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PubMed1999

Do morning report format changes affect educational content?

OBJECTIVE: The goal of this prospective observational study was to determine if format changes in a paediatric morning report conference affected the educational content presented. DESIGN: Case presentations during paediatric morning report at the University of Iowa during the academic years 1995-96 (Format 1 - informal presentations) and 1996-97 (Format 2 - formal presentations) were analysed for demographics, clinical venues where patients were encountered, case diagnoses and ensuing discussion. A 10-item questionnaire of participants' attitudes was conducted during May 1997. SETTING: Department of Pediatrics, University of Iowa. SUBJECTS: Paediatric residency trainees. RESULTS: The number of cases was significantly decreased from 4 to 3.2 per morning report session. A wide variety of patient ages and all clinical venues were represented in both formats. Case diagnoses covered 30 of 31 Pediatrics Review and Education Program (PREP, American Academy of Pediatrics, Elk Grove Village, IL, USA) Content Specification Headings during Format 1 vs. 29 of 31 during Format 2. The most common headings were the same. Patient evaluations were discussed less and patient clinical presentations and pathophysiology were discussed more during Format 2. Participants rated Format 2 higher; 54% of participants wanted to continue this format and 16% wanted a combination of both formats. CONCLUSIONS: This research suggests that while format changes in paediatric morning report resulted in a 20% decrease in the number of cases presented, only relatively small changes in the overall educational content occurred. Changes in educational programmes should be accompanied by educational evaluation.

Curriculum↗
PubMed1987

Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2.

In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E2 (PGE2) synthesis. The EC50 values for stimulation of PGE2 synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-[gamma-thio]triphosphate stimulated PGE2 synthesis and InsP formation, and guanosine-5'-[beta-thio]diphosphate inhibited both PGE2 synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE2 synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE2 synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE2 synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited bradykinin-stimulated PGE2 synthesis but was without effect on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with [3H]choline, the phospholipase A2 products lysophosphatidylcholine and glycerophosphocholine were generated. In cells pretreated with dexamethasone, lysophosphatidylcholine and glycerophosphocholine formation induced by bradykinin were inhibited. Treatment of cells with phorbol ester enhanced bradykinin-induced formation of these metabolites. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A2 and that phospholipase A2 is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.

Animals↗
PubMed1988

Transforming growth factor beta inhibits formation of osteoclast-like cells in long-term human marrow cultures.

Transforming growth factor beta (TGF-beta), a polypeptide present in abundant amounts in bone matrix, was examined for its effects on osteoclast formation by using a human bone marrow culture system in which multinucleated cells (MNCs) with osteoclast characteristics form. TGF-beta strongly inhibited MNC formation at concentrations as low as 1 ng/ml. TGF-beta also completely suppressed the effects of osteotropic factors known to stimulate MNC formation. The inhibitory effect of TGF-beta on osteoclast-like cell formation was more pronounced during the first week of culture, which corresponds to the period of proliferation of mononuclear osteoclast precursors. To examine whether the inhibitory effects of TGF-beta on MNC formation could be due to inhibition of the granulocyte/macrophage progenitor cell [colony-forming unit granulocyte/macrophage (CFU-GM)], the probable precursor for MNC, we tested the effects of TGF-beta on CFU-GM formation in presence of a source of colony-stimulating factor. Unexpectedly, TGF-beta at concentrations (0.1-1 ng/ml) that were inhibitory for MNC formation enhanced day 7 CFU-GM colony formation. This increase in CFU-GM colony formation seen in cultures containing TGF-beta resulted from significantly more granulocytic colonies being formed in the cultures, suggesting that TGF-beta may induce CFU-GM to differentiate preferentially to cells of the granulocytic lineage. Differentiation of CFU-GM to granulocytes rather than osteoclast precursors in response to TGF-beta would result in inhibition of MNC formation by depleting the precursor pool for MNC. These data suggest that inhibition of osteoclast-like cell formation by TGF-beta may be an important mechanism of control of local bone resorption.

Bone Marrow↗
PubMed1996

Tumor board formats: "fascinating case" versus "working conference".

BACKGROUND AND METHODS: To test the hypothesis that a "working conference" (WC; tumor board format in which any case requiring multispecialty input is presented for discussion) was preferable to a "fascinating case" (FC; only "interesting" or "unusual" cases presented) format, tumor board format at the Portland Veterans Affairs Medical Center was changed from FC to WC. RESULTS: The number of cases presented in tumor board reflected this change. For example, for the period from 1990 to 1991 (prior to the change), 63 of 557 registered cases (11%) were presented in tumor board. By 1992-1993, 206 of 547 registered cases (38%) were presented. This increase was highly significant (p < 0.001). Three years after the format change, the authors surveyed 22 regular participants of the tumor board, many of whom had been participants prior to the change. Of those surveyed, 77% preferred the WC to the FC format, and 18% preferred a combined format. Most of those surveyed felt that the WC format was more helpful to the provider and to patient care (86% and 100%, respectively). Eighty percent felt that the WC format facilitated protocol enrollment, and 100% of the respondents were satisfied with the quality of case discussion that resulted from the WC format. CONCLUSIONS: The change to a "working case" format increased provider satisfaction with tumor board quality compared with the previous "fascinating case" format.

Case Management↗
PubMed1997

Formation of platelet-leukocyte conjugates in whole blood.

The purpose of this investigation was to obtain information on platelet-leukocyte conjugate formation in whole blood and on factors that affect it. We also measured platelet and leukocyte activation by quantitating the expression of CD62P and CD11b. In both cases a flow cytometric approach was used. The results show that platelet-monocyte and platelet-polymorphonuclear leukocyte (PMNL) conjugate formation is enhanced by simply stirring blood, with optimum conjugate formation occurring after 10 min. In the case of monocytes,conjugate formation was enhanced by adenosine diphosphate (ADP). Both monocyte and PMNL conjugate formation was enhanced by phorbol myristate acetate (PMA), but L-formyl methionyl lysyl proline (FMLP) was either without effect (monocytes) or inhibitory (PMNL). EDTA also inhibited conjugate formation (implying involvement of divalent cations), as did dextran sulphate (implying involvement of P-selectin = CD62P). Interestingly GR144053F, which acts at GpIIb-IIIa on platelets to interfere with fibrinogen binding, and also glycyl prolyl arginyl proline (GPRP), a peptide that interferes with the interaction between CD11c on leukocytes and fibrinogen, did not inhibit platelet-monocyte conjugate formation, but did inhibit the platelet-PMNL interaction; this indicates that GpIIb-IIIa on platelets and CD11c on leukocytes and fibrinogen are involved in mediating the interaction between platelets and PMNL but not platelets and monocytes. Surprisingly arginyl-glycyl aspartyl serine (RGDS) inhibited the formation of both types of conjugate but this may be because it also inhibited both platelet and leukocyte activation as measured by CD62P and CD11b exposure and/or interferes with the binding of adhesion molecules other than fibrinogen. The results show that a flow cytometric procedure can be effective in obtaining rapid information on platelet-leukocyte conjugate formation in whole blood and on factors that are involved in its regulation. It is suggested that the technique may be applicable to the study of platelet-leukocyte conjugate formation in whole blood in disease, and also to study the effects of drugs interfering with conjugate formation.

Journal Article↗
PubMed1995

Fibrin II induces endothelial cell capillary tube formation.

We studied the formation of capillary tubes by endothelial cells which were sandwiched between two fibrin gels under serum-free conditions. After formation of the overlying fibrin gel, the endothelial cell monolayer rearranged into an extensive net of capillary tubes. Tube formation was apparent at 5 h and was fully developed by 24 h. The capillary tubes were vacuolated, and both intracellular and intercellular lumina were present. Maximal tube formation was observed with fibrin II (which lacks both fibrinopeptide A and B), minimal tube formation with fibrin I (which lacks only fibrinopeptide A), and complete absence of tube formation with fibrin 325 (which lacks the NH2-terminal beta 15-42 sequence, in addition to fibrinopeptides A and B). The inability of fibrin 325 to stimulate capillary tube formation supports the idea that beta 15-42 plays an important role in this process, and its importance was confirmed by the finding that exogenous soluble beta 15-42 inhibited fibrin II-induced capillary tube formation. This effect was specific for fibrin, since beta 15-42 did not inhibit tube formation by endothelial cells sandwiched between collagen gels. The interaction of the apical surface of the endothelial cell with the overlying fibrin II gel, as opposed to the underlying fibrin gel upon which the cells were seeded, was necessary for capillary tube formation. These studies suggest that the beta 15-42 sequence of fibrin interacts with a component of the apical cell surface and that this interaction plays a fundamental role in the induction of endothelial capillary tube formation.

Amino Acid Sequence↗
PubMed2000

Role of neurotropins in rat embryonic testis morphogenesis (cord formation).

The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 = plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, P0) with an antibody to p75/LNGFR. The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and P0 testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development. The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.

Animals↗
PubMed2004

Preferred retinal locus and reading rate with four dynamic text presentation formats.

BACKGROUND: Electronic display devices hold the potential to improve access to written material by people with low vision. For those with central field loss, the optimal form of electronic text presentation may vary according to the location of the preferred retinal locus, but this has never been investigated. In this study, we examined the relationship between preferred retinal locus location and reading rate for four electronic display formats (rapid serial visual presentation, horizontal scroll, vertical scroll, and page). METHODS: Short sentences were presented in each format to 35 low-vision (most with central field loss) and 14 age-matched control subjects. Subjects read aloud to determine maximum oral reading rate and read silently to determine preferred silent reading rate. RESULTS: With the exception of page format, maximum oral reading rates were faster than silent preferred reading rates for both groups of subjects. For the low-vision group, there was no significant difference in maximum oral reading rates between the four display formats; and when reading at a preferred silent rate, page format was faster than the other three formats. Though page format was read more quickly, half of the low-vision subjects preferred the horizontal-scroll format. Contrary to our predictions, there was no significant effect of preferred retinal locus location (vertical vs. lateral) on reading rate and no significant interaction between preferred retinal locus location and display format. CONCLUSIONS: The differences between maximum oral and preferred silent reading rates and the lack of a relationship between reading rates and preferred display format reinforce the importance of patient preference in the evaluation and selection of a device or display format during rehabilitation.

Adult↗
PubMed1994

Upstream introns influence the efficiency of final intron removal and RNA 3'-end formation.

For all intron-containing pre-mRNAs of higher eukaryotes that have been examined using either living cells or cell-free extracts, a functional 3' splice site within the 3'-terminal intron is required for efficient RNA 3'-end formation. The mechanism by which intron sequences facilitate RNA 3'-end formation, which is achieved by endonucleolytic cleavage and polyadenylation, is not understood. We report here that in intact cells the efficiency of RNA 3'-end formation correlates with the efficiency of final intron removal, even when the intron is normally a 5'-terminal or internal intron. Therefore, the influence of the 3'-terminal intron on 3'-end formation is likely to be attributable to the determinants of splicing efficiency, which include but are not limited to the 3' splice site. Quantitative RNase mapping and methods that couple reverse transcription and the polymerase chain reaction were used to assess the consequence to RNA 3'-end formation of intron deletions within the human gene for triosephosphate isomerase (TPI). Results indicate that the formation of TPI RNA 3' ends requires TPI gene introns in addition to the last intron, intron 6, to proceed efficiently. These additional TPI gene introns are also required for the efficient removal of intron 6. When introns 1 and 5 were engineered to be the final intron, they were found, as was intron 6, to function in RNA 3'-end formation with an efficiency that correlated with their efficiency of removal. The simultaneous deletion of the 5' and 3' splice sites of intron 6 reduced the efficiencies of both RNA 3'-end formation and the removal of intron 5, which constituted the 3'-most functional intron. Deletion of only the 3' splice site of intron 6 precluded RNA 3'-end formation but had no effect on the efficiency of intron 5 removal. Deletion of only the 5' splice site of intron 6, which resulted in exon 6 skipping (i.e., the removal of intron 5, exon 6, and intron 6 as a single unit), had no effect on the efficiencies of either RNA 3'-end formation or the removal of intron 5-exon 6-intron 6. These results indicate that sequences within the 3'-terminal intron are functionally coupled to both RNA 3'-end formation and removal of the penultimate intron via a network of interactions that form across the last two exons and, most likely, between RNA processing factors.

Base Sequence↗
PubMed1979

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration.

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H(2)O(2) became "available" during the oxidation of [1-(14)C]glycolate was sufficient to account for the breakdown of the intermediate [1-(14)C]glyoxylate to formate (C(1) unit) and (14)CO(2). Under aerobic conditions formate production closely paralleled (14)CO(2) release from [1-(14)C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H(2)O(2) production was increased artificially by addition of an active preparation of fungal glucose oxidase.When [(14)C]formate was added to these preparations it was oxidized directly to (14)CO(2) by the peroxidatic action of peroxisomal catalase; however, the breakdown of formate was slow relative to the rate of formate production. For example, when [(14)C]formate was generated from [2-(14)C]glycolate it was not readily oxidized to (14)CO(2) in these organelles. Because the activity of formate-NAD(+) dehydrogenase in cell-free leaf extracts was low compared with that of formyl tetrahydrofolate synthetase it is suggested that most of the formate produced during glycolate oxidation could be metabolized via the one carbon pool and not oxidized directly to CO(2).At 25 C the rate of release of (14)CO(2) from [2-(14)C]glycolate in leaf discs was 40 to 50% of the rate from [1-(14)C]glycolate. Isonicotinyl hydrazide inhibited (14)CO(2) release from both [1-(14)C]- and [2-(14)C]glycolate; but this inhibitor was more effective in blocking (14)CO(2) release from [2-(14)C]glycolate. It is argued that the oxidation of the methylene carbon group of glycolate does not occur as a direct consequence of formate (C(1) unit) breakdown, but is a product of the further metabolism of formate and glycine, possibly, via serine.

Journal Article↗
PubMed1986

Stimulation of delta-Aminolevulinic Acid Formation in Algal Extracts by Heterologous RNA.

Formation of the chlorophyll and heme precursor delta-aminolevulinic acid (ALA) from glutamate in soluble extracts of Chlorella vulgaris, Euglena gracilis, and Cyanidium caldarium was stimulated by addition of low molecular weight RNA derived from greening algae or plant tissue. Enzyme extracts were prepared for the ALA formation assay by high-speed centrifugation, partial RNA depletion, and gel filtration through Sephadex G-25. RNA was extracted from greening barley epicotyls, greening cucumber cotyledon chloroplasts, and growing cells of Chlorella, Euglena, Chlamydomonas reinhardtii, and Anacystis nidulans, freed of protein, and fractionated on DEAE-cellulose to yield an active component corresponding to the tRNA-containing fraction. RNA from homologous and heterologous species stimulated ALA formation when added to enzyme extracts, and the degree of stimulation was proportional to the amount of RNA added. Algal enzyme extracts were stimulated by algal RNAs interchangeably, with the exception of RNA prepared from aplastidic Euglena, which did not stimulate ALA production. RNA from greening cucumber cotyledon chloroplasts and greening barley epicotyls stimulated ALA formation in algal enzyme incubations. In contrast, tRNA from Escherichia coli, both nonspecific and glutamate-specific, as well as wheat germ, bovine liver, and yeast tRNA, failed to reconstitute ALA formation. Moreover, E. coli tRNA inhibited ALA formation by algal extracts, both in the presence and absence of added algal RNA. Chlorella extracts were capable of catalyzing aminoacyl bond formation between glutamate and both the activity reconstituting and nonreconstituting RNAs, indicating that the inability of some RNAs to stimulate ALA formation was not due to their inability to serve as glutamyl acceptors. The first step in the ALA-forming reaction sequence has been proposed to be activation of glutamate via aminoacyl bond formation with a specific tRNA, analogous to the first step in peptide bond formation. Our results suggest that the RNA that is required for ALA formation may be functionally distinct from the glutamyl-tRNA species involved in protein synthesis.

Journal Article↗
PubMed1995

Specificity of different organic nitrates to elicit NO formation in rabbit vascular tissues and organs in vivo.

1. In the present study we assessed the formation of nitric oxide (NO) from classical and thiol-containing organic nitrates in vascular tissues and organs of anaesthetized rabbits, and established a relationship between the relaxant response elicited by nitroglycerin (NTG) and NO formation in the rabbit isolated aorta. Furthermore, the effect of isolated cytochrome P450 on NO formation from organic nitrates was investigated. 2. Rabbits received diethyldithiocarbamate (DETC; 200 mg kg-1 initial bolus i.p. and 200 mg kg-1 during 20 min, i.v.) and either saline, or one of the following organic nitrates: nitroglycerin (NTG, 0.5 mg kg-1), isosorbide dinitrate (ISDN), N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 3672), S-carboxyethyl-N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 5185), at 10 mg kg-1 each. After 20 min the animals were killed, blood vessels and organs were removed, and subsequently analyzed for spin-trapped NO by cryogenic electron spin resonance (e.s.r.) spectroscopy. 3. In the saline-treated control group, NO remained below the detection limit in all vessels and organs. In contrast, all of the nitrates tested elicited measurable NO formation, which was higher in organs (liver, kidney, heart, lung, spleen) (up to 4.8 nmol g-1 20 min-1) than in blood vessels (vena cava, mesenteric bed, femoral artery, aorta) (up to 0.7 nmol g-1 20 min-1). Classical organic nitrates (NTG, ISDN) formed NO preferentially in the mesenteric bed and the vena cava, while the SPM compounds elicited comparable NO formation in veins and arteries. 4. Using a similar spin trapping technique, NO formation was assessed in vitro in phenylephrine-precontracted rabbit aortic rings. The maximal relaxation elicited by a first exposure (10 min) to NTG (0.3 to 10 microM) was positively correlated (r = 0.8) with the net increase (NTG minus basal) of NO spin-trapped during a second exposure to the same concentration of NTG in the presence of DETC. 5. Cytochrome P450 purified from rabbit liver enhanced NO formation in a NADPH-dependent fashion from NTG, but not from the other nitrates, as assessed by activation of purified soluble guanylyl cyclase. 6. We conclude that the vessel selective action of different organic nitrates in vivo reflects differences in vascular NO formation. Thus, efficient preload reduction by classical organic nitrates can be accounted for by higher NO formation in venous capacitance as compared to arterial conductance and resistance vessels. In contrast, NO is released from cysteine-containing nitrates (SPMs) to a similar extent in arteries and veins, presumably independently of an organic nitrate-specific biotransformation. Limited tissue bioavailability of NTG and ISDN might account for low NO formation in the aorta, while true differences in biotransformation seem to account for differences in NO formation in the other vascular tissues.

Animals↗
PubMed1962

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS I. : General Properties of the Formate-Exchange Reaction.

McCormick, N. G. (University of Washington, Seattle), E. J. Ordal, and H. R. Whiteley. Degradation of pyruvate by Micrococcus lactilyticus. I. General properties of the formate-exchange reaction (J. Bacteriol. 83:887-898. 1962.-At an alkaline pH, extracts of Micrococcus lactilyticus(2) catalyze the phosphoroclastic degradation of pyruvate to formate and acetyl phosphate and the rapid exchange of formate into the carboxyl group of pyruvate. At an acid pH, hydrogen, carbon dioxide, and acetyl phosphate are produced, and carbon dioxide is exchanged into the carboxyl group of pyruvate. A concentration of approximately 1 m phosphate is required for the phosphoroclastic reaction and formate exchange; the production of carbon dioxide and hydrogen is greatly inhibited by high concentrations of phosphate. Formate exchange requires a divalent metal ion and is stimulated by reducing agents and an atmosphere of hydrogen. Inhibition by p-chloromercuribenzoate, Zn(++), Cd(++), and arsenite indicates that sulfhydryl groups on the enzyme are involved in the reaction; the inhibition by arsenite and Cd(++) may be relieved by 2,3-dimercaptopropanol, suggesting that vicinal dithiols may be required. Inhibition by hypophosphite may reflect a competition with formate for a site on the enzyme. At an alkaline pH, alpha-ketobutyrate is degraded to propionate and formate, whereas alpha-ketoglutarate is fermented to succinate, propionate, carbon dioxide, hydrogen, and formate. Formate is exchanged into the carboxyl groups of alpha-ketobutyrate and alpha-ketoglutarate under these conditions. Only traces of alpha-ketovalerate and alpha-ketoisovalerate are fermented at an alkaline pH and the exchange of formate into these compounds is very low.The addition of viologen dyes under the conditions used for formate exchange causes a reduction of pyruvate, alpha-ketobutyrate, alpha-ketovalerate, and alpha-ketoisovalerate to the corresponding alpha-hydroxy acids.

Journal Article↗
PubMed1987

Mechanism of NaCl transport-stimulated prostaglandin formation in MDCK cells.

Recently we have found that stimulation of NaCl transport in high-resistance MDCK cells enhances their prostaglandin formation. In the present study, we investigated the mechanisms by which prostaglandin formation could be linked to the ion transport in these cells. We found that stimulation of transport caused a transient stimulation of prostaglandin formation lasting 5-10 min. The rise in prostaglandin formation was paralleled by a rise of free intracellular arachidonic acid. Analysis of membrane lipids revealed that the rise of free arachidonic acid was paralleled by a loss of arachidonic acid from polyphosphoinositides. We failed to obtain indications for the stimulation of calcium-dependent phospholipase A2. However, we did obtain evidence that the incorporation of arachidonic acid into phospholipids was diminished during stimulation of ion transport, indicating a decreased rate of reesterification. Despite the fact that there was no significant fall in total cellular ATP on stimulation of ion transport, we found a high and transient rise of lactate production of the cells on stimulation of the ion transport indicating an alteration of the ADP/ATP ratio. Moreover, prostaglandin formation and lactate formation were linearly correlated in this situation. When glucose utilization was inhibited by mannoheptulose, the rise in lactate formation was abolished, whereas that of PG formation was unaltered, indicating that lactate formation and prostaglandin formation were not causally linked on stimulation of ion transport. Our results suggest that an increase in the rate of sodium chloride transport by MDCK cells stimulates formation by an inhibition of reesterification of free arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Presence of chloride-formate exchange in vascular smooth muscle and cardiac cells.

The presence of chloride-formate anion exchange in vascular smooth muscle cells (VSMCs) and cardiac myocytes was investigated. Imposing an outward chloride gradient in sarcolemmal microsomes isolated from canine aorta stimulated [14C]formate uptake compared with the absence of a chloride gradient (24.3 +/- 2.33 versus 9.8 +/- 1.41 pmol/mg protein for 30 seconds, P < .03) and induced transient uphill [14C]formate uptake. The chloride-formate exchange was significantly inhibited in the presence of 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or furosemide (57% and 61%, respectively). Incubation of rat cultured VSMCs in a medium containing [14C]formate resulted in uptake of formate that was significantly DIDS and furosemide sensitive (79.34 +/- 2.47, 43.03 +/- 2.37, and 44.65 +/- 1.68 pmol/mg protein for 4 minutes in control, DIDS, and furosemide groups, respectively). Preincubation of the VSMCs in chloride-free medium significantly reduced the DIDS-sensitive (36.31 versus 16.85 pmol/mg protein for 4 minutes, P < .001) and furosemide-sensitive (34.72 versus 8.78 pmol/mg protein for 4 minutes, P < .001) [14C]formate uptake. These results are compatible with the presence of chloride-formate exchange in VSMCs. Influx of [14C]formate into sarcolemmal vesicles isolated from canine heart was significantly higher in the presence of an outward chloride gradient than in its absence (18.1 +/- 2.3 versus 9.6 +/- 1.7 pmol/mg protein for 30 seconds, P < .03). The chloride-formate exchange was significantly inhibited in the presence of 1 mmol/L DIDS or furosemide (41% and 52%, respectively). We conclude that the distribution of chloride-formate exchange may be more universal than previously suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Mouse osteoblastic cells (MC3T3-E1) at different stages of differentiation have opposite effects on osteoclastic cell formation.

Using our new culture system for multinucleate cells (MNCs) that have many characteristics of osteoclasts, we examined the effects of factors produced by osteoblastic cells on osteoclastic cell formation. Conditioned medium (CM) from undifferentiated osteoblastic MC3T3-E1 cells during their growth phase inhibited MNC formation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Diluted CM (1:81) from differentiated cells obtained after cultivation for more than 20 days stimulated MNC formation, but at lower dilutions inhibited their formation. Dialyzed CM (greater than 2000 mol wt) from the differentiated cells was more stimulatory than undialyzed CM and showed no inhibitory effect on MNC formation. The inhibitory effect was observed with filtered (less than 3000 mol wt) CMs and was specific for osteoblastic cell CM. Prostaglandin E2 (PGE2) was detected in the CM from undifferentiated or differentiated MC3T3-E1 cells at concentrations (317 +/- 66 and 1287 +/- 179 pg/ml, respectively) sufficient to inhibit MNC formation, and this inhibition was partially abolished with CM (at 3-fold dilution) in indomethacin-treated cells (PGE2, less than 20 pg/ml), suggesting PGE2-mediated inhibition of MNC formation and the presence of another factor(s) besides PGE2 that influenced MNC formation. In contrast to day 3 CM plus 1,25-(OH)2D3, day 60 CM plus 1,25-(OH)2D3 induced MNC formation even in the absence of GM-CSF, and this induction was inhibited by an antibody to GM-CSF. Secondary colony formation assays showed the presence of a GM-CSF-like factor in the day 60 CM. These findings indicate that osteoblastic cells are involved in the process of osteoclastic cell formation, with at least two soluble factors produced by osteoblasts, a GM-CSF-like factor, which is stimulatory, and PGE2, which is inhibitory. The effects of CMs also differed depending on the stage of osteoblast differentiation.

Animals↗
PubMed2004

Intravenous pamidronate prevents femoral bone loss and renal stone formation during 90-day bed rest.

UNLABELLED: Long-term bed rest has potential risks of bone loss and renal stone formation. We examined the effects of resistive exercise and intravenous pamidronate on BMD, bone turnover, urinary calcium, and renal stone formation in 25 healthy males during 90-day bed rest. Pamidronate prevented femoral bone loss and renal stone formation, but resistive exercise showed little effects. INTRODUCTION: Long-term bed rest increases the risks of bone loss and urinary stone formation. Resistive exercise increases bone formation, and bisphosphonates reduce bone resorption. However, the effects of muscle exercise and bisphosphonates have not been examined side-by-side. The objectives of this study are to compare the effects of pamidronate with resistive exercise on BMD and renal stone formation during prolonged bed rest. MATERIALS AND METHODS: Twenty-five male white volunteers, 26-45 years of age, were randomly assigned to the control (n = 9), exercise (n = 9), and pamidronate (n = 7) groups and underwent 90-day 6 degrees head-down tilt bed rest. Exercise group performed squats and heel raises on a flywheel device for 30 minutes every 3 days. Pamidronate (60 mg) was administered intravenously 14 days before bed rest. BMD of the head, forearm, lumbar spine, and proximal femur; biochemical bone markers; calcium (Ca) metabolism; and abdominal radiographs were examined during 90 days of bed rest and 360 days of reloading. RESULTS: In controls, proximal femoral BMD decreased, and bone resorption markers and urinary Ca increased during bed rest, along with development of renal stones in two of nine subjects. Resistive exercise increased bone formation but was unable to prevent femoral BMD decrease and increases in bone resorption and urinary Ca during bed rest, with formation of renal stones in four of nine subjects. Pamidronate maintained femoral BMD, reduced bone resorption and urinary Ca, and completely prevented renal stone formation. CONCLUSIONS: Resistive exercise increased bone formation but could not reduce bone resorption and the risk of renal stones. In contrast, inhibition of bone resorption by pamidronate could preserve bone mineral and reduce the risk of renal stone formation during prolonged bed rest.

Adult↗
PubMed2002

MLO-Y4 osteocyte-like cells support osteoclast formation and activation.

Osteocytes are terminally differentiated cells of the osteoblast lineage that have become embedded in mineralized matrix and may send signals that regulate bone modeling and remodeling. The hypothesis to be tested in this study is that osteocytes can stimulate and support osteoclast formation and activation. To test this hypothesis, an osteocyte-like cell line called MLO-Y4 and primary murine osteocytes were used in coculture with spleen or marrow cells. MLO-Y4 cells support osteoclast formation in the absence of 1,25-dihydroxyvitamin D3 [1,25(OD)2D3] or any other exogenous osteotropic factor. These cells alone stimulate osteoclast formation to the same extent or greater than adding 1,25(OH)2D3. Coaddition of 1,25(OH)2D3 with MLO-Y4 cells synergistically increased osteoclast formation. Optimal osteoclast formation and pit formation on dentine was observed with 200-1,000 MLO-Y4 cells per 0.75-cm2 well. No osteoclast formation was observed with 2T3, OCT-1, or MC3T3-E1 osteoblast cells (1,000 cells/well). Conditioned media from the MLO-Y4 cells had no effect on osteoclast formation, indicating that cell contact is necessary. Serial digestions of 2-week-old mouse calvaria yielded populations of cells that support osteoclast formation when cocultured with 1,25(OH)2D3 and marrow, but the population that remained in the bone particles supported the greatest number of osteoclasts with or without 1,25(OH)2D3. To examine the mechanism whereby these cells support osteoclast formation, the MLO-Y4 cells were compared with a series of osteoblast and stromal cells for expression of macrophage colony-stimulating factor (M-CSF), RANKL, and osteoprotegerin (OPG). MLO-Y4 cells express and secrete large amounts of M-CSF. MLO-Y4 cells express RANKL on their surface and their dendritic processes. The ratio of RANKL to OPG mRNA is greatest in the MLO-Y4 cells compared with the other cell types. RANK-Fc and OPG-Fc blocked the formation of osteoclasts by MLO-Y4 cells. These studies suggest that both RANKL and OPG may play a role in osteocyte signaling, OPG and M-CSF as soluble factors and RANKL as a surface molecule that is functional in osteocytes or along their exposed dendritic processes.

Animals↗