Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

Summary Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth. This work was supported in part by the National Institutes of Health, Colorado and American Lung Associations, Colorado and American Heart Associations, the Council for Tobacco Research, and the Kroc, Hill, Swan and Kleberg Foundations. Dr. Bowman is a Clinician Scientist Awardee of the American Heart Association.     

Genetic structure of natural populations of the red-spotted newt,Notophthalmus viridescens

Genetic variation is described at 15 loci in 2 neotenic and 12 nonneotenic populations of red-spotted newts. Though high levels of genetic similarity (I=0.990) were found among all populations, allele frequencies at six of the eight most polymorphic loci show significant heterogeneity across populations. Change in allele frequencies at two of these loci (Pep-2 and Ldh-1) is significantly correlated with latitude. Interspecific homologies are established for newt peptidases based on substrate specificities and lactate dehydrogenases based on tissue distribution, thermal stability, and kinetic properties. Nonneotenic populations are highly variable (H=0.157) and neotenic populations are only slightly, but significantly, less variable (H=0.120). The high levels of heterozygosity detected in nonneotenic populations may result from large effective population size and/or environmental heterogeneity. The unexpectedly high heterozygosity values obtained for the neotenic populations may indicate adult dispersal or the presence of some previously undetected red efts at these localities. In any case, a major change in life history has apparently had little effect on the genetic structure of these populations.This research was supported by grants from the Blakeslee Fund of Smith College.     

Life history variation within a parthenogenetic population of Daphnia parvula (Crustacea: Cladocera)

Summary Evidence for genetically determined life history variability within a population or a species is rare. In this three year experimental examination of a parthenogenetically reproducing population of the planktonic crustacean Daphnia parvula, we found evidence for a succession of clones or groups of clones that exhibited distinctive body size and reproductive differences that were maintained after numerous generations under standardized conditions in the laboratory. The D. parvula population reached maximum density in the fall and maintained relatively high densities through the winter and spring. Isolates from this fall-winter-spring period all had a larger body size at death and higher fecundity when compared with summer isolates under natural food and temperature conditions. These differences could not be accounted for by differences in temperature and food abundance among the seasons. An additional difference in these experiments was a shift in reproductive effort by the summer isolate which produced a higher proportion of its offspring in the first two broods. The shift in life history characteristics and a summer decline of the Daphnia parvula population was correlated with both an increase in inedible and perhaps toxic blue-green algae and an increase in a dipteran predator Chaoborus. Comparison of the survivorship curves for all of the seasonal life history experiments indicated that D. parvula survivorship was not lower during the summer discounting a toxic effect from blue-green algae. Positive population growth on natural food in the laboratory at this time indicated food was not limiting and that predation was the probable cause of the population decline.Laboratory life history experiments under standardized food and temperature conditions were run with D. parvula isolates from the spring and summer plankton. Genetically based differences as determined in these experiments were smaller body size, lower fecundity, smaller brood size, and shorter life span for the summer animals relative to spring animals. Thirty seven percent of the summer animals also reproduced at an earlier age under standardized conditions. The shift in reproductive effort to earlier broods by summer animals rnder natural conditions appeared to be a phenotypic response as the summer isolate did not produce a higher proportion of its offspring in early broods under standardized conditions.When estimates of predatory mortality were added to the life tables of the standardized experiments, the earlier reproduction of some of the summer animals allowed a population increase under a regime of intense predation. Life tables for the spring animals predicted a population decline under these circumstances. Predictable seasonal changes in biotic factors such as predation suggest a mechanism whereby diverse life history patterns with corresponding differences in r may be maintained within a population.     

Effect of Lipid Structure on the Capacity of Myelin Basic Protein to Alter Vesicle Properties: Potent Effects of Aliphatic Aldehydes in Promoting Basic Protein-Induced Vesicle Aggregation

The capacity of myelin basic protein or of poly-L-lysine to promote leakage of carboxyfluorescein from vesicles or the aggregation of vesicles was studied. The vesicles were composed of phosphatidylcholine as the sole or major lipid component. Addition of 10% sphingomyelin, 10% phosphatidylglycerol, 10% egg or bovine brain phosphatidylethanolamine, or 30% dodecanal had relatively little effect on the extent of carboxyfluorescein release in the presence of either myelin basic protein or poly-L-lysine. In contrast with these results, the extent of vesicle aggregation was very sensitive to lipid composition. Addition of 10% phosphatidylglycerol induced more aggregation than the other phospholipids tested. Admixing 10% of a partially degraded sample of bovine brain phosphatidylethanolamine also led to a large amount of aggregation induced by the myelin basic protein. This latter aggregation appeared more specific for the basic protein, as it occurred to a much smaller extent with poly-L-lysine. In general, the effects of the myelin basic protein on either carboxyfluorescein release or vesicle aggregation were similar to, although somewhat greater than, that of poly-L-lysine. The aggregation of vesicles containing degradation products of phosphatidylethanolamine can be ascribed largely to the presence of aliphatic aldehydes. The effect of aliphatic aldehydes was specific in that the aliphatic alcohol, hexadecanol, or the short-chain aldehydes, acetaldehyde or butyraldehyde, did not promote myelin basic protein-induced vesicle aggregation. In addition, poly-L-lysine was less effective than the basic protein in aggregating vesicles containing aliphatic aldehydes. (ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of Homovanillic Acid and 5-Hydroxyindoleacetic Acid in Cerebrospinal Fluid from Human Infants in the Perinatal Period

To assess maturation of central serotonin and catecholamine pathways at birth, we measured lumbar CSF homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), stable acid metabolites of dopamine and serotonin, using HPLC with electrochemical detection. CSFs from 57 neonates (38 premature and 19 at term) and 13 infants 1-6 months old were studied. HVA levels increased with maturity (p less than 0.05; ANOVA), whereas 5-HIAA levels were similar in all these subjects. HVA/5-HIAA ratios increased markedly from 1 +/- 0.12 in the most premature neonates to 1.98 +/- 0.17 in the older infants (p less than 0.01; t test). There were no sex differences for these values.     

Metastatic phenotype of human prostate tumor cells in athymic nude mice: Alteration by exposure to ethyl methanesulfonate and “reversion” by 5-azacytidine

Summary The human prostate tumor subline 1-LN-PC-3-1A (1-LN) is reproducibly metastatic in adult athymic nude mice. Cells surviving a brief in vitro exposure to ethyl methanesulfonate (EMS) exhibited a profound decrease in capacity for experimental lung metastasis in nude mice. Thirty days after EMS treatment, 1×10⁶ uncloned EMS-treated 1-LN cells (1-LN-EMS-10) were injected IV into groups of 6 to 8-week-old male athymic nude mice (BALB/cAnBOM). A median of 8.5 colonies/lung was observed among 20 1-LN-EMS-10-injected mice, which was significantly different from the median of 51 colonies/lung produced among 14 1-LN-injected mice (P=0.0002). This altered phenotype remained stable during 150 days of continuous culture. However, the 1-LN-EMS-10 cells were tumorigenic in 10/10 nude mice injected SC. Single lung tumor colonies recovered from 1-LN-EMS-10-injected mice and reinjected IV into nude mice produced medians of 32–63 colonies/lung. The altered metastatic phenotype resulting from treatment of 1-LN with EMS was reversed by exposure to a noncytotoxic dose of 5-azacytidine, but unaffected by a second exposure to EMS. Collectively these data demonstrate that the metastatic phenotype of these human tumor cells in athymic nude mice can be heritably altered by in vitro exposure to EMS and 5-azacytidine. Analysis of the mechanisms underlying these phenotypic changes may provide insight into parts of the complex process of tumor cell evolution.     

Effect of methylation of histidine-48 on some enzymatic and pharmacological activities of snake venom phospholipases A2

The effects on some pharmacological and enzymatic properties were determined following methylation of histidine at the enzymatic active site of the basic relatively toxic $\text{Naja}$$\text{nigricollis}$ and the acidic relatively non-toxic $\text{Naja}$$\text{naja}$$\text{atra}$ phospholipases A₂. Following methylation a very low residual enzymatic activity (0.4 -- 1% of control) was accompanied by a parallel loss in intraventricular lethality, anticoagulant potency, direct hemolytic action and ability to block directly and indirectly evoked contractions of the mouse phrenic nerve-diaphragm preparation. Since methylation does not impair the enzyme's ability to bind monomeric of micellar substrates or Ca²⁺, the results suggest that the pharmacologicallly active region of the molecule is different from the micellular substrate binding site but strongly influenced by the invariant histidine-48 located at the enzymatic active site.     

Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex

Recessive lethal mutations in the T/t-complex of the mouse characteristically show defective genetic complementation, even when they affect very different stages of embryogenesis and are known to be nonallelic. To address the question of their genetic or functional relationship, we have applied the cis-trans test, using several well defined recombinant t-chromosomes that carry two or more lethal mutations, and others that are devoid of specific lethals. We show here that the defective complementation that occurs between different t-lethals is a specific result of the trans configuration; thus these genes, which may map as much as 15 cM apart, constitute a functional unit. Some speculations are presented to interpret this enigma in terms of DNA plasticity.     

The chimpanzee M blood-group antigen is a variant of the human M-N glycoproteins

Chimpanzee erythrocytes express strong M but weak, occasional N blood-group activity, as detected by anti-M and anti-N reagents. We have found that the M activity is carried by a major membrane glycoprotein that is similar but not identical to the human MM glycoprotein (glycophorin A). We have isolated and characterized this glycoprotein from erythrocyte membranes of four individual chimpanzees. The purified glycoproteins strongly inhibited agglutination of M cells by rabbit anti-human M sera and only weakly inhibited the agglutination of N cells by rabbit anti-human N sera. They also displayed medium-to-strong inhibitory activity against chimpanzee iso- and crossimmune antisera tested with chimpanzee erythrocytes of various V-A-B-D and W^c specificities, which are known as chimpanzee extensions of the human type M-N system and the Miltenberger counterpart, respectively. Each glycoprotein was cleaved with CNBr into three fragments, whose size, solubility, and composition were analogous to those obtained by similar treatment of the human M-N antigens. The amino-terminal fragment was found to be a glycooctapeptide whose amino acid composition and partial sequence indicated that it is an intermediate form of the human M and N glycooctapeptides. Its carbohydrate content comprised two threonine-linked saccharide units that, although similar in composition to the human threonine-linked units, were fewer in number than the three units found in the corresponding human glycooctapeptides. Structural similarities to the human antigens strongly suggest that the amino terminus bears the major antigenic determinants of the molecule, and the occurrence in this region of numerous, albeit rare, variants among humans and in chimpanzees indicates that the corresponding coding sequence of the structural gene is particularly susceptible to mutational events. We conclude that the chimpanzee M gene product is a variant of the human type and that the chimpanzee gene is an allele of the human polymorphic M-N locus.This research was supported by National Institutes of Health Grants GM 16389 and HL 19011 and March of Dimes Grant 1-661.