Protease-Dead Separase Is Dominant Negative in the C. elegans Embryo

Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic C. elegans line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage.     

Effects of tin and lead on organ levels of essential minerals in rabbits

The effect of tin and lead on levels of essential metals (Zn, Cu, Ca, Fe) in rabbit tissues was compared in relation to the route of administration. Animals received intraperitoneally, or per os, SnCl2 (2 mg Sn/kg) or Pb(CH3COO)2 (3.5 mg Pb/kg) every day for 5 d or for 1 mo. Copper, zinc, iron, and calcium were determined by AAS in the liver, kidneys, spleen, brain, bone marrow, and blood; lead and tin concentration were measured in the blood of animals. Tin and lead administered per os caused either no changes or the decreased concentration of endogenous metals in several tissues. The other route of administration (ip) of both metals generally contributed to the increased storage of essential elements. Blood tin levels of tin treated animals were only about less than or equal to 1/10 of blood lead concentrations of rabbits exposed to lead.     

The codon 408 mutation associated with haplotype 2 is predominant in Polish families with phenylketonuria

Summary The incidence of phenylketonuria (PKU) in the western part of Poland is 1 in 5000 live births. Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase locus have been analysed in 46 Polish families with PKU. Among 43 fully-informative families 16 RFLP haplotypes were identified. Haplotype 2 is the most frequently (62%) associated with Polish PKU alleles, and the codon 408 mutation is in complete linkage disequilibrium with this haplotype in Poland. This finding is in agreement with observations in other eastern European countries (German Democratic Republic, Czechoslovakia, and Hungary) and in contrast to the genotype distribution observed in western European countries. The present observation suggests the spread of classical PKU, due to the codon 408 mutation associated with haplotype 2, from east to west in European populations. Perhaps more important for genetic counselling, 62% of all PKU chromosomes in the Polish population can now be detected using only one mutantspecific oligonucleotide probe.     

Mating-induced loss of sex pheromone and sexual receptivity in insects with emphasis on Helicoverpa zea and Lymantria dispar

Mating in most species of insects leads to a transient or permanent loss in sexual receptivity of the females. Among moths, this loss of receptivity is often accompanied with a loss of the sex pheromone in the absence of calling, which also could be temporary or permanent. Most of the earlier work on changes in reproductive behavior after mating was done with Diptera in which sperm and/or male accessory gland secretions were shown to be responsible for termination of receptivity. In the corn earworm moth, Helicoverpa zea, mated females become depleted of pheromone and become nonreceptive to further mating attempts, but only for the remainder of the night of mating. A pheromonostatic peptide isolated from the accessory glands of males may be responsible for the depletion of pheromone, while the termination of receptivity is independently controlled. In the gypsy moth, Lymantria dispar, the changes in behavior following mating are permanent. In this species, the switch from virgin to mated behavior involves three steps: a physical stimulation associated with mating, transfer of viable sperm to the spermatheca, and commencement of oviposition. Signals generated by these factors operate through neural pathways and, unlike in H. zea, accessory gland factors seem not to be involved. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Lactate dehydrogenase in abdominal muscle of crayfishOrconectes limosus and shrimpcrangon crangon (Decapoda: Crustacea): Properties and evolutionary relationship

In crayfishOrconectes limosus and shrimpCrangon crangon abdominal muscle, lactate dehydrogenase (LDH, EC 1.1.1.27) is encoded by one locus. No polymorphism was detected. The enzymes were purified to homogeneity. The specific activities for purified crayfish and shrimp LDHs were 472 and 414 μmol NADH min⁻¹ mg⁻¹, respectively, at 30°C. Their physicochemical and kinetic properties did not resemble fish (Gadus morhua) LDH-A₄ isoenzyme. Their amino acid composition indicated greater similarity to fish LDH-C₄ isoenzymes.     

Immunological properties of low molecular-weight proteins binding heavy metals in rat kidney and liver

Two homogenous fractions of hepatic metallothioneins ((Cd,Zn) MT-1 and (Cd,Zn) MT-2) and renal metal binding proteins ((Bi,Cu) BP-1 and (Bi,Cu) BP-2) were isolated from rats exposed to heavy metals and specific antisera to them were produced in rabbits.These antisera were tested by immunodiffusion and immunoelectrophoresis for their ability to bind different fractions of hepatic Cd,Zn -metallothionein and renal (Bi,Cu)-, (Hg,Cu)- and (Cd,Cu)-binding proteins. It was found that anti (Bi,Cu) BP antisera did not cross-react with hepatic (Cd,Zn) MT-1 and (Cd,Zn) MT-2. Strong immunological cross-reactions were detected between anti (Bi,Cu) BP antisera and individual forms of (Cd,Cu)-, (Hg,Cu)- and (Bi,Cu)-binding proteins isolated from rat kidneys.     

Effects of oleate,palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3-$\text{OHB}\text{AcAc}$) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Effect of photoperiod on development and fecundity in the flour moth, Ephestia kuehniella

Examination was made of the effect of alternating light and darkness (LD 12:12) and constant light (LL) conditions on length of development and fecundity of the moth, Ephestia kuehniella. It was found the conditions applied cause acceleration of larval development on an average by 3 days in comparison with development under constant darkness conditions (DD). The fecundity of the insects examined decreases considerably under the experimental conditions.When pharate adults are kept in light for 18 hr per 24-hr period on the 6th to 7th day of their development, males incapable of fertilizing females are produced.     

Effect of phenethyl alcohol and other organic substances on cellulase production

Cellulase can be produced from growth in noncellulosic substrate if the growth rate of the producing organism is restricted. Phenethyl alcohol (PEA) is a growth inhibitor and was used to control the growth of M. verrucaria in attempts to obtain increased cellulase production. Cellulase yield was found to be increased without a restriction in growth rate when PEA was present in low concentrations (0.03% v/v). The effect was observed for other organisms but notably L. trabea, which produced considerable enzyme from a small quantity of mycelium. Here increased cellulase synthesis was concomitant with restricted growth. Other chemicals with PEA-like structure (e.g. benzyl alcohol) resulted in similar or more extensive cellulase synthesis. Of the substances tried, propyl alcohol was most effective, followed by acetone. PEA causes a swelling of cell walls and inhibits spore formation. This and other data given suggest that PEA affects the cytoplasmic membrane or the cell wall or both. Cellulase synthesis is considered to take place in the membrane and wall region of the cell.