Changes in calmodulin and its mRNA accompany reentry of quiescent (G0) cells into the cell cycle

Release of CHO-K1 cells from plateau or stationary phase and reentry into the cell cycle is specifically and reversibly blocked at two distinct sites by the anticalmodulin drug W13. The first block occurs early during release while the cells are still at G0/G1, whereas the second occurs later in reentry during early S phase. As determined by radioimmunoassay, calmodulin levels undergo changes at three distinct steps in plateau-phase entry and release. First, the entry of exponentially growing cells into plateau phase is accompanied by an increase in the calmodulin level. The second change is a reduction in the calmodulin content of cells within the first hour following release from plateau phase. The third change is the subsequent increase in calmodulin levels, which precedes entry of the cells into S phase. Analysis of calmodulin mRNA levels by dot-blot hybridization demonstrates that the changes in calmodulin protein are preceded by changes in calmodulin mRNA. Furthermore, whereas a decrease in CaM mRNA is observed within the first hour following plateau release, no such decrease is observed for beta-actin mRNA, suggesting that this decrease may be selective for calmodulin. This selectivity is further substantiated by the fact that identical changes in calmodulin and calmodulin mRNA are observed in cells released from plateau by two different techniques. Taken together, these data suggest that calmodulin may play an important role in the reentry of cells into the cell cycle.     

The pharmacological actions of 5-hydroxytryptamine,FMRF-amide and substance P and their possible occurrence in the heart of the snail Helix aspersa L.

The actions of 5-hydroxytryptamine (5-HT), FMRF-amide and substance P were tested on the isolated heart of Helix aspersa. All three compounds were found to produce positive inotropic and chronotropic effects, the order of potency being 5-HT > substance P > FMRF-amide. However, whereas the responses to 5-HT and FMRF-amide were maximal within a few seconds, the response to substance P had a longer latency. Two other similar undecapeptides, eledoisin and physalaemin, were also tested and were found to have very similar actions to those of substance P. The effects of 5-HT and FMRF-amide could be separated using the 5-HT blockers methysergide and ketanserin, which had relatively little effect on the response to FMRF-amide.Immunohistochemical staining was carried out on sections through the Helix auricle and ventricle for 5-HT, FMRF-amide and substance P. Substantial 5-HT-like and substance P-like immunoreactivity was observed, apparently concentrated in nerve endings, but the level of FMRF-amide-like immunoreactivity was considerably lower. The fluorescence produced by all three compounds was evenly distributed throughout the heart tissue. HPLC analysis of heart tissue extract demonstrated a high level of 5-HT (about 8 μg/g wet weight) but a negligible catecholamine content.     

Skeletal changes associated with vascular malformations

Five hundred and eighty birthmarks were reviewed; 356 were hemangiomas and 224 were malformations. Bony alterations occurred in association with only 1 percent of hemangiomas, in contrast with 34 percent of patients with vascular malformations. These alterations in bone development were classified according to size, shape, and density changes. Hypertrophy and distortion were typical of lymphatic malformations. Hypoplasia and demineralization were characteristic findings in the extremity venous malformations. Destructive and intraosseous changes were more commonly noted in the arterial or high-flow lesions. Possible mechanisms of altered skeletal growth include mechanical, physiological, and developmental processes.     

Field and laboratory observations of spawning periodicity and behavior of a northern population of the Atlantic silverside,Menidia menidia (Pisces: Atherinidae)

Synopsis The spawning periodicity and mating behavior of a northern population of the Atlantic silverside, Menidia menidia, was studied both in the field (Salem Harbor, Massachusetts) and in laboratory experiments. Spawning in the field coincided with new and full moons. Under conditions of unlimited food availability in artificial outdoor pools that received natural celestial illumination or in indoor aquaria receiving no evening illumination, spawning occurred every 1–3 days rather than fortnightly. These results suggest that tidal influences are a primary factor that synchronizes spawning in M. menidia. Published reports of diel time of spawning in both marine and freshwater species of Menidia indicate a generic tendency to spawn during mid-morning. This pattern coupled with high tides that cover suitable spawning substrates during mid-morning only at fortnightly intervals could account for the semilunar spawning cycle in marine populations of Menidia. Although general characteristics of the reproductive ecology of M. menidia in Massachusetts were similar to a thoroughly studied population in South Carolina, several differences were evident. The breeding season was shorter in Massachusetts (late; April–June), and occurred over a much lower range of temperatures (9–21°C). Intensity and frequency of spawning was correlated with the height of high tide. Eggs were deposited only on mats of intertidal, filamentous algae rather than on roots or stems of Spartina alterniflora or other intertidal vegetation. Promiscuous spawning occurred in small, highly male-dominated groups of fish, primarily after the tide had begun to recede, and often at the water's edge. No predation on spawning adults was observed but the mummichog, Fundulus heteroclitus, fed daily on developing embryos during high tide. Contribution No. 81, Massachusetts Cooperative Fisheries Research Unit, Contribution No. 349, Marine Sciences Research Center, State University of New York at Stony Brook.     

Measurement of serum gentamycin levels by rapid tests

Gentamicin is a very useful antimicrobial agent for the treatment of serious infections caused by gram-negative bacteria. However, it's low therapeutic index and potential ototoxic and nephrotoxic side effects necessitate frequent determinations of serum concentration to assist in maintaining therapeutic levels and avoiding toxic levels. Two bioassays and a latex agglutination inhibition card (LAIC) test were evaluated to determine gentamicin levels in nearly 100 patient sera. Results were compared with a radioimmunoassay (RIA). Two bioassays, the Bio-Monitor and the GentaSak, gave correlation coefficients of 0.987 and 0.982, respectively. The correlation coefficient for the LAIC test was 0.987. All three tests compared well with RIA in accurately detecting gentamicin levels in patient as well as simulated sera. The LAIC test, however, was more rapid, giving results within half an hour whereas bioassays required 6–8 hours for completion. The LAIC test was also found to be more economical. It provides a suitable alternative to RIA procedures in small laboratories and for performing stat tests since batching is not necessary.     

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.     

Effect of vasotocin on locomotor activity in bullfrogs varies with developmental stage and sex

Although neurohypophysial peptides are present in many regions of the developing and adult bullfrog (Rana catesbeiana) brain, the function of these peptides remains unclear. To investigate possible behavioral actions, we examined locomotor activity following peptide injection in bullfrogs at various developmental stages. An intraperitoneal (ip) injection of arginine vasotocin (AVT) in tadpoles (stages V, X, or XVII) produced an immediate and dose-dependent inhibition of locomotor activity. On the other hand, AVT stimulated activity when administered ip to juvenile or adult female bullfrogs, but did not influence activity in juvenile or adult males. The minimum effective dose of AVT, when injected directly into the brain of tadpoles, was 100-fold less than that observed when injected ip, suggesting a central nervous system site of action for this peptide. A vasopressin receptor antagonist (d(CH2)5[Tyr(Me)2]AVP administered ip or icv) significantly increased locomotor activity in tadpoles, compared to controls. Oxytocin, vasopressin, and AVP4-9 inhibited activity in tadpoles while mesotocin, des Gly(NH2)AVP, and pressinoic acid had no significant effect. Injection of PGF2 alpha also significantly decreased activity levels in tadpoles. However, pretreatment of tadpoles with indomethacin, a prostaglandin synthesis inhibitor, did not prevent the behavioral effects of AVT, suggesting that prostaglandin synthesis is not required for this response. In summary, AVT influenced locomotor activity in bullfrog tadpoles and female frogs. This effect shifted during development from an inhibitory action in tadpoles to a stimulatory effect in metamorphosed female frogs. The effect of AVT on juvenile and adult frog locomotion was sexually dimorphic, as this peptide altered female behavior but not male behavior.