Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene.

To test the hypothesis that agents activating receptors negatively coupled to adenylyl cyclase (AC) can stimulate cell proliferation, we have expressed a human alpha 2-adrenergic receptor (alpha 2-C10) in CCL39 cells and studied the effects of alpha 2-agonists on reinitiation of DNA synthesis in quiescent cells. We report that the alpha 2-agonists epinephrine and clonidine stimulate [3H]-thymidine incorporation in synergy with fibroblast growth factor and that the alpha 2-antagonist yohimbine efficiently inhibits this response. Epinephrine- and clonidine-stimulated DNA synthesis is completely blocked by pertussis toxin and correlates well with the inhibition of prostaglandin E1-stimulated AC. Thus, their action closely resembles the action of serotonin in the same cell system, which is mediated through 5-HT1b receptors. In fact, serotonin- and epinephrine-stimulated DNA synthesis reinitiation is not additive, suggesting that both agents act through a common pathway. Interestingly, alpha 2-agonists also induced a moderate release of inositol phosphates, indicating that alpha 2-adrenergic receptors can interact both with the AC and phospholipase C messenger system. Activation of phosphoinositide (PI) turnover by epinephrine leads to a significant stimulation of Na+/H+ exchange but is insufficient to trigger a mitogenic response in CCL39 cells, as will be discussed. We found no evidence for epinephrine-induced activation of Na+/H+ exchange by a mechanism independent of PI breakdown.Our data show that alpha 2-adrenergic receptors can play a role in the regulation of cell proliferation in an appropriate context; also, the data support the hypothesis that receptors negatively coupled to AC must be taken into account as mediators of growth factor action in fibroblasts, in particular when activated in parallel with receptor tyrosine kinases.     

Nefiracetam (DM-9384) reverses apomorphine-induced amnesia of a passive avoidance response: Delayed emergence of the memory retention effects

Nefiracetam is a novel pyrrolidone derivative which attenuates scopolamine-induced learning and post-training consolidation deficits. Given that apomorphine inhibits passive avoidance retention when given during training or in a defined 10–12h post-training period, we evaluated the ability of nefiracetam to attenuate amnesia induced by dopaminergic agonism. A step-down passive avoidance paradigm was employed and nefiracetam (3 mg/kg) and apomorphine (0.5 mg/kg) were given alone or in combination during training and at the 10–12h post-training period of consolidation. Co-administration of nefiracetam and apomorphine during training or 10h thereafter produced no significant anti-amnesic effect. However, administration of nefiracetam during training completely reversed the amnesia induced by apomorphine at the 10h post-training time and the converse was also true. These effects were not mediated by a dopaminergic mechanism as nefiracetam, at millimolar concentrations, failed to displace either [³H]SCH 23390 or [³H]spiperone binding from D₁ or D₂ dopamine receptor subtypes, respectively. It is suggested that nefiracetam augments molecular processes in the early stages of events which ultimately lead to consolidation of memory.     

Redesigning the hydrophobic core of a four-helix-bundle protein.

Rationally redesigned variants of the 4-helix-bundle protein Rop are described. The novel proteins have simplified, repacked, hydrophobic cores and yet reproduce the structure and native-like physical properties of the wild-type protein. The repacked proteins have been characterized thermodynamically and their equilibrium and kinetic thermal and chemical unfolding properties are compared with those of wild-type Rop. The equilibrium stability of the repacked proteins to thermal denaturation is enhanced relative to that of the wild-type protein. The rate of chemically induced folding and unfolding of wild-type Rop is extremely slow when compared with other small proteins. Interestingly, although the repacked proteins are more thermally stable than the wild type, their rates of chemically induced folding and unfolding are greatly increased in comparison to wild type. Perhaps as a consequence of this, their equilibrium stabilities to chemical denaturants are slightly reduced in comparison to the wild type.     

Does suppressing luteinising hormone secretion reduce the miscarriage rate? Results of a randomised controlled trial.

OBJECTIVE--To determine whether prepregnancy pituitary suppression of luteinising hormone secretion with a luteinising hormone releasing hormone analogue improves the outcome of pregnancy in ovulatory women with a history of recurrent miscarriage, polycystic ovaries, and hypersecretion of luteinising hormone. DESIGN--Randomised controlled trial. SETTING--Specialist recurrent miscarriage clinic. SUBJECTS--106 women with a history of three or more consecutive first trimester miscarriages, polycystic ovaries, and hypersecretion of luteinising hormone. INTERVENTIONS--Women were randomised before conception to receive pituitary suppression with a luteinising hormone releasing hormone analogue followed by low dose ovulation induction and luteal phase progesterone (group 1) or were allowed to ovulate spontaneously and then given luteal phase progesterone alone or luteal phase placebo alone (group 2). No drugs were prescribed in pregnancy. MAIN OUTCOME MEASURES--Conception and live birth rates over six cycles. RESULTS--Conception rates in the pituitary suppression and luteal phase support groups were 80% (40/50 women) and 82% (46/56) respectively (NS). Live birth rates were 65% (26/40) and 76% (35/46) respectively (NS). In the luteal phase support group there was no difference in the outcome of pregnancy between women given progesterone and those given placebo pessaries. Live birth rates from an intention to treat analysis were 52% (26/50 pregnancies) in the group given pituitary suppression and 63% (35/56) in the controls (NS). CONCLUSIONS--Prepregnancy suppression of high luteinising hormone concentrations in ovulatory women with recurrent miscarriage and hypersecretion of luteinising hormone does not improve the outcome of pregnancy. The outcome of pregnancy without pituitary suppression is excellent.     

Pathways,pathway tubes,pathway docking,and propagators in electron transfer proteins

The simplest views of long-range electron transfer utilize flat one-dimensional barrier tunneling models, neglecting structural details of the protein medium. The pathway model of protein electron transfer reintroduces structure by distinguishing between covalent bonds, hydrogen bonds, and van der Waals contacts. These three kinds of interactions in a tunneling pathway each have distinctive decay factors associated with them. The distribution and arrangement of these bonded and nonbonded contacts in a folded protein varies tremendously between structures, adding a richness to the tunneling problem that is absent in simpler views. We review the pathway model and the predictions that it makes for protein electron transfer rates in small proteins, docked proteins, and the photosynthetic reactions center. We also review the formulation of the protein electron transfer problem as an effective two-level system. New multi-pathway approaches and improved electronic Hamiltonians are described briefly as well.     

Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of escherichia coli

The glucose metabolism of an Escherichia coli strain bearing mutations abolishing both acetyl phosphotransferase (PTA) and acetate kinase (ACK) activities was studied under aerobic and anaerobic conditions. These studies were conducted in a complex medium with the mutant carrying no plasmid, the mutant carrying the common cloning vector pUC19, and the mutant carrying a plasmid bearing the "pet" operon that encodes Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase activities. The mutant carrying no plasmid showed lower specific growth and glucose uptake rates relative to the parent wild-type strain (K-12), Lactic acid was produced at higher levels than the wild type, and considerable amounts of pyruvic acid were secreted as an unusual byproduct. Analysis of other fermentation products showed low but significant amounts of acetic acid, no accumulation of formic acid, and lower secretion of succinate and ethanol. The maintenance of the plasmid pUC19 in the mutant negatively affected metabolism. Expression of the pet operon overcame the metabolic stress caused by the plasmid, enhancing growth and glucose uptake rates to the values observed in the plasmidfree mutant. Also, expression of the pet operon allowed consumption of pyruvate accumulated during the first hours of fermentation.     

Specific recognition of the neuronal cell surface by an antiserum raised plasma membrane preparations of immature rat cerebellum

A brain specific antiserum was prepared by immunizing rabbits with a crude membrane fraction from 8-day old rat cerebella. In immunofluorescence studies the antiserum labeled the perikarya and processes of cultured cerebellar neurones. In contrast, other cell types, encountered in cerebellar cultures including astrocytes, endothelial cells and fibroblasts, were consistently unstained. The antiserum when used in crossed immunoelectrophoresis with Triton X-100 solubilized brain extracts reacted predominantly with one antigen that could be identified as the D2 protein.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Application of arabinofuranosyl cytosine in the kinetic analysis and quantitation of DNA repair in human cells after ultraviolet irradiation.

We have developed a technique whereby 3-h pulses of arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) are used to analyze the kinetics of repair with time after ultraviolet irradiation in human fibroblasts. We demonstrate that this technique offers a significant improvement over existing repair assays in its ability to visualize between 57 and 100% of all sites undergoing repair in a given period of time. In addition, kinetic analyses of repair are more easily made and yield more information than techniques such as repair replication or unscheduled DNA synthesis. We have also examined the nature of the inhibition event by ara-C and have determined that repair breaks accumulate in the presence of ara-C and HU only up to a certain time beyond which no further breaks appear. The time needed to reach this saturation point depends on the number of sites undergoing repair during the treatment time. This observation is discussed with respect to a possible mechanism of excision repair inhibition by ara-C and HU.     

Flocculation of Chlorella vulgaris by Lactobacillus casei

Summary The flocculation of Chlorella vulgaris by Lactobacillus casei was studied to determine whether the latter could act as a suitable flocculant for the removal of Chlorella from algal ponds. The flocculating activity of the Lactobacilli was caused by the bacterial cells themselves, and not by diffusible products of bacterial metabolism. Diffusible products of algal metabolism inhibited flocculation. For algae resuspended in water, the best flocculation occurred at pH values less than 3.5 where the charges on the bacterial and algal cells were opposite. For flocculation at least one bacterium was required for every algal cell; in terms of cell concentrations,10 mg/l of bacteria were required to flocculate an algal suspension of 1,000 mg/l. The mechanism of flocculation implied by the results is that positively charged cells of L. casei adsorb to the surface of negatively charged cells of C. vulgaris neutralizing the charge and thus destabilizing the algal suspension. Because of the low pH required and because diffusible products of algal metabolism inhibit the flocculation, it is unlikely that L. casei could be usefully employed as a flocculant of Chlorella from algal ponds.     

ERYTHROID AND GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN PRE-NATAL 'STEEL' (SlJ/SlJ) ANAEMIC MICE

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Sl^J/Sl^J mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Sl^J/Sl^J foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when ‘normal’ livers contain approximately 6 × 10⁵ erythroid colony forming cells/liver. In Sl^J/Sl^J fetuses the maximum reached is only 1 × 10⁵. Granulocyte-macrophage colony forming cells (CFU_C) in Sl^J/Sl^J fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Sl^J/Sl^J mice in the 2–3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Sl^J/Sl^J mice, but their production in the Sl^J/Sl^J pre-natal spleen appears unaffected.