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Biomedical subjects

G L Rossi

Publications and source records attributed to G L Rossi.

At least 37 records · Page 2Linked to original sources
PubMed1996

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

Amino Acid Sequence↗
PubMed1996

Protein function in the crystal.

Protein crystals contain wide solvent-filled channels that allow for traffic of metabolites and intramolecular motility. Ligand binding, catalysis and allosteric regulation occur in the crystalline environment but intermolecular interactions may hinder function-associated transitions and alter activity with respect to solution. Lattice constraints have, however, provided the opportunity to isolate and characterize conformational states that are poorly populated in solution. New methods are being developed to initiate reactions rapidly and synchronously throughout the crystal and to monitor their time course. A model consistent with kinetics in the crystal is necessary to interpret the results of time-resolved macromolecular crystallography.

Allosteric Regulation↗
PubMed1995

Monovalent cations affect dynamic and functional properties of the tryptophan synthase alpha 2 beta 2 complex.

Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits. In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit. Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations. The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature. We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold. The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗
PubMed1995

LH, FSH, PRL and ACTH cells in pituitary glands of cows with ovarian cysts.

We investigated morphometrically and densitometrically LH, FSH, PRL, and ACTH cells in pituitary glands in cows with follicular cysts and normal animals. Three groups were considered: cows in diestrus, cows in periestrus and cows with follicular cysts. The periestrous group was obtained by merging pro- and metaestrous groups, since the two did not differ when evaluated separately. The attribution to groups was confirmed by plasma progesterone measurements and by postmortem examination of the ovaries. After immunocytochemical labeling the pituitary cells were counted, and their area and total immunoreactivity were measured. The results suggest hypofunction of the LH cells (P < 0.05) and hyperfunction of the ACTH cells (P < 0.05) from the cows of the follicular cyst group. The FSH and PRL cells were similar in the three groups. These changes are consistent with the reduced LH release reported by most authors in cows with follicular cysts and should be relevant to the pathogenesis of the bovine follicular cysts. The subnormal activity of LH cells might be secondary to the activation of the ACTH cells.

Journal Article↗
PubMed1995

Structure and oxygen affinity of crystalline desArg141 alpha human hemoglobin A in the T state.

The correlation of a protein structure determined crystallographically to its functional properties determined in solution can be an extremely complex problem due to potential differences of protein conformational flexibility in the two physical states. A more direct approach to the correlation of structure with function is to examine both the structure and the function of a protein in the same crystalline environment. In this paper, the structural and functional properties of T state desArg hemoglobin (human hemoglobin modified by removal of the alpha-chain COOH-terminal residue, Arg141 alpha) have been studied in the same crystal form by high resolution X-ray diffraction methods and by polarized absorption microspectrophotometry. Specifically, the crystal structure of deoxygenated desArg human hemoglobin has been refined at a 2.1 A resolution using crystals grown at low salt concentration from solutions of polyethylene glycol. The loss of Arg141 alpha and all of the salt bridges in which it participates is associated with subtle structural perturbations of the alpha-chains which include an increase in the conformational flexibility of both the NH2 and COOH-terminal peptides. Although the heme pockets appear unchanged and even the side-chain of Tyr140 is oriented nearly as in HbA, the functional characterization by microspectrophotometric measurements indicates that crystals of desArg hemoglobin bind oxygen with an affinity which is roughly 15-fold greater than that of crystals of human hemoglobin A. There is no alkaline Bohr effect or effect of chloride ions, but an acid Bohr effect is observed. The oxygen affinities measured along two principal axes of the crystals differ by 25%, indicating heterogeneity in the affinities of the oxygen binding sites. This finding and the measured Hill coefficient of unity suggest significant cooperativity in the binding of oxygen in these crystals. The origins of the observed heterogeneity and the implied cooperativity are unknown.

Arginine↗
PubMed1995

The reduction of circulating growth hormone and prolactin in streptozocin-induced diabetic male rats is possibly caused by hypothalamic rather than pituitary changes.

To gain further information on diabetes-related disorders in the somatotrophic and lactotrophic axes, we undertook a functional, morphometrical and densitometrical study of the arcuate nucleus (AN), median eminence (ME) and anterior pituitary gland of adult male rats one month after streptozocin-induced diabetes (STZ-D). The basal secretory activity of somatotrophs and lactotrophs was tested by the reverse haemolytic plaque assay (RHPA) and plasma GH and prolactin (PRL) levels were determined by RIA. The number of GH-releasing factor (GRF)-labelled axons and the amount of axonal tyrosine hydroxylase (TH)-immunoreactivity increased in STZ-D. There were no significant differences in any of the other densitometrical measurements performed on GRF-, somatostatin-, thyrotropin-releasing hormone- and TH-labelled ME axon cross-sections as well as those on tuberoinfundibular-dopaminergic neurones of the AN in STZ-D compared with control rats. Plasma GH and PRL levels and measurements on anterior pituitary GH- and PRL-labelled structures were decreased in STZ-D. However, the GH and PRL plaque areas were increased after RHPA implying that the secretory capacity of somatotrophs and lactotrophs was not impaired. Taken together, these results suggest that the accumulated GRF in the ME is due to reduced GRF release. This could account for the reduced amplitude and/or frequency of GH secretory pulses. The increased axonal TH-immunoreactivity may indicate an increased dopamine synthesis. If coupled to increased release this could, in turn, be partly responsible for the reduced plasma and anterior pituitary PRL concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Antagonistic binding of substrates to 3-phosphoglycerate kinase monitored by the fluorescent analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate.

The analogue of ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), binds tightly to pig muscle 3-phosphoglycerate kinase. A dissociation constant Kd of 0.0095 +/- 0.0015 mM was determined by fluorimetric titration on the basis of 1:1 stoichiometry. TNP-ATP is a strong competitive inhibitor towards MgATP and MgADP with a Ki of 0.008 +/- 0.001 mM for both substrates. It is also a mixed-type inhibitor towards 3-phosphoglycerate with similar inhibition constants. Binding of TNP-ATP to 3-phosphoglycerate kinase is accompanied by a tenfold intensity increase and a blue shift of about 20 nm in its fluorescence emission spectrum and a shift of the pK of its trinitrophenyl group towards a more acidic pH. These findings suggest that the negatively charged trinitrophenyl group of TNP-ATP significantly contributes to the binding of the analogue. By stepwise replacement of the fluorescent TNP-ATP, the dissociation constants (Kd) for ADP and MgADP binding were determined and found to be 0.78 +/- 0.08 and 0.048 +/- 0.006 mM respectively, which are consistent with the values previously determined by equilibrium dialysis [Molnár and Vas (1993) Biochem J. 293, 595-599]. In similar competitive-titration experiments, ATP and MgATP did not completely substitute for TNP-ATP. For the fraction of the analogue that could be substituted, the dissociation constants for MgATP and ATP were estimated to be 0.27 +/- 0.09 and 0.33 +/- 0.15 mM respectively, close to the values determined by equilibrium dialysis. Using the same method, a significant weakening of binding of both (Mg)ADP and (Mg)ATP could be detected in the presence of 3-phosphoglycerate: their respective Kd values became 0.34 +/- 0.04 and 0.51 +/- 0.22 mM. The reciprocal effect, i.e. weakening of 3-phosphoglycerate binding in the presence of the nucleotide substrates, has been observed previously [Vas and Batke (1984) Eur. J. Biochem. 139, 115-123]. Similarly, a much weaker binding of (Mg)ATP could be observed in the presence of 1,3-bisphosphoglycerate (Kd = 2.30 +/- 0.68 mM). The possible reason for the mutual weakening of substrate binding is discussed in the light of the available structural data.

Adenosine Diphosphate↗
PubMed1994

Pituitary gland changes in canine hypoadrenocorticism: a functional and immunocytochemical study.

Pituitary gland changes were evaluated immunocytochemically in three dogs with primary hypoadrenocorticism (HAC) and one dog with secondary HAC and related to the clinical, endocrinological and pathological findings. In primary HAC, the adrenocorticotrophic hormone (ACTH) immunoreactivity was increased in the pars distalis of the pituitary gland and all cortical layers of the adrenal gland were severely atrophied. This increase would be expected to reflect a high level of ACTH synthesis in the corticotrophs due to a lack of negative cortisol feed-back. In contrast, in the dog with secondary HAC the ACTH immunoreactivity was decreased in the pituitary gland, the basal plasma ACTH concentration was low and the zona fasciculata and reticularis were atrophied, but the zona glomerulosa was well preserved. These findings are consistent with a diminished synthesis of ACTH. Thus, pituitary gland changes differ in primary and secondary HAC and are consistent with the clinical, endocrinological and histopathological findings.

Adrenal Cortex Diseases↗
PubMed1994

Secretory disorders in pituitary thyrotropes of streptozocin-diabetic male rats.

Pituitary cells transport secretory products from deep cytoplasmic portions towards the subplasmalemmal region (SPL) by two mechanisms: (a) the regulated, secretagogue-dependent and (b) the secretagogue-independent constitutive pathway. Since thyrotropin (TSH) secretion is impaired in diabetes, we studied both pathways in the thyrotropes of control and streptozocin (STZ)-treated male rats. By electron microscopy, we measured the nucleus and cytoplasm area and counted the secretory granules. In 4 selected SPL (500 nm deep) areas per cell we also counted the marginated secretory granules and measured their area. Moreover, after TSH indirect immunogold labelling we counted immunogold particles on (a) the secretory granules of the SPL and (b) the intergranular SPL cytoplasm. To check the background staining we also counted immunogold particles on nonthyrotropic cells. In diabetic compared with control thyrotropes we found: (a) cytoplasmic atrophy and increased number of secretory granules per cytoplasm unit area, (b) reduced number of secretory granules in the SPL (blunted regulated secretion), (c) larger secretory granules in the SPL (abnormal secretory granule maturation), (d) decreased immunogold labelling of secretory granules in the SPL (reduced TSH transported by each secretory granule towards the cell membrane) and (e) decreased immunogold labelling of the intergranular SPL cytoplasm (blunted constitutive secretion). Our data suggest reduced regulated and constitutive TSH secretion and may thus contribute to a better understanding of the hypothyroidism in STZ-diabetic rats.

Animals↗
PubMed1994

Somatotropic dysfunction in growth hormone-releasing hormone-deprived neonatal rats: effect of growth hormone replacement therapy.

In a previous work, we reported that passive immunization with anti-growth hormone-releasing hormone (GHRH) antibodies (GHRH-Ab) in neonatal rats caused disruption of somatotropic function that was still present 60 d posttreatment. We studied the reversibility of this condition by growth hormone (GH) replacement therapy. Neonatal rats received GHRH-Ab (50 microL/rat, s.c.) or normal rabbit serum every second day from birth up to postnatal d 10 and received hGH (0.4 microgram/g body weight, s.c., b.i.d.) or vehicle in a 2 x 2 factorial design. Animals were studied on d 11 of age. In GHRH-Ab-treated rats, GH therapy 1) counteracted the reduced body weight and low plasma IGF-I levels; 2) failed to modify the reduced pituitary weight and GH content; 3) further reduced the low plasma GH levels; 4) partially restored the defective GH responsiveness to GHRH; 5) failed to modify the reduced hypothalamic somatostatin and increased GHRH gene expression in the hypothalamus; and 6) reverted the decreased pituitary somatostatin binding. Morphologic and morphometric evaluation of the pituitary gland from GHRH-AB+GH pups showed that the number of GH-labeled structures was lower than in normal rat serum-GH-treated pups, whereas the total GH immunoreactivity per unit surface, an index of intracellular hormone concentration, was slightly higher than in vehicle-GH or GHRH-Ab pups. As determined by electron microscopy, somatotropes from GHRH-Ab+GH pups had morphologic features of high cellular activity. It appears that in GHRH-deprived pups GH replacement therapy can normalize most but not all altered indices of the somatotropic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

An "in vitro" model for combined functional and morphological investigations on the rat pituitary gland.

We have developed an alternative model for combined "in vitro" functional and morphological studies on the rat pituitary gland. To test its use for biochemical investigations we studied some aspects of the secretion in pituitary thyrotrophs under basal conditions and after stimulation with TRH. After stabilisation of basal TSH release, the stimulation with TRH significantly increased the TSH secretion. To test the usefulness of our model for morphological investigations the tissue was processed for conventional and immunocytochemical light and electron microscopy at the end of the incubation. Different types of pituitary cell resulted to be well preserved. Our model allows combined biochemical and morphological studies on the pituitary gland and thus reduces the number of animal groups, eliminates the extrapolation of results from different groups and replaces the "in vivo" by an "in vitro" experiment.

Animal Testing Alternatives↗
PubMed1993

Retinol bound to cellular retinol-binding protein is a substrate for cytosolic retinoic acid synthesis.

Retinol bound to cellular retinol-binding protein (CRBP) was found to be oxidized to retinoic acid by a soluble activity from calf liver. Cytosolic retinoic acid synthesis from retinol-CRBP was strictly dependent on the exogenous supply of either NAD or NADP. NAD-supported reactions carried out in the presence or in the absence of dimethyl sulfoxide yielded apparent Km and Vmax values for the retinol-CRBP complex of 3.5 +/- 0.6 microM, 611 +/- 49 pmol h-1 (mg of protein)-1, and 0.84 +/- 0.12 microM, 601 +/- 38 pmol h-1 (mg of protein)-1, respectively. The corresponding values for the oxidation of free retinol, dissolved in dimethyl sulfoxide, were 7.1 +/- 0.3 microM and 948 +/- 47 pmol h-1 (mg of protein)-1. Since the dissociation constant of the bovine retinol-CRBP complex is less than 10(-8) M, whereas the Km for retinol-CRBP is of the same order as the Km for free retinol, synthesis of retinoic acid from retinol-CRBP does not rely on prior dissociation of retinol. ApoCRBP proved to be a specific inhibitor of retinoic acid synthesis from CRBP-bound retinol. Its inhibitory effect was indistinguishable from the dilution of the radioactive retinol-CRBP substrate that was obtained by the addition of unlabeled holoCRBP. In contrast, the oxidation of CRBP-bound retinol was not inhibited by the addition of other retinoid binding proteins nor by the addition of either free retinol or retinol complexed with proteins distinct from CRBP. These results indicate that the protein moiety of holoCRBP is specifically recognized by the cytosolic enzyme system that catalyzes retinoic acid synthesis from CRBP-bound retinol.

Animals↗
PubMed1993

Sexually dimorphic effects of aging on tuberoinfundibular dopaminergic neurons and lactotropes of rats.

The effect of aging on plasma prolactin (PRL) levels, hypothalamic tuberonifundibular dopaminergic (TIDA) neurons and pituitary lactotropes was evaluated in prolactinoma-free young (5-month-old) and old (23- to 24-month-old) Long-Evans rats of either sex. The young female rats were in diestrus, the old ones in recurrent pseudo-pregnancy. The tyrosine hydroxylase (TH)-labelled neurons in the arcuate nucleus (AN) and axons in the median eminence (ME) as well as the PRL-labelled lactotropes in the pituitary gland were studied by morphometry and densitometric immunohistochemistry. Further, we investigated the secretory function of isolated lactotropes by reverse hemolytic plaque assay (RHPA) and by cell culture in comparable animal groups. Compared with young animals, the plasma PRL levels of old rats of both sexes were similar or reduced. All morphometric and densitometric measurements of the AN neurons, ME axons (except number) and pituitary lactotropes were comparable in young and old female rats. In old male rats the AN and ME measurements were mostly decreased, while the lactotropes remained almost unchanged. The RHPA generally showed a reduced PRL release from lactotropes of old animals of both sexes. The PRL release from the cultured lactotropes, on the contrary, was greatly increased in old female rats and unchanged in old male rats. Our functional and morphological data suggest that the in vivo function of lactotropes in old prolactinoma-free female and male rats does not seem to be strongly influenced by the mildly reduced TIDA neuron activity, yet emphasize the differences of the aging process in the two sexes.

Aging↗
PubMed1993

Effect of chloride on oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure.

Oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure has been investigated by polarized absorption microspectrophotometry. These crystals were grown from poly(ethylene glycol) solutions containing low concentrations of salt. In the absence of chloride, they have a significantly higher oxygen affinity than crystals of human hemoglobin A grown in a similar manner, and exhibit Hill coefficients lower than 1. There is no Bohr effect from pH 6 to 9. We have found that chloride decreases the oxygen affinity of Hb Rothschild crystals, an effect which is absent in crystals of HbA. This dependence of affinity on chloride is almost certainly associated with the chloride binding sites which have been localized crystallographically at the mutant arginine residues (Kavanaugh et al., 1992). Since chloride binding appears to lower the oxygen affinities of both the alpha and beta chains, the linkage between the binding of oxygen and the dissociation of chloride results in significant cooperativity in oxygen binding to the crystals.

Amino Acid Sequence↗
PubMed1993

Oxygen binding by single crystals of hemoglobin.

Reversible oxygen binding curves for single crystals of hemoglobin in the T quaternary structure have been measured using microspectrophotometry. Saturations were determined from complete visible spectra measured with light linearly polarized parallel to the a and c crystal axes. Striking differences were observed between the binding properties of hemoglobin in the crystal and those of hemoglobin in solution. Oxygen binding to the crystal is effectively noncooperative, the Bohr effect is absent, and there is no effect of chloride ion. Also, the oxygen affinity is lower than that of the T quaternary structure in solution. The absence of the Bohr effect supports Perutz's hypothesis on the key role of the salt bridges, which are known from X-ray crystallography to remain intact upon oxygenation. The low affinity and absence of the Bohr effect can be explained by a generalization of the MWC-PSK model (Monod, Wyman, & Changeux, 1965; Perutz, 1970; Szabo & Karplus, 1972) in which both high- and low-affinity tertiary conformations, with broken and unbroken salt bridges, respectively, are populated in the T quaternary structure. Because the alpha and beta hemes make different projections onto the two crystal axes, separate binding curves for the alpha and beta subunits could be calculated from the two measured binding curves. The approximately 5-fold difference between the oxygen affinities of the alpha and beta subunits is much smaller than that predicted from the crystallographic study of Dodson, Liddington, and co-workers, which suggested that oxygen binds only to the alpha hemes.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization↗
PubMed1993

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c-type cytochrome.

A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c-type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C222(1) with cell dimensions a = 148.81 A, b = 68.85 A, and c = 187.18 A. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper-free apo-amicyanin. The diffraction data were collected at 2.75 A resolution from the former and at 2.4 A resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD-MADH) is an H2L2 hetero-tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero-octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers--i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c55li--are presented.

Bacterial Proteins↗