Apparent independence of the spawning and moulting cycles in female Antarctic krill (Euphausia superba Dana)

Summary Sixty female Antarctic krill (Euphausia superba Dana) spawned in shipboard experiments and the interval between egg-laying and ecdysis was noted. The number of eggs laid per female ranged from 263–3662, most females produced only one batch of eggs before moulting, and the post-spawn ovaries of all females contained few, if any, mature oocytes. As reported in other studies, the total number of eggs produced per female was not well correlated with body size. Females appeared to spawn at all times during the moulting cycle and although no diurnal rhythm in spawning was observed, moulting occurred mainly at night-time despite the animals being kept in near-constant darkness. No evidence of synchronous moutling was detected.     

Potentiation of interleukin-2 production and its binding by monoclonal antibodies to the gangliosides GD3 and GD2

Summary Previous studies have shown that monoclonal antibodies (mAbs) against certain gangliosides, which induced remissions in patients with melanoma, also potentiated the response of lymphocytes to a variety of stimuli, including lectins, interleukin-2 (IL-2) and antigens. The present studies have investigated the mechanism of these effects on lymphocytes. Although the mAbs potentiated phytohemagglutinin (PHA)-induced IL-2 production at high concentrations of mAbs and of PHA, this did not appear to explain their potentiation of the proliferative responses of lymphocytes. Hence, although IL-2 production was minimal or absent from the CD8⁺ subset the latter showed the highest degree of augmentation. Furthermore, addition of IL-2 to PHA-stimulated cultures did not produce similar augmentation of mitogenic responses to that produced by the mAb to GD3 or GD2. The augmented and normal mitogenic responses were, however, dependent on IL-2, as shown by their inhibition with mAbs against IL-2. The antiganglioside mAbs did not have significant effects on IL-2 receptor expression measured by mAbs to Tac. However, the mAbs appeared to increase the affinity of binding of radiolabelled IL-2 to IL-2 receptor and increased internalization of the latter. These results suggest that the effects of the mAbs on IL-2 production may be distinguished from their effects on the proliferative responses of T cells and that the latter were associated with changes in affinity and internalization of ¹²⁵I-IL-2. Whether the latter is a direct cause of the increased proliferative response remains unknown. The ability of mAbs to GD2 and GD3 to increase IL-2 production and to enhance IL-2-dependent proliferative responses suggests the may have valuable clinical roles as immunopotentiating agents.     

Carbon metabolism in Bradyrhizobium japonicum bacteroids

Abstract Carbon metabolism in Bradyrhizobium japonicum bacteroids is reviewed. Additionally, the bacteroid tricarboxylic acid (TCA) cycle and its regulation under oxygen-limited conditions is considered, with emphasis on possible sites of TCA cycle rate-limiting reactions. Furthermore, we consider other adaptive pathways that may be employed by these organisms while in symbiosis. These pathways include: (1) a poly-β-hydroxy-butyrate shunt, (2) a malate-aspartate shuttle, (3) an α-ketoglutarate-glutamate shunt, (4) a modified dicarboxylic acid cycle, and (5) fermentation pathways leading to lactate, acetaldehyde and ethanol. The effects of oxygen limitation on host carbon metabolism are also considered briefly.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Two-step purification of full-length nerve growth factor receptor and maintenance of receptor-specific binding activity

A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification.     

An endochitinase gene expressed at high levels in the stylar transmitting tissue of tomatoes

A gene (pMON9617; Chi2;1) identified by screening a tomato pistil cDNA library has been found to encode a protein similar in sequence to class II chitinases. Using a baculovirus expression system we show that the Chi2;1 protein is an active endochitinase. The Chi2;1 protein is most similar in sequence to a previously described stylar chitinase (SK2) isolated from potato. Both proteins lack the diagnostic N-terminal cysteine-rich regions and the C-terminal vacuolar targeting signals of class I chitinases and appear to define a novel type of class II endochitinases associated with flowers. Chi2;1 is expressed predominantly in floral organs and its expression within these organs is temporally regulated. The highest level of expression is found in the transmitting tissue of the style where Chi2;1 mRNA begins to accumulate just prior to anthesis. In vegetative tissue, low levels of Chi2;1 mRNA were detected, and these levels did not increase in response to wounding or treatment with ethephon. mRNA from Chi2;1 orthologs was not detected in most other angiosperms tested, even including some members of the Solanaceae, and it is therefore unlikely that Chi2;1 is essential for stylar function. A fragment containing 1.4 kilobase pairs of 5-flanking DNA from the Chi2;1 gene was shown to drive high-level expression of an attached reporter gene in the styles of transgenic tomatoes. This fragment has utility for engineering expression of other coding regions in styles.