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C Ho

Publications and source records attributed to C Ho.

At least 55 records · Page 3Linked to original sources
PubMed1999

Conformation of the C1 phorbol-ester-binding domain participates in the activating conformational change of protein kinase C.

The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.

Binding Sites↗
PubMed1999

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.

2,3-Diphosphoglycerate↗
PubMed1999

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.

Amino Acid Substitution↗
PubMed1999

Inactivation of CACNA1G, a T-type calcium channel gene, by aberrant methylation of its 5' CpG island in human tumors.

Using a newly developed PCR-based technique called methylated CpG island amplification, we have identified several DNA fragments that are aberrantly methylated in a colon cancer cell line. One of the fragments, termed MINT31, mapped to human chromosome 17q21, where frequent loss of heterozygosity is detected in various human tumors. By characterizing the genomic sequence around this area, we identified a gene encoding a T-type calcium channel, CACNA1G, as a target for hypermethylation in human tumors. By reverse transcriptase-PCR we detected expression of CACNA1G in normal colon and bone marrow, but expression was absent in the five tumor cell lines in which methylation was found. After treatment with the methylation inhibitor 5-deoxyazacytidine, the expression of CACNA1G was restored in all five cell lines. Detailed methylation mapping of the 5' CpG island by bisulfite-PCR revealed that methylation of a region 300-800 bp upstream of the translation initiation site closely correlated with the inactivation of CACNA1G. This region contained the transcription start site, as determined by 5' rapid amplification of cDNA ends analysis. Aberrant methylation of CACNA1G was also examined in various human primary tumors and was detected in 17 of 49 (35%) colorectal cancers, 4 of 16 (25%) gastric cancers, and 3 of 23 (13%) acute myelogenous leukemia cases. Inactivation of CACNA1G may play a role in cancer development by modulating calcium signaling, which potentially affects cell proliferation and apoptosis.

Bone Marrow Cells↗
PubMed1999

Adsorption properties and breakthrough model of 1,1-dichloro-1-fluoroethane on activated carbons.

Hydrochlorofluorocarbons (HCFCs), among the major replacements for chlorofluorocarbons (CFCs) and chlorinated solvents, are now considered to the prime contribution to the stratospheric ozone depletion. This paper reports the adsorption equilibrium of 1,1-dichloro-1-fluoroethane (HCFC-141b) vapor in air streams on the commercial activated carbons PCB and BPL, which were made from coconut shell and bituminous coal, respectively, at 283, 293, 303, and 313 K. The experimental results show that within the experimental conditions, the adsorption capacities of the two adsorbents for HCFC-141b in dry air streams can be well-fitted by the Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) adsorption equations. The physical properties of the adsorbents are consistent with the isotherm parameters obtained from the adsorption results. In addition, available experimental data of adsorption breakthrough at 283 K were used in conjunction with the Yoon and Nelson model to generate theoretical breakthrough curves based on the obtained values of parameters. In each case, calculated theoretical breakthrough curves are in agreement with corresponding experimental data.

Journal Article↗
PubMed1999

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.

Adult↗
PubMed1999

Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.

2,3-Diphosphoglycerate↗
PubMed1999

Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.

CpG island methylation has been linked to tumor suppressor gene inactivation in neoplasia and may serve as a useful marker to clone novel cancer-related genes. We have developed a novel PCR-based method, methylated CpG island amplification (MCA), which is useful for both methylation analysis and cloning differentially methylated genes. Using restriction enzymes that have differential sensitivity to 5-methyl-cytosine, followed by adaptor ligation and PCR amplification, methylated CpG rich sequences can be preferentially amplified. In a model experiment using a probe from exon 1 of the p16 gene, signal was detected from MCA products of a colorectal cancer cell line but not in normal colon mucosa. To identify novel CpG islands differentially methylated in colorectal cancer, we have applied MCA coupled with representational difference analysis to the colon cancer cell line Caco2 as a tester and normal colon mucosa as a driver. Using this strategy, we isolated 33 differentially methylated DNA sequences, including fragments identical to several known genes (PAX6, Versican, alpha-tubulin, CSX, OPT, and rRNA gene). The association of hypermethylation of the clones obtained and transcriptional suppression in colorectal cancer was confirmed by examining the Versican gene, which we found to be silenced in methylated cell lines and reactivated by the methylation inhibitor 5-aza-2'-deoxycytidine. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.

5-Methylcytosine↗
PubMed1999

Synergistic activation of protein kinase Calpha, -betaI, and -gamma isoforms induced by diacylglycerol and phorbol ester: roles of membrane association and activating conformational changes.

Protein kinase Calpha (PKCalpha) has been shown to contain two discrete activator sites with differing binding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiated level of activity [Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D. , Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancement of activity was examined with respect to the Ca2+ dependences of membrane association and accompanying conformational changes that lead to activation. The association of PKCalpha with membranes containing 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1, 2-dioleoylglycerol (DAG), determined from tryptophan to dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca2+ levels (</=1 microM). However, PKCalpha was found to be inactive even though membrane association was complete at these Ca2+ levels and further titration of Ca2+ to a concentration of approximately 100 microM was required for activation. This increase in Ca2+ concentration also led to a further increase in RET, which was due to a Ca2+-induced activating conformational change, as verified by an accompanying increase in the PKCalpha tryptophan fluorescence anisotropy. Coaddition of DAG and TPA resulted in a reduction in the Ca2+ levels required for both the conformational change and enzyme activation. Also, it was found that incubation of the enzyme with TPA alone resulted in a time-dependent increase in the Ca2+-independent PKCalpha activity, the rate and extent of which was further enhanced upon coaddition with DAG. Tauhe results suggest that the enhanced level of activity induced by coaddition of DAG and TPA involves both Ca2+-dependent and Ca2+-independent activating conformational changes which result in active conformers of PKCalpha distinct from those formed by interaction with either activator separately.

Anisotropy↗
PubMed1999

Early perfusion after controlled cortical impact in rats: quantification by arterial spin-labeled MRI and the influence of spin-lattice relaxation time heterogeneity.

Early posttraumatic cerebral hypoperfusion is implicated in the evolution of secondary damage after experimental and clinical traumatic brain injury (TBI). This is the first report of cerebral blood flow (CBF) measurement by continuous arterial spin-labeled magnetic resonance imaging (MRI) early after TBI in rats using the controlled cortical impact (CCI) model. CCI reduced CBF globally at approximately 3 hr (versus normal), with 85% and 49% reductions in a contused cortical region and contralateral cortex, respectively. In contrast, a prior MRI study from this laboratory showed at 24 hr post trauma a focal CBF reduction restricted to the injury site. In vivo spin-lattice relaxation time (T(1obs)), which is used in CBF quantification, was spatially heterogeneous early after CCI, a time when edema is developing in injured brain tissue. At 4.7 T, T(1obs) values are increased 29% in the contusion (versus normal), consequently reducing CBF quantification to a similar degree. MRI should facilitate coupling posttraumatic CBF with long-term functional outcome. Magn Reson Med 42:673-681, 1999.

Animals↗
PubMed1999

Practitioners' guide to meeting the vitamin B-12 recommended dietary allowance for people aged 51 years and older.

In response to research findings that 10% to 30% of people aged 51 years and older may have protein-bound vitamin B-12 malabsorption, the National Academy of Sciences' Institute of Medicine recommends that these people consume a majority of the new Recommended Dietary Allowance (RDA) of 2.4 micrograms/day in its synthetic form rather than in its food form. Protein-bound vitamin B-12 malabsorption in older adults has been attributed to reduced pepsin activity and gastric acid secretion, which interfere with cleavage of vitamin B-12 from dietary protein before absorption. Unlike patients with pernicious anemia, most people with protein-bound vitamin B-12 malabsorption produce intrinsic factor and have the ability to absorb synthetic vitamin B-12 normally. Early diagnosis is necessary to prevent the untoward effects of vitamin B-12 deficiency. A thorough assessment of vitamin B-12 status entails measurement of multiple biochemical assessment indexes, including serum vitamin B-12, methylmalonic acid, and homocysteine concentrations. Dietitians and other health care professionals should be aware of the prevalence of vitamin B-12 deficiency in older adults and be familiar with sources of synthetic vitamin B-12 to facilitate implementation of the new RDA.

Absorption↗
PubMed1999

Detection of single mammalian cells by high-resolution magnetic resonance imaging.

This study reports the detection of single mammalian cells, specifically T cells (T lymphocytes) labeled with dextran-coated superparamagnetic iron oxide particles, using magnetic resonance microscopy. Size amplification due to sequestration of the superparamagnetic particles in vacuoles enhances contrast in localized areas in high-resolution magnetic resonance imaging. Magnetic resonance images of samples containing differing concentrations of T cells embedded in 3% gelatin show a number of dark regions due to the superparamagnetic iron oxide particles, consistent with the number predicted by transmission electron microscopy. Colabeling of T cell samples with a fluorescent dye leads to strong correlations between magnetic resonance and fluorescence microscopic images, showing the presence of the superparamagnetic iron oxide particles at the cell site. This result lays the foundation for our approach to tracking the movement of a specific cell type in live animals and humans.

Animals↗
PubMed1999

Cardiac function of transplanted rat hearts using a working heart model assessed by magnetic resonance imaging.

BACKGROUND: A direct correlation between graft rejection and cardiac contractile function in small-animal models has been difficult to establish because (i) the conventional non-working heart model is greatly different from the orthotopic heart in terms of left ventricular work and (ii) it is difficult to obtain hemodynamic data in situ. We have used magnetic resonance imaging (MRI) techniques to obtain noninvasive, in-situ quantitation of ventricular volume after heterotopic cardiac transplantation. METHODS: Infra-renal heterotopic cardiac transplantation was performed on rats using syngeneic and allogeneic untreated donors in both working and non-working left heart models. An occluding balloon catheter in the inferior vena cava was used to vary the pre-load to the graft. An arteriovenous fistula was created to raise inferior caval oxygen saturation. Magnetic resonance imaging measurements were carried out at day 3, 4, and 5 post-transplantation. Left ventricle end-diastolic and end-systolic volumes were calculated using a biplanar ellipsoid model. RESULTS: Stroke volume was significantly increased in the working heart model as compared to the non-working model. At day 4 post-transplant, the diastolic pressure-volume relationship in the allograft group was significantly shifted, indicative of decreased myocardial distensibility, whereas the indices of systolic function were preserved. CONCLUSIONS: We have developed a heterotopic transplant working rat heart model and have used it to assess in-situ cardiac function by MRI. Sensitive indices of diastolic contractile function can be obtained in this rodent transplant model that correlate well with histologic evidence of early rejection.

Animals↗
PubMed1999

Simultaneous high-performance liquid chromatographic analysis for famotidine, ranitidine HCl, cimetidine, and nizatidine in commercial products.

A high-performance liquid chromatographic (HPLC) procedure for the simultaneous determination of famotidine (FMT), ranitidine HCl (RNT), cimetidine (CMT), and nizatidine (NZT) was developed using a two-level, full-factorial design with three variables (volume of methanol, percentage of triethylamine, and concentration of phosphate buffer) to select an acceptable mobile phase. A column (15 cm x 4.6 mm ID) of Inertsil ODS-2 (5 microns) was used, and 0.04 M aqueous sodium dihydrogen phosphate/acetonitrile/methanol/TEA at a proportion of 345/20/35/0.7 (v/v/v/v) was the selected mobile phase (1 ml/min). The detection wavelength was set at 230 nm, and procaine HCl was used as the internal standard. Precision and linearity of the method were assessed. None of the commercial samples was found to be outside the compendial limits of 90.0% to 110.0% of the claim amount.

Algorithms↗
PubMed1999

Paecilomyces lilacinus fungemia in an adult bone marrow transplant recipient.

Paecilomyces lilacinus is a rare fungal pathogen in humans. We report a case of fungemia caused by P. lilacinus in a non-neutropenic adult, 120 days after bone marrow transplant. The patient's primary risk factor was the presence of an indwelling vascular catheter. Her initial clinical course was characterized by fever, chills, and rigors. Blood cultures from the central line and peripheral veins were positive, as was a peripheral specimen drawn after removal of the catheter. Two initial peripheral specimens were positive for P. lilacinus only by blind subculture and/or sustained incubation. She developed peripheral pulmonary nodules following the fungemia, thus raising the possibility of disseminated disease, but definitive diagnosis was confounded by Pseudomonas bacteremia. The nodules cleared and she recovered following removal of the central line and treatment with amphotericin B and 5-fluorocytosine, despite in vitro resistance to these antifungal drugs. This case underscores the increasing importance of P. lilacinus as a human pathogen capable of producing disease in immunocompetent, as well as in immunocompromised hosts. Also of note is that blood culture systems may require extended incubation or subcultures in order to detect fungi.

Adult↗
PubMed1999

An acquired factor V inhibitor: clinical and laboratory features.

A sixty-five year old white male presented with an acquired Factor V inhibitor after an episode of cholecystitis and cefotaxime therapy. Plasma Factor V activity was less than 1%. He developed lower gastrointestinal bleeding a week after onset of coagulopathy, and was treated with plasmapheresis, fresh frozen plasma, oral cyclophosphamide, and prednisone. The coagulopathy resolved within four days of treatment, and within two weeks of presentation. Laboratory studies revealed an IgG inhibitor to Factor V that closely mimicked the more commonly encountered lupus anticoagulant. We would like to alert clinicians to this entity because, in contrast to a lupus anticoagulant, the acquired Factor V inhibitor can be associated with clinical bleeding as in our patient, and requires therapy prior to any surgical procedures.

Aged↗
PubMed1998

Inhibition of membrane lipid-independent protein kinase Calpha activity by phorbol esters, diacylglycerols, and bryostatin-1.

The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKCalpha, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKCalpha also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKCalpha were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this "non-membrane" PKC activity was inhibited by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCalpha. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4alpha-TPA (4alpha-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4alpha-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4alpha-TPA also displaced SAPD from the isolated C1 domain. These results show that although high and low affinity phorbol ester-binding sites are found on non-membrane-associated PKC, the phorbol ester binding properties change significantly upon association with membranes.

Allosteric Regulation↗