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G K Smith

Publications and source records attributed to G K Smith.

At least 19 recordsLinked to original sources
PubMed1998

Purification and characterization of a membrane bound neutral pH optimum magnesium-dependent and phosphatidylserine-stimulated sphingomyelinase from rat brain.

Sphingomyelin hydrolysis and ceramide generation catalyzed by sphingomyelinases (SMase) are key components of the signaling pathways in cytokine- and stress-induced cellular responses. In this study, we report the partial purification and characterization of the membrane bound, neutral pH optimal, and magnesium-dependent SMase (N-SMase) from rat brain. Proteins from Triton X-100 extract of brain membrane were purified sequentially with DEAE-Sephacel, heparin-Sepharose, ceramic hydroxyapatite, Mono Q, phenyl-Superose, and Superose 12 column chromatography. After eight purification steps, the specific activity of the enzyme increased by 3030-fold over the brain homogenate. The enzyme hydrolyzed sphingomyelin but not phosphatidylcholine and its activity was dependent upon magnesium with an optimal pH of 7.5 and a native pI of 5.2. Delipidation of the enzyme through chromatographic purification or by extraction with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid followed by gel filtration revealed that the enzyme became increasingly dependent on phosphatidylserine (PS). Up to 20-fold stimulation was observed with PS whereas other lipids examined were either ineffective or only mildly stimulatory. The Km of the enzyme for substrate sphingomyelin (3.4 mol %) was not affected by PS. The highly purified enzyme was inhibited by glutathione with a >95% inhibition observed with 3 mM glutathione and with a Hill number calculated at approximately 8. The significance of these results to the regulation of N-SMase is discussed.

Animals↗
PubMed1998

Increases in neutral, Mg2+-dependent and acidic, Mg2+-independent sphingomyelinase activities precede commitment to apoptosis and are not a consequence of caspase 3-like activity in Molt-4 cells in response to thymidylate synthase inhibition by GW1843.

Thymidylate synthase (TS) inhibition causes cell death, and this enzyme is the target for the important chemotherapy regime 5-fluorouracil/leucovorin. GW1843 (1843U89) is a potent and specific folate analog TS inhibitor in clinical development. Because of the importance of TS as a chemotherapy target, we are studying the mechanism of TS inhibition-induced cell death by GW1843. Ceramide is a regulatory lipid generated by the action of sphingomyelinase and is believed to signal apoptosis. The role of the ceramide in apoptotic signaling was studied in Molt-4 human T-cell leukemia cells undergoing cell death after treatment with GW1843. In response to GW1843, Molt-4 cells undergo apoptosis with both acidic pH, Mg2+-independent sphingomyelinase (ASMase) and neutral pH, Mg2+-dependent sphingomyelinase (NSMase) activities elevated as early steps in the initiation of apoptosis before Molt-4 commitment to death. These activities lead to ceramide production with kinetics consistent with a role as an effector molecule signaling the initiation of apoptosis in Molt-4 cells. These changes were found to be independent of caspase 3-like (CPP32/apopain) activity and DNA degradation, but were not separable from membrane blebbing or cell lysis in this cell line. In this report, kinetic evidence is provided for a role of ceramide in initiating GW1843-induced cell death of Molt-4 T-cell leukemia cells.

Apoptosis↗
PubMed1998

Reliability of the hip distraction index in two-month-old German shepherd dogs.

OBJECTIVE: To determine whether distraction index (DI), a measure of passive hip joint laxity, at 2 months of age was predictive of DI at 4 or 12 months of age in German Shepherd Dogs. DESIGN: Prospective cohort study. ANIMALS: 45 German Shepherd Dogs. PROCEDURE: DI was measured at 2, 4, and 12 months of age. At the same times, a standard ventrodorsal radiographic projection of the pelvis with the hip joints extended was obtained and examined for evidence of degenerative joint disease (DJD). To facilitate radiographic positioning, dogs were sedated or anesthetized. RESULTS: DI at 2 months of age was not significantly correlated with DI at 4 or 12 months of age. However, DI at 4 months of age was correlated with DI at 12 months of age. The proportion of dogs with DI > or = 0.3 at 12 months of age that had radiographic evidence of DJD by 12 months of age (13/22; 59%) was significantly greater than the proportion of dogs with DI < 0.3 at 12 months of age that had radiographic evidence of DJD by 12 months of age (1/9; 11%). CLINICAL IMPLICATIONS: For German Shepherd Dogs, DI at 2 months of age was not sufficiently reliable to predict DI at 4 and 12 months of age; however, DI at 4 and 12 months of age were comparable. We recommend that, for German Shepherd Dogs, DI not be measured before 4 months of age and that particularly for breeding dogs, DI be remeasured after maturity to confirm DI obtained at earlier ages. Studies including other breeds of dogs should be done to determine the youngest reliable age to initiate hip joint screening.

Age Factors↗
PubMed1998

Correlation between longitudinal bone growth, growth hormone, and insulin-like growth factor-I in prepubertal dogs.

OBJECTIVE: To determine the association between longitudinal bone growth and concentrations of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in serum from prepubertal dogs. Animals-6 male 14-week-old German Shepherd Dogs. PROCEDURE: Blood was obtained every 30 minutes for 14 consecutive days. Concentrations of GH and IGF-I in serum were determined, using a canine-specific radioimmunoassay and conventional radioimmunoassay after acid-ethanol extraction, respectively. Simultaneous biplanar radiography was performed daily to measure bone growth. Spectral analysis was used to estimate specific features of GH secretion during an extended period. Multiple linear regression with different lag times between independent and dependent variables was used to determine the strongest predictors of bone growth. RESULTS: The power spectra of GH concentrations in serum had a primary peak at a frequency of 0.02 cycles/h or a periodicity of 50 h/cycle. A significant determinant of longitudinal bone growth was a lag time of 1 day in concentration of GH in serum. The relationship between IGF-I concentration in serum and bone growth was not significant. CONCLUSIONS: The primary frequency of GH secretion is outside the time frame of a single day and the concentration of GH in serum is a primary determinant of bone growth. CLINICAL RELEVANCE: A better understanding of the components of bone growth provide discernment to improved diagnosis and treatment of abnormal bone growth.

Animals↗
PubMed1997

Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate.

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.

Animals↗
PubMed1997

Advances in diagnosing canine hip dysplasia.

The DI method was developed, at least in part, because of perceived variations among radiologists in regard to subjective hip scores assigned to radiographs. It also was recognized that the prevalence of CHD among many dog breeds was disturbingly high, despite efforts to lower the prevalence using subjective score as a selection criterion. The DI method was developed on the basis of results of mechanical testing of cadaver hip joints, which accomplished 3 principal objectives. First, it showed that when the hip joint is in the standard, hip-extended, internally rotated position, passive laxity is minimized. Second, it showed that passive laxity was maximized when the hip joint is in the neutral position. Third, it demonstrated the sigmoidal nature of the load/displacement curve for the hip joint in the neutral position, which suggested that high repeatability from examiner to examiner in regard to DI could be expected without the need to standardize applied force. The mean and range of DI were shown to vary from 1 breed to the next, but within an individual dog, DI appears to remain constant (within limits of scientific acceptability and clinical applicability) from 16 weeks of age. Passive hip laxity measured on the distraction view is, on average, 2.5 to 11 times greater than that measured on the standard, hip-extended radiographic view. Performance Borzois and Greyhounds, which have an extremely low prevalence of CHD, uniformly have tight hip joints (DI < 0.3), and mean DI for dog breeds that have a high prevalence of CHD is significantly greater than mean DI for Borzois and Greyhounds. However, individual dogs that have DI < 0.3, even though members of breeds prone to CHD, have a low risk of developing DJD. Some, but not all, dogs with DI > 0.3 will develop DJD by 3 years of age, and DI has been shown to be the principal risk factor for development of DJD. Susceptibility to development of DJD appears to be breed-specific. For example, given equivalent DI, German Shepherd Dogs are more at risk for developing DJD than are Rottweilers. Heritability of DI is higher than that of the subjective hip score; thus, selection pressure based on DI should result in faster genetic change than selection pressure based on subjective hip scores. Finally, the DI method has been performed on approximately 14,000 dogs, some of which were evaluated multiple times. It is apparent that this method is no more harmful than the standard, hip-extended radiographic method or palpation performed as part of a routine orthopedic examination.

Animals↗
PubMed1997

Synovial fluid cavitation during distraction radiography of the coxofemoral joint in dogs.

OBJECTIVE: To determine risk factors for, and prevalence and short- and long-term effects of synovial fluid cavitation during distraction radiography. DESIGN: Multicenter prevalence survey. ANIMALS: 6,649 purebred dogs comprising 129 breeds. PROCEDURE: Radiographs from the PennHIP (University of Pennsylvania Hip Improvement Program) Laboratory were subjectively evaluated for evidence of cavitation. Multiple logistic regression was used to determine whether sex, breed, age, weight, distraction index (DI), or examining veterinarian was associated with cavitation. Short-term effects of cavitation were assessed by comparing DI for the hip with cavitation with DI for the contralateral hip in dogs with unilateral cavitation. Long-term effects of cavitation were assessed by comparing DI before and after cavitation was detected. RESULTS: Cavitation was detected in 279 (4.2%) of the radiographs analyzed. Male dogs, Golden Retrievers, and heavier dogs were at a decreased risk for cavitation. Irish Wolfhounds, Irish Setters, Rhodesian Ridgebacks, and Weimaraners had an increased risk for cavitation. Age and DI were not risk factors for cavitation. Mean DI was 0.08 greater in hips with cavitation than in paired hips without cavitation. Significant differences were not detected between DI before and after cavitation, but only 7 dogs were included in this analysis. CLINICAL IMPLICATIONS: Cavitation is rare during distraction radiography and can increase measured DI. Radiographs should be routinely examined to ensure accurate reporting of DI.

Animals↗
PubMed1997

Five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs.

OBJECTIVE: To examine the effects of limited food intake on frequency and severity of osteoarthritis in coxofemoral joints of labrador Retrievers. DESIGN: Dogs were paired according to gender and body weight, within each litter at 8 weeks of age. One dog of each pair was fed ad libitum. The limit-fed pairmate was fed 75% of the amount eaten the previous day by the ad libitum-fed counterpart. ANIMALS: 48 Labrador Retrievers. PROCEDURE: All dogs received the same diet. Radiographic evaluation of coxofemoral joints for frequency and severity of osteoarthritis were made when dogs were 4 and 6 months and 1, 2, 3, and 5 years old. RESULTS: Radiographic evaluation for osteoarthritis indicated greater frequency and more severity of osteoarthritis in the ad libitum-fed group of dogs. CLINICAL IMPLICATIONS: Analysis of data suggested that limit feeding of dogs over a 5-year period minimizes development of osteoarthritis in the coxofemoral joints.

Animals↗
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↗