Subunit interactions of class I histocompatibility antigens

The kinetics of dissociation of iodinated beta 2-microglobulin (beta 2m) from the papain-solubilized class I histocompatibility antigen HLA-B7 have been investigated. In the presence of unlabeled beta 2m, most of the HLA dissociates according to a single rate constant, whereas in the absence of unlabeled beta 2m, the system approaches an equilibrium dependent upon the initial HLA concentration. When iodinated beta 2m is incubated with unlabeled HLA-B7, the rate of incorporation of beta 2m into the complex is much less dependent on the concentration than is expected for a simple association/dissociation system; instead, the system behaves as if the "activity" (in a thermodynamic sense) of the HLA heavy-chain intermediate cannot surpass a critical concentration. The dissociation rate for each class I specificity is a function of temperature, ionic strength, pH, and the status of the heavy chain (papain solubilized vs. detergent solubilized). High temperature, high ionic strength, and extremes of pH promote dissociation. The intact molecule dissociates about 10 times more slowly than the papain-solubilized molecule. In contrast, the rate of dissociation of all papain-solubilized class I antigens tested falls within the range of about a factor of 2. The presence of the carbohydrate has no effect on the rate of dissociation. The possibility that HLA class I antigen dissociation may occur in vivo within acidic internal vesicles is discussed.     

Relative developmental success of interspecific Lepomis hybrids as an estimate of gene regulatory divergence between species

The developmental success of interspecific Lepomis hybrids is used as an index of gene regulatory divergence between the green sunfish, L. cyanellus, and each of three other parental species, longear sunfish, L. megalotis, warmouth, L. gulosus, and bluegill, L. macrochirus. This gene regulatory divergence is compared to the degree of structural gene divergence among these four species (genetic distance [Nei, '78], D, ranged from 0.206 to 0.586). The developmental success of the hybrid embryos at the level of morphogenesis was higher than expected from the genetic distance between the parental species. The rates of morphogenesis of the hybrid embryos were the same as that for the green sunfish embryos. The percentage of embryos that hatched was relatively high in all crosses. However, two of the hybrid crosses resulted in enhanced percentages of hatched embryos. Slight increases in the extent of morphological abnormalities were observed in hybrids from crosses between more distantly related parental species. The schedules and levels of enzyme locus expression of the hybrids, assessed spectrophotometrically and electrophoretically for nine enzyme systems (encoded in a total of 14 loci), were different from each other and from those of the green sunfish embryos. Alterations in the time of first enzyme appearance and in the time of first increase in enzyme activity in the developing hybrid embryos were not correlated with genetic distance between parental species. However, the extents of alteration of enzyme activities over the entire period of hybrid embryogenesis were correlated with the genetic distance. We attribute the morphological and molecular anomalies observed in the hybrids to gene regulatory incompatibilities between species. Although the exact number of mutational differences and their relative developmental impacts are not known, some inferences can be drawn about the degree of divergence in gene regulation between species. It appears that an uncoupling of the rates of structural and regulatory gene evolution can occur between species of some taxa, an observation that has implications for the roles of gene regulatory differences in organismic evolution.     

Quantity and quality of seston in an impounded and a free-flowing river in Virginia,U.S.A.

Seston in the impounded North Anna River (NAR) was analyzed and compared to that in the free-flowing South Anna River (SAR) in Virginia, U.S.A. A wet filtration technique was used to separate seston into five size classes. The overall quantity of organic seston was much lower in the NAR than in the SAR. The seston in the NAR was composed of more living organisms, in particular zooplankters in the medium large (234–864 µm) and small (105–234 µm) size classes and diatoms and other algae in the fine (43–105 µm) and very fine (25–43 µm) size classes. The percentage of zooplankton declined sharply before reaching the downstream study site (32 km). The seston in the NAR tended to consist of slightly larger particles, but 80–85% of the seston in both rivers was in the ultrafine (0.45–25 µm) size class, which was almost entirely composed of detritus. One indication of seston food quality, the organic/inorganic ratio, was considerably higher for the medium large and small size classes in the NAR immediately below the dam and for the fine and very fine size classes farther downstream. Another indication of food quality, the usable caloric content, showed that the seston in the NAR contained considerably less total assimilable energy as far as 32 km downstream from the dam, but that the medium large and small size classes were rich in assimilable energy immediately below the dam because of zooplankton released from the reservoir. Thus, the overall quantity and quality of seston was lower in the NAR, but the quality of the seston, for at least a short distance below the dam, was higher in a well-defined size range that can be effectively utilized by certain filter feeders.     

Genetic Variation for the Sex Ratio in NASONIA VITRIPENNIS

We detected genetic variation for the sex ratio in the parasitoid wasp Nasonia vitripennis by analysis of inbred lines and with an artificial selection experiment. Sex ratios differed significantly among five independently isolated lines. Furthermore, sex ratio in broods produced by single females in single hosts shifted from 80-90% female to 50-55% female in 13 to 15 generations in each of two replicate selection lines. The final sex ratios of both selection lines were significantly lower than any of the inbred line sex ratios. Backcrosses revealed that the selection response was due to nuclear genes acting through the female parent. In light of known facultative sex ratio behavior and major genes affecting sex ratio in Nasonia, our results suggest that population and individual sex ratios in this species are molded by processes at both genetic and behavioral levels.     

Genetical analysis of chromosome 5A of wheat and its influence on important agronomic characters

Summary Chromosome 5A of bread wheat, Triticum aestivum carries the major gene, Vrnl, which is one of the main determinants of the winter/spring growth habit polymorphism in this species. Genetical analysis of this chromosome has been carried out using single-chromosome recombinant lines to establish the pleiotropic effects of this locus and two other major genes, q determining ear morphology and bl determining the presence of awns, on important agronomic characters. The three major genes were located on the long arm of chromosome 5A with a gene order of: centromere -bl-q-Vrnl. Analysis of quantitative characters from a winter sowing revealed pleiotropic effects of Vrnl or the effects of closely linked loci on the characters plant height, tiller number and spikelet number. However effects on ear emergence time were not associated with Vrnl but with q as were effects on spikelet number and ear length. In addition a locus determining yield/plant was located between Vrnl and q. Independant loci determining height and ear length were apparent on the short arm of chromosome 5A. From a spring sowing, however, there was a large pleiotropic effect of Vrnl on ear emergence time, as well as the effects previously detected. In addition, associated with q were effects on plant height and grain size which were not expressed from the winter sowing.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

A transient inward current elicited by hyperpolarization during serotonin activation in Xenopus oocytes

Activation of serotonin, glutamate or muscarinic receptors, incorporated into the membrane of Xenopus oocytes following injection of messenger RNA from rat brain, caused the development of a transient inward (Tin) current when the membrane was hyperpolarized. A detailed study was made of the Tin current induced during serotonin activation. The current is due principally to efflux of chloride ions, and is presumably activated by an influx of calcium ions, because it was blocked by removal of calcium from the bathing medium, by addition of manganese, cobalt or lanthanum, or by intracellular injection of EGTA. During application of serotonin, the amplitude of the Tin current increased slowly, and after washing it persisted for longer than the direct serotonin-induced current. The amplitude of the Tin current was sensitive to temperature and pH, and was abolished at pH 6.5 or by cooling to 12 degrees C. The Tin current may be of importance in regulating the excitability of neurons in the central nervous system.     

Osmotic reflection coefficient for total plasma protein in lung microvessels

The osmotic reflection coefficient (sigma) for total plasma proteins was estimated in 11 isolated blood-perfused canine lungs. Sigma's were determined by first measuring the capillary filtration coefficient (Kf,C in ml X min-1 X 100g-1 X cmH2O-1) using increased hydrostatic pressures and time 0 extrapolation of the slope of the weight gain curve. Kf,C averaged 0.19 +/- 0.05 (mean +/- SD) for 14 separate determinations in the 11 lungs. Following a Kf,C determination, the isogravimetric capillary pressure (Pc,i) was determined and averaged 9.9 +/- 0.5 cmH2O for all controls reported in this study. Then the blood colloids in the perfusate were either diluted or concentrated. The lung either gained or lost weight, respectively, and an initial slope of the weight gain curve (delta W/delta t)0 was estimated. The change in plasma protein colloid osmotic pressure (delta IIP) was measured using a membrane osmometer. The measured delta IIP was related to the effective colloid osmotic pressure (delta IIM) by delta IIM = (delta W/delta t)0/Kf,C = sigma delta IIP. Using this relationship, sigma averaged 0.65 +/- 0.06, and the least-squares linear regression equation relating Pc,i and the measured IIP was Pc,i = -3.1 + 0.67 IIP. The mean estimate of sigma (0.65) for total plasma proteins is similar to that reported for dog lung using lymphatic protein flux analyses, although lower than estimates made in skeletal muscle using the present methods (approximately 0.95).     

Effects of prolonged elevated microvascular pressure on lung fluid balance in sheep

Experiments were conducted in seven chronically instrumented unanesthetized sheep to estimate the osmotic reflection coefficient (sigma d) for total proteins and the solvent-drag reflection coefficients (sigma f) for six endogenous protein fractions. We measured the lymph-to-plasma ratio of total proteins (CL/CP) and six protein fractions during base-line conditions and after left atrial pressure elevations of 24-26 h per elevation. We also monitored pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, and lung lymph flow at the various levels of pulmonary microvascular pressure. Our results indicate the CL/CP may require up to 24 h to reach a true steady state. It was found that sigma d is at least 0.89 for total proteins and sigma f is at least 0.84, 0.87, 0.86, 0.92, 0.95, and 0.96 for protein fractions with effective molecular radii of 36, 39.5, 44, 66, 105, and 123 A, respectively. In addition, the sigma f values for various protein fractions obtained from this investigation are compared with the predicted values of various mathematical models of the lung microcirculation.     

Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the adrenal medulla of the rat

Summary The pre- and postnatal development of the adrenal medulla was examined in the rat by immunohistochemistry and by assay of catecholamines. Immunohistochemistry involved the use of antibodies to noradrenaline (NA), adrenaline (A) and the biosynthesizing enzymes dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Adrenal glands were obtained from animals from the 16th day of gestation to the 7th postnatal day at daily intervals, and at the 14th postnatal day, and from adult rats. Tissues were fixed in ice-cold, 4% paraformaldehyde, buffered at pH 7.3. Cryostat sections (7 m) were stained with the indirect immunofluorescence technique. Adrenals from the same developmental stages were assayed for the presence of DA (dopamine), NA and A by ion-pair reversed-phase liquid chromatography with electrochemical detection.In adult adrenals the majority of the medullary cells (approximately 80%) were highly immunoreactive to A and moderately immunoreactive to NA. They also showed immunoreactivity to both DBH and PNMT, i.e., they are synthesizing and storing A. The remaining cell clusters were only stained by antibodies to DBH and NA (NA-synthesizing and -storing cells). These findings correlate well with the relative concentrations of A and NA as determined by assay.Three developmental phases could be distinguished. In the first phase, the 16th and 17th prenatal day, medullary cells were only immunoreactive to DBH and NA, and only very small amounts of A as compared to NA were found. During the second period, from the 18th prenatal day to 2 or 3 days after birth, all medullary cells were immunoreactive to DBH, NA, PNMT and A, and during this phase the adrenaline concentration increased daily and became the predominant amine on the 20th day of gestation. Adrenaline represented 75% of total catecholamine on the 1st to 3rd day after birth. The third phase started at the 2nd or 3rd postnatal day and was characterized by the presence of an increasing number of medullary cells solely immunoreactive to DBH and NA, hence synthesizing and storing NA. The remaining cells were immunoreactive to DBH, NA, PNMT and A. Postnatally, the relative concentration of A continued to rise reaching 79% by the 4th postnatal day. These results indicate that initially the adrenal medullary cells are synthesizing and storing almost exclusively NA. Probably, adrenaline synthesis begins at the 16th–17th day of gestation and the cells are then capable of synthesizing and storing both NA and A (mixed cell type) with A synthesis and storage rapidly becoming predominant. Finally, after birth, separate NA-synthesizing and -storing cell types are formed and the so-called A cells stored predominantly (probably >90%) adrenaline with a small proportion of noradrenaline.In the medullary blastema and in the sympathetic ganglia of prenatal animals two cell types, only immunoreactive to DBH and NA, were observed. Presumably, these cells represent developing sympathetic neurons and extra-adrenal chromaffin cells; the latter cell type occasionally invades the adrenal gland. Thus, prospective medullary cells are able to synthesize and store NA before they have made contact with the cortical blastema but A-synthesizing cells are found only within the adrenal gland.Low but significant amounts of DA were found in the adrenal before birth and during the first two postnatal weeks but in the adult animal this accounted for less than 0.1% of total catecholamine.Preliminary reports of this study were made to the American Association of Anatomists (Anat. Rec. 196; 196A, 1980), the Dutch Anatomical Society (Acta Morphol. Neerl. Scand. 19; 330, 1981, and the XIIIth Acta Endocrinologica Congress (Acta Endocrinol. 97: Suppl. 243, 285, 1981)