Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Daily relaxation modifies serum and salivary immunoglobulins and psychophysiologic symptom severity

This study investigated the effect of daily relaxation on concentrations of serum immunoglobulins A, G, and M and secretion rates of salivary immunoglobulin A (S-IgA). Twenty-four volunteers were randomly assigned to practice a relaxation technique daily for 3 weeks and 16 to a waiting list control condition. Blood and saliva samples were collected before and after a supervised 20-min relaxation session at the beginning and end of the 3-week practice period. S-IgA secretion rate increased significantly (p<.001) after 20 min of relaxation. A longer-term practice effect also occurred in that the increase in secretion rate in before to after relaxation samples was higher (p=.014) in subjects who had practiced relaxation once a day for 3 weeks than in waiting list control subjects practicing for the first time. Serum IgA (p<.001), IgG (p<.001), and igM (p<.05) increased significantly over the 3-week practice period. Relaxation may be a self-regulating strategy affecting both humoral and cellular divisions of the immune system.Parts of this paper were presented at the annual meeting of the Biofeedback Society of America, March 1987. Materials for the IgA assays were provided by Cooper Biomedical, Malvern, Pennsylvania.     

Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G

The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.     

Efficient extraction of RNA from mammalian tissue

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.     

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

Effect of Ethnicity and Geographical Location on Body Weight,Dietary Restraint,and Abnormal Eating Attitudes

Previous studies have examined the effect of ethnicity on obesity, concerns about shape and weight, and attitudes about eating. We hypothesized that geographical location would also influence these variables, and that students growing up in the northern part of the United States and attending northern colleges would differ from students from the South. To examine this, we studied a random sample of 275 African-Americans (AA) and 224 white college students in the entering class of two northern colleges (University of Pittsburgh or University of Massachusetts) or two southern colleges (Augusta or Paine College). All subjects were weighed and completed the Revised Restraint Scale and the EAT-26. AA women were heavier than white women, with no differences due to geographical location. Despite being thinner, white women reported more dietary restraint than AA women. This difference between AA and white women was apparent in both northern and southern college students. In contrast, geographical location was the strongest determinant of bulimic attitudes; both men and women at northern colleges reported higher bulimia scores than those at southern schools. Thus ethnicity appears to be a major determinant of body weight and attitudes about shape and dieting, whereas geographical location appears to exert greater influence on bulimic attitudes.     

BLUE-GREEN ALGAL MATS IN A SMALL STREAM1

Bedrock erosional features in a small stream (Little Schultz Creek, Bibb County, Alabama) created a variety of habitats for epilithic growth. One suck habitat was illustrated by the occurrence of small falls (<0.3 m) in the main channel of the stream and blue-green algal mats associated with them. The cohesive, laminar algal mats were found at 15 such sites along a 250-m reach of the stream. The primary mat matrix consisted of the blue-green alga Oscillatoria submembranacea Ardissone and Strafforella. The uppermost portion of each mat consisted of a thin (<1 mm thick) green layer of biologically active filaments. The lower layers were thicker (up to 2 cm thick) and consisted of brown laminae of Oscillatoria filaments, and associated sediments. In addition, numerous diatoms mere associated with the mat surface. Some were loosely attached (e.g. Achnanthes); others (Cymbella tumida (Bréb.) V. H.) were stalked. These mats were present throughout the year and showed a bimodal annual distribution with maxima hi February and July. In February, total mat coverage was higher than in July. This winter maximum may have been related to a mode of growth dependent upon sedimentation from storm events and subsequent upward growth of the alga. Mat primary productivity on an areal basis (432 mg C · m^?2· d^?1 in March and 907 mg C · M^?2· d^?1 in April) was 2–12 times the maxima measured on epizoic and cobbles surfaces and other bedrock surfaces in the same stream. The limited areal coverage of the mats, when compared to other surfaces available for algal colonization, made them less important than other epilithic and epizoic surfaces in terms of total primary production in this stream reach. However, we propose that the combination of their unique structure and high primary productivity may make these algal mats sites of high algal and bacterial metabolic activity, which may include anaerobic processes in midchannel, where such activity would not be expected to occur.     

Adrenocortical suppression in workers manufacturing synthetic glucocorticoids.

A man who had worked for 16 years in the manufacture of a potent corticosteroid was found to be suffering from chronic adrenocortical insufficiency attributed to chronic absorption of the glucocorticoid. Eleven other symptom-free workers were therefore screened. Two of these workers, like the first patient, gave grossly abnormal responses to the Synacthen (tetracosactrin) test; one had been employed for only seven months. All 12 men had facial plethora, suggesting absorption of the drug in spite of their having adhered to the safety precautions. All workers manufacturing potent steroids should therfore be screened regularly by measurement of their plasma cortisol concentrations and should be moved regularly to processing other drugs.