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K McGinnis

Publications and source records attributed to K McGinnis.

18 recordsLinked to original sources
PubMed2001

Inhibition of p38 kinase mimics survival signal-linked protection against apoptosis in rat cerebellar granule neurons.

The mitogen-activated protein kinase (MAPK) cascades are thought to be important mediators in the transduction of extracellular signals into cellular responses. The p38 kinase, a member of the MAPK superfamily, is activated by a wide variety of extracellular stimuli and has been implicated in neuronal apoptosis induced by glutamate. In this study we have examined the role of p38 kinase in the potassium deprivation model of apoptosis in rat cerebellar granule neurons (CGN). An increase in p38 kinase activity was observed with a 15-minute potassium deprivation when compared to the basal level. We also found that SB203580 and PD169316, specific p38 kinase inhibitors, significantly attenuated apoptosis in potassium-deprived cells in a dose dependent manner. A decrease in caspase-3 mediated DEVD-MCA, substrate hydrolysis and the appearance of the 120 kDa-spectrin breakdown product in cells treated with SB203580 further suggests that the p38 kinase acts upstream of caspase-3 in the apoptosis cascade. The data provides evidence for an essential role of p38 kinase in mediating apoptotic cell death in CGN and the inhibition of p38 kinase mimics the suppression of apoptosis provided by natural survival signals.

Animals↗
PubMed2000

Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.

Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo.

Animals↗
PubMed1999

The use of delayed computerized tomography in the evaluation of blunt abdominal trauma: a preliminary report.

This purpose of this study was to analyze the use of abdominal computed tomography (CT) imaging in patients with possible blunt abdominal trauma. A retrospective analysis of all trauma patients over a 1-year period (1993-1994) was conducted, with prospective study protocol in 52 patients using serial abdominal exam and hematocrits (Hcts) instead of abdominal CT for evaluation of blunt abdominal trauma. Urgent abdominal CT was used as the initial diagnostic test for evaluation of blunt abdominal trauma in 813 patients over this 1-year period. CT was obtained in 379 (46.6%) of these patients who arrived hemodynamically stable (admission systolic blood pressure > or = 90), had a Glasgow Coma Scale > 13, and had admission Hct > or = 35 because of distracting injuries, possible traumatic brain injury, or alcohol/drug use, which might render the abdominal physical exam unreliable. Only 47 CT scans (12.4%) were positive, and three patients (0.8%) required laparotomy. In an effort to more efficiently use abdominal CT, we performed a prospective study in 52 patients with possible blunt abdominal trauma, admission systolic blood pressure > or = 90, Hct > or = 35, Glasgow Coma Scale > 13, and a normal abdominal exam on admission. These patients were followed with serial abdominal examinations and Hcts every 6 hours for 24 hours, and delayed CT, when applicable. CT was obtained in seven patients (13.5%) for evaluation of fall in Hct or abnormal abdominal examination; all were negative for abdominal injury. A protocol using serial abdominal exams, Hcts, and delayed abdominal CT imaging may be useful in select patients to decrease the high number of negative routine abdominal CTs that are obtained in the evaluation of blunt abdominal trauma.

Abdominal Injuries↗
PubMed1998

The five cysteine residues located in the active site region of bovine aspartyl (asparaginyl) beta-hydroxylase are not essential for catalysis.

In previous chemical modification studies on bovine aspartyl (asparaginyl) beta-hydroxylase, cysteines were implicated as critical catalytic residues. Using site-directed mutagenesis, the five cysteine residues located in a highly conserved region of the enzyme identified as the active site were individually mutated to alanine. Substitutions at cysteine 637, 644, 656, 681, and 696 resulted in active mutant enzymes indicating that these residues are not required for catalysis.

Alanine↗
PubMed1998

Simultaneous degradation of alphaII- and betaII-spectrin by caspase 3 (CPP32) in apoptotic cells.

The degradation of alphaII- and betaII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both alphaII- and betaII-subunits. Major C-terminal alphaII-spectrin BDPs were detected at approximately 150, 145, and 120 kDa (alphaII-BDP150, alphaII-BDP145, and alphaII-BDP120, respectively); major C-terminal betaII-spectrin BDPs were at approximately 110 and 85 kDa (betaII-BDP110 and betaII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that alphaII-BDP150 and alphaII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For betaII-spectrin, a major caspase site was detected at DEVD1457*S1458, and both betaII-BDP110 and betaII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for betaII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both alphaII- and betaII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.

Amino Acid Sequence↗
PubMed1997

Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells.

We characterized the activation of interleukin-1beta-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.

Antibody Specificity↗
PubMed1996

Effects of ICE-like protease and calpain inhibitors on neuronal apoptosis.

Both ice-like protease and calpain have been shown to be involved in apoptosis in non-neuronal cells. Cultured rat cerebellar granule neurons undergo apoptosis when exposed to low potassium-containing medium. Calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and N-acetyl-Leu-Leu-Met-CHO (calpain inhibitor II) as well as interleukin-beta 1 converting enzyme (ICE)-like protease inhibitor Z-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) protect against such apoptotic death. They also reduce DNA laddering and the number of apoptotic nuclei. Staurosporine treatment also evokes apoptosis in human neuroblastoma SH-SY5Y. While Z-D-DCB is again anti-apoptotic, calpain inhibitors only provide modest effects in this model. Our results suggest that ICE-like protease plays a critical role in neuronal apoptosis whereas the contributions of calpain are more cell-type dependent.

Animals↗
PubMed1996

Site-directed mutagenesis of residues in a conserved region of bovine aspartyl (asparaginyl) beta-hydroxylase: evidence that histidine 675 has a role in binding Fe2+.

The roles in catalysis of several residues in bovine aspartyl (asparaginyl) beta-hydroxylase that are located in a region of homology among alpha-ketoglutarate-dependent dioxygenases were investigated using site-directed mutagenesis. Previous studies have shown that when histidine 675, an invariant residue located in this highly conserved region, was mutated to an alanine residue, no enzymatic activity was detected. A more extensive site-directed mutagenesis study at position 675 has been undertaken to define the catalytic role of this essential residue. The partial hydroxylase activity observed with some amino acid replacements for histidine 675 correlates with the potential to coordinate metals and not with size, charge, or hydrophobic character. Furthermore, the increase in Km for Fe2+ observed with the H675D and H675E mutant enzymes can account for their partial activities relative to wild type. No significant changes in the Km for alpha-ketoglutarate (at saturating Fe2+) or Vmax were observed for these mutants. These results support the conclusion that histidine 675 is specifically involved in Fe2+ coordination. Further site-directed mutagenesis of other highly conserved residues in the vicinity of position 675 demonstrates the importance of this region of homology in catalysis for Asp (Asn) beta-hydroxylase and, by analogy, other alpha-ketoglutarate-dependent dioxygenases.

Amino Acid Sequence↗
PubMed1994

A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases.

The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC 1.14.11.16) specifically hydroxylates one aspartic or asparagine residue in certain epidermal growth factor-like domains of a number of proteins. The expression in Escherichia coli, purification, characterization of a fully active catalytic domain, and evidence for the identification of an active-site region of this enzyme are described. Sequence alignment analyses among the vertebrate alpha-ketoglutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant aspartyl (asparaginyl) beta-hydroxylase involved in substrate binding and/or catalysis. Based upon these studies, an alignment of the C-terminal regions of prolyl and lysyl hydroxylase and of aspartyl (asparaginyl) beta-hydroxylase is proposed. When histidine-675, an invariant residue located in a region of homology within this alignment, was mutated to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H675A), no enzymatic activity was detected. Chemical modification studies show that the wild-type protein is protected from iodo[14C]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.

Amino Acid Sequence↗
PubMed1993

Dimerization of Thermomonospora fusca beta-1,4-endoglucanase E2.

Unboiled Thermomonospora fusca endoglucanase E2 electrophoresed on SDS-polyacrylamide gels migrated in the range of 80-90 kDa, but when boiled it migrated in the 40-42-kDa range. Sedimentation equilibrium centrifugation as well as chemical cross-linking experiments confirmed that E2 is a dimer. The dimer was reversibly dissociated at low pH. The E2 dimer was stable up to 70 degrees C, but began to dissociate at this temperature after a 30-60-min incubation. A nondimerizing mutant was obtained using region-specific chemical mutagenesis. DNA sequencing of this mutant revealed a single base change that substituted Gly for Glu-263. Chemical modification of carboxylic acid residues in E2 disrupted the dimer interaction.

Actinomycetales↗
PubMed1993

Disulfide arrangement and chemical modification of beta-1,4-endoglucanase E2 from Thermomonospora fusca.

Thermomonospora fusca endoglucanase E2 contains six cysteine residues scattered along the protein sequence. Four of the cysteine residues were shown to participate in two disulfide bonds while the last two form a third disulfide bond. Neither full reduction of the disulfides nor complete carboxymethylation of all six cysteines totally destroys enzymatic activity, but the activity of the reduced enzyme is much lower than the native enzyme and the iodoacetamide-modified enzyme has very low activity. Reduction of only the accessible disulfides drastically decreases the enzyme's thermostability. One disulfide linkage joins Cys80 to Cys125, another joins Cys232 to Cys267, and the third joins Cys315 to Cys407. The first two bonds are similar to those in cellobiohydrolase II, which also belongs to cellulase family B (Rouvinen et al., 1990; Lao et al., 1991; Henrissat et al., 1989). Direct evidence for the involvement of carboxyl groups in catalysis by E2 was demonstrated by chemical modification with carbodiimide.

Actinomycetales↗
PubMed1993

Disulfide arrangement and functional domains of beta-1,4-endoglucanse E5 from Thermomonospora fusca.

Thermomonospora fusca cellulase E5 contains six cysteine residues. The number and location of the disulfide bonds and the effect of reduction of the disulfides and modification of the resulting half-cystine residues on enzymatic activity were determined. No free sulfhydryl groups were found in E5. Reduction and subsequent labeling with iodoacetamide of E5 and of an enzymatically active 32-kDa proteolytic derivative of E5 (E5cd) showed that one of the three disulfides is accessible to reduction under nondenatured conditions while the other two are not accessible. Full reduction of the disulfides and complete carboxymethylation of the six cysteines decrease the specific activity of E5 on CMC by more than half, but reduction of only the exposed disulfide bond does not affect enzymatic activity or binding of E5 to cellulose. A 14-kDa proteolytic fragment of E5 containing 120 amino acids from the N-terminus of the protein was shown to bind to crystalline cellulose. This confirms earlier evidence that the cellulose binding domain of E5 is located at the N-terminus of the protein. This 14-kDa fragment contains the accessible disulfide bond involving Cys93 and Cys100. The location of the two disulfide bonds in the other fragment (E5cd) was determined by cleaving it with cyanogen bromide under conditions that left the disulfide bonds intact. The resulting peptides were separated under both nonreducing and reducing conditions using RP-HPLC. Amino acid analysis of peptide peaks indicated that one disulfide linkage in E5cd joins Cys138 to Cys143 while the other joins Cys166 to Cys406.

Actinomycetales↗
PubMed1990

Expanded indications for laser-assisted balloon angioplasty in peripheral arterial disease.

Successful recanalization of isolated femoropopliteal occlusions by laser-assisted balloon angioplasty has been widely reported. However, the utility of this approach in other sites remains undetermined. The potential of laser-assisted balloon angioplasty in the treatment of a broad spectrum of lower extremity atherosclerosis was explored. Seventy-seven limbs in 69 patients were treated with laser-assisted balloon angioplasty by means of a percutaneous approach. Technical success was achieved in 62/77 (81%), with 52/77 (68%) open at discharge. At a mean of 8.6 months' follow-up, cumulative patency was 50% at 6 months and 42% at 9 months. In 10 cases initial guide wire placement was not possible; primary laser probe passage was technically successful in six, with two open at latest follow-up. Eleven patients underwent laser therapy without balloon angioplasty of distal sites with technical success in eight; at most recent follow-up five were open at the site of sole laser therapy. Complications were frequent. Bleeding requiring transfusion occurred in six, with three patients requiring emergency surgery; worsened ischemia necessitating urgent arterial bypass grafting developed in five patients. In this series laser angioplasty carried significant complications and a high failure rate. Widespread application of this procedure should await further clinical investigation.

Aged↗
PubMed1987

Biochemical and spectroscopic characterization of the blue-green photoreceptor in Halobacterium halobium.

Spectroscopic evidence indicates the presence of a second sensory receptor sR-II in Halobacterium halobium, which causes a repellent response to blue-green light. Reactions with hydroxylamine and NaCNBH3 and reconstitution of the bleached pigment with retinal show that it is very similar to the other retinylidene pigments bacteriorhodopsin, halorhodopsin, and especially the earlier-discovered phototaxis receptor, sensory rhodopsin, renamed sR-I587. The second sensory receptor, sR-II480, has an absorbance maximum at 480 nm and undergoes a cyclic photoreaction with a half-time of approximately 200 msec. Its predominant photocycle intermediate absorbs maximally near 360 nm. The receptor can be detected spectroscopically in the presence of sR-I587 and quantitated through its transient response to 450-nm excitation. It is selectively bleached by low hydroxylamine concentrations that are insufficient to bleach sR-I587 significantly. Its photochemical and phototactic activities can be restored by addition of retinal. The mobility of the receptor, on NaDodSO4/polyacrylamide gels, was similar or identical to that of sR-I587 and slightly faster than bacteriorhodopsin, yielding an apparent molecular mass of 23-24 kDa.

Bacteriorhodopsins↗
PubMed1980

A murine C4 molecule with reduced hemolytic efficiency.

C4 functional activity and antigenic levels were determined in H-2-congenic mouse strains. In strains with the H-2w7 haplotype, the C4 hemolytic activity per unit of residual Ss antigenic activity, after depletion of the nonfunctional Slp-positive molecules was 25-33% that found with other H-2 haplotypes. This reduced hemolytic efficiency was not the result of either a more labile C4 molecule or of the presence of inhibitors. Moreover, other strains with comparable antigenic concentrations of Ss (C4) and Slp has three- to fourfold higher levels of C4 hemolytic activity. Based on these data and previously reported structural differences between C4 molecules from the H-2w7 haplotype compared with other standard H-2 haplotypes, the reduced hemolytic efficiency of this molecule is probably secondary to alterations in the structure of its alpha-chain.

Animals↗
PubMedDate not supplied

Higher ploidy in Saccharomyces cerevisiae supports enhanced hepatitis B virus S cloned gene expression at the pilot scale.

The effect of host strain ploidy on the production of hepatitis B surface antigen (HBsAg) in Saccharomyces cerevisiae was evaluated at the pilot scale (75 L). We found that the accumulation of HBsAg normalized to cell protein was 2-fold higher for the diploid strain compared to its isogenic haploid. No detectable differences in many fermentation parameters were observed (e.g., rate of fermentation, growth rate, final cell yield). However, the enhancement of productivity in the diploid strain appeared to be associated with a slower rate of plasmid shedding (2 microns element) and, thus, a higher average copy number (2-fold at stationary phase) compared to those of the haploid strain.

Cloning, Molecular↗