GENETIC RELATEDNESS OF TWO POPULATIONS OF THE SOUTHERN ELEPHANT SEAL, MIROUNGA LEONINA

Blood samples from southern elephant seals ( Mirounga leonina ) from Heard and Macquarie Islands were surveyed electrophoretically for protein variation. Thirty proteins encoded by a minimum of 35 loci were screened, four of which were found to be polymorphic. Statistically significant differences in allele frequencies were found between the two populations at three loci. Heterozygosity estimates for the Heard and Macquarie island populations were 0.034 ± 0.020 (mean ± standard error) and O.029 ± 0.017 respectively, with a Nei distance of 0.007. The findings suggest that the two populations may have diverged genetically and very limited gene flow exists between the islands, a finding consistent with limited information from mark-recapture studies.     

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.     

Heat treatment of ripening apples: Differential effects on physiology and biochemistry

Apples ( Malus domestica Borkh.) were heated for 4 days at 38°C immediately after harvest and then placed at 20°C for 7–10 days. Protein synthesis, ethylene production and fruit softening were reversibly inhibited by the heat treatment. Fruit respiration, membrane permeability and chlorophyll degradation in the fruit peel were enhanced during the treatment. The heat-treated apples ripened normally but more slowly than untreated apple We hypothesize that heat treatment differentially affects processes which normally increase simultaneously during fruit ripening, by inhibiting those processes which require tie novo protein synthesis and enhancing those that do not.     

Selection and characterization of sethoxydim- tolerant maize tissue cultures

`Black Mexican Sweet' (BMS) maize (Zea mays L.) tissue cultures were selected for tolerance to sethoxydim. Sethoxydim, a cyclohexanedione, and haloxyfop, an aryloxyphenoxypropionate, exert herbicidal activity on most monocots including maize by inhibiting acetyl-coenzyme A carboxylase (ACCase). Selected line B10S grew on medium containing 10 micromolar sethoxydim. Lines B50S and B100S were subsequent selections from B10S that grew on medium containing 50 and 100 micromolar sethoxydim, respectively. Growth rates of BMS, B10S, B50S, and B100S were similar in the absence of herbicide. Herbicide concentrations reducing growth by 50% were 0.6, 4.5, 35, and 26 micromolar sethoxydim and 0.06, 0.5, 5.4, and 1.8 micromolar haloxyfop for BMS, B10S, B50S, and B100S, respectively. Sethoxydim and haloxyfop concentrations that inhibited ACCase by 50% were similar for BMS, B10S, B50S, and B100S. However, ACCase activities were 6.01, 10.7, 16.1, and 11.4 nmol HCO₃⁻ incorporated per milligram of protein per minute in extracts of BMS, B10S, B50S, and B100S, respectively, suggesting that increased wild-type ACCase activity conferred herbicide tolerance. Incorporation of [¹⁴C]acetate into the nonpolar lipid fraction was higher for B50S than for BMS in the absence of sethoxydim providing further evidence for an increase in ACCase activity in the selected line. In the presence of 5 micromolar sethoxydim, [¹⁴C]acetate incorporation by B50S was similar to that for untreated BMS. The levels of a biotin-containing polypeptide (about 220,000 molecular weight), presumably the ACCase subunit, were increased in the tissue cultures that exhibited elevated ACCase activity indicating overproduction of the ACCase enzyme.     

Dilator actions of endothelin in coronary resistance vessels and the abdominal aorta of the guinea pig

Endothelin has been characterized as a potent constricting factor. The purpose of this study was to investigate possible dilator effects of this peptide and to examine whether dilator responses occur through an endothelium-mediated mechanism in guinea pig coronary resistance vessels and isolated aortic rings. Changes in perfusion pressure after bolus injections of endothelin were measured using a constant-flow modified Langendorff preparation with a transducer between the flow pump and the heart. An immediate fall in perfusion pressure, averaging 6 mmHg, was observed after injection of endothelin (10(-14)-10(-12) moles). This effect was maximal at 1 minute and tended to return toward baseline levels within 4 minutes. In response to endothelin (10(-9) M), isolated aortic rings relaxed 35% after being contracted with prostaglandin F2 alpha (10(-7) M). In both preparations, dilation was converted to constriction after endothelium damage by oxygen radicals or endothelium removal (mechanical rubbing). Dilator responses to endothelin were blocked by pretreatment for 30 minutes with indomethacin (14 microM) in the presence of an intact endothelium in coronary resistance vessels, whereas in the abdominal aorta they were not. We conclude that endothelin has significant dilator properties and that this effect is opposed by its constrictor action at higher doses. In addition, dilator responses to endothelin require an intact endothelium in both coronary vessels and abdominal aorta. Finally, endothelin-induced dilation in coronary resistance vessels appears to occur through a cyclooxygenase product-mediated mechanism.     

Purification and partial characterization of recombinant human differentiation-stimulating factor

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to greater than 95% purity from Chinese hamster ovary cells. RhD-factor is a glycoprotein with an apparent molecular weight of 45.6 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration in 6 M guanidine-hydrochloride, rhD-factor elutes with an apparent molecular weight of 21.5 kDa; it elutes with an apparent molecular weight of 44.8 kDa under neutral pH (native) conditions. The amino-terminal sequence (12 residues) is consistent with the expected sequence derived from the genomic DNA sequence. Recombinant D-factor is heavily glycosylated with 30% by weight neutral sugar and 12% sialic acid. The ED50 for rhD-factor was 0.25 ng/ml. Trifluoromethanesulfonic acid-deglycosylated rhD-factor has a biological activity comparable to that of the native recombinant protein (ED50 = 0.40 ng/ml). The biological activity of rhD-factor was stable at pH 1 for 40 h, in 6 M guanidine-HCl containing buffers with or without reducing agent, and in 1% SDS. Carboxymethylation of D-factor after reduction totally destroyed biological activity.     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Adrenergic and cholinergic interaction in central ventilatory control

The ventrolateral medulla, which functions as integrator of cardiorespiratory control, contains cholinergic and adrenergic neurons. Exogenously administered cholinergic and adrenergic agents affect both ventilation and circulation. It is not clear whether these agents act in an independent or coordinate manner. beta-Adrenergic and alpha 2-adrenergic agents stimulate and depress the cardiorespiratory system, respectively. beta-Adrenergic and alpha 2-adrenergic agents stimulate and depress the production of adenosine 3',5'-cyclic monophosphate (cAMP), respectively. Increased intracellular cAMP may facilitate the release of acetylcholine (ACh). This work seeks to answer the following questions: 1) Are the cardiorespiratory effects of adrenergic agents secondary to possible changes in ACh release? 2) Does cAMP production have an intermediate role? By means of ventriculocisternal perfusion in anesthetized (pentobarbital sodium, 30 mg/kg) spontaneously breathing dogs, isoproterenol (ISO) increased ventilation (VE) 75% (P less than 0.05); heart rate and cardiac output were also increased (P less than 0.05). Esmolol (a beta-antagonist) blocked both the cardiovascular and ventilatory effects of ISO. Atropine (a cholinergic antagonist) blocked the ventilatory effects of ISO, but the circulatory changes persisted. Forskolin (a direct activator of adenylate cyclase) increased VE 51% (P less than 0.05), and its effect was also blocked by atropine. Clonidine decreased VE 42% (P less than 0.05); heart rate and cardiac output were also decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular tools in marine ecology

Molecular tools have diverse applications in marine ecology. In microbial systems, DNA sequences of rRNA and other genes have identified a variety of novel lineages of bacteria inhabiting marine environments that have resisted traditional culture methods. However, relatively few natural populations have been characterized due to the rather labor-intensive methodologies employed. Recent technological developments such as in situ PCR and flow cytometry promise to greatly enhance the speed at which microbial taxa can be identified and enumerated in field collected water and substrate samples; such advances will allow future work to employ the spatial and temporal field sampling required to monitor the impact of natural and anthropogenic changes in the environment. This approach also holds promise for examining physiological status of field collected cells, garnering information on such elusive parameters as growth rates and the extent of nutrient limitation under natural conditions. Studies of macrobiota have similarly benefited from the use of molecular approaches to species identification. This has been particularly true with regard to distinguishing among larval forms of closely related taxa which are nearly identical morphologically. Genetic variation within species assayed by molecular tools has been useful in examining the stability of populations through time and in assessing patterns of recruitment to geographically separated populations. Enhanced understanding of these ecological problems will also require intensive spatial and temporal monitoring of both larval and adult populations. Often, the newer techniques based on DNA sequence variation have practical advantages over allozyme techniques: e.g., PCR allows assay of minute quantities of DNA that may come from ethanol preserved samples. However, when ample allozyme variation exists to address a given issue, these older techniques may be favored on a variety of criteria, including speed and cost. Hence, choice of methodology should be based on the expected efficiency of a given approach to a specific problem rather than the apparent sophistication of the method itself.     

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups. Present address: Department of Pathology, Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112 Correspondence to: M.A. Batzer