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G K Smith

Publications and source records attributed to G K Smith.

At least 37 records · Page 2Linked to original sources
PubMed1997

Within- and between-examiner repeatability of distraction indices of the hip joints in dogs.

OBJECTIVE: To evaluate in vivo repeatability of the distraction index method of evaluating hip joint laxity in dogs. ANIMALS: 31 two-year-old Labrador Retrievers. PROCEDURE: Each dog was anesthetized and radiographically evaluated for hip joint laxity 4 times: twice by an experienced examiner and twice by an examiner who had no previous knowledge of or training in the technique prior to the first day of testing. Distraction indices (DI) were determined from the radiographs and intraclass correlation coefficients were calculated to evaluate the repeatability of DI measurements between and within examiners. RESULTS: Intraclass correlation coefficients were high (range, 0.85 to 0.94). Lower limits of the 95% confidence intervals for the intraclass correlation coefficients ranged from 0.75 to 0.89. CONCLUSIONS: Between- and within-examiner repeatabilities of DI measurements were high, suggesting that the technique is clinically reliable. CLINICAL RELEVANCE: Distraction index is a reliable measure of hip joint laxity and a good predictor of the risk of development of degenerative joint disease associated with hip dysplasia in dogs. Establishment of high repeatability of DI measurements suggests that the stress-radiographic method may be used by multiple examiners with the expectation of comparable and consistent results.

Acetabulum↗
PubMed1997

Biomechanical characterization of passive laxity of the hip joint in dogs.

OBJECTIVE: To investigate the in vitro load/displacement characteristics of the hip joints in dogs as a function of joint position. SAMPLE POPULATION: 10 hip joints from 5 healthy dogs. PROCEDURE: A material test system was used to generate load/displacement curves for each joint. Joints were mounted in a custom-designed jig that held the joint in fixed anatomic orientations while plotting displacement and corresponding applied loads. All hips were cycled between 40 N of compression and 80 N of distraction. Each hip was tested at 10 degrees increments from 30 degrees flexion to 70 degrees extension. RESULTS: When the hips were in a neutral orientation (approximately a standing position), load/displacement curves were characteristically sigmoidal (tri-phasic), indicating that, in this position, displacement was not highly dependent on load. The curves had a central low-stiffness region in which most of the lateral displacement took place. In contrast, when hips were positioned at the extremes of flexion and extension, this central, low-stiffness region was less distinct, and load/displacement curves were more linear, indicating a proportional relation between load and displacement. The load/displacement curve of 1 hip joint in the study deviated markedly from the others in a pattern consistent with cavitation of the synovial fluid. CONCLUSIONS: When the hip joint is positioned in a neutral position, load-displacement behavior is sigmoidal, whereas when the hip joint is in an extended position, load/displacement behavior is more linear. CLINICAL RELEVANCE: Establishing load/displacement behavior of the hip joints in dogs was an important exercise in establishing the position for and estimating the repeatability of a clinical stress-radiographic method for quantitating joint laxity in dogs.

Animals↗
PubMed1996

Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies.

A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Genetically unstable CXG repeats are structurally dynamic and have a high propensity for folding. An NMR and UV spectroscopic study.

Recent molecular genetics studies have revealed a correlation between spontaneous, progressive expansion of several DNA trinucleotide repeats and certain hereditary neurodegenerative diseases. Triplet repeat (TR) sequences may be present in structured forms that can mediate the processes interrupting normal cellular replication, transcription, or repair activities, eventually leading to gene mutation. Using high resolution NMR spectroscopy and other biophysical methods, we probed the solution structures and properties of single-stranded TR sequences. These studies have led to the discovery of a new duplex motif (e-motif), present in CCG repeats, and to the elucidation of the structure of the (CTG)3 duplex. In this paper we provide a global picture of the solution behavior of the human disease-related CXG (X = A, C, G, or T) and the comparison GXC (X = A, or T) TR sequences. All six triplet repeats form antiparallel duplexes. The mismatched bases in CAG and CGG repeat duplexes are rather flexible and they do not appear to form stable, paired conformations. By comparison, GAC repeat duplexes and their mismatched A residues are well-structured. Most interestingly, the structures of the disease-related CXG repeats exhibit a propensity for folding at chain lengths as short as 12 residues. Furthermore, the energy barrier for the formation of homo-duplexes from the corresponding complementary hetero-duplexes are much lower for the CXG TR sequences than for the GAC or GTC TR sequences. These results provide insights into the conformation and physiochemical properties of TR sequences. Thus, a basis is provided for further studies of the behavior of long TR sequences in an effort to elucidate the molecular mechanisms of in vivo expansion and function of TR sequences.

Base Composition↗
PubMed1996

Glucocorticoids regulate inducible nitric oxide synthase by inhibiting tetrahydrobiopterin synthesis and L-arginine transport.

The cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) plays an important role in the immune response to some pathogens. Within the heart, increased activity of NOS2 in cardiac microvascular endothelial cells (CMEC) also can diminish the contractile function of adjacent cardiac myocytes. Glucocorticoids, which are known to suppress cytokine induction of NOS2 in many cell types, caused only a moderate (approximately 20%) decline in NOS2 protein content and maximal activity measured in homogenates of cytokine-treated CMEC, but almost completely inhibited synthesis of nitrogen oxides (NOx) by intact cells. To determine whether glucocorticoids were inhibiting cellular NOx production by limiting the availability of NOS co-factors or substrate, the effect of dexamethasone on tetrahydrobiopterin (BH4) and L-arginine availability in cytokine-treated CMEC was examined. Dexamethasone prevented the coordinate induction of GTP cyclohydrolase I with NOS2 after exposure to interleukin-1beta and interferon-gamma and also the increase in intracellular BH4 content in cytokine-treated CMEC. Addition of BH4 overcame dexamethasone-mediated suppression of nitrite production. Dexamethasone also prevented a cytokine-mediated increase in L-arginine uptake into CMEC by suppressing the induction of the high affinity cationic amino acid transporters CAT-1 and CAT-2B and the low affinity CAT-2A transporter. In addition, dexamethasone also inhibited cytokine induction in CMEC of argininosuccinate synthase, the rate-limiting enzyme for the de novo synthesis of arginine from citrulline. Thus, glucocorticoids regulate NOx production following cytokine exposure in cardiac microvascular endothelial cells primarily by limiting BH4 and L-arginine availability.

Animals↗
PubMed1996

Expression and characterization of human pancreatic preprocarboxypeptidase A1 and preprocarboxypeptidase A2.

We are investigating the potential utility of human carboxypeptidases A in antibody-directed enzyme prodrug therapy (ADEPT). Hybridization screening of a human pancreatic cDNA library with cDNA probes that encoded either rat carboxypeptidase A1 (rCPA1) or carboxypeptidase A2 (rCPA2) was used to clone the human prepro-CPA homologs. After expression of the respective pro-hCPA cDNA in Saccharomyces cerevisiae, the enzymes were purified to homogeneity by a combination of hydrophobic and ion-exchange chromatography. Purified hCPA1 and hCPA2 migrate as a single protein band with M(r) 34,000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Kinetic studies of the purified enzymes with hippuryl-L-phenylalanine resulted in kcat/Km values of 57,000 and 19,000 M-1 s-1 for hCPA1 and hCPA2, respectively. Using the ester substrate, hippuryl-D, L-phenyllactate, we found unique esterase/ peptidase specific activity ratios among hCPA1, hCPA2, rCPA1, and bovine CPA (bCPA) ranging from 13 to 325. Two potential ADEPT substrates, methotrexate-alpha-phenylalanine (MTX-Phe) and methotrexate-alpha-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. The kcat/Km values for MTX-Phe were 440,000 and 90,000 M-1 s-1 for hCPA1 and hCPA2, respectively, and for MTX-naphthylAla these values were 1400 and 1,400,000 M-1 s-1 for hCPA1 and hCPA2, respectively. The kinetic data show that hCPA2 has a larger substrate binding site than the hCPA1 enzyme. Differences between hCPA1 and hCPA2 were also observed in thermal stability experiments at 60 degrees C where the half-life for thermal denaturation of hCPA2 is eightfold longer than that for hCPA1. These experiments indicate that hCPA1 and hCPA2 are potential candidates for use in a human-based ADEPT approach.

Amino Acid Sequence↗
PubMed1996

Diagnostic probes targeting the major sperm protein gene that may be useful in the molecular identification of nematodes.

Discrimination of closely related nematode species is typically problematic when traditional identification characteristics are prone to intraspecific variation. In this study, a molecular approach that can distinguish Pratylenchus penetrans and P. scribneri is described. The approach uses universal primers in conjunction with polymerase chain reaction (PCR) to amplify equivalent fragments of the major sperm protein (msp) gene from any nematode. This gene fragment typically includes an intron of variable sequence. The presence of this highly variable segment in an otherwise conserved gene sequence allows P. penetrans and P. scribneri to be distinguished by either a species-specific amplification or by dot-blot hybridization. The approach is potentially of general utility in species-specific identification of nematodes.

Animals↗
PubMed1995

Enhanced antitumor activity for the thymidylate synthase inhibitor 1843U89 through decreased host toxicity with oral folic acid.

The purpose of this investigation was to determine whether antitumor selectivity of the third generation thymidylate synthase inhibitor 1843U89 could be enhanced by a combination of the drug with folic acid. The effects of folic acid on toxicity of 1843U89 to the dog and mouse and on antitumor efficacy of 1843U89 in the mouse were studied. These data were compared to the effect of folic acid on the in vitro cell culture antitumor activity of 1843U89. The sensitivity of eight cancer cell lines (three ovarian, one colon, one ileocecal, one epidermoid, one osteosarcoma, and one breast line) to 1843U89 was tested in vitro in the presence and absence of folic acid. Folic acid concentrations greater than 100 microM were required to decrease 1843U89 activity in seven of the cell lines. Only the activity in HCT-8, the ileocecal line, was reserved at folic acid concentrations below 100 microM. Oral folic acid given 30 min prior to an i.v. dose of 1843U89 increased the maximally tolerated dose and the lethal dose of 1843U89, both in dogs and in thymidine-depleted mice. In mice, oral folic acid produced little or no effect upon the antitumor efficacy of 1843U89 in two of three tumor cell lines in vivo. HCT-8, the line that was sensitive to folate reversal in vitro, was also sensitive in vivo. The results show that an oral dose of folic acid 30 min prior to i.v. 1843U89 can block mouse and dog intestinal toxicity without decreasing efficacy of 1843U89 in two of three human tumor lines in the nude mouse. Thus, the data reported here indicate that the antitumor selectivity of 1843U89 may be enhanced through a combination of 1843U89 with oral folic acid.

Animals↗
PubMed1995

DNA CTG triplet repeats involved in dynamic mutations of neurologically related gene sequences form stable duplexes.

DNA triplet repeats, 5'-d(CTG)n and 5'-d(CAG)n, are present in genes which have been implicated in several neurodegenerative disorders. To investigate possible stable structures formed by these repeating sequences, we have examined d(CTG)n, d(CAG)n and d(CTG).d(CAG)n (n = 2 and 3) using NMR and UV optical spectroscopy. These studies reveal that single stranded (CTG)n (n > 2) forms stable, antiparallel helical duplexes, while the single stranded (CAG)n requires at least three repeating units to form a duplex. NMR and UV melting experiments show that the Tm increases in the order of [(CAG)3]2 < [(CTG)3]2 << (CAG)3.(CTG)3. The (CTG)3 duplex is stable and exhibits similar NMR spectra in solutions containing 0.1-4 M NaCl and at a pH range from 4.6 to 8.8. The (CTG)3 duplex, which contains multiple-T.T mismatches, displays many NMR spectral characteristics similar to those of B-form DNA. However, unique NOE and 1H-31P coupling patterns associated with the repetitive T.T mismatches in the CTG repeats are discerned. These results, in conjunction with recent in vitro studies suggest that longer CTG repeats may form hairpin structures, which can potentially cause interruption in replication, leading to dynamic expansion or deletion of triplet repeats.

Base Composition↗
PubMed1995

Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

Amino Acid Oxidoreductases↗
PubMed1995

Evaluation of risk factors for degenerative joint disease associated with hip dysplasia in dogs.

Passive coxofemoral joint laxity of dogs, as quantitated by a distraction-stress radiographic method, may have important prognostic value in determining susceptibility to hip dysplasia. Data from 151 dogs, representing 13 breeds, were included in a logistic regression model to evaluate the contribution of factors such as age, breed, weight, sex, distraction index, and Norberg angle to the risk of developing degenerative joint disease (DJD) of the coxofemoral joint. Of the factors studied, the amount of passive hip laxity, as quantitated by the distraction index, was the most significant (P < 0.0001) determinant of the risk to develop DJD of the coxofemoral joint. In the longitudinal and cross-sectional components of the study, distraction index was a significant (P < 0.001) risk factor for DJD, irrespective of age at evaluation (4, 12, or 24 months). The strength of the hip laxity:DJD correlation increased with the age of dog. In contrast, the Norberg angle, a measure of hip laxity on the standard hip-extended radiograph, was not found to be a significant risk factor for DJD, either in the longitudinal or cross-sectional analyses. Breed-specific probability curves of DJD susceptibility indicated that German Shepherd Dogs had a significantly (P < 0.05) greater risk of developing DJD than did the pool of non-German Shepherd Dogs. The information derived from this statistical model will help to scientifically characterize the role of passive hip laxity as a component in the pathogenesis of DJD of the coxofemoral joint.

Age Factors↗
PubMed1995

Comparison of susceptibility for hip dysplasia between Rottweilers and German shepherd dogs.

Passive laxity of the coxofemoral joints, as measured quantitatively by radiographing the joints under stress, has been shown to be an accurate measure of the risk for developing degenerative joint disease (DJD) of the coxofemoral joints. Seventy-four Rottweilers between 12 and 40 months old were evaluated subjectively for radiographic evidence of DJD, using the ventrodorsal view of the pelvis with the coxofemoral joints fully extended and the knees internally rotated (standard hip-extended view). Effect of age, sex, weight, and distraction index on the risk of developing DJD was evaluated by use of a logistic regression model. Results were compared with those from a group of German Shepherd Dogs. Results indicated that in Rottweilers the distraction index was the only statistically significant predictor of the risk of developing DJD of the coxofemoral joint. When German Shepherd Dogs were included in the model, they had a significantly greater risk of developing DJD than did Rottweilers. This finding provides further support for the theory that there are differences in disease susceptibility among breeds and emphasizes the need to develop disease susceptibility curves for all breeds affected by hip dysplasia to facilitate accurate, scientifically based recommendations for breeding or treatment.

Age Factors↗
PubMed1995

Insights to primitive replication derived from structures of small oligonucleotides.

Available information on the structure of small oligonucleotides is surveyed. It is observed that even small oligomers typically exhibit defined structures over a wide range of pH and temperature. These structures rely on a plethora of non-standard base-base interactions in addition to the traditional Watson-Crick pairings. Stable duplexes, though typically antiparallel, can be parallel or staggered and perfect complementarity is not essential. These results imply that primitive template directed reactions do not require high fidelity. Hence, the extensive use of Watson-Crick complementarity in genes rather than being a direct consequence of the primitive condensation process, may instead reflect subsequent selection based on the advantage of accuracy in maintaining the primitive genetic machinery once it arose.

Base Composition↗
PubMed1994

Thienyl and thiazolyl acyclic analogues of 5-deazatetrahydrofolic acid.

Analogues of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino] benzoyl]-L-glutamic acid (5-DACTHF), in which the phenylene group is replaced by either a thienoyl or a thiazolyl group were synthesized. These compounds were prepared by reductive amination of suitably protected pyrimidinylpropionaldehyde with the aminoaroyl glutamates. These glutamates were in turn synthesized from the corresponding nitroaroyl carboxylic acids by condensation with protected glutamic acid followed by catalytic reduction. The compounds were tested as inhibitors of methotrexate uptake as a measure of binding to the reduced folate transport system, as inhibitors of glycinamide ribonucleotide transformylase, as substrates for folylpolyglutamate synthetase, and as inhibitors of tumor cell growth in cell culture. The thiophene analogue was found to be equal in activity to 5-DACTHF in the MCF-7 cell growth inhibition assay while the thiazole analogue was 9-fold more active. Indeed this thiazole was over 4 times more active in the MCF-7 cell line than the clinically investigated compound 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).

Adenocarcinoma↗
PubMed1994

Benzo[f]quinazoline inhibitors of thymidylate synthase: methyleneamino-linked aroylglutamate derivatives.

Syntheses of several new inhibitors of thymidylate synthase (TS) structurally related to folic acid are described in which the pterin portion of the folate molecule is replaced by a benzo[f]quinazoline moiety, but which retain the natural methyleneamino link to the benzoylglutamate side chain. The effect on enzyme activity and cytotoxicity of various changes in the structure of the (p-aminobenzoyl)glutamate side chain are reported. Replacement of the benzamide portion of the (p-aminobenzoyl)glutamate moiety with 2-fluorobenzamido, 2-isoindolinyl, 1,2-benzisothiazol-2-yl, and 2-thenamido moieties varied in effect from a 9-fold diminution of TS activity to a 5-fold enhancement, while cytotoxic potency on SW-480 and MCF-7 tumor lines showed increases ranging from 3.6- to 450-fold. The detrimental effect on enzyme activity and cytotoxicity of alkyl substitution on the PABA nitrogen is confirmed for these compounds, in contrast with several series of previously reported quinazoline antifolates (2). Substitution of a C3-methyl substituent for 3-amino had little effect on TS activity but was beneficial in terms of solubility and cytotoxicity. The excellent combination of TS inhibitory activity, FPGS substrate activity, and affinity for the reduced folate transport system in the most potent of these derivatives, 3e, resulted in IC50 values of 0.2-0.8 nM against these tumor lines.

Adenocarcinoma↗