Biomedical subjects

R Green

Publications and source records attributed to R Green.

At least 199 records · Page 11Linked to original sources

PubMed1995

Properties of mitochondrial DNA polymerase in mitochondrial DNA synthesis in yeast.

Mitochondrial DNA polymerase from Saccharomyces cerevisiae, purified 3500 fold, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three polypeptides. The major 150 kDa polypeptide was probably the catalytic subunit of the mitochondrial (mt) DNA polymerase and the other two polypeptides could be either proteolytic cleavage products of the polymerase, other subunits of the enzyme or protein contaminants. The mtDNA polymerase preferred an A+T-rich DNA template and did not require any RNA primer for DNA synthesis, at least under in vitro reaction conditions. It showed higher processivity on a double-stranded linear DNA template than on a single-stranded circular DNA template, and was capable of synthesizing at least about 1200 nucleotide primer-extended products without any major pause on a double-stranded DNA template.

Base Sequence↗

PubMed1994

Crystallization of the malonyl coenzyme A-acyl carrier protein transacylase from Escherichia coli.

The malonyl coenzyme A-acyl carrier protein transacylase, a single polypeptide chain of 358 amino acid residues and a molecular mass of 32 kDa, is a key component of the fatty acid synthase multienzyme complex. The elucidation of its three-dimensional structure will help in the understanding of the molecular basis of the biosynthesis of fatty acids, as well as of polyketides and related biologically active molecules. Three X-ray-quality crystal forms of the Escherichia coli fabD gene product encoding for malonyl coenzyme A-acyl carrier protein transacylase have been obtained using the hanging-drop method and ammonium sulfate as precipitant. Two are tetragonal and each contains two molecules in the asymmetric unit (form I: space group P4(3(1))2(1)2 with a = b = 83.9 A, c = 166.5 A and form II: space group P4 with a = b = 132.64 A, c = 38.85 A), whereas the third form belongs to the hexagonal system and contains one molecule in the asymmetric unit (space group P6(1(5)) with a = b = 68.52 A, c = 117.71 A). In each case, the diffraction pattern extends to approximately 2.0 A resolution using CuK alpha radiation from a rotating anode source.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed1994

Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of beta-1,4-D-glycanases.

The crystal structure of the 32-kDa catalytic domain of the Streptomyces lividans xylanase A was solved by molecular isomorphous replacement methods and subsequently refined at 2.6-A resolution to a conventional crystallographic R factor of 0.21. This is the first successful structure determination of a member of the F family of endo-beta-1,4-D-glycanases. Unlike the recently determined xylanases of the G family (Wakarchuk, W. W., Campbell, R. L., Sung, W. L., Davoodi, J., and Yaguchi, M. (1994) Protein Sci. 3, 467-475), where the catalytic domains have a unique beta-sheet structure, the 32-kDa domain of the S. lividans xylanase A is folded into a complete (alpha/beta)8 barrel, the first such fold observed among beta-1,4-D-glycanases. The active site is located at the carbonyl end of the beta barrel. The crystal structure supports the earlier assignment of Glu-128 and Glu-236 as the catalytic amino acids (Moreau, A., Roberge, M., Manin, C., Shareck, F., Kluepfel, D., and Morosoli, R. (1994) Biochem. J., in press).

Binding Sites↗

PubMed1994

Effect of formate and oxalate on fluid reabsorption from the proximal convoluted tubule of the anaesthetized rat.

1. It has recently been suggested that both formate and oxalate may be involved in the reabsorption of chloride from the proximal convoluted tubule (PCT). Reabsorption is achieved via a mechanism coupling Na(+)-H+ exchange to Cl(-)-anion exchange thus facilitating net NaCl transport. This possible mechanism was investigated further by observing the effect of luminal addition of formate and oxalate on proximal tubule transport in anaesthetized rats, previously prepared for in vivo microperfusion. 2. Addition of formate (0.25 mmol l-1) to the luminal perfusate resulted in a significant stimulation of fluid reabsorptive rate. 3. Oxalate was found to have a concentration-dependent effect on fluid reabsorption. Luminal oxalate concentrations below 0.05 mmol l-1 stimulated fluid reabsorption, while an oxalate concentration of 0.5 mmol l-1 inhibited fluid reabsorption. 4. The presence of formate (0.25 mmol l-1) and oxalate (0.001 mmol l-1) in the same luminal perfusate stimulated fluid reabsorption from the proximal tubule lumen, but the effects of formate and oxalate were not additive. 5. The removal of bicarbonate from the luminal perfusate and its replacement with phosphate had no significant effect on fluid reabsorptive rate. An alteration in perfusate pH was also ineffective. Addition of oxalate at high and low pH failed to stimulate fluid reabsorption compared with pH-matched control perfusions. 6. The experimental data suggest the involvement of both formate and oxalate in the reabsorption of NaCl from the proximal tubule lumen, though their influence may not be exerted through the same mechanisms.

Absorption↗

PubMed1994

Crystallization of thioesterase II from Escherichia coli.

X-ray quality single crystals of the Escherichia coli thioesterase II have been obtained. The protein used for crystallization was overexpressed in parent organism. The crystals are orthorhombic, space group C222(1) with axial lengths a = 99.0 A, b = 121.1 A, c = 166.6 A. A complete homotetramer (120,000 Da) of four polypeptide chains, each 286 residues long, occupies the asymmetric unit. The diffraction pattern extends to approximately 2.4 A resolution using CuK alpha radiation from a rotating anode source. The elucidation of the three-dimensional structure of this unusual bacterial thioesterase will provide basis for the analysis of its unique catalytic mechanism and substrate specificity.

Crystallization↗

PubMed1994

In vitro genetic analysis of the hinge region between helical elements P5-P4-P6 and P7-P3-P8 in the sunY group I self-splicing intron.

Modeling of the group I intron RNA suggests that its catalytic core is primarily composed of two extended structural elements (stacked helices P5-P4-P6 and P7-P3-P8) whose relative orientation is partially determined by base-triple interactions between paired regions P4 and P6, and single-stranded joining regions J6/7 and J3/4, respectively. In vitro genetic selection was used to isolate functional sequence variants of the proposed triple helical domain of the sunY intron. Comparative sequence analysis of the selected variants provided supporting evidence for the two previously established base-triples between P4 and J6/7 and provided the first experimental evidence for an interaction between P6(1) and J3/4(3). Sequence covariations also indicated that a simple relationship exists between the length of a single-stranded joining region, J3/4, and the identity of a particular base-pair, P4(1). Selected variants based on a core structure with an extra nucleotide inserted in J3/4 revealed two different responses to this structural perturbation: a base-triple interaction and an intrahelical bulged pyrimidine. Chemical modification analysis supported the existence of these alternative structures. The function of this region of the ribozyme can therefore be fulfilled by at least three different structures.

Animals↗

PubMed1994

Serum cobalamin deficiency is uncommon in multiple sclerosis.

OBJECTIVES: To determine the frequency of serum cobalamin (Cbl) deficiency and to clarify the biologic importance of low screening Cbl levels in patients with multiple sclerosis (MS) and idiopathic myelopathy (MYL). BACKGROUND: A significant association between Cbl metabolism and MS has been postulated based on the observations that patients with MS have lower serum Cbl levels, higher unsaturated Cbl binding capacities, and a higher prevalence of macrocytosis than do normal controls. Whether such observations have biologic importance as documented by abnormal accumulation of metabolites that would result from Cbl deficiency has not yet been determined. METHODS: Serum Cbl and folate levels were determined in 208 consecutively evaluated patients seen in an outpatient MS clinic setting during a 7-month period. Necessary blood samples were obtained for 165 of these patients. One hundred twenty-five patients had clinically definite MS, 31 had clinically probable MS, and nine had MYL. Serum methylmalonic acid (MMA) and homocysteine (HCY) concentrations, which rise in biologically severe Cbl deficiency, were subsequently determined in all patients whose Cbl levels were lower than 301 pg/mL. RESULTS: A Cbl level lower than 301 was found in 32 of 156 patients with either clinically definite MS or clinically probable MS but in none of the patients with MYL. Elevated MMA or HCY levels were found in seven of 32 patients with either clinically definite MS or clinically probable MS, six of whom had an elevated HCY level and one of whom had elevated HCY and MMA levels. CONCLUSIONS: Despite the observation that 32 (19.4%) of 165 of these patients with MS and MYL had screening Cbl levels less than 301 pg/mL, only seven (4.2%) of 165 had elevated MMA or HCY levels. The frequency of biologically severe Cbl deficiency in these patients with MS and MYL was very low.

Adult↗

PubMed1994

Differential effects of transforming growth factor-beta 1 on distinct developmental stages of murine megakaryocytopoiesis.

The effect of transforming growth factor-beta 1 (TGF beta 1) on three developmental stages of megakaryocytopoiesis was investigated. Using a murine bone marrow agar culture system, titrated doses of TGF beta 1 were added to cultures assaying primitive high proliferative megakaryocyte progenitors, committed megakaryocyte precursors, and nondividing, endoreduplicating megakaryocytes. The growth of high proliferative megakaryocyte colony-forming cells (HPP-CFU-Mk) that require the growth factors interleukins-1, 3 and 6 (IL-1 + IL-3 + IL-6) for colony detection was abrogated by the addition of 1 ng TGF beta 1/ml. The sensitivity of committed megakaryocyte progenitors (colony-forming unit-megakaryocyte, CFU-Mk) to TGF beta 1 depended on the growth factor combination. TGF beta 1 (1 ng/ml) completely inhibited megakaryocyte colony formation from CFU-Mk only in cultures stimulated by low doses of IL-3. TGF beta 1 (> 10 ng/ml) could only marginally inhibit megakaryocyte colony formation generated in the presence of either high doses of IL-3 or the combination of low dose IL-3 + IL-6. TGF beta 1 inhibited both IL-3-dependent and IL-6-dependent megakaryocyte growth but tenfold higher doses of TGF beta 1 were required to inhibit growth generated by the combination of IL-3 + IL-6. The data showed that the capacity of TGF beta 1 to inhibit distinct differentiation stages of the megakaryocytopoietic lineage depended on the concentration and combination of growth factors involved.

Animals↗

PubMed1994

Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar.

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 A, b = 128.9 A, c = 78.3 A, beta = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2(1)2(1)2(1), with a = 79.8 A, b = 115.2 A, c = 73.0 A) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form.

Crystallization↗

PubMed1994

Prenatal gender preference of mothers of feminine and masculine boys: relation to sibling sex composition and birth order.

A mother's (prenatal) wish for a girl has, at times, been implicated as an etiological factor for boyhood femininity or its DSM-III-R diagnostic equivalent, the Gender Identity Disorder of Childhood. In one sample of 52 feminine and 52 nonclinical control boys from Los Angeles, the proportion of mothers who recalled a wish for a girl during the pregnancy did not differ significantly between groups (26.9% vs. 19.2%, respectively); however, in both groups, the maternal wish for a girl was significantly more common in sibships in which all of the proband's older siblings were male. In a second sample of 103 feminine boys from Toronto, the maternal wish for a girl was significantly higher than that found in the Los Angeles sample (43.7% vs. 26.9%), but a control group was not available; as in the Los Angeles sample, the maternal wish for a girl was significantly more common in sibships in which all of the proband's older siblings were male. Implications of these findings for further study of maternal influences on boyhood femininity are discussed.

Birth Order↗

PubMed1994

Rat cluster of differentiation 1 molecule: expression on the surface of intestinal epithelial cells and hepatocytes.

BACKGROUND/AIMS: Cluster of differentiation 1 (CD1) is a family of nonpolymorphic, major histocompatibility complex class I-like molecules with prominent expression in the liver and intestinal epithelium of humans and mice. Models of intestinal and hepatobiliary inflammation and experimental liver transplantation are conducted in the rat, yet nothing is known of CD1 expression in this species. METHODS: Monoclonal antibodies against murine CD1 were used to identify a rat CD1 homologue. Tissue messenger RNA expression was confirmed by Northern blot analysis with a murine CD1 complementary DNA probe. RESULTS: Immunoperoxidase staining detected CD1 in intestinal epithelial and liver cells but not in the thymus. Immunofluorescence of isolated hepatocytes and a rat fetal intestinal cell line, SLC-44, showed cell surface expression of CD1. Metabolic labeling and immunoprecipitation of the SLC-44 cell line and a mouse intestinal epithelial cell line, MODE-K, showed a protein of 55 kilodaltons. Immunoblotting of CD1 in isolated intestinal epithelial cells and hepatocytes showed a molecule of 55 kilodaltons. Northern blot analysis showed a single message of approximately 2.2 kilobases in hepatocytes. CONCLUSIONS: A CD1 homologue exists in the rat. Expression in the rat intestine and liver are similar to those in the human and mouse. The rat may be useful as a model for the study of CD1 function.

Animals↗

PubMed1994

Hepatotoxicity related to total parenteral nutrition: comparison of low-lipid and lipid-supplemented solutions.

PURPOSE: Because it is unclear whether or not the lipid or carbohydrate component of total parenteral nutrition solutions determines the development of cholestasis during total parenteral nutrition, a prospective randomized clinical trial of a predominantly carbohydrate solution (group I) versus one with isocaloric substitution of 30% nonprotein calories with lipid (group II) was performed. METHODS: Twelve patients in group I, 4 female and 8 male, and 14 patients in group II, 8 female and 6 male, were studied. Each subject received 100% to 150% of the basal energy needs, determined by the Harris-Benedict equation. Amino acids were supplied to provide a protein:kilocalorie ratio of 1:150 to 200. There were no differences between the groups in terms of mortality, nitrogen balance, and maintenance of weight. Weekly serum aspartate aminotrasferase (AST) and alkaline phosphatase (AP) levels were obtained. The change from baseline values was calculated. RESULTS: The observed difference between the two groups for AST was 5.7 (higher for group II), P = .55, and for AP, 39.1 (higher for group I), P = .23. A power calculation was performed to determine the number of patients required for a statistically significant difference in AP between the two groups with an alpha of 0.05 and a power of 0.85:67 patients. CONCLUSIONS: With these statistical considerations, we conclude that there was probably no statistically significant difference between the groups for an increase in AST or AP levels during total parenteral nutrition.

Adult↗

PubMed1994

Identification of the cysteine in the steroid-binding site of human corticosteroid binding globulin by site-directed mutagenesis and site-specific chemical modification.

Binding of corticosteroids by human corticosteroid binding globulin (hCBG) is thought to involve interaction with one of its two cysteine residues (Cys60 and Cys228). To identify which of the two cysteine residues mediates steroid binding, we have produced mutant hCBGs containing serine or alanine in place of Cys228 by site-directed mutagenesis. Alteration of Cys228 to serine or alanine does not change the steroid binding affinity of hCBG, demonstrating that Cys228 is not involved in the binding interaction. This finding strongly suggests that Cys60 is the functionally important cysteine. By modifying the wild-type and mutant hCBGs with the sulfhydryl-specific reagents N-ethylmaleimide, iodoacetamide, and sodium tetrathionate, we have demonstrated that Cys60 is present at the steroid binding site, and that it may be directly involved in steroid binding. This result also identifies Cys60 as the accessible cysteine reported in previous studies.

Base Sequence↗

PubMed1994

Renal tubular function in the gravid rat.

Pregnancy in the rat is accompanied by enhanced reabsorption of salt and water throughout most, if not all, of the gestational period. Many mechanisms have been suggested but definitive answers are still awaited. The major area of controversy centres around the detection of changes at term. There is general agreement that, at least in mid-gestation, the increase in reabsorption can be attributed to increases in the proximal tubules, the loop of Henle and the collecting duct. The contribution of the proximal tubule to the increased reabsorption at term is still uncertain. Enhanced salt and water reabsorption is demonstrated in distal nephron segments irrespective of the stage of gestation. Micropuncture and microperfusion experiments have identified increased reabsorption of water, sodium and chloride in the loop of Henle, but it appears that there is net addition of glucose, urea and potassium to the tubular fluid in this segment which, at least for potassium and glucose, offsets to some extent increased reabsorption by the proximal tubule. Altered renal handling of other solutes (uric acid, calcium and magnesium) also occurs throughout pregnancy but the mechanisms responsible and nephron sites involved remain to be investigated. Attempts to attribute altered reabsorption to direct renal effects of changes in maternal hormones are inconclusive. Prolactin mimics some of the pregnancy-associated increases in reabsorption following chronic administration to male and non-pregnant female rats. These effects might be due to a direct renal action of the hormone or even to the volume expansion following its dipsogenic action.

Absorption↗

PubMed1994

An unusual buried polar cluster in a family of fungal lipases.

The stability of globular proteins arises largely from the burial of non-polar amino acids in their interior. These residues are efficiently packed to eliminate energetically unfavorable cavities. Contrary to these observations, high resolution X-ray crystallographic analyses of four homologous lipases from filamentous fungi reveal an alpha/beta fold which contains a buried conserved constellation of charged and polar side chains with associated cavities containing ordered water molecules. It is possible that this structural arrangement plays an important role in interfacial catalysis.

Amino Acid Sequence↗

PubMed1994

Current progress in crystallographic studies of new lipases from filamentous fungi.

Lipases from filamentous fungi have been studied extensively over many years. They exhibit properties attractive for industrial applications, e.g. in laundry detergents, tanning and paper industries and stereospecific organic synthesis. Enzymes from the fungi Rhizomucor miehei and Geotrichum candidum have been among the first neutral lipases to be characterized structurally by X-ray diffraction methods. In this paper we report a preliminary account of crystallographic studies of three other fungal lipases homologous to that from R. miehei and obtained from Humicola lanuginosa, Penicillium camembertii and Rhizopus delemar. These newly characterized structures have important implications for our understanding of structure-function relationships in lipases in general and the molecular basis of interfacial activation.

Amino Acid Sequence↗

PubMed1994

Longitudinal research on adolescent voice change in males.

Many of the characteristics and processes associated with adolescence are reasonably well understood; others are not. Still unanswered are questions about adolescent voice change. For example, when does the average child start the process; what changes result and what is their extent; when is it complete? Further, how closely do shifts in vocal level parallel, or even predict, the adolescent process? This project was carried out in order to obtain data which would complement available information on the subject and provide additional specificity about voice change and pubescence--at least for boys. To that end, a longitudinal study was conducted in which 48 males were tracked over a 5-year period. Voice measurements were made--including speaking fundamental frequency (SFF) and phonational frequency range (PFR)--as were others relating to physical size (height, weight, and six body dimensions). It was found that (1) stable adolescent voice change (AVC) measurements could be obtained, (2) maturation patterns appeared predictable on the basis of AVC changes, and (3) both the onset and duration of voice change extended over a longer period of time than had been previously thought.

Adolescent↗

PubMed1994

Epiphysiodesis in juvenile hallux abducto valgus. A preliminary retrospective study.

Juvenile hallux abducto valgus manifested prior to physeal plate closure was treated by lateral epiphyseal stapling and modified McBride bunionectomy in nine children (15 feet) from 1984 to 1992. The prevalent preoperative symptoms were significantly reduced postoperatively. The intermetatarsal angle decreased an average of 6.6 degrees (initial decrease was 2.7 degrees with an additional 3.9 degrees occurring over time). The hallux abducto valgus angle decreased an average of 19.6 degrees (initial decrease was 10.7 degrees with an additional 8.9 degrees occurring over time).

Adolescent↗