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Biomedical subjects

J G Joshi

Publications and source records attributed to J G Joshi.

At least 37 records · Page 2Linked to original sources
PubMed1989

Neurotensin regulation of macrophage colony-stimulating factor-stimulated in vitro myelopoiesis.

Neurotensin, at less than or equal to 10(-9) M, in the presence of an optimal concentration of macrophage CSF (CSF-1), stimulated a dose-dependent enhancement of colony formation by murine marrow-derived mononuclear phagocyte progenitor cells. The additional colonies arose from the cell cycle and Ia Ag-positive subpopulation previously identified as two-signal-dependent progenitors. Two-signal colony formation diminished when the peptide was added at concentrations greater than 10(-9) M. Neurotensin binds specifically to two distinct receptors, a high affinity receptor (KD approximately 10(-9) M) and a lower affinity (KD approximately 10(-7) M) receptor identified as the tuftsin receptor. Rat liver ferritin and an inhibitory tuftsin analog. (ALA1)-tuftsin, which inhibit two-signal colony formation stimulated by tuftsin and tuftsin-like peptides in combination with CSF-1, did not inhibit colony formation stimulated by CSF-1 and 10(-9) M neurotensin. Both inhibitors, however, reversed the loss of two-signal colony growth in the presence of higher neurotensin concentrations. Neurotensin fragment 1-6, unlike ferritin and (ALA1)-tuftsin, inhibited two-signal colony formation stimulated by 10(-9) M neurotensin. However, like ferritin and (ALA1)-tuftsin, fragment 1-6 permitted full expression of two-signal colony formation in the presence of CSF-1 and 10(-7) M neurotensin. The data indicated that occupancy of both receptors at neurotensin concentrations greater than 10(-9) M might be responsible for the diminished progenitor response. The data further support a potential role for neurotensin as an inflammatory mediator. In addition to direct effects on mature phagocytic leukocytes, neurotensin, at least in vitro can influence the production of new mononuclear phagocytes.

Animals↗
PubMed1989

Ribonuclease inhibitor from pig brain: purification, characterization, and direct spectrophotometric assay.

The ribonuclease inhibitor from pig brain has been purified 1,500-fold by a combination of ammonium sulfate fractionation, ion-exchange chromatography, hydroxylapatite chromatography, and gel filtration. The inhibitor has a Mr 50,000. It is a noncompetitive inhibitor for pancreatic ribonuclease A with a Ki of 1 nM, forming a 1:1 complex. Both ribonuclease A and B, but not ribonuclease U1 and T1, are inactivated by the inhibitor. The inhibition capacity was abolished by sulfhydryl reagents such as p-chloromercuribenzoate. Incubation of the enzyme-inhibitor complex with the sulfhydryl reagent caused dissociation into active ribonuclease and inactive inhibitor. Dithiothreitol was required during purification to retain the activity of the inhibitor.

Amino Acids↗
PubMed1989

Time-dependent inactivation of glucose-6-phosphate dehydrogenase from yeast by aluminum.

Aluminum inhibited yeast glucose-6-phosphate dehydrogenase (EC 1.1.1.49) by a pseudo-first-order reaction. The inhibition was proportional to the incubation time and the concentration of aluminum. Double reciprocal plots gave a straight line with a kinact of 8.3 min-1 and indicated the presence of a binding step prior to inhibition. The kinetic study showed that 1 mol of aluminum was bound per mol of enzyme subunit.

Aluminum↗
PubMed1989

Inactivation of glucose-6-phosphate dehydrogenase isozymes from human and pig brain by aluminum.

Prolonged intake of low levels of aluminum from the drinking water has been found to increase the aluminum content in rat brain homogenates and to reduce the activity of hexokinase and glucose-6-phosphate dehydrogenase (G6PD). To determine the interaction of G6PD with aluminum in the brain, we have recently purified two isozymes of G6PD (isozymes I and II) from human and pig brain. Unlike isozyme I, isozyme II also had 6-phosphogluconate dehydrogenase (6-PGD) activity. We report here that G6PD isozymes I and II from human and pig brain purified to apparent homogeneity are inactivated by aluminum. Aluminum did not affect the 6-PGD activity of isozyme II. The aluminum-inactivated enzyme contained 1 mol of aluminum/mol of enzyme subunit. The protein-bound metal ion was not dissociated by exhaustive dialysis at 4 degrees C against 10 mM Tris-HCl (pH 7.0) containing 0.2 mM EDTA. Preincubation of aluminum with citrate, NADP+, EDTA, NaF, ATP, and apotransferrin protected the G6PD isozymes against aluminum inactivation. However, when the G6PD isozymes were completely inactivated by aluminum, only citrate, NaF, and apotransferrin restored the enzyme activity. The dissociation constants for the enzyme-aluminum complex of the isozymes varied from 2 to 4 microM, as measured by using NaF, a known chelator for aluminum. Inhibition of G6PD by low levels of aluminum further strengthens the suggested role of aluminum toxicity in the energy metabolism of the brain.

Aluminum↗
PubMed1989

T2 values in the human brain: comparison with quantitative assays of iron and ferritin.

Magnetic resonance (MR) imaging with a whole-body imager was performed in 10 fresh, unfixed whole human brains selected randomly from cadavers. All subjects were neurologically intact before death. T2 time constants were measured within the caudate nucleus, putamen, globus pallidus, cortical gray matter, subcortical white matter, and optic radiation. These regions were then excised, and T2 values were measured again with a 1.5-T MR spectrometer. Quantitative assays of iron, ferritin, and protein from these areas were then performed. Iron concentration varied significantly among brain regions, whereas ferritin and protein concentrations were constant among brain regions and among individuals. Neither iron nor ferritin concentration showed any consistent correlation with T2 values. Histologic examination of brain micro-sections with iron- and ferritin-specific stains of demonstrated poor correlation with biochemical assays of ferritin and iron concentrations. Results indicate that T2 values correlate poorly with iron and ferritin concentrations found in neurologically intact brains.

Adolescent↗
PubMed1988

Substance P augmentation of CSF-1-stimulated in vitro myelopoiesis. A two-signal progenitor restricted, tuftsin-like effect.

The inflammatory neuropeptide substance P acted as a costimulant for macrophage CSF-1-induced clonal proliferation of murine marrow-derived two signal-dependent mononuclear phagocyte progenitors. Substance P had no effect on clonal proliferation by progenitors responding solely to CSF-1. Substance P fragment 2-11 had no costimulatory activity; however, SP fragment 1-4 retained the full activity of the parent undecapeptide. Fragment 1-4 (ARG-PRO-LYS-PRO), a peptide containing a PRO residue between two positive charges, is a tuftsin-like (THR-LYS-PRO-ARG) tetrapeptide, and tuftsin exerted an identical costimulatory effect. Substance P, SP:1-4, and tuftsin were optimally effective as costimulants at 10(-7) to 10(-6) M. (ALA1)-tuftsin, an inhibitory analog of tuftsin, was a potent negative regulator of two signal-dependent colony formation. (ALA1)-tuftsin at concentrations less than or equal to 10(-9) M exerted dose-dependent inhibition of the positive effects of optimal concentrations of all of the co-stimulants tested, including bacterial LPS. The inhibitory tetrapeptide was equivalent in activity to ferritin, an established inhibitor of two signal-dependent colony formation. The results indicated that SP may influence myelopoiesis in addition to its other inflammatory and immunopotentiating properties. In addition, a potentially valuable modulator of SP and LPS responses in this system, (ALA1)-tuftsin, was identified.

Amino Acid Sequence↗
PubMed1988

Ferritin: an expanded role in metabolic regulation.

Synthesis of ferritin, a constitutive protein, is increased by iron. This protein is well recognized as a protein which detoxifies, stores and transports iron. The 24 subunits of ferritin assemble to form a protomer of Mr 480,000. This protein shell can sequester up to 4500 g atoms of iron as ferrichydroxyphosphate. Ferritin in vitro and in vivo binds other metal ions such as Cu, Zn, Cd, Pb, Be and Al. Next to Fe it binds large quantities of Be. Therefore, in vitro ferritin protects against and reverses the inhibition by Be of enzymes susceptible to this metal ion. Also, rats pretreated with Fe survive otherwise toxic levels of either pulmonary or intravenous exposure of Be. Liver ferritin from rats injected with Zn contains some of the injected metal ion. Incubation of such ferritin-zinc complex with zinc-requiring apoenzymes restores their activity. Fe(III) of ferritin is released only after its reduction to Fe(II) by a reductant. Incubation of phosphoglucomutase, a phosphoserine containing enzyme with ferritin and a reductant causes irreversible inactivation of the enzyme and removes 70% of its phosphate. Some other phosphoproteins are similarly inactivated but without the loss of the bound phosphate. Thus, uncontrolled release of iron from ferritin, in the presence of a reductant and oxygen can modify several biomolecules and can affect metabolic processes. A subclass of ferritin, acidic isoferritins, have been implicated in leukemia-associated inhibitory activity and has been suggested to inhibit production of Ia+ macrophage progenitors.

Animals↗
PubMed1988

Effect of long-term feeding of aluminium chloride on hexokinase and glucose-6-phosphate dehydrogenase in the brain.

Rats were fed 100 microM AlCl3 for 1 year in their drinking water, then killed and their brains homogenized in 0.1 M Tris (pH 7.4). The 800 g supernatants were assayed for Al3+ and the activities of acetylcholine esterase (ACE), hexokinase and glucose-6-phosphate dehydrogenase (G6PDH). The concentrations of Al in the homogenates, as computed on the original brain for the control and Al fed group were 40 ng and 80 ng/g wet wt, respectively. The activity of ACE was the same in both groups but that of hexokinase and G6PDH in the Al-fed group was about 73% and 80%, respectively, of the control. Dialysis restored the G6PDH but increased the hexokinase of the control group 2-fold and that of Al-fed group 2.7-fold. Thus at this elevated level it was same in both groups. The contribution of Al from the undialysed homogenates during assay was too low to account for the inhibition. It is therefore suggested that a dialyzable inhibitor for hexokinase is normally present in the brain and that Al feeding increases its concentration to further inhibit the utilization of glucose.

Acetylcholinesterase↗
PubMed1988

Ferritin: an iron storage protein with diverse functions.

Ferritin is the major protein for iron storage and iron detoxification. Since non-ferrous metals, such as aluminum, beryllium and zinc, are bound both in vivo and in vitro, ferritin is implicated as a general metal ion donor and detoxicant. The role of ferritin in Al and Be toxicity is discussed. During iron release ferritin produces free radicals which are involved in phosphoprotein inactivation, lipid peroxidation and, possibly, the general aging process. Conversely, during iron loading oxidative energy in the form of electrons and protons is given off. The different subunit compositions of ferritin, termed isoferritins, are, at least in part, involved with the multifunctionality of this protein.

Aluminum↗
PubMed1987

Isolation and characterization of ferritin from soyabeans (Glycine max).

Ferritin from the soyabean Glycine max was isolated and characterized. The protein has many features in common with ferritin from mammalian systems, including extensive sequence homology, as determined by two-dimensional peptide mapping. No immunocross-reactivity between the plant and animal proteins was detected. The ferritin isolated by MgCl2 precipitation has a single subunit of 28 kDa, whereas the ferritin remaining in the supernatant exhibits marked heterogeneity, with a main subunit of 22 kDa. This form of the protein appears to be the result of specific proteolytic processing that is not affected by serine protease inhibitors, and appears only after the seeds have been soaked long enough to induce germination. The appearance of the 22-kDa form corresponds to the appearance of "crystalline arrays" of ferritin in the amyloplasts of the plant cotyledons and may represent a plant form of hemosiderin. In support of this hypothesis, the 22-kDa protein appears to be incompletely assembled, as determined by sucrose gradient centrifugation and iron uptake studies. Although ferritin is normally quite resistant to proteolysis, the 22-kDa protein is easily generated from the 28-kDa form by treatment with subtilisin, suggesting the presence of a specific, protease-sensitive sequence on the protein's surface, possibly used to mark the phytoferritin for conversion to hemosiderin and construction of ferritin crystalline arrays.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Differential loss of enzyme activity by vitC and iron containing proteins.

Our earlier studies showed that rabbit muscle phosphoglucomutase was irreversibly inactivated by exposure to a mixture of vitamin C, FeCl3 and O2. The enzyme lost about 70% of its phosphate (V.V. Desphande and J.G. Joshi, J. Biol. Chem. 260, 754-764, 1985). The present report shows that several other iron proteins can substitute for FeCl3 to a varying degree. The rate of inactivation by FeCl3 greater than ferritin greater than hemoglobin = hemerythrin greater than transferrin = ferridoxin = vitamin C. These iron compounds also produced dephosphoenzyme but did not dephosphorylate ATP, ADP, AMP or phospholipids.

Animals↗
PubMed1987

Ferritin: isolation of aluminum-ferritin complex from brain.

Ferritin was isolated from the livers and brains of two groups of rats, one of which was fed aluminum chloride (100 microM) for 1 year in the drinking water. Brain tissue contained about one-third of the amount of ferritin found in the liver. While brain ferritin from normal rats contained 42.1 +/- 14.3 mol of aluminum, that from the aluminum-fed group contained 115.4 +/- 48.3 mol of aluminum per mol of ferritin. Liver ferritin from both groups contained similar amounts of both aluminum and iron, and the amounts were less than that found associated with brain ferritin. Ferritin isolated from the brains of patients who died of Alzheimer disease contained more aluminum and more iron than that from age-matched controls. Human brain ferritin is composed of two types of subunits--about 70% heavy chain (Mr, 22,000) and 30% light chain (Mr, 19,500). The isoelectric focusing pattern of human brain ferritin was considerably different from that of human liver. Only 5 of the 20 brain ferritin bands migrated similarly to the acidic isoferritins from the liver, and the major component of brain ferritin, representing 30% of the total ferritin, had a pI of 8.0.

Administration, Oral↗
PubMed1986

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin.

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.

Animals↗
PubMed1986

Ferritin and in vivo beryllium toxicity.

Beryllium (Be+2), a divalent metal ion, is toxic to both man and animal. Although the molecular basis for its toxicity is unknown, it is well established that micromolar concentrations of beryllium specifically inhibit certain enzymes. Previous in vitro studies have shown that the presence of ferritin, an iron-storage protein, reactivated these enzymes by sequestering beryllium (Price and Joshi, 1984). In the present study we demonstrate in vivo that beryllium and zinc are bound by ferritin in greater amounts than Pb+2, Cu+2, and Cd+2. Beryllium did not induce the synthesis of metallothionein. In animals pretreated with an iron salt (ferric ammonium citrate, 40 mg/kg body wt), liver ferritin was elevated approximately five times and the toxicity of intravenously injected beryllium was significantly attenuated. Excretion and deposition studies suggested that iron salt treatment resulted in a reduction of liver beryllium. Thus the protection against beryllium toxicity by ferric ammonium citrate may be due to increased production of ferritin which binds beryllium and its subsequent elimination in the feces.

Animals↗
PubMed1985

Vit C.Fe(III) induced loss of the covalently bound phosphate and enzyme activity of phosphoglucomutase.

Rabbit muscle phosphoglucomutase was irreversibly inactivated upon preincubation with vitamin C (Vit C). Fe(III), NADH.NADH oxidase.Fe(III), or ferritin.Vit C. Substrate, glucose 1-phosphate and Mg2+ afforded partial protection. No altered amino acid could be detected in the inactive enzyme. Enzyme so inactivated was more susceptible to trypsin. More importantly, during inactivation, the enzyme lost up to 70% of its enzyme-bound phosphate; the completely inactivated enzyme retained the remainder of the bound phosphate which was isolatable as phosphoserine residing in the 22-amino acid long tryptic peptide. Free phosphoserine as well as those in phosphorylase alpha and phosphocasein were resistant to the oxidizing system, suggesting that the phosphoserine of phosphoglucomutase is uniquely vulnerable to these treatments. Alternatively, a fraction of the total 1 mol of phosphate in the phosphoform of phosphoglucomutase may not be associated with phosphoserine. Phosphoglyceromutase, which has phosphohistidine at its active site, was also inactivated by the oxidizing system. However, it did not release any of the bound phosphate.

Amino Acids↗
PubMed1985

Insulin or increased concentration of zinc in liver, kidney or muscle does not alter the phosphoglucomutase activity in vivo.

Injection of insulin into mice and rabbit has been reported to convert the latent form of phosphoglucomutase (E X Zn) to an active form (E X Mg) without any net increase in the protein associated with this enzyme (Hashimoto et al., Biochem. Biophys. Res. Commun., 27 (1967) 369 and Peck et al., J. Biol. Chem. 242 (1971) 1160). Because the injection of Cd in rats causes a 2-fold increase in Zn concentration in the liver, it appeared that such treatment in mice would increase the concentration of the latent enzyme and thereby enhance the insulin induced activation of the enzyme. The results however show that insulin or Cd treatment, alone or in combination do not alter the concentrations of the 2 forms of enzyme in liver, kidney or muscle of normal or fasted mice. In all instances the conversion of the latent enzyme to its activated form required preincubation with a chelating agent. Fractionation of the tissue homogenates showed that upon Cd injection the concentration of Zn in the fractions of Mr less and 1000 increased 6-fold in liver and 3-fold in kidney, that in muscle remained unchanged.

Animals↗
PubMed1984

Ferritin: protection of enzymatic activity against the inhibition by divalent metal ions in vitro.

Ferritin binds a large quantity of Be2+ (Price D.J. and Joshi, J.G. J. Biol. Chem. 258 (1983) 10873) as well as other divalent metal ions. Therefore the ability of this protein to protect enzymes against or reverse the inhibition by metal ions was studied. Evidence presented here shows that the inhibition by Be2+ of the enzymes Na+K+ATPase, alkaline phosphatase and phosphoglucomutase is reversed by ferritin. Be2+ can be transferred reversibly between phosphoglucomutase and ferritin depending upon the relative concentrations of the 2 proteins. Ferritin also reactivated phosphoglucomutase inhibited by Zn2+, Cu2+, or Cd2+. Incubation of ferritin containing Be2+ with 4-10 fold molar excess of phosphoglucomutase (with respect to Be2+) removed 90% of the Be2+ from ferritin. The rates of inactivation of phosphoglucomutase by Be2+ donated by apoferritin or ferritin were identical. Based upon these observations it is suggested that Be2+ bound to the protein shell and to the iron core are in equilibrium with each other with the equilibrium favoring ferritin-Be2+ complex.

Alkaline Phosphatase↗