Bernt T. Walther

The major structural proteins of cod (Gadus morhua) eggshells and protein crosslinking during teleost egg hardening

The highly hydrophobic protein aggregate which constitutes the fish eggshell has for the first time been quantitatively solubilized. This study shows that the nonactivated eggshell from cod is composed primarily of only three protein monomers, designated alpha (74 kDa) beta (54 kDa) and gamma (47 kDa). Protein extraction studies of the eggshells before and after egg activation demonstrate that egg hardening is accompanied by a 10-fold decline in total protein solubility, which is due to nonextraction of the alpha, beta, and gamma chains. When present during the egg activation process monodansylcadaverine (MDC-a fluorescent lysine analog) inhibits eggshell hardening and at the same time becomes covalently incorporated into the eggshell. This MDC incorporation is calcium-dependent and suggests the induction of a perivitelline transglutaminase activity after egg activation. (Transglutaminases catalyze the formation of an amide bond (isopeptide bond) between the gamma-carbonyl group of glutamine and the epsilon-amino group of lysine with release of ammonia. Crosslinks between proteins are generated when the two amino acid residues are located on different proteins.) Protein solubilization studies and NaDodSO4 gel analysis of the eggshell proteins from eggs subjected to 5 mM MDC during egg activation, reveal that when eggshell hardening is blocked by MDC, the three main eggshell proteins remain extractable even after egg activation. Simultaneously we observed a covalent incorporation of MDC into the gamma protein.

Purification and characterization of chymotrypsin, trypsin and elastase like proteinases from cod (Gadus morhua L.)

1. Chymotrypsin, trypsin and elastase have been purified from the pyloric caeca of cod. 2. The enzymes were separated by affinity/hydrophobic chromatography on phenyl-butyl-amine (PBA) substituted sepharose. Chymotrypsin eluted in two separate isozyme fractions whereas trypsin and elastase eluted in separate fractions consisting of two closely-related polypeptide chains as revealed by SDS-polyacrylamide electrophoresis and isoelectric focusing. 3. The cod enzymes consist of single polypeptide chains with apparent molecular weights of about 27,000 Da as shown by denaturing polyacrylamide gel electrophoresis. 4. The cod proteinases were retarded on gel filtration media. The retardation increased with increasing pressure. 5. Isoelectric focusing analysis shows that the cod enzymes have isoelectric points in the range between 5 and 7. 6. The cod proteinases are rapidly inactivated when stored at low pHs.

Plasma levels of vitellogenin and eggshell zona radiata proteins in 4-nonylphenol and o,p′-DDT treated juvenile Atlantic salmon (Salmo salar)

Abstract Induction of vitellogenin (Vtg) in males and juveniles of oviparous vertebrates has been used as a biomarker for xenoestrogens. Recently, we have demonstrated that synthesis of eggshell zona radiata proteins (Zrp) or zonagenesis is an integral aspect of fish oogenesis (Oppen-Berntsen et al. (1994) Journal of Experimental Zoology 268, 59–70), and that Zrp synthesis is a sensitive biomarker for nonylphenol (Arukwe et al. (1997) Environmental Health Perspective 105, 418–422). This study compares the responses of Vtg and Zrp in plasma of juvenile Atlantic salmon (Salmo salar) treated with 4-nonylphenol (NP) and o,p′-DDT (both at 25 mg kg−1, singly and in combination). Validated ELISA and immunoblot analysis show that Vtg and Zrp respond significantly stronger to NP treatment alone and in combination with o,p′-DDT compared to control and o,p′-DDT treatment alone. However, a slight reduction in NP-induced Zrp levels was indicated when NP was injected in combination with o,p′-DDT.

Behavioral effects after ibotenic acid, 6-OHDA and electrolytic lesions in the central amygdala nucleus of the rat

Selective lesions of central amygdaloid neurons with ibotenic acid and electrolytic destruction of the nucleus both led to marked increases in open field activity and activity during passive avoidance conditioning. However, electrolytic lesions of both neurons and fibers resulted in the most pronounced passive avoidance impairments and it is suggested that this lesion effect should be attributed to a combined destruction of intrinsic neurons and neurons located outside the central amygdala nucleus. The 6-OHDA lesions resulted in no significant changes in the behavioral parameters under investigation or in plasma corticosterone levels. The lack of reduced corticosterone levels in any of the lesioned groups do not indicate that general fear arousal is critically dependent on intact central amygdala neurons in the rat. The behavioral data are, however, still compatible with a hypothesis of a temporary reduction in fear arousal during the initial phase of the passive avoidance conditioning.

Hatching in zebrafish (Danio rerio) embryos exposed to a 50 Hz magnetic field.

Zebrafish embryos were exposed intra ovo to a 50 Hz AC magnetic field of 1000 microT rms, and the progress of asynchronous hatching was monitored. A statistically significant delay was observed when field exposure started 48 h after fertilization. In contrast, when exposure started 2 h after fertilization, no statistically significant effect was seen. When field exposure was administered together with submaximal doses of progesterone at 48 h postfertilization, the two treatments appeared to delay hatching in an additive manner. Evaluating the progress of hatching in zebrafish embryos seems relevant for exploration of EMF effects on reproduction.

Brief uncontrollable stress and psychological parameters influence human plasma concentrations of IgM and complement component C3.

Thirty-eight men participated in a study of immunological, hormonal, and psychological parameters before and after acute stress situations. A brief but acute stress was repeated daily for 4 days. This exposure caused the plasma levels of IgM and C3 to increase from Basal Day to Experimental Day 4. Significant correlations between endocrine and immunological parameters, and also between psychological measures and immunological parameters, were found. Use of psychological defense was related both to endocrine and immunological changes. The authors concluded that psychological stress may influence immunological functions both indirectly, by hormonal changes, and directly, by nervous regulation during brief but acute stress periods.

Effect of central noradrenaline depletion on corticosterone levels and gastric ulcerations in rats.

Effects of central noradrenergic depletion on the stress responses of rats were explored using the new selective neurotoxin (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)). Noradrenergic depletion using DSP-4 was followed by a reduction in basal corticosterone levels after 7 days. Three weeks after DSP-4 treatment, animals exhibited less severe and fewer gastric ulcerations than control animals following 23 h immobilization stress, but stress levels of corticosterone were similar for the two groups. No differences could be found in the peripheral gastric levels of noradrenaline between experimental and control animals, while central noradrenaline was reduced to approximately 30% of control levels. The data support previous findings using other methods that central noradrenaline is an important factor in stress-induced gastric ulceration. The peripheral mechanisms for the protective effects of DSP-4 remain to be elucidated, and studies of these may cast light on the efferent pathways between the central nervous system and gastric mucosa which are involved in stress-induced gastric pathology.

Isolation and characterization of growth hormone from Atlantic cod (Gadus morhua).

Growth hormone was purified from cod pituitary extract by a simple two-step procedure involving gel filtration and reversed-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by rpHPLC, SDS-polyacrylamide gel electrophoresis, and immunoblotting using anti-chum salmon growth hormone (GH) antiserum. The yield of purified hormone was 1.3 mg/g pituitary. Cod GH was found to exist in two monomeric forms (Mr = 20K and 22K) and dimeric forms (Mr = 40K and 42K). The two monomeric forms have a pI of 5.8, an identical amino acid composition, histidine as the N-terminal residue, and an identical lysyl endopeptidase peptide map. Staining with concanavalin A was observed on the 20K component only, but analysis for total reducing sugar did not confirm these results. Cod GH was found to be a potent stimulator of growth in juvenile rainbow trout which received intraperitoneal injections of the hormone. The partial amino acid sequence has been determined.

Relations between environmental problems, psychology and health among shift-workers in the Norwegian process industry

Abstract A group of 89 male shift-workers, mean age 38 years, was examined for health complaints, subjective experience of their work environment, psychological defence strategies, and immunological factors. They worked in a process industry, controlling a complex chemical process partly by instruments and partly by direct checking of mechanical devices such as valves. Shifts were eight hours long and changed every second week. The main problem areas identified were problems with the management, colleagues and superiors, feelings of being under-valued, time pressure, fear of making mistakes, and lack of influence and communication. The main health complaints were sleep disturbance and gastro-intestinal problems. Shift workers also complained about allergies, breathing difficulties, tension, anxiety and depression. There were no pathological levels of plasma immunoglobulins (Ig) or Ig complements. However, levels did correlate with experience of work-related problems and with measures of defence mechanisms...

Free amino acids in live freshwater zooplankton and dry feed: Possible importance for first feeding in Atlantic salmon fry (Salmo salar)

Abstract Feeding preference and free amino acids (FAA) contents were determined in live and dry food, and also correlated with salmon growth rate. A possible signalling effect of FAA during feeding is supported by the rapid leakage of FAA from available food. Feeding response experiments indicated that salmon fry show a preference for daphnids of high rather than low nutritional value. It is proposed that the observed FAA leakage may explain this observation. The data are consistent with FAA being sensed when the fish make olfactory and gustatory choices, and thus the salmon fry may economize its efforts at prey capture as well as prey digestion. Certain FAA may have nutritional and pharmacological effects on the salmon fry, independent of the metabolic and protein synthetic roles of amino acids derived from food proteins. Hence the FAA content of food should be varied independently of general amino acid nutritional requirements in order to evaluate the suitability of a given food for salmon fry growth.