Detailed pathological examination of the male genital tract was carried out. In animals examined at days 3 and 4 after infection, replication of EHV-1 was detected bilaterally in mesothelial and endothelial cells of the parietal and visceral vaginal tunics. The mesothelial infection had resolved by day 12 after infection, with no evidence of direct extension to deeper genital organs. None of the four colts showed significant scrotal or testicular swelling.

Animals↗
PubMed1993

The identification of 5-hydroxy-L-norvaline in cultures of pyridoxine auxotrophs of Escherichia coli B.

Gc-ms and hplc have been employed to identify 5-hydroxy-L-norvaline (L-2-amino-5-hydroxypentanoic acid) in the culture fluids of pyridoxine-starved cultures of two pyridoxine auxotrophs of Escherichia coli B. The production of 5-hydroxynorvaline is shown to be secondary to pyridoxine starvation of the cultures, and the quantities of 5-hydroxynorvaline are similar to those of the common protein amino acids present in these cultures. This is the first time that 5-hydroxynorvaline has been identified as a product of bacterial metabolism.

Amino Acids↗
PubMed1993

An immunohistological study of the uterus of mares following experimental infection by equid herpesvirus 1.

Twelve Welsh Mountain pony mares in late gestation were infected intranasally with EHV-1 (AB4 isolate) at dose rates from 10(3) to 10(7.3) TCID50. This resulted in 3 cases of paresis, at Days 9, 10 and 12 after inoculation, and 5 abortions, at Days 6, 9, 18, 19 and 20. Euthanasia was performed between Days 6 and 21, with collection of uterine specimens for histopathology, virus isolation and immunoperoxidase staining from the pregnant horn, non-pregnant horn and body. EHV-1 replication in endometrial vessels was detected as early as Day 6 and was maximal at Days 9-11, when widespread thromboischaemic damage was present. By Days 15-19 in mares remaining pregnant, EHV-1 antigen expression in the endometrium was sparse, despite residual lesions but little associated thrombosis. Endometrial vascular pathology varied considerably in degree and extent, and no consistent predilection sites for replication within the uterus were apparent.

Abortion, Veterinary↗
PubMed1993

Nutrient effects on growth and the production of 5-hydroxy-4-oxonorvaline by Streptomyces akiyoshiensis.

5-Hydroxy-4-oxonorvaline (HON) was produced optimally by Streptomyces akiyoshiensis in media containing starch and casein supplemented with high concentrations of phosphate and magnesium sulfate; cultures grown at initial pH values between 6.3 and 6.6 yielded HON titres of 13-14 mM. Physiological analysis of HON production provided evidence that the excess inorganic constituents in this medium played an important role in optimizing production. In simple defined media buffered at pH 5.5-6.0 and inoculated with either a spore suspension or washed vegetative mycelium, formation of HON was less than one-third the amount produced in the starch-casein-salts medium. Production was markedly affected by the carbon and nitrogen sources used; media containing starch and potassium nitrate or aspartate supported the highest yields of HON. In starch-nitrate media, production was suppressed by excess nitrogen source, and the HON titre decreased 3-fold as the phosphate content was increased from 0.5 to 5 mM; growth was not appreciably altered. The results suggest that the high level of HON production in the complex starch-casein-salts medium is associated with sequestration of metabolic ammonium and reduced availability of phosphate through formation of the poorly soluble magnesium ammonium phosphate.

Aminolevulinic Acid↗
PubMed1992

Modeling the anti-CEA antibody combining site by homology and conformational search.

A model for an antibody specific for the carcinoembryonic antigen (CEA) has been constructed using a method which combines the concept of canonical structures with conformational search. A conformational search technique is introduced which couples random generation of backbone loop conformations to a simulated annealing method for assigning side chain conformations. This technique was used both to verify conformations selected from the set of known canonical structures and to explore conformations available to the H3 loop in CEA ab initio. Canonical structures are not available for H3 due to its variability in length, sequence, and observed conformation in known antibody structures. Analysis of the results of conformational search resulted in three equally probable conformations for H3 loop in CEA. Force field energies, solvation free energies, exposure of charged residues and burial of hydrophobic residues, and packing of hydrophobic residues at the base of the loop were used as selection criteria. The existence of three equally plausible structures may reflect the high degree of flexibility expected for an exposed loop of this length. The nature of the combining site and features which could be important to interaction with antigen are discussed.

Amino Acid Sequence↗
PubMed1992

Involvement of RecB-mediated (but not RecF-mediated) repair of DNA double-strand breaks in the gamma-radiation production of long deletions in Escherichia coli.

Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12. Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear. Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts. In addition, about 80% of the spontaneous deletions were absent in the recB21 strain. A recC1001 mutation, which confers a 'hyper-Rec' phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts. A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts. A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions. Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations. However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions. A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.

Bacterial Proteins↗
PubMed1992

Properties of R-plasmid pEB017, which confers both enhanced UV-radiation resistance and mutability to wild-type, recA and umuC strains of Escherichia coli K12.

The R-plasmid, pEB017, restored recombination ability to recA56 and conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability to wild-type, recA56 and umuC36 strains of Escherichia coli K12. Comparatively, pEB017 enhanced UV-radiation mutability in a umuC strain, and also enhanced UV-radiation and nitrofuran mutability in a wild-type strain several-fold more than did another R-plasmid, pKM101. Plasmid pEB017 also mediated about a 3-fold enhancement of the SOS induction of beta-galactosidase synthesis in a recA strain, compared with the normal recA+ gene of E. coli. A BamHI fragment of pEB017 DNA was cloned into plasmid vector pBR322 to yield pEB021. The BamHI fragment in pEB021 (3.5 kb) is about 170 bp longer between the BamHI and PstI sites on the left end of the recA-like fragment, compared with published data on a similarly cloned recA gene from E. coli. Plasmid pEB021 conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability in wild-type and recA strains, and restored recombination ability in a recA strain. The introduction of pEB021 into a umuC strain made the cells slightly more resistant to killing by UV-irradiation, and promoted a small amount of UV-mutability in an otherwise nonmutable strain. These results suggest that R-plasmid pEB017 has a recA-like gene that mediates the enhanced resistance to UV-radiation and enhanced UV-radiation mutability, but which seems different in several important aspects from the normal recA gene in E. coli.

Cloning, Molecular↗
PubMed1992

Spontaneous mutagenesis: experimental, genetic and other factors.

Spontaneous mutations are "the net result of all that can go wrong with DNA during the life cycle of an organism" (Glickman et al., 1986). Thus, the types and amounts of spontaneous mutations produced are the resultant of all the cellular processes that are mutagenic and those that are antimutagenic. It is not widely appreciated that the types and frequencies of spontaneous mutations change markedly with subtle changes in experimental conditions. All types of mutations are produced spontaneously, i.e., base substitutions, frameshifts, insertions and deletions. However, very few papers have appeared that are devoted exclusively to the study of the mechanisms of spontaneous mutagenesis, and of the subtle experimental factors that affect the types and frequencies of spontaneous mutations. This is unfortunate because spontaneous mutagenesis appears to play a major role in evolution, aging, and carcinogenesis. This review emphasizes subtle experimental variables that markedly affect the results of a spontaneous mutation experiment. A thorough understanding of these variables eliminates the need for a theory of "directed" mutagenesis. The intrinsic instability of DNA, and the types of normal metabolic lesions that are produced in DNA that lead to mutations via errors made in replication, repair, and recombination are reviewed, as is the genetic control of spontaneous mutagenesis. As with spontaneous mutagenesis, spontaneous carcinogenesis can also be considered to be the net result of all that can go wrong with DNA during the life of an organism.

Cell Transformation, Neoplastic↗
PubMed1992

An immunoperoxidase method applied to the diagnosis of equine herpesvirus abortion, using conventional and rapid microwave techniques.

An indirect immunoperoxidase (IMP) technique was applied to cryostat and paraffin sections of liver from ten aborted equine foetuses. Equid herpesvirus type 1 (EHV-1) had been isolated from seven of them and EHV-4 from one: the remaining two were virologically negative and were not used as controls. In the eight virus-infected cases the immunostaining revealed foci of cells exhibiting a distinct brown cytoplasmic and inclusion body pigmentation. No specific signal was present in the non-infected controls. The method also was adapted for incubation in a microwave oven, which allowed the total length of the procedure to be reduced to a minimum of 35 mins for cryostat sections, without significant loss of signal.

Abortion, Veterinary↗
PubMed1992

Abortion of virologically negative foetuses following experimental challenge of pregnant pony mares with equid herpesvirus 1.

From 1988 to 1991, 51 pregnant pony mares were challenged intranasally or by aerosol with an isolate of EHV-1 (AB4) originally recovered from a quadriplegic mare. This resulted in 32 abortions, occurring from 9 to 29 days after infection. In 14 of the early abortions (Days 9-14), EHV-1 was not demonstrated in the foetal tissues by virus isolation or immunostaining despite no other non-viral cause for the abortion being evident. Application of the polymerase chain reaction to foetal tissues from 9 of these cases also proved negative. One of the 14 mares was destroyed immediately after abortion, and post-mortem examination revealed severe and widespread vasculitis, thrombosis and secondary ischaemic damage in the endometrium with replication of EHV-1 in endothelial cells. These findings suggest that EHV-1 abortion can occur due to endometrial damage without the establishment of a foetal infection.

Abortion, Veterinary↗
PubMed1990

UV photoreceptors in the compound eye of Daphnia magna (Crustacea, Branchiopoda). A fourth spectral class in single ommatidia.

The spectral sensitivities of individually stimulated ommatidia in the compound eye of Daphnia magna were measured using a fast spectral scan voltage-clamp technique with extracellular recording. Chromatic adaptation was used to reveal the contributions of individual spectral classes of photoreceptors to the ommatidial sensitivity. Ommatidia in the dorsal and ventral regions of the compound eye were tested. Four spectral classes of photoreceptors were found in each ommatidium, among them a previously undetected class with peak sensitivity in the ultraviolet. The wavelengths of peak sensitivity were at 348, 434, 525, and 608 nm for the dorsal ommatidia. The three longer wavelength classes agreed well with those found previously by intracellular recording (Schehr 1984). Only small differences in wavelength and magnitude of peak sensitivity were found between the four classes in the dorsal versus ventral ommatidia.

Animals↗
PubMed1989

Discontinuous DNA replication in a lig-7 strain of Escherichia coli is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil.

After pulse-labeling with 3H-thymidine for 30 s at 42 degrees C, the newly-synthesized DNA from uvrB5 lig-7, uvrB5 lig-7 ung-1 (or ung152), uvrB5 lig-7 mutL218 (or mutS215), and uvrB5 lig-7 ung-1 mutL218 (or mutS215) cells sedimented very slowly in alkaline sucrose gradients. The bulk of these DNA molecules were smaller than 2,000 nucleotides long (i.e., about the size of Okazaki fragments), and none of the 3H-radioactivity was found to sediment as high-molecular-weight DNA. These results indicate that the apparent discontinuous DNA replication observed in lig-7 strains is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil.

DNA Repair↗
PubMed1989

recA-dependent DNA repair processes.

UV-radiation-induced lesions in DNA result in the formation of: (1) excision gaps (i.e. a lesion is excised, leaving a gap), (2) daughter-strand gaps (i.e. a lesion can be skipped during replication, leaving a gap), and (3) double-strand breaks (i.e. the DNA strand opposite a gap can be cut). In Escherichia coli, the recA gene product is involved in repairs of all three types of lesions--repair of daughter-strand gaps (2) and double-strand breaks (3) constitutes post-replication repair. The evidence suggests, furthermore, that recA-dependent repair of excision gaps (1) produced in DNA replicated prior to UV irradiation (pre-replication repair) appears to occur by similar mechanisms.

DNA Repair↗
PubMed1989

Articular cartilage and intervertebral disc proteoglycans differ in structure: an electron microscopic study.

Articular cartilage and the intervertebral disc tissues have different material and biological properties and different patterns of aging and degeneration. To determine if the proteoglycans of these tissues differ in structure, we used the electron microscopic monolayer technique to compare baboon articular cartilage proteoglycans with baboon annulus fibrosus, transition zone, and nucleus pulposus proteoglycans. Intervertebral disc and articular cartilage proteoglycans differed significantly. Articular cartilage contained large proteoglycan aggregates formed from hyaluronic acid central filaments, multiple monomers, and large nonaggregated monomers. These molecules were identical to those of nasal cartilage, growth plate cartilage, chondrosarcomas, or menisci. In contrast, the intervertebral disc tissues contained only nonaggregated proteoglycan monomers and clusters of monomers without apparent central filaments. Intervertebral disc nonaggregated monomers were shorter and more variable in length than those from articular cartilage, and nucleus pulposus nonaggregated monomers were even shorter and more variable in length than transition zone and annulus fibrosus monomers. These observations suggest that significant differences in proteoglycan metabolism exist between articular cartilage and intervertebral disc.

Animals↗
PubMed1989

The roles of RecBCD, Ssb and RecA proteins in the formation of heteroduplexes from linear-duplex DNA in vitro.

The formation of heteroduplexes from linear duplex DNA, where one molecule possesses a DNA double-strand break, was assayed by agarose gel electrophoresis. Using unlabeled whole-length linear duplex DNA and 3H-labeled half-length linear duplex DNA (obtained from plasmid pACYC184), the appearance of 3H-labeled DNA that migrated as whole-length linear DNA was taken as evidence for formation of heteroduplex DNA. When the DNA mixtures were incubated with RecA, RecBCD, or Ssb proteins, or any double or triple combination of these proteins under a variety of reaction conditions, no heteroduplex DNA was detected. However, heteroduplex DNA was detected when the DNA mixtures were first incubated briefly with the RecBCD and Ssb proteins under reaction conditions that allow unwinding to proceed, and then the MgCl2 concentration was raised such that renaturation could proceed. The inclusion of the RecBCD and Ssb proteins was sufficient to catalyze the slow formation of heteroduplex DNA, but the presence of RecA protein greatly increased the kinetics. The roles of the RecBCD, Ssb and RecA proteins in heteroduplex formation in vitro are discussed.

DNA Repair↗