Search PubMed⌕ Search

Biomedical subjects

B T Walther

Publications and source records attributed to B T Walther.

At least 19 recordsLinked to original sources
PubMed2000

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.

Animals↗
PubMed1999

A sensitive zonagenetic assay for rapid in vitro assessment of estrogenic potency of xenobiotics and mycotoxins.

Mounting evidence confirms that hepatic biosynthetic processes are essential for female sexual maturation in fish, which is directly controlled by estrogens. These oogenetic events (zonagenesis and vitellogenesis) are induced in both sexes by estrogens. In this paper, we report the induction of zona radiata (zr) proteins and vitellogenin in primary hepatocytes from Atlantic salmon (Salmo salar L.) exposed to xenoestrogens and mycotoxins. Cells were treated with doses of 1, 5, and 10 microM 4-nonylphenol (4-NP), o, p'-DDT, lindane ([gamma]-HCH), and bisphenol A (BPA), which all induced zr proteins and vitellogenin in an approximate dose-dependent manner. Hepatocytes were also treated with combinations of xenoestrogens at 1 or 2 microM, resulting in elevated levels of both zr proteins and vitellogenin, compared to single treatment. The estrogenic activity of the mycotoxin zearalenone (ZEA) and its metabolites [alpha]-ZEA) and ss-zearalenol (ss-ZEA)], with regard to zonagenesis and vitellogenesis, was assessed in this assay system. Mycotoxins were used at concentrations of 10, 100, or 1,000 nM. All induced zr proteins and vitellogenin, with [alpha]-ZEA being the strongest inducer. When cells were treated with xenoestrogens or mycotoxins in combination with an estrogen receptor inhibitor (ICI 182,780), the induction of both zr proteins and vitellogenin was inhibited in all cases. Thus, the reported estrogen effects are bonafide estrogen responses. Zona radiata proteins were more responsive than vitellogenin to both xenoestrogens and mycotoxins. The versatility and sensitivity of the hepatocyte assay demonstrates that biosynthesis of zr proteins provides a new supplementary method for estimating xenoestrogenicity and mycotoxin action.

Animals↗
PubMed1998

Differential sensitivity of zonagenesis and vitellogenesis in Atlantic salmon (Salmo salar L) to DDT pesticides.

In Atlantic salmon (Salmo salar L) female sexual maturation entails both zonagenesis and vitellogenesis, both of which are controlled by increasing levels of estradiol-17 beta (E2). Antibodies against salmon zona radiata proteins (eggshell zr-proteins) and vitellogenin were used to monitor induction of oogenesis in juvenile salmon. Molecular weights of zr-monomers were estimated to about 66, 61, and 55 kDa, and to about 180 kDa for vitellogenin. Xenobiotics such as the pesticide DDT impair biological reproduction. The o,p'-DDT (1,1,1-trichloro-2[2-chlorophenyl]-2-[4-chlorophenyl]ethane) isomer seems to be a xenoestrogen. Serum levels of zr-proteins and vitellogenin, and hepatocytic biosynthesis of these components, were determined after in vivo treatment of salmon with DDT (technical, p,p'-(1,1,1-trichloro-2,2-bis[4-chlorophenyl]ethane) or o,p'-DDT) or E2. Exposing fish to frequent doses of o,p'-DDT (25 mg/kg b.w. (body weight) twice a week, six times totally) resulted in induction of all three zr-protein monomers, but not of vitellogenin. In contrast, three weekly injection of 10 mg/kg b.w. of either of the three DDT preparations did not induce typical zr-proteins or vitellogenin in serum. In vivo studies with combined DDT + E2 injections showed that none of the DDT preparations influenced E2-induced biosynthesis of zr-proteins or vitellogenin. E2 induction of these oogenetic processes was not blocked even by a high concentration (125 mg/kg b.w.) of o,p'-DDT. Furthermore, pretreatment of salmon with o,p'-DDT for 2 weeks, followed by one injection of E2, did not antagonize biosynthesis of zr-proteins, but serum concentration of vitellogenin was decreased. The data indicate that in juvenile salmon o,p'-DDT may be xenoestrogenic with regard to zonagenesis, but weakly anti-(xeno)estrogenic with regard to vitellogenesis. These findings suggest new complexities in fish reproductive toxicology of xenoestrogens. Compared to vitellogenesis, zonagenesis is a more sensitive parameter for monitoring reproductive effects of xenoestrogens.

Animals↗
PubMed1998

Oogenesis in Atlantic salmon (Salmo salar L.) occurs by zonagenesis preceding vitellogenesis in vivo and in vitro.

Fish oogenesis represents pleiotropic cytodifferentiative programs including hepatic synthesis of the molecular components for both the eggshell and the oocytic energy deposits. Both hepatic processes are directly controlled by plasma levels of estradiol (E2), and injected E2 induces both biogenetic processes in prepubertal fish of both sexes. This work compares the temporal pattern of E2-induced biosynthesis of zona radiata proteins (zr-proteins) and vitellogenin in Atlantic salmon (Salmo salar L.) in vivo and in vitro. We monitored the presence of plasma zr-proteins and vitellogenin, using homologous polyclonal antiserum to zr-proteins and a monoclonal antibody to vitellogenin. Zr-proteins were induced by all E2 concentrations (0.001-1.1 mg/kg body weight (bw)) within one week of exposure while vitellogenin was not induced until two weeks post-injection and then only in plasma from fish injected with high E2 concentrations (0.4 mg or 1.1 mg/kg bw). After E2 treatment, hepatocytes isolated from male fish synthesized zr-proteins and vitellogenin in vitro. However, zr-proteins were secreted into the medium two days before vitellogenin, as measured by ELISA. The data indicate a preferential induction of zr-proteins compared with vitellogenin, both with regard to E2 sensitivity and response time to E2 treatment. These findings suggest an obligate sequence in salmon oogenesis. During sexual maturation low E2 levels at first induce only zonagenesis, while increasing levels of E2 subsequently induce both zonagenesis and vitellogenesis. In nature, the interval between zonagenesis and vitellogenesis may, therefore, be considerable. The data suggest new control mechanisms in fish oogenesis.

Animals↗
PubMed1993

Development of hatchability in halibut (Hippoglossus hippoglossus) embryos.

Halibut (Hippoglossus hippoglossus) eggs raised in darkness hatched between days 14.5 and 16 after fertilization. Eggs incubated in white light (3.2 microE/sr1/m2) remained unhatched, so that time of intra ovo development could be doubled. Photo-arrest of hatching was non-diapausal since embryonic growth continued. Transfer of photo-arrested eggs to darkness induced rapid and synchronous hatching. This procedure allowed analysis of development of hatchability. Hatching was not observed prior to day 14. Nonsynchronous hatching over three days was seen when eggs were induced on day 14 + 1 h, or on day 14 + 9 h. However, darkness-induction on day 14 + 22 h produced synchronous hatching within 140 min. This high rate of inducibility persisted until day 18, before declining slowly. Hatching-induction was not observed beyond day 22. Low hatchability in long-term photo-arrested embryos apparently reflects a loss of the anatomical prerequisites for the rim-hatching mechanism. Altered hatchability and morphogenesis after prolonged intra ovo development indicate that hatching in halibut is possible only at an early, defined ontogenetic stage.

Animals↗
PubMed1992

Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties.

The enzymatic basis for cod digestive lipolysis has been investigated. Lipase activity was found in aqueous extracts from pyloric caeca as well as in pancreatic tissue surrounding the caeca and the bile duct. A bile salt-dependent lipase (BSDL) was purified from either defatted powder of cod pyloric caeca or aqueous pancreatic extracts by combined affinity chromatography on cholate-Sepharose and gel filtration on Sephacryl S-200 HR. By SDS-PAGE analysis the molecular weight of purified cod BSDL was estimated to 60 kDa. The enzyme was totally dependent on bile salts for hydrolysis of insoluble fatty acid esters. Antiserum raised against purified cod BSDL reacted specifically with selected mammalian pancreatic BSDLs by Western blot analysis. Results presented in this paper strongly suggest that the bile salt-dependent lipase is the only pancreatic enzyme involved in lipid digestion in cod. The enzyme has been characterized and compared to human pancreatic BSDL with respect to substrate specificity, temperature- and pH-dependence and inhibitors. Both soluble and insoluble fatty acid esters were hydrolysed and the enzyme was 1,3-specific in hydrolysis of triolein. The enzyme was inhibited by di-isopropyl fluorophosphate and phenyl boronic acid, but not significantly by phenyl methyl sulfonyl fluoride. The cod BSDL is probably homologous to mammalian pancreatic BSDLs.

Amino Acids↗
PubMed1992

Zona radiata proteins are synthesized by rainbow trout (Oncorhynchus mykiss) hepatocytes in response to oestradiol-17 beta.

The present study delineates the origin of the three major proteins constituting the rainbow trout (Oncorhynchus mykiss) zona radiata. Intraperitoneal administration of oestradiol-17 beta induced the appearance in the blood from juvenile fish (both sexes) of proteins immunoreactive to rabbit antisera against zona radiata proteins (zr proteins). These proteins had similar molecular weights to the zr proteins (alpha, 60 kDa; beta, 55 kDa; and gamma, 50 kDa). Primary hepatocyte cultures from fish treated with oestradiol-17 beta incorporated radioactive [35S] methionine into four major proteins with molecular weights of 160, 60, 55 and 50 kDa. Only the latter three proteins were specifically immunoprecipitated with antibodies to zr proteins. Furthermore, our data demonstrate that in such cultures the biosynthetic mole ratios of these secreted proteins (60, 55 and 50 kDa) are close to one. Control cultures from fish that had not been treated with oestradiol-17 beta failed to produce immunoreactive proteins. The data support the hypothesis that zr proteins are synthesized in a concerted manner in the liver during teleostean oogenesis and transported by the blood for deposition in ovarian follicles.

Animals↗
PubMed1992

Eggshell zona radiata-proteins from cod (Gadus morhua): extra-ovarian origin and induction by estradiol-17 beta.

Using Atlantic cod (Gadus morhua) as a model organism, the aim of this report was to delineate whether teleostean eggshell zona radiata proteins have their origin, i.e., site of synthesis, in gonadal or somatic tissues. Estradiol-17 beta was administered intraperitoneally to one-year-old cod (Gadus morhua) with either undeveloped gonads or with differentiated gonads. By immunoblotting procedures estradiol-dependent protein induction was investigated using specific rabbit antisera directed against cod eggshell proteins and brown trout vitellogenin. No immunological cross-reactions were observed between the two antisera, and eggshell proteins and vitellogenin were detected in blood plasma and somatic tissues only in estradiol-treated cod. Three plasma-components were immunoreactive to antiserum directed against eggshell proteins, and these proteins possessed molecular weights of 78, 54 and 47 kDa, identical to the molecular weights of the cod eggshell alpha, beta and gamma zona radiata-proteins. These three immunoreactive plasma-components were observed after administration of estradiol-17 beta to both sexes, also in males having reached spermiation, and in juveniles of either sex without developed gonads. The data are interpreted to signify that cod eggshell zona radiata-proteins originate in an extra-ovarian tissue and are transported in the blood for deposition in the ovaries. We propose that oogenesis involves estradiol-17 beta regulation of both eggshell zona radiata-proteins and vitellogenin synthesis.

Animals↗
PubMed1991

Oestradiol-17 beta induces the major vitelline envelope proteins in both sexes in teleosts.

During growth of the ovarian follicle, the teleost oocyte becomes surrounded by an acellular coat, the vitelline envelope. The nature, origin and number of the vitelline envelope proteins in fish appear to vary with species. In this work, polyclonal antibodies directed against vitelline envelope proteins from rainbow trout, brown trout and turbot were used to show that oestradiol-17 beta induces the major vitelline envelope proteins in juveniles, both males and females, from different species. The fact that males can synthesize vitelline envelope constituents shows that the origin of these proteins is not confined to the ovary. The vitelline envelope of rainbow trout eggs consists of three major proteins, designated alpha (60 kDa), beta (55 kDa) and gamma (50 kDa). The amino acid composition of each of the three proteins indicated that the three proteins are alike and the suggestion that these proteins represent a separate class of structural proteins is sustained.

Amino Acids↗
PubMed1991

The hatching mechanism in Atlantic halibut (Hippoglossus hippoglossus).

In general fish larvae emerge from the protective egg after secreting a hatching enzyme (HE) from diffusely located hatching gland cells (HGCs). This proteolytic enzyme is distributed over the entire inner part of the eggshell (zona radiata). In a marine flatfish halibut, (Hippoglossus hippoglossus), we have found a more specialized hatching process. A strategic location of the HGCs in a narrow belt on the anterior part of the yolk sac leads to restricted degradation of the eggshell resulting in cleavage of the eggshell into two distinct rigid parts. This hatching process--termed "rim-hatching"--results in an empty eggshell with a lid approximately 1/4 the size of the bottom shell. During the hatching process the yolk sac is reshaped. The posterior part of the yolk sac contracts and the yolk mass is squeezed forward before hatching. This mechanism ensures close contact between the HGCs and the eggshell during the release of the hatching enzyme, which is a prerequisite for restricted degradation of the eggshell.

Animals↗
PubMed1990

Paradoxical DSP-4 effects: protection against gastric erosions and depletion of mucosal glycoproteins.

We have previously reported that rats pretreated with the central noradrenergic neurotoxin DSP-4 are protected against 23 h restraint-induced gastric erosions. To elucidate the peripheral mechanisms of this protection, we undertook direct biochemical analyses of the gastric mucosa in terms of glycoproteins and proteins. A simple method for preparation of gastric mucosa devoid of the muscular stomach wall tissue is described. A restraint-induced decrease in gastric mucosal wet weight and mucosal glycoprotein content was revealed. Restraint had no effect on mucosal protein content, and no changes were found in gastric wall glycoprotein or protein content. Despite showing protection against restraint-induced gastric erosions, unrestrained DSP-4-treated animals exhibited reduced mucosal wet weight and mucosal glycoprotein content when compared to unrestrained controls. After the stress period, no significant differences on mucosal weight or glycoproteins could be detected between control and DSP-4-treated animals. The results indicate that the protective effect of DSP-4 in this paradigm is not due to enhanced gastric mucosal protection against erosive factors. We suggest that an additional effect of central nervous NA depletion by DSP-4 may be elimination of aggressive factors which precipitate overt ulcers.

Animals↗
PubMed1990

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.

Amino Acids↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

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 pH's.

Animals↗
PubMed1973

A quantitative assay for intercellular adhesion.

Intercellular adhesion is measured by a new method based on determination of the rates of attachment of single cells to confluent cell monolayers. The procedure is simple, rapid, and reproducible. Specific and nonspecific intercellular adhesions can be quantitated and distinguished from each other, and from the adhesion to glass (or plastic). The rate of adhesion of single cells to the monolayer is characteristic of more than 80% of the single-cell population. This method, therefore, provides a means for study of the molecular basis of intercellular adhesion.

Animals↗