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D Leonard

Publications and source records attributed to D Leonard.

At least 55 records · Page 3Linked to original sources
PubMed1993

Identification of a novel protein with GDP dissociation inhibitor activity for the ras-like proteins CDC42Hs and rac I.

We have recently cloned the human cDNA for a gene, denoted D4, that encodes a protein 67% identical to the bovine rhoGDI protein, a GDP dissociation inhibitor (GDI) for the ras-related rho-subtype proteins. We now present data on the cloning and structural analysis of the murine D4 cDNA and confirm its preferential expression in hematopoietic tissues. The predicted murine and human D4 proteins are almost 90% identical, indicating that D4 and rhoGDI are different genes and that they are probably members of a related family of genes. Functional studies with the human D4 protein demonstrate that D4 has GDI activity against the CDC42Hs and rac I proteins, but binds to these proteins with a significantly weaker affinity than does the rho-subtype GDI. These data suggest that D4, which will in subsequent communications be denoted as GDI.D4, might be a GDI for other known or as yet unidentified ras-like GTP-binding proteins. Alternatively, D4 could have other biochemical functions. During murine embryogenesis, D4 transcripts are detected in yolk-sac cells, where the earliest hematopoietic precursors are found. When these precursors undergo proliferation and differentiation in vitro, a dramatic increase in D4 expression is seen. D4 probably has a significant function during the growth and development of hematopoietic precursors.

Amino Acid Sequence↗
PubMed1993

Improving vaccination coverage: the experience of the Expanded Programme on Immunization in Vanuatu.

This paper looks at the success of measures adopted to improve vaccination coverage of infants in the Republic of Vanuatu. In 1982 the Department of Health introduced an Expanded Programme on Immunization (EPI). Since the republic has over 80 inhabited islands, a scattered population, rough terrain and a lack of transport and communications infrastructure, achieving a high vaccination coverage rate proved difficult. Coverage of infants remained low until 1987. From that year onwards various strategies were employed to increase coverage, including 1) adopting the WHO-recommended diphtheria-pertussis-tetanus (DPT) and oral polio vaccine (OPV) vaccination schedule (6, 10 and 14 weeks) instead of the former schedule (3, 6 and 9 months); 2) improving the training, support and supervision of staff delivering maternal and child health (MCH) services; and 3) improving community involvement through social mobilization activities in areas of low coverage. Data on vaccination coverage of infants for the period 1984 to 1990 were compared. Over this period coverage of infants with 3 doses of DPT rose from 29% to 76%, with 3 doses of OPV from 29% to 78%, and with measles vaccine from 19% to 66%. These dramatic improvements have largely occurred since 1987. The results demonstrate the success of the measures adopted, and the experience of Vanuatu offers lessons in improving vaccination coverage for other countries in the region.

Community Participation↗
PubMed1992

The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein.

The ras-related protein, CDC42Hs, is a 22-kDa GTP-binding protein which is the human homolog of a Saccharomyces cerevisiae yeast-cell-division cycle protein. In attempting to isolate and biochemically characterize mammalian proteins capable of regulating various activities of CDC42Hs, we have identified an activity in bovine brain cytosol which effectively inhibits the dissociation of [3H]GDP from the platelet- or the Spodoptera frugiperda-expressed CDC42Hs protein. The purification of this activity was achieved by a series of steps which included ammonium sulfate fractionation, DEAE-Sephacel, Mono-Q, and Mono-S chromatographies. The purified CDC42Hs regulatory protein has an apparent molecular weight of 28,000, and cyanogen bromide-generated peptide sequences of this protein were identical to sequences from the carboxyl-terminal portion of rho-GDP-dissociation inhibitor (rho-GDI) (Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H., Araki, S., Ueda, T., Kikuchi, A., and Takai, Y. (1990) Oncogene 5, 1321-1328). In addition, an Escherichia coli-expressed, glutathione S-transferase-rho-GDI fusion protein fully substitutes for the GDI which we have purified from bovine brain in its ability to inhibit GDP dissociation from CDC42Hs. These findings suggest either that a common regulatory protein (GDI) is capable of inhibiting GDP dissociation from the rho and CDC42Hs proteins or that these two GTP-binding proteins interact with GDI proteins of very similar structure. The purified brain GDI protein shows little ability to inhibit GDP dissociation from the E. coli-expressed CDC42Hs and is capable of only a very weak inhibition of the dissociation of [35S]guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) from the Spodoptera frugiperda-expressed CDC42. However, brain GDI very effectively inhibits the ability of the human dbl oncogene product to catalyze GDP dissociation from CDC42Hs. In addition to influencing guanine nucleotide association with CDC42Hs, the purified brain GDI protein also appears to catalyze the dissociation of CDC42Hs from the plasma membranes of human placenta and human epidermoid carcinoma (A431) cells. This effect by the GDI protein is observed whether the membrane-associated CDC42Hs is preincubated with GDP, GTP gamma S, or no guanine nucleotides, and occurs over a similar concentration range as that necessary for the inhibition of the intrinsic GDP dissociation.

Amino Acid Sequence↗
PubMed1992

A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs.

Members of the family of Ras-related guanosine triphosphate (GTP) binding proteins appear to take part in the regulation of a number of biological processes, including cell growth and differentiation. Three different classes of proteins that regulate the GTP binding and GTP hydrolytic activities of the Ras family members have been identified. These different regulatory proteins inhibit guanosine diphosphate (GDP) dissociation (designated as GDIs), stimulate GDP dissociation and GDP-GTP exchange (designated as GDSs), or stimulate GTP hydrolysis (designated as GAPs). In the case of the Ras-like protein CDC42Hs, which is the human homolog of a Saccharomyces cerevisiae cell division cycle protein, the GDI protein also inhibited both the intrinsic and GAP-stimulated hydrolysis of GTP. These findings establish an additional role for the GDI protein--namely, as a guanosine triphosphatase (GTPase) inhibitory protein for a Ras-like GTP binding protein.

Animals↗
PubMed1992

Evaluation of nonionic nitroxyl lipids as potential organ-specific contrast agents for magnetic resonance imaging.

Considering their intrinsic properties of accumulation in the hepatic tissue, we have synthesized nitroxyl-containing lipids as potential organ-specific contrast agents for magnetic resonance imaging (MRI). Their resistance to reduction by ascorbate and in liver homogenates, and their relaxivity in different media were investigated and compared to those of free carboxyl-Proxyl (3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl) and Tempamaine (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl). With respect to the reduction rates by ascorbate, the lipid derivatives show the same well-known order of reactivity as carboxy-Proxyl and Tempamine, the five-membered nitroxyls being more stable than the six-membered compounds. However the binding of the piperidinoxyl compounds to the fatty acids confers to those lipid derivatives a markedly increased stability. Similarly, in liver homogenates, the nitroxyl lipids remained unchanged more than 20 min, contrarily to carboxy-Proxyl and Tempamine. The measurements of spin-lattice relaxation time (T1) in biological media have demonstrated a higher relaxivity of nitroxyl lipids, which can be related to their interaction with proteins. Tested in vivo, one of the synthesized compounds (0.75 mmol/kg) produced an enhancement of 44 +/- 12% of the hepatic signal 5 min after intraportal injection in T1-weighted images. The potential applicability of the other nitroxyl lipids as contrast agents for MRI was limited in the in vivo studies by an unexpected toxicity. Work is currently in progress to improve the therapeutic index of the present class of nitroxyl lipids.

Animals↗
PubMed1992

Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins.

Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form. This receptor-mediated event occurs in the cytoplasm, with subsequent translocation of the activated factor to the nucleus. ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma. ISGF3 gamma serves as the DNA recognition subunit, while ISGF3 alpha, which appears to consist of three polypeptides, is a target for alpha interferon signaling and serves as a regulatory component whose activation is required to form ISGF3. ISGF3 gamma DNA-binding activity was identified as a 48-kDa polypeptide, and partial amino acid sequence has allowed isolation of cDNA clones. ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells. Sequencing of ISGF3 gamma cDNA clones revealed significant similarity to the interferon regulatory factor (IRF) family of DNA binding proteins in the amino-terminal 117 residues of ISGF3 gamma. The other IRF family proteins bind DNA with a specificity related to but distinct from that of ISGF3 gamma. We note sequence similarities between the related regions of IRF family proteins and the imperfect tryptophan repeats which constitute the DNA-binding domain of the c-myb oncoprotein. These sequence similarities suggest that ISGF3 gamma and IRF proteins and the c-myb oncoprotein use a common structural motif for DNA recognition. Recombinant ISGF3 gamma, like the natural protein, interacted with HeLa cell ISGF3 alpha to form the mature ISGF3 DNA-binding complex. We suggest that other IRF family members may participate in signaling pathways by interacting with as yet unidentified regulatory subunits analogous to ISGF3 alpha.

Amino Acid Sequence↗
PubMed1992

Postsplenectomy leukocystosis: physiologic or an indicator of infection?

The records of 42 patients who underwent splenectomy following blunt abdominal trauma were reviewed to determine whether white blood cell (WBC) count is a useful indicator of acute infection in the postoperative period. Patients, ranging in age from 14 to 54 years, were divided into two groups: 1) infected (n = 18) and 2) noninfected (n = 24). Group average daily WBC counts (thousands/mm3) and differential counts (polymorphonuclear leukocyte/bands) were evaluated for the first 14 postoperative days. The infected group had a mean WBC of 21.2 and a mean differential count of 73/8. The average daily WBC count began to rise on Day 4 (WBC = 10.8) and peaked on Day 7 at 27.8. There were 30 episodes of infection: 14 pneumonias and 16 others, including sinusitis, empyema, subphrenic abscess, urinary tract infection, and bacteremias. The noninfected group had a mean WBC count of 14.2 and a mean differential of 73/5. For this group, the average WBC peaked by the postoperative Day 2 (WBC = 15.9), then the trend was downward. Thus, for patients with traumatic splenic injuries, WBC count may predict infection in the postsplenectomy period. In the noninfected group, the authors observed no average daily WBC count above 16; whereas, in the infected group, no average daily WBC count less than 16 was observed. The differential counts were not significantly different between the two groups.

Abdominal Injuries↗
PubMed1991

Should survivors with an injury severity score less than 10 be entered in a statewide trauma registry?

The necessity of including survivors with minor (ISS less than 10) injuries in a statewide trauma registry with a quality assurance focus was evaluated. During a 3-month period, data for 3,594 admissions to 28 trauma centers were entered into the registry. Of these admissions 1,696 patients (50.8% of patients studied) had an ISS less than 10. Of those, 10 (0.6%) were nonsurvivors and 67 (3.9%) had severe disability (66) or were in a persistent vegetative state (PVS) (1) at hospital discharge. Five nonsurvivors were 65 years of age or older. Four were injured in falls and one was an injured pedestrian. The disabled subset included a high percentage of older patients (61.2% greater than or equal to 55). Minor falls, including those from a bed or chair or from the same level accounted for nearly one half (46.2%) of the disabling injuries. Fifty-one disabled patients had isolated extremity or pelvic fractures. Their hospital stays ranged from 1 to 42 days and averaged 13.0 days. The proportion of elderly in the United States is increasing substantially. Because of the significant risk of death or serious disability to elderly patients, even with minor injuries, we conclude it is appropriate to include data for elderly patients with an ISS less than 10 who meet other registry inclusion criteria. We also recommend the entry of data for patients with an ISS less than 10 and significant disability at discharge who qualify by other criteria. Exclusion of remaining patients with an ISS less than 10 would reduce qualifying cases by 38%.

Adolescent↗
PubMed1989

Glutamine transport by rat basolateral membrane vesicles.

Glutamine, a neutral amino acid, is unlike most amino acids, has two amine moieties which underlies its importance as a nitrogen transporter and a carrier of ammonia from the periphery to visceral organs. The gastrointestinal tract utilizes glutamine as a respiratory substrate. The intestinal tract receives glutamine from the luminal side and from the arterial side through the basolateral membranes of the enterocyte. This study characterizes the transport of glutamine by basolateral membrane vesicles of the rat. Basolateral membranes were prepared by a well validated technique of separation on a percoll density gradient. Membrane preparations were enriched with Na+/K+-ATPase and showed no 'overshoot' phenomena with glucose under sodium-gradient conditions. Glutamine uptake represented transport into the intravesicular space as evident by an osmolality study. Glutamine uptake was temperature sensitive and driven by an inwardly directed sodium gradient as evident by transient accumulation of glutamine above the equilibrium values. Kinetics of glutamine uptake under both sodium and potassium gradients at glutamine concentrations between 0.01 and 0.6 mM showed saturable processes with Vmax of 0.39 +/- 0.008 and 0.34 +/- 0.05 nmol/mg protein per 15 s for both sodium-dependent and sodium-independent processes, respectively. Km values were 0.2 +/- 0.01 and 0.55 +/- 0.01 mM, respectively. pH optimum for glutamine uptake was 7.5. Imposition of negative membrane potential by valinomycin and anion substitution studies enhanced the sodium-dependent uptake of glutamine suggesting an electrogenic process, whereas the sodium-independent uptake was not enhanced suggesting an electroneutral process. Other neutral amino acids inhibited the initial uptake of glutamine under both sodium-dependent and sodium-independent conditions. We conclude that glutamine uptake by basolateral membranes occurs by carrier-mediated sodium-dependent and sodium-independent processes. Both processes exhibit saturation kinetics and are inhibited by neutral amino acids. The sodium-dependent pathway is electrogenic whereas the sodium-independent pathway is electroneutral.

Animals↗
PubMed1989

Burn injury alters intestinal glutamine transport.

Several studies have established that intestinal glutamine (GLN) metabolism is altered during catabolic states. It remains unclear whether these alterations are due to a defect in metabolism or in transport of the amino acid. The present study examines the kinetics of GLN transport across basolateral membrane vesicles (BLMV) of enterocytes obtained from control rats and rats subjected to 20% full-thickness scald burn, 48 hr previously. BLMV were prepared from freshly isolated enterocytes using differential centrifugation with separation on a Percoll density gradient. BLMV were enriched (10- to 12-fold) with Na+-K+-ATPase while markers for brush border membranes were impoverished. Previous studies from our laboratory indicated that, in this preparation, GLN transport is into an osmotically sensitive space, dependent on GLN concentration, linear up to 30 sec, and both temperature and Na+ dependent. Our results indicate that in thermal injury, initial rates of GLN uptake were depressed (y = 3.67 + 0.435X for burned rats vs y = 18.7 + 0.907X for controls, P less than 0.01). Kinetic analysis of GLN uptake showed a marked decrease in transport Vmax (81.8 +/- 15 nmole/mg protein/15 sec for burned rats vs 185 +/- 17 nmole/mg protein/15 sec for controls, P less than 0.001). Transport Km also decreased from 0.25 +/- 0.004 mM for controls to 0.08 +/- 0.03 mM glutamine for burned rats (P less than 0.001). Kinetic studies performed at GLN levels greater than 0.6 mM showed that GLN uptake proceeded by a nonsaturable process in both the control and burned rats. No significant alteration in this nonsaturable component was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Calcium transport by plasma membranes of enterocytes during development: role of 1,25-(OH)2 vitamin D3.

Calcium transport across the intestinal enterocytes represents an entry process at the brush border membranes and an ATP-dependent exit process located at the basolateral membranes. Both processes exhibit developmental changes. The present studies were designed to define the role of vitamin D in calcium transport during maturation. Brush border and basolateral membranes from vitamin D-deficient suckling and adolescent rats were used to study calcium entry and exit. 1,25-(OH)2 vitamin D3 administration enhanced calcium entry at the brush border membranes of suckling and adolescent rats. The increase in calcium uptake in both age groups was secondary to an increase in maximal transport capacity (Vmax) rather than in Km. In suckling rat brush border membranes, 1,25-(OH)2 vitamin D3 treatment increased the Vmax from 1.0 +/- 0.1 to 1.8 +/- 0.2 nmol/mg protein/7 s (p less than 0.01), whereas in adolescent rats, Vmax increased from 1.5 +/- 0.1 to 2.5 +/- 0.3 nmol/mg protein/7 s (p less than 0.01). Km values were not altered. Similarly, 1,25-(OH)2 vitamin D3 administration enhanced ATP-dependent calcium exit at the basolateral membranes of both suckling and adolescent rats. Vmax of ATP-dependent calcium uptake by basolateral membranes of suckling rats increased from 0.5 +/- 0.05 to 0.81 +/- 0.06 nmol/mg protein/20 s (p less than 0.01) whereas in adolescent rats, Vmax increased from 0.3 +/- 0.03 to 0.6 +/- 0.04 nmol/mg protein/20 s (p less than 0.001). Km values were not altered. The current studies indicate that 1,25-(OH)2 vitamin D3 stimulates calcium entry and exit across the enterocytes during maturation.

Animals↗
PubMed1987

Effects of pregnenolone-16 alpha-carbonitrile on drug metabolizing enzymes in hypophysectomized female rats.

Treatment of intact and hypophysectomized female rats with pregnenolone-16 alpha-carbonitrile (PCN) resulted in a significant increase in hepatic aryl hydrocarbon hydroxylase (AHH) activity. However, the total cytochrome P-450 concentration, as measured by CO difference spectra, was increased to a greater extent in hypophysectomized rats than in intact rats. Total cytochrome P-450 was found to be 0.82 +/- 0.16 vs 2.43 +/- 0.31 nmoles/mg protein for control and PCN-treated hypophysectomized rats, respectively, and 0.68 +/- 0.23 vs 1.28 +/- 0.05 nmoles/mg protein for control and PCN-treated intact rats respectively. The concentration of metyrapone complex in microsomes from intact control and PCN-treated rats was found to be 0.4 +/- 0.11 vs 1.88 +/- 0.23 M respectively. Treatment of hypophysectomized rats with PCN resulted in an approximate 10-fold increase in the concentration of the metyrapone complex (0.42 +/- 0.15 M for control and 4.46 +/- 0.44 M for PCN-treated). Microsomal NADPH and NADPH cytochrome c reductase activities were also altered by PCN-treatment. Aminopyrine demethylase activity was stimulated approximately three-fold by PCN treatment in both intact and hypophysectomized rats. Benzphetamine demethylase activity was not significantly affected by PCN treatment. The results of these studies suggest that the absence of the pituitary gland can markedly influence PCN induction of cytochrome P-450 in the liver in female rats. PCN also differentially affects microsomal mixed-function oxidase activities associated with drug and xenobiotic metabolism.

Aminopyrine N-Demethylase↗
PubMed1987

Recovery of muscle from tetrodotoxin-induced disuse and the influence of daily exercise. 1. Contractile properties.

The extent of recovery in rat gastrocnemius muscles which have undergone atrophic changes due to 4 weeks of inactivity caused by sciatic nerve superfusion of tetrodotoxin, was investigated. Control and tetrodotoxin-treated female Sprague-Dawley rats were subjected to either a daily program of grid-climbing or swim-training for 4 weeks, or benefited from cage activity only. In situ contractile properties of the gastrocnemius were then measured. Twenty-eight days of recovery was insufficient to allow a complete reversal of the changes produced by 28 days of inactivity. The gastrocnemius remained atrophied (25%) and tetanically weaker than normal in the recovered animals. The maximal rate of rise of the twitch was partially recovered whereas that developed during a maximal contraction was normal. Maximal rate of rise expressed relative to the tension output was elevated, however, for both twitch (%Pt/ms) and maximal contraction (%Po/ms). Swimming did not influence the recovery of muscle size or strength. Grid-climbing, on the other hand, produced heavier and stronger muscles and a faster recovery of %Pt/ms. This study clearly illustrates the importance of muscle load in regulating muscle size and strength.

Animals↗
PubMed1986

Regional blood flow and neurohormonal responses to milrinone in congestive heart failure.

We measured systemic hemodynamics, regional blood flow, and neurohormonal parameters in 13 patients with severe chronic congestive heart failure before and after 1 month of therapy with oral milrinone, a bipyridine cardiotonic agent. After milrinone there were significant reductions in pulmonary wedge pressure (27 +/- 2 to 19 +/- 3 mm Hg; P less than 0.02) and systemic vascular resistance (1866 +/- 152 to 1393 +/- 93 dyne X sec/cm5; P less than 0.05) that were associated with increases in cardiac index (1.85 +/- 0.15 to 2.47 +/- 0.20 L/min/m2; P less than 0.02). There was a marked improvement in forearm blood flow (1.98 +/- 0.14 to 3.02 +/- 0.16 ml/min/dl; P less than 0.01) and a reduction in forearm vascular resistance (45 +/- 3 to 30 +/- 3 U; P less than 0.01). Overall there was no significant change in renal blow flow, renal vascular resistance, or glomerular filtration rate. However, there was a heterogeneous response of renal blood flow and glomerular filtration rate, such that both were directly correlated with the magnitude of increase of cardiac index (r = 0.587 [P less than 0.05] and r = 0.721 [P less than 0.01], respectively). After milrinone there were no significant overall or subgroup changes in urinary sodium excretion, blood volume, plasma renin activity, urinary aldosterone levels, plasma or platelet vasopressin levels, or plasma norepinephrine levels. Thus 1 month of therapy with milrinone improves systemic and forearm hemodynamics, but its effects on renal blood flow and function were heterogeneous. These heterogeneous effects on regional blood flow may depend on the relative vasodilator and inotropic effects of milrinone.

Aged↗
PubMed1986

Age-dependent inhibition of pentobarbital sleeping time by ozone in mice and rats.

The effect of age on the metabolism of pentobarbital in mice and rats was investigated following exposure to 0.3 ppm of ozone for 3.75 hr. Young animals were 2.5 months of age and the mature were 18 months. The pentobarbital sleeping time was significantly prolonged following the ozone exposure in both the mice and rats when compared with an air control. No ozone effect on sleeping time was found in the young animals. The results indicate that there may be an age-related sensitivity to the occurrence of ozone-related inhibition of pentobarbital metabolism.

Aging↗