Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” seque?ce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.     

Target-specific nerve regeneration through a nerve guide in the rat

Nerve regeneration across a gap in peripheral nerve has been achieved through various nonneural nerve guides in both lower and primate species. This technique can only be useful if the regenerated nerve cable grows specifically to and reinnervates the appropriate distal target. In this study, the proximal peroneal fascicle of rat sciatic nerve was inserted into the proximal limb of a Y-shaped nerve guide. Distal peroneal and tibial fascicles were placed within the two distal limbs of the same Y. The proximal peroneal nerve grew preferentially by a 2:1 ratio to the appropriate distal peroneal fascicle suggesting that target-specific reinnervation is possible through a nerve guide.     

Seasonal and size-related changes in the food of the short-finned eel,Anguilla australis in Lake Ellesmere,Canterbury, New Zealand

Synopsis An approximately monthly sampling programme in Lake Ellesmere, Canterbury, New Zealand, from January 1974 to April 1976 yielded 487 eels. The stomachs were fixed in 10% neutralised formalin and the contents examined. Preliminary analysis indicated that the mollusc Potamopyrgus antipodarum, the isopod Austridotea annectens, the mysid Tenagomysis chiltoni, the amphipod Paracalliope fluviatilis, the midge larva Chironomus zealandicus and the teleosts Retropinna retropinna, Galaxias maculatus and Gobiomorphus cotidianus together made up the bulk of the diet. The pre-ingested dry weight (i.e. the reconstructed weight) of the most important of these prey species was obtained by relating the length of a digestion resistant part to actual dry weight in field collected specimens. Regression equations for this relationship in each season enabled the reconstructed dry weight of each stomach item to be calculated. In some instances reconstructed weight was less than the actual digested dry weight of the prey specimen. In every case the larger value was used. This method is referred to as Combination Dry Weight (CDW) and is believed to be new. These data, used in conjunction with the energy content of the species concerned, enabled the caloric dietary contribution of each prey species to be determined. Comparison of relative contribution to eel diet between CDW and energy values calculated from CDW and bomb calorimetry revealed large differences. Marked variations in diet between ⩽40 cm, 40.1–50 cm, and>50.1 cm size classes were also shown. Eels ≤40 cm feed primarily on invertebrates and become progressively more piscivorous as they grow. Eels >50.1 cm are almost entirely piscivorous. Seasonal differences in diet also exist within each size class examined.     

Enzymic saccharification of sugarcane bagasse pretreated by autohydrolysis-steam explosion

Pretreatment of bagasse by autohydrolysis at 200 degrees C for 4 min and explosive defibration resulted in the solubilization of 90% of the hemicellulose (a heteroxylan) and in the production of a pulp that was highly susceptible to hydrolysis by cellulases from Trichoderma reesei C-30 and QM 9414, and by a comercial preparation, Meicelase. Saccharification yields of 50% resulted after 24 h at 50 degrees C (pH 5.0) in enzymic digests containing 10% (w/v) bagasse pulps and 20 filter paper cellulase units (FPU). Saccharifications could be increased to more than 80% at 24 h by the addition of exogenous beta-glucosidase from Aspergillus niger. The crystallinity of cellulose in bagasse remained unchanged following autohydrolysis-explosion and did not appear to hinder the rate or extent of hydrolysis of cellulose. Autohydrolysis-exploded pulps extracted with alkali or ethanol to remove lignin resulted in lowere conversions of cellulose (28-36% after 25 h) than unextracted pulps. Alkali extracted pulps arising from autohydrolysis times of more than 10 min at 200 degrees C were less susceptible to enzymic hydrolysis than unextracted pulps and alkali-extracted pulps arising from short autohydrolysis times (e.g., 2 min at 200 degrees C). Autohydrolysis-explosion was as effective a pretreatment method as 0.25M NaOH (70 degrees C/2 h) both yielded pulps that resulted in high cellulose conversions with T. reesei cellulase preparations and Meicelase. Supplementation of T. reesei C-30 cellulose preparations with A. niger beta-glucosidases was effective in promoting the conversion of cellulose into glucose. A ration of FPU to beta-glucosidase of 1:1.25 was the minimum requirement to achieve more than 80% conversion of cellulose into glucose within 24 h. Other factors which influenced the extent of saccharification of autohydrolysis-exploded bagasse pulps were the enzyme-substrate ratio, the substrate concentration, and the saccharification mode.     

Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides

beta-Tubulin subunits isolated from chicken brain tissue and erythrocytes are distinguishable as unique biochemical species by electrophoretic and peptide mapping procedures. 1) The subunits of beta-tubulin exhibit major differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels that vary according to the pH and ionic strength of the gel. 2) The isoelectric points of urea-denatured beta subunits from brain tissue and erythrocytes are pH 5.1 and 5.4, respectively, whereas those of both alpha subunits are approximately pH 5.2.3) Two-dimensional peptide maps prepared with alpha-chymotrypsin or V8 protease show that alpha-tubulin peptides are indistinguishable, whereas beta-tubulin peptides are very different. Only one-third of the 15 major tyrosine-containing beta-tubulin peptides prepared with alpha-chymotrypsin are common to both beta-tubulin species. The data indicate that the beta-tubulin subunits of brain tissue and erythrocytes are biochemically distinct and may be different gene products. The presence of tubulin variants in brain tissue and erythrocytes may indicate special requirements for microtubule assembly and function in different cell types.     

Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria

We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two- dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min- 1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.     

Significance of the delta 5 and delta 4 steroidogenic pathways in the hamster preovulatory follicle

Isolated hamster granulosa cells and theca from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroid substrates. The purpose of the experiments was to determine, in theca, the relative activities of the delta 5 and delta 4 pathways under controlled conditions, and to compare the ability of granulosa cells and theca to form progesterone from exogenous pregnenolone. The results of the experiments show that the delta 5 pathway in theca predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from delta 5 to delta 4 as the major metabolic pathway. Progesterone formation from exogenous pregnenolone is 7 to 10 times greater in unstimulated granulosa cells than in theca. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theca. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone.     

Sexual behavior and reproduction ofCercocebus albigena johnstonii in Kibale forest,Western Uganda

Gray-cheeked mangabeys live in multimale social groups. Two groups of these monkeys were studied extensively over a period of 22 months, and a further eight groups were observed opportunistically. Data on the occurrence of sexual swelling infernales were collected and the different phases of swelling and deflation are described in detail together with data on their duration. The data were found to agree broadly with those presented by other workers on captive animals. Pre and postpartum swellings are described. The majority of copulations was observed to occur at peak swelling and a specific gesture, the head flag, was noted as a solicitation. The data on initiation of copulations show that the males initiated more copulations than the females. However, female-initiated copulations ended in ejaculation more often than male-initiated copulations. The behaviors of the mangabeys during precopulation and copulation were found to be broadly similar to those of the macaque. Copulations were observed most often on the day of peak swelling;however, adult males were the only animals to copulate prior to peak swelling. Subadult male copulations were most often after peak swelling. Masturbation was observed to ejaculation on two occasions. The data show gestation periods of between 184 and 189 days. A mean interbirth interval was calculated to be 33.33 ± 15.87 months.