Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

Heterogeneity of glucagon receptors of rat hepatocytes: a synthetic peptide probe for the high affinity site

A glucagon analog with the following sequence has been synthesized: His- Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg -Leu-Gln-Glu-Phe-Leu-Gln-Trp-Ala-Leu-Gln-Thr. When interacting with rat hepatocytes, the analog mimics, in part, the activities of glucagon in receptor binding and inhibition of carbohydrate incorporation into glycogen. Comparison of the binding of the analog with that of glucagon demonstrates the existence of two distinct homogeneous populations of glucagon receptors. The synthetic analog acts as a specific probe for those receptors that have a high affinity for glucagon.     

Direct effect of gossypol on TR-ST cells: perturbation of rhodamine 123 accumulation in mitochondria

Gossypol has deleterious effects directly on TR-ST cells originating from a rat testicular tumor. Exposure of TR-ST cells to gossypol (5 micrograms/ml) decreases their rate of protein synthesis approximately 30% within 1 h and 65% by greater than 10 h, causes intracellular vacuolation, changes cell shape from cobblestone to a rounded conformation and inhibits cell proliferation. Yet, these gossypol-treated cells remain viable, as assessed by their ability to hydrolyze fluorescein diacetate. Gossypol also perturbs mitochondrial transmembrane potential in TR-ST cells, as demonstrated by marked changes in rhodamine 123 staining. Mitochondria of control TR-ST cells avidly accumulate rhodamine 123, but those in cells exposed to gossypol (greater than or equal to 5 micrograms/ml) for greater than 1 h fail to sequester the fluorochrome. Instead, the cell cytoplasm shows a light and diffuse staining with rhodamine 123. Rat spermatozoa show a similar response. Conversely, at concentrations of 20 micrograms/ml, gossypol has minimal effects on rhodamine 123 accumulation by primary cultures of hepatocytes and by rat spermatogenic cells, including primary spermatocytes and spermatids (Steps 1-12). Moreover, TR-ST cells exhibit reduced mitochondrial staining with gossypol at an ED50 of 7.6 micrograms/ml, while those for the nontesticular Rat-1, AnAn, 3T3 and PtK2 cell lines are 13.1, 21.5, 28.5 and 26.4 micrograms/ml, respectively.     

Expression of the SOS response following simultaneous treatment with methyl-nitrosoguanidine and mitomycin C in Escherichia coli

Simultaneous treatment of Escherichia coli cultures with methyl-nitrosoguanidine and mitomycin C induces recA-dependent inhibition of respiration but not inhibition of cell division. This pattern of SOS functions expression is the same as that is found following treatment with methyl-nitrosoguanidine alone and contrary to the pattern induced after mitomycin C addition. The same result is obtained when a culture of E. coli RecA441 (formerly tif) is shifted to 42 degrees C and treated simultaneously with methyl-nitrosoguanidine. The suppressor effect of this compound over the pattern of SOS functions expression induced by mitomycin C or high temperature in recA441 mutants is directly related to the increase in its dose. Moreover, the division temperature-sensitive mutant ftsA treated with methyl-nitrosoguanidine and high temperature does not show any decrease in its normal filamentous growth when cultured at 42 degrees C. This indicates that the effect of methyl-nitrosoguanidine on the recA-independent inhibition of cell division is not due to any indiscriminate effect of this compound over the division process. These results suggest that the specific kind of lesion caused in DNA is very important in determining which SOS function is induced.     

Antagonism of diazepam-induced feeding in rats by antisera to opioid peptides

Diazepam-induced feeding in rats is antagonized not only by the opiate antagonist naloxone but also intraventricular administration of specific antisera to the endogenous opioid peptides met-enkephalin or beta-endorphin. Pituitary beta-endorphin is probably not implicated in the diazepam effect since blockade with the glucocorticoid dexamethasone of the release of beta-endorphin from the anterior pituitary does not modify the diazepam-induced feeding, which is however prevented by TRH, a suggested physiological antagonist of some of the effects of opioid peptides. The possible central participation of both beta-endorphin and met-enkephalin in the ingestive behavior induced by diazepam gives further support to the postulated physiological role of endogenous opioids in appetite regulation.     

Inhibition of gastric acid secretion by intracerebral injection of calcitonin gene related peptide in rats

Calcitonin gene related peptide (CGRP), a 37 amino acid peptide recently characterized from the rat brain, injected into the cisterna magna or the lateral hypothalamus, inhibited gastric acid secretion and increased serum gastrin levels in conscious pylorus-ligated rats. CGRP suppressed pentagastrin- and histamine-induced stimulation of gastric secretion in urethane-anesthetized gastric fistula rats. Peptide action was dose-dependent, not modified by adrenalectomy and peripheral sympathectomy induced by guanethidine pretreatment and reversed by vagotomy. These results demonstrate that intracisternal or intralateral hypothalamic injection of CGRP inhibits stimulated gastric acid secretion. The mechanism of action is vagal dependent and unrelated to modification of gastrin secretion or activation of sympathetic outflow.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

Bidentate peptides: highly potent new inhibitors of enkephalin degrading enzymes

Three series of bidentates bearing an hydroxamic or an N-Acyl-N-hydroxy amino group on structures related to Phe-Gly or Phe-Ala exhibit strong inhibitory potency against purified enkephalinase with IC50 values in the 4 to 15 nM range. As with thiol-containing inhibitors, such as thiorphan, the most active compounds are those in which a methylene spacer separates the benzyl P1' moiety from the Zn coordinating residue. Formation of a bidentate complex with the metal enzyme is clearly demonstrated by a loss of potency of three order of magnitude following the removal of one component of the bidentate group. All the compounds studied are unable to interact with angiotensin converting enzyme (IC50 greater than 10,000 nM). Moreover, compounds of the general formula HONHCO-CH2-CH(CH2 phi)-CONH-CH(R)-COOH belonging to the most active series of enkephalinase blockers (IC50 approximately 4 nM) behave also as highly potent and competitive inhibitors (IC50 approximately 10 nM) of a Tyr-Gly releasing dipeptidylaminopeptidase purified from rat brain. The pure steroisomer [(R)-3-(N-hydroxy)carboxamido-2-benzylpropanoyl]-L-alanine designated kelatorphan, exhibits also a relatively good inhibitory potency against aminopeptidases (IC50 approximately 10 microM) and can be considered as the first virtually complete inhibitor of enkephalin metabolism. This very interesting property of inhibiting all three enzymes of enkephalin metabolism could enhance the required selectivity for a possible clinical use of these inhibitors as new analgesic and psychoactive drugs.