Biomedical subjects
B Malone
Publications and source records attributed to B Malone.
Differential regulation of three catalytic activities of platelet-activating factor (PAF)-dependent transacetylase.
We have previously established that PAF-dependent transacetylase (TA) purified to apparent homogeneity from rat kidney membranes and cytosol contains three separate catalytic activities, namely PAF lysophospholipid transacetylase (TAL), PAF sphingosine transacetylase (TAS), and PAF acetylhydrolase (AH). In the present investigation, we studied the biochemical factors and mechanism(s) that differentially regulate these three TA activities of the purified enzymes. We found that only the TAS activity of the TA purified from the membranes was stimulated by phosphatidyl-serine (PS) with optimal concentration of activation occurring at 25 microM. Other acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP), are partially effective, while diacylglycerol and free fatty acids had no effect on the TAS activity. PS exerted its effect on the TAS activity through the increases of both Km and Vmax. In addition, N-ethylmalimide (NEM) and dithiobis-(2-nitro-5-thiobenzoic acid) (DTNB) strongly inhibited the TAS activity and partially decreased the TAL and AH activities of the purified membrane enzyme in a dose-dependent manner. The addition of PS, but not by its substrate, sphingosine, could prevented the inhibition by NEM on the basal level of TAS. On the other hand, the inhibition of TAL by NEM and DTNB were partially protected by the substrate, lysoplasmalogens. Furthermore, PAF fully protects the inhibition of AH, partially protects the inhibition of TAL, and does not protect the inhibition of TAS by NEM. These results suggested that the three individual catalytic activities of TA have different dependencies on the thiol-containing residue(s) of the enzyme, i.e., cysteine. Furthermore, the nonresponsiveness of the purified cytosolic TAS to PS activation is consistent with our previous notions that membrane and cytosolic TA are posttranslationally distinct.
Solution fluorometric method for deoxynivalenol in grains.
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Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases.
Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.
Fas-mediated apoptosis eliminates B cells that acquire self-reactivity during the germinal center response to NP.
C57Bl/6 mice with the lpr mutation of Fas (CD95) were tested for deviation from the genetically restricted antibody response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). lambda1+ germinal centers (GC) with the canonical v186.2 V(H) gene element develop in lpr/lpr mice with the same time course as in wild-type (+/+) mice. In contrast to +/+ mice, however, lambda1+ GC persist in the spleens of lpr/lpr mice 25 days after immunization. Virtually all of the lambda1+ GC are reactive with NP 10 days after immunization. Sixteen days after immunization, however, many of the lambda1+ GC are not reactive with NP, and few of the lambda1+ GC are reactive with NP 25 days after immunization. The V(H) gene elements of three lambda1+NP- GC 25 days after immunization are derived by somatic mutation of v186.2, but have lost reactivity with NP. The mutated VDJs from these GC react with cells in spleen sections from +/+ and lpr/lpr mice, indicating that they represented secondary antibody responses induced by self antigens that are available as presented antigen. These data indicate that Fas-mediated apoptosis serves to eliminate a (limited) population of B cells that acquire reactivity to "self antigens" by somatic mutation of VDJs in the GC.
BTK mutations in patients with X-linked agammaglobulinemia: lack of correlation between presence of peripheral B lymphocytes and specific mutations.
X-linked agammaglobulinemia (XLA) is a human antibody deficiency that results from mutation of the tyrosine kinase btk. We tested the hypothesis that XLA patients who varied from the classic phenotype of XLA by presence of normal or near normal number of peripheral B lymphocytes would have a set of mutations of BTK that is different from the mutations found in patients without peripheral B lymphocytes. The mutations of BTK we found in two patients with normal numbers of peripheral B lymphocytes have been previously identified in patients without peripheral B lymphocytes. A third patient, without peripheral B cells, was found to express normal levels of wild type btk. Exmination of the mutations of the BTK gene in patients in the BTKbase who were identified as having peripheral B lymphocytes found that these same mutations, or mutations of the same protein domains, were also present in patients identified as lacking peripheral B lymphocytes. Analysis of mutations in BTK has previously led to the conclusion that severity of disease in XLA cannot be predicted from the specific mutation of BTK. The results of this study suggest that whether an XLA patient will develop peripheral B lymphocytes cannot be predicted from the specific mutation of BTK.
A tribute to Beverly Malone. Interview by Val J. Halamandaris.
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Beverly Malone. Interview by Ian McMillan.
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Chlorinated hydrocarbon contaminants in polar bears from eastern Russia, North America, Greenland, and Svalbard: biomonitoring of Arctic pollution.
Adipose tissue samples from polar bears (Ursus maritimus) were obtained by necropsy or biopsy between the spring of 1989 to the spring of 1993 from Wrangel Island in Russia, most of the range of the bear in North America, eastern Greenland, and Svalbard. Samples were divided into 16 regions corresponding as much as possible to known stocks or management zones. Concentrations of dieldrin (DIEL), 4,4'-DDE (DDE), sum of 16 polychlorinated biphenyl congeners (sigma PCB), and sum of 11 chlordane-related compounds and metabolites (sigma CHL) were determined. In order to minimize the effect of age, only data for adults (320 bears age 5 years and older) was used to compare concentrations among regions. Concentrations of sigma PCB were 46% higher in adult males than females, and there was no significant trend with age. Concentrations of sigma CHL were 30% lower in adult males than females. Concentrations of sigma PCB, sigma CHL, and DDE in individual adult female bears were standardized to adult males using factors derived from the least-square means of each sex category, and geometric means of the standardized concentrations on a lipid weight basis were compared among regions. Median geometric mean standardized concentrations (lipid weight basis) and ranges among regions were as follows: sigma PCB, 5,942 (2,763-24,316) micrograms/kg; sigma CHL, 1,952 (727-4,632) micrograms/kg; DDE, 219 (52-560) micrograms/kg; DIEL, 157 (31-335) micrograms/kg. Geometric mean sigma PCB concentrations in bears from Svalbard, East Greenland, and the Arctic Ocean near Prince Patrick Island in Canada were similar (20,256-24,316 micrograms/kg) and significantly higher than most other areas. Atmospheric, oceanic, and ice transport, as well as ecological factors may contribute to these high concentrations of sigma PCB. sigma CHL was more uniformly distributed among regions than the other CHCs. Highest sigma CHL concentrations were found in southeastern Hudson Bay, which also had the highest DDE and DIEL concentrations. In general, concentrations of sigma CHL, DDE, and DIEL were higher in eastern than western regions, suggesting an influence of North American sources. Average sigma PCB concentrations in bears from the Canadian Arctic were similar to those in 1982-84, while average sigma CHL and DDE concentrations were 35-44% lower and DIEL was 90% lower. However, the significance of these temporal trends during the 1980s is not conclusive because of the problems of comparability of data.
International focus: feeling the buzz. Interview by Ian McMillan.
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Transcription of germline VH gene elements by normal human fetal liver.
Transcription of the gene elements that form the variable region of immunoglobulin heavy chains has been proposed to represent the process that controls access for the recombination enzymes in their sequential steps of catalysis. Evidence for germline transcription of VH gene elements, as part of VH to DJH recombination, has been limited to transcripts of only a few gene elements. We have examined normal fetal liver mRNA by Northern blotting and present evidence for germline transcripts from six human VH gene families. The candidate VH4 transcripts have been confirmed as germline transcripts by hybridization with 3' flanking sequences that would have been removed by recombination from mature VHDJH genes. The candidate transcripts for VH1, VH3, VH4 and VH6 have been confirmed by polymerase chain reaction amplification with primers from the 3' flanking sequences of these gene families and determination of the sequence of these products. Determination of sequence from two clones of VH1, VH3 and VH4 indicates that more than one gene from each of these families is transcribed. PCR amplification of VH4 and VH6 with primers specific for the leader sequence (exon 1) and 3' flanking sequence indicate that these transcripts are spliced, representing RNA processing. Germline transcripts from these families are also present in normal human bone marrow. These results indicate that transcriptional activation of germline VH gene elements is a general phenomenon in tissues undergoing V to DJ recombination.
Teagle LPN to BSN Initiative: a success story.
Success was ultimately reflected by the lack of difference in the performance of the generic BSN nursing student and the LPN to BSN student. While there were slight variations in NCLEX-RN examination pass rates-LPNs, 100 percent; control group of generic students, 97 percent-this initiative showed the enormous potential of a predominantly female population of health care providers who have frequently been discounted by baccalaureate nursing education. Some of the critical factors leading to success in the Teagle Initiative seem to be: an interested, supportive faculty, counseling support, curricula revised to acknowledge the LPNs' prior learning, and tuition and resource support. These success stories prove that, by nursing doing the unexpected and continuing to open its educational arms to those in the health care provider family who require additional knowledge, skills and abilities, the health care environment benefits.
Biosynthesis of N-acetylsphingosine by platelet-activating factor: sphingosine CoA-independent transacetylase in HL-60 cels.
We have previously identified a novel CoA-independent transacetylase in the membrane fraction of HL-60 cells that transfers the acetate group from platelet activating factor (PAF) to a variety of lysophospholipid acceptors (Lee, T.-c., Uemura, Y., and Snyder, F. (1992) J. Biol. Chem. 267, 19992-20001). In the present study, we demonstrate that a similar transacetylase can transfer the acetate group from PAF to sphingosine forming N-acetylsphingosine (C2-ceramide). The chemical structure of the reaction product, C3-ceramide, was established by its identical Rf value with authentic C2-ceramide standard on thin-layer plate, sensitivity to acid treatment, resistance to alkaline hydrolysis, and ability to form the C2-ceramide dibenzoate derivative. Nonspecific transfer of the acetate from PAF to sphingosine in the absence of enzyme and nonlinearity of the reaction rates were rectified by complexing sphingosine to bovine serum albumin in a 1:1 molar ratio. Under these conditions, the apparent Km for PAF is 5.4 microM, which is in the same range as the Km (12.0 microM) when lysoplasmalogen is the acetate acceptor. PAF:sphingosine transacetylase has a narrow substrate specificity and strict stereochemical configuration requirements. Ceramide, sphingosylphosphocholine, stearylamine, sphingosine 1-phosphate, or sphingomyelin are not substrates, whereas sphinganine has a limited capacity to accept the acetate from PAF. Also, only the naturally synthesized D-erythroisomer but not the synthetic L-erythro-, D-threo-, or L-threosiomers of sphingosine can serve as a substrate. PAF transacetylase activity is widely distributed among several tissues and may involve histidine and cysteine for its catalytic activity due to inhibitory effects to the enzyme by diethyl pyrocarbonate and N-ethylmaleimide, respectively. C2-ceramide is produced via PAF:sphingosine transacetylase, and physiological levels of C2-ceramide are detected in both undifferentiated and differentiated intact HL-60 cells. Collectively, because C2-ceramide has many biological activities that differ from that of PAF and sphingosine, the CoA-independent, PAF-dependent transacetylase serves as a modifier of PAF, and sphingosine functions by generating a variant lipid mediator, C2-ceramide.
Shared visions & hallucinations: acquiring a taste for chaos.
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Strengthening nursing through partnerships.
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The power of nursing education.
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Creating an environment for positive patient outcomes: an interview with Beverly Malone. Interview by Karen E. Schardin.
With health care reform initiatives sweeping America, nurses must now more than ever strive to create environments to enhance positive patient outcomes. In this interview, Beverly Malone, PhD, RN, FAAN, dean of the School of Nursing at North Carolina A & T State University in Greensboro, NC, describes nurses' roles in today's managed care climate. Dr. Malone, who will deliver the keynote address at ANNA's 26th National Symposium on June 10 in Philadelphia, discusses future recruitment of students, minority issues, transforming the nursing work force, validating nursing's worth, and the future of nursing.
Differentiation induced increase of platelet-activating factor acetylhydrolase in HL-60 cells.
Platelet-activating factor (PAF) acetylhydrolase catalyzes the conversion of PAF to lyso-PAF and acetate. In this study we show that induced cellular differentiation of HL-60 cells grown in chemically defined media by dimethylsulfoxide (DMSO) to granulocytic cells increases the acetylhydrolase activity with a concomitant increased secretion of the enzyme into the media. This increase in acetylhydrolase activity is blocked by the presence of actinomycin D (1 microM) or cycloheximide (1-2 microM) in the culture media. Acetylhydrolase is located both in the cytosolic and particulate fractions; the relative distribution of acetylhydrolase activity in the particulate fraction and cytosol increases and decreases respectively, as the differentiation progresses. The addition of an intracellular protein transport inhibitor, monensin, causes further accumulation of acetylhydrolase activity in the particulate fraction and a decrease in the media, with no effect on the acetylhydrolase activity in the cytosol. Acetylhydrolase in differentiated HL-60 cells acquires properties similar to those of the plasma acetylhydrolase in that it becomes less sensitive to 5,5'-dithiobis-2-nitrobenzoic acid and p-bromophenacylbromide inhibition than the acetylhydrolase in undifferentiated cells. The acetylhydrolase secreted into the media by the differentiated cells was almost totally insensitive to these inhibitors, whereas the acetylhydrolase from the particulate fraction gave an intermediate response; the cytosolic acetylhydrolase was sensitive to both inhibitors. However, the acetylhydrolase secreted by differentiated HL-60 cells has a different electrophoretic mobility, temperature sensitivity, and association with lipoproteins when compared to that of human plasma acetylhydrolase. Collectively, these results indicate cellular differentiation induces intracellular acetylhydrolase activity through a mechanism involving both transcriptional and translational events. Furthermore, the acetylhydrolase synthesized during the DMSO-induced differentiation of HL-60 cells is then secreted into the media via the intracellular membrane transport system for proteins. Based on results obtained with HL-60 cells as a cell model, it is likely that more than one isoform of acetylhydrolase exists in the extracellular milieu.