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Biomedical subjects

M Pines

Publications and source records attributed to M Pines.

At least 109 records · Page 6Linked to original sources
PubMed1986

Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain.

We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the alpha subunit (39 kDa) of a G protein of unknown function, termed Go, as well as with the beta subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the alpha subunits of the stimulatory (Gs) or inhibitory (Gi) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affinity-purified antiserum showed that RV3 specifically recognizes the Go alpha subunit and the beta subunit in crude brain membranes. Using RV3, we found approximately equal amounts of Go in brain membranes from frog, chicken, rat, cow, and man. Quantitative immunoblotting gave Go alpha subunit/ beta subunit ratios approximately equal to 1 in cerebral cortex, raising the possibility that free Go alpha subunit (unassociated with beta subunit) may exist in brain. The concentration of Go alpha subunit in cortex is about 5 times that of Gi alpha subunit. The results show that Go is an immunochemically distinct, highly conserved protein distributed throughout the brain, with particularly high concentrations in forebrain.

Animals↗
PubMed1986

Polyacrolein microspheres as a new solid phase for radioimmunoassay.

Polyacrolein (PA) microspheres contain reactive aldehyde groups through which ligands containing primary amino groups such as proteins and drugs can be covalently bound in a single step at physiological pH. Antibodies against cyclic-AMP, digoxin and rabbit serum were thus coupled to PA microspheres. The immuno-microspheres were kept in suspension or freeze-dried, with insignificant decrease in their binding capacity. The conjugates were used in the respective radioimmunoassay (RIA) systems to facilitate the separation of the free and the antibody-bound 125I ligands, in comparison with precipitation of Protein A of Staphylococcus aureus. Cyclic-AMP was assayed using PA microspheres coupled either with the primary antibody or with anti-rabbit serum as a secondary antibody, in a buffer system, in chick plasma, in urine and in media in which avian dispersed kidney cells had been stimulated by various agents. The results obtained using the immuno-microspheres and the bacterial separation methods were indistinguishable. Other 125I-ligands, such as digoxin in buffer system or thyroxine and triiodothyronine in chick plasma, were assayed in the picogram range. Owing to the solubility of non crosslinked microspheres conjugates in toluene-based scintillation fluids, both the free and the bound fractions could be counted when using 3H-ligands. Corticosterone was assayed using this technique.

Acrolein↗
PubMed1986

The inhibitory guanine nucleotide regulatory protein modulates agonist-stimulated cAMP production in rat osteosarcoma cells.

We identified the subunits of the stimulatory and inhibitory guanine nucleotide binding proteins (Gs and Gi, respectively) associated with adenylate cyclase in rat osteosarcoma (ROS) cells. Pertussis toxin catalyzed ADP-ribosylation of Gi alpha in ROS cells increased agonist (PTH and isoproterenol)-stimulated, but not basal, cAMP production. The effect of pertussis toxin was dose and time dependent, and slowly reversible (T 1/2 approximately 30 h) during continued culture without toxin. Pertussis toxin treatment of ROS cell lines (17/2.8 and 24/l) with markedly different agonist responsiveness increased agonist-stimulated cAMP production in proportion to the response without toxin treatment. Pertussis toxin treatment further increased cAMP response to PTH in dexamethasone treated cells. We conclude that ROS cells contain functional Gi which modulates agonist-stimulated cAMP formation. Alterations in ROS cAMP responsiveness caused by steroids, and the reduced responsiveness of the 24/1 cell line, however, are unlikely to be due to changes in Gi.

Adenylate Cyclase Toxin↗
PubMed1985

The tryptic and chymotryptic fragments of the beta-subunit of guanine nucleotide binding proteins in brain are identical to those of retinal transducin.

The 35-kDa beta-subunit of transducin purified from rod outer segment membranes is cleaved into 2 major fragments by trypsin, and 7 major fragments by chymotrypsin. Identical fragments are visualized by immunoblotting with transducin-beta specific antisera after proteolysis of rod outer segment membranes, purified brain guanine nucleotide binding proteins, and brain membranes. The results indicate that the beta-subunits of transducin and of brain guanine nucleotide binding proteins are not only similar structurally, but are also similarly oriented in membranes with respect to accessibility to proteolytic enzymes.

Animals↗
PubMed1985

Antibodies against the carboxyl-terminal 5-kDa peptide of the alpha subunit of transducin crossreact with the 40-kDa but not the 39-kDa guanine nucleotide binding protein from brain.

We tested 18 antisera showing reactivity against the alpha subunit of transducin, the guanine nucleotide binding protein from rod outer segment, for crossreactivity against the 40- and 39-kDa guanine nucleotide binding proteins purified from bovine brain. A single antiserum, CW6, showed crossreactivity, and this was predominantly against the 40-kDa protein. Immunoblots of the tryptic fragments of transducin alpha subunit with multiple antisera raised against that subunit showed that only CW6 recognizes a COOH-terminal 5-kDa peptide that includes the site of pertussis toxin ADP-ribosylation. Antibodies against the 5-kDa peptide, affinity-purified from CW6, specifically react with the 40-kDa brain protein on immunoblots. The results show that the 39- and 40-kDa guanine nucleotide binding proteins from brain differ immunochemically and that the COOH-terminal 5-kDa peptide of transducin alpha subunit is homologous to a region in the 40-kDa brain protein. We speculate that this homologous region may be in a domain that confers specificity for receptor interactions of guanine nucleotide binding proteins.

Adenosine Diphosphate Ribose↗
PubMed1985

Cyclic AMP response to various haemopoietic regulators.

Diffusible inhibitors and stimulators are involved in the regulation of bone marrow pluripotent stem cell (CFU-S) proliferation. We have previously shown the existence of CFU-S inhibitors in foetal calf marrow and liver and have started their purification. The lack of a simple and time-saving test to determine the kinetic state of CFU-S and the activity of the inhibitors led us to explore the possibility of a biochemical proliferation marker that could be used for screening purpose. Since it was shown that cyclic AMP was implicated in the regulation of CFU-S proliferation, it was of interest to study the variations in cAMP levels after stimulation and inhibition of CFU-S entry into cycle. The results of in vitro experiments showed that the increase in cAMP levels observed in bone marrow cells after incubation with different haemopoietic stimulators was specific neither for bone marrow cells nor for the various haematopoietic regulators. In the in vivo experiments, an increased cAMP level was observed 8 hr after one injection of Ara-C at the time when CFU-S are recruited into S phase. However, no modification of cAMP levels has been observed after injection of CFU-S inhibitors in the Ara-C-treated mice. Although cAMP does not seem to be a suitable marker for testing the activity of inhibitory fractions during the purification process, this work has contributed to the study of CFU-S stimulators.

Animals↗
PubMed1984

Neutralization of heat-labile toxin of E. coli by antibodies to synthetic peptides derived from the B subunit of cholera toxin.

Antibodies elicited by six synthetic peptides corresponding to various fragments of B subunit of cholera toxin (CT) were evaluated for their cross-reactivity with heat-labile toxin (LT) of Escherichia coli. The antiserum directed towards the peptide CTP3 (residues 50-64) was found highly cross-reactive with the LT, in radioimmunoassay and immunoblotting. This peptide was also the most cross-reactive with intact CT. The antiserum against CTP1 (residues 8-20) was also cross-reactive with the two toxins, although to a much lower extent. Antisera to both CTP1 and CTP3, which are inhibitory towards CT, were found equally effective in neutralizing the biological activity of the E. coli LT. This was manifested by inhibition of both adenylate cyclase activity and fluid secretion into ligated ileal loops of rats. These results might indicate the potential of such synthetic peptides as the basis for a general vaccine against several types of infectious diarrhea.

Adenylyl Cyclases↗
PubMed1984

Isolation and purification of avian parathyroid hormone using high performance liquid chromatography, and some of its properties.

A peptide has been extracted in acetic acid from the parathyroid glands of chicks (Gallus domesticus) raised on a vitamin D-deficient diet. The peptide was purified by gel filtration and high-performance liquid chromatography (HPLC). Its degree of purity was ascertained also by SDS-polyacrylamide gel electrophoresis. The avian hormone had the same SDS electrophoretic mobility as bovine parathyroid hormone (bPTH) and therefore a similar molecular weight. The avian peptide was lower than bPTH in basic amino acids and considerably higher in glycine. Evidence for the identity of the peptide as avian parathyroid hormone relies on its source, the similarity of its molecular weight to that of bPTH, and its high potency in stimulating cyclic AMP production in kidney cells in vitro and in vivo.

Animals↗
PubMed1984

Both cholera toxin-induced adenylate cyclase activation and cholera toxin biological activity are inhibited by antibodies against related synthetic peptides.

The immune response against six synthetic peptides corresponding to various segments of the B subunit of cholera toxin was evaluated. Conjugates in which the peptides were covalently linked to tetanus toxoid served for immunization of rabbits. As previously reported, four of these conjugates elicited antibodies cross-reactive with intact cholera toxin. We report here that antisera against two of these synthetic peptides inhibit the entire spectrum of activities of the intact cholera toxin. This is manifested both on the biochemical level (adenylate cyclase induction) and on the biological effect (intestinal fluid secretion). These results indicate that these peptides may serve as suitable candidates for preparation of a synthetic anticholera vaccine.

Adenylyl Cyclases↗
PubMed1983

Urinary cyclic AMP excretion in birds: dependence on parathyroid hormone activity.

The cyclic AMP response of avian kidney to parathyroid activity has been evaluated both in vitro and in vivo. The production of cyclic AMP by dispersed avian kidney cells was stimulated by bovine parathyroid hormone or by an extract of avian parathyroid glands. Intravenous infusion of bovine parathyroid hormone resulted in increased urinary excretion and plasma concentration of cyclic AMP, as well as increased plasma calcium and urinary phosphorus excretion. The increases in plasma and urinary cyclic AMP preceded those of plasma calcium and of phosphorus excretion. EDTA infusion resulted in a decrease in plasma calcium and an increase in urinary cyclic AMP excretion. After 10 days on a low-calcium diet, chickens exhibited a 4.5-fold increase in urinary cyclic AMP excretion. The results suggest that urinary cyclic AMP in chickens reflects PTH activity similar to some mammalian species.

Animals↗
PubMed1983

Simulation of calcium homeostasis: modeling and parameter estimation.

The system that regulates plasma calcium in the bird has been formalized into a model based on a series of differential equations and solved by computer simulation. Bone, kidney, and intestine have been considered as the control subsystems, with parathyroid hormone and 1,25-dihydroxycholecalciferol as the regulating hormones. The parameters used in the simulation model have been computed either from published results or by specifically designed experiments described here. For the estimation of parameters, an iterative procedure has been developed that was designed to minimize the sum of square errors between observed and system-simulated values. Parameters of 1,25-dihydroxycholecalciferol metabolism were experimentally obtained from the kinetic behavior of the 3H-labeled hormone in rachitic birds after a single dose. Model parameters have been adjusted using the results of in vivo calcium loading and validated by an EDTA infusion experiment. The simulation model has been used to study the hierarchy of the activities of the three control subsystems and of the regulating hormones, at different calcium intakes. Positive or negative errors in plasma calcium resulted in an asymmetry in the activities of the controlling systems, bone and kidney, whereas the intestine is characterized by its relatively long response time.

Animals↗
PubMed1983

The Foundations' Fund for Research in Psychiatry and the growth of research in psychiatry.

For over a quarter century, until it disbursed its remaining funds in 1981, the Foundations' Fund for Research in Psychiatry (FFRP) aided hundreds of researchers in fields related to mental health. The fund was established by a private donor, the late Charles B. G. Murphy. Much of the research it sponsored during its early years was psychoanalytically oriented. In the 1960s it shifted to a more biological and social orientation. Its influence was greatest during its first decade, when its research grants, fellowships, and support to departments of psychiatry helped to launch the modern era of psychiatric research. This review analyzes FFRP's activities and examines its achievements.

Foundations↗
PubMed1983

The amino acid requirements of growing turkeys. 1. Model construction and parameter estimation.

A model has been formulated to calculate the protein and amino acid requirements for growing turkeys. A requirement was taken as the sum of the needs for maintenance, for gain in carcass weight without feathers, and for the feathers. Parameters used in the model were obtained from a study of carcass composition along the growth period and from two balance trials with adult turkeys. The maintenance requirement was considered to be the sum of the needs for replacing skin and intestinal protein losses and for the obligatory creatinine excretion in the urine. Nitrogen retention was determined at 2.3 g/day in protein-fed adult (17 kg) male turkeys as an estimate for the replacement needs for skin protein loss. Creatinine excretion in these birds was 123 mg/day. When fed an N-free diet, 16.5 kg adult turkeys excreted a total of 3.5 g/day amino acids as a result of intestinal losses. The protein requirement for maintenance was estimated at 32 mg/day/g2/3 of body weight by summing up the different needs and dividing by .85, the coefficient of protein absorption. Protein and amino acid analyses of nude carcass and feathers and the growth curve were used to calculate the requirements for growth. These were then related to energy intake predicted by equations, which included previously determined coefficients for maintenance and growth (Hurwitz et al., 1980a), and the results of carcass lipid analysis. The calculated amino acid requirements were similar to those of the National Research Council (1977).

Amino Acids↗