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Atlantic salmon hatching fluid (HF) contains numerous polypeptides. A component unidentified by proteomics, was cloned from tryptic peptides and characterized as lectin-like (LL-) proteins in the tectonin-family. Purified salmon LL-proteins elicited high-titre, LL-specific polyclonal antibodies. This study aims to delineate the cellular and genetic basis of fish embryonic LL-expression. LL-proteins were detected in salmon, cod, rainbow trout and zebrafish HFs. LL-immunoreactive cells were numerous in salmon and rainbow trout embryos, but fewer in zebrafish, cod and halibut. Peridermal salmon LL-positive cells (lectocytes) corresponded to non-eosinophilic cells stained by PAS-reagent. Northern blots revealed two transcripts in salmon and zebrafish embryos, and LL-transcripts were detected specifically in lectocytes. Dual in situ hybridization distinguished lectocytes from hatching glands. BAC-library screening yielded salmon Leukolectin’s gene-structure with 4 introns, 5 exons, TATA-box, multiple upstream putative transcription-factor binding-sites, and polyadenylation site. Sequence-analysis indicated zebrafish LL’s conserved nt-sequences and gene-structure, which exhibited mature and truncated LL-transcripts. Zebrafish LL-expression was detected at 6 hpf (yolk syncytium) and 19 hpf (lectocytes and PVF). In dermal mucus, Leukolectins with TECPR-domains may function as pathogen-recognition receptors in first-line innate immunity defence.SUMMARY STATEMENT:At hatching, embryos lose maternal chorions, their first-line innate immuno-protection. Novel leukolectin-genes specifically expressed in non-eosinophilic peridermal cells (lectocytes) help explain how embryos develop innate immuno-competency to survive as larvae.

Fish embryonic hatching glands (HGs) secrete choriolysin-zymogens, which when activated degrade the chorion, allowing larval exit. Thus, hatching encompasses two dissimilar choriolysin-processes: pre-choriolysis including activated choriolysins accessing the perivitelline space (PVS), followed by choriolysis. Discovery of serine-proteolytic zonase in Atlantic salmon hatching fluid (HF) raises questions concerning its participation in hatching. This work aims to identify salmon choriolysins, and evaluate their role and that of zonase during hatching. Precocious salmon hatching occurs under alkaline conditions, producing an HF containing similar metallo- and serine- proteolytic activities. Purified zonase is selectively inhibited by peFabloc, whose MW (580 Da) allows diffusion through the chorion into the PVS. Without apparent toxicity, brief peFabloc-pretreatment (2 mM) of salmon eggs reduced precocious hatching substantially, compatible with a zonase-relevance for hatching. Atlantic salmon differed from other fishes since their HGs were not immuno-stained by polyclonal antibodies against pike choriolysins. However, cloning and sequencing experiments revealed a single low choriolytic enzyme (LCE) of 69% identity to pike LCE. Similar experiments with high choriolytic enzymes (HCEs) revealed two types (HCE-1 and HCE-2) with respectively 71% and 91% identity to pike HCE-1HCE-2. In situ hybridization revealed typical HGs. However, zebrafish-choriolysis is achieved by HCE-class choriolysins alone. Another zebrafish choriolysin (HE2) was not expressed. Peptide-bond hydrolysis by non-choriolytic zonase mimicks HCE-action generating hydrophilic sites for LCE-choriolytic catalysis. Ultimately, precise definitions of choriolytic and pre-choriolytic catalysis requires developmental genetics. Our data are compatible with a complex pre-choriolytic hatching-stage in Atlantic salmon, before LCE-choriolysis degrades the chorion.

Zonagenesis (zona radiata protein synthesis) and vitellogenesis (yolk protein synthesis) are two reproductive responses that are integral aspects of fish oogenesis. This study examines the responses of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) to five environmental pollutants; 4-nonylphenol (NP) and o,p'-DDT [both at 25 mg/kg], lindane (gamma-HCH) [10 mg/kg], a technical PCB mixture (Aroclor 1254; A1254) and bisphenol A (BPA) [both at 5 mg/kg] in juvenile Atlantic salmon (Salmo salar). Fish were given intraperitoneal injections of o,p'-DDT, gamma-HCH, A1254 or BPA; singly, in combination with NP, and as a cocktail of all five chemicals, and later compared to NP-treated and untreated fish. In a separate experiment, fish were exposed to BPA in a dose-response manner (1, 5, 25 and 125 mg/kg fish). Based on previous studies, blood and liver samples were collected 2 weeks after injection. Zrp and Vtg levels were analyzed in plasma using immunoblotting and enzyme-linked immunosorbent assay. Liver cytochrome P4501A was analyzed by 7-ethoxyresorufin O-deethylase (EROD) activity. NP caused pronounced elevations in plasma Zrp and Vtg levels. In comparison, when NP was given in combination with gamma-HCH, Vtg levels was significantly reduced, compared to NP treatment alone. Using o,p'-DDT, A1254 and BPA, significant elevations of plasma Zrp and Vtg were seen when chemicals were given in combination with NP, but not when administered by themselves. An apparent dose-response induction of Vtg and Zrp levels were observed in BPA treated juvenile salmon. In immunoblots, one component of molecular weight approximating the Zrp-beta was detected when either o,p'-DDT, gamma-HCH, A1254 or BPA were given singly. A1254 significantly induced hepatic EROD activity when administered alone. However, when given as a mixture with all the other xenobiotics, reduction of EROD activity was observed. The data suggest a pattern of xenobiotics action which may complicate assessment of their reproductive effects. Zrp (the beta monomer) was more responsive to the xenoestrogens than Vtg, and provides a sensitive means of detecting exposure to environmental estrogens.

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.

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. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

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.

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.

Hatching occurs in all vertebrates, yet knowledge about the control of this developmental transition is incomplete. Incubation of halibut Hippoglossus hippoglossus eggs in light results in sustained inhibition of hatching. The inhibitory efficacy of light is affected both by its intensity and its colour. Transfer of hatching-arrested eggs to darkness (0.1 lux and below) reverses such inhibition and causes hatching of all eggs between 80–140 min later. Darkness is only required during the first 60 min of this induced hatching and initiates a signal which makes hatching irreversible and darkness independent. The darkness-generated signal ultimately causes release of hatching enzyme, which digests the zona radiata so that the eggshell may be mechanically opened by the larva. We conclude that light serves as a natural cue for hatching in halibut eggs, and that halibut larvae in nature hatch in darkness. © 1992 Wiley-Liss, Inc.

An alkaline deoxyribonuclease (DNase) from cod pancreatic tissue has been characterized. The enzyme is a DNase I type endonuclease and hydrolyzes effectively both native and denatured DNA. Monomeric actin inhibits the enzyme reaction. The enzyme obeys Michaelis-Menten kinetics and the apparent Km value for native linear duplex DNA is 33 µg/ml. The cod DNase opens supercoiled plasmid DNA, by introducing adjacent nicks in both strands, possibly separated by 5-10 nucleotides. DNA hydrolyzed by cod DNase functions as substrates both for DNA polymerase and ligase, and the nicks therefore contain 5'-phosphoryl and 3'-hydroxyl groups. Optimum concentrations of divalent cations are 5 mM Mg(2+), 0.63 mM Mn(2+) and 0.075 mM Ca(2+). However, Ca(2+) is apparently not essential for the enzymatic functions. The enzyme has a narrow temperature optimum at 42°C and is thermolabile above 50°C; however, Mn(2+) shifts the optimum slightly to 45°C by causing increased temperature stability. The cod DNase reaction is inhibited by the DNA intercalating compounds actinomycin D and ethidium bromide. Histidine-modifying reagents such as tosyl phenylalanyl chloromethylketone and diethyl pyrocarbonate inhibit the enzyme activity, but the cod DNase is insensitive to disulfide-reducing agents.

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.

A deoxyribonuclease (DNase) of pancreatic origin has been purified from extracts of the pyloric caeca from Atlantic cod (Gadus morhua L.). The crude extract was prepared by mincing frozen caeca tissue in equal volumes of buffer. The enzyme was isolated from the supernatant after streptomycin sulfate precipitation and centrifugation. The purification scheme further included chromatography on Q-Sepharose Fast Flow and hydroxyapatite columns. Affinity adsorption chromatography of the hydroxyapatite fraction on 8-(6-aminohexyl)-amino-5'-AMP-Sepharose, revealed an apparently homogeneous protein with molecular weight of 35,000 Da as judged by NaDodSO4-PAGE. In sum a 644-fold enzymatic enrichment and 3.5% total enzyme recovery was achieved. The cod enzyme resembles DNase I-type enzymes with an alkaline pH activity optimum and shows dependency for Mg(2+). The pI of the enzyme is 6.5 as determined by isoelectric focusing and DNase-zymography. Our findings suggest that the nuclease is a member of the cod's digestive enzymes secreted from the connective tissue surrounding the caeca.

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.

The halibut hatching gland (HG) cells are first observed as a cellular disc in front of the embryonic head around the midpoint of intra ovo development. The disc is subsequently transformed into a loop of increasing diameter as the HG cells migrate over the anterior part of the yolk sac. When the HG disc is transformed into a loop, the density of HG cells is highest at the migratory front. Some HG cells lag behind the migrating front at the early stages of HG development. At maturity, all cells are contained in a narrow belt which is about 10 cells wide. The HG belt structure consists of a monolayer of HG cells, and is maintained while the cells migrate between the two epidermal cell layers. Migration is halted about 2 days before normal hatching when the HG cells reach a destination at about a right angle to on the embryonic axis. Under the scanning electron microscope, the differentiating HG cells protrude as a ridge the yolk sac surface. The HG cells immunostain with antiserum to hatching enzyme when the HG is observed as a crescent structure around the embryonic head. By counting the number of immunostaining cells in composite photos of the entire yolk sac membrane, we found that the HG belt consists of approximately 2000 secretory cells at maturity. This cell number stays fairly constant throughout the period of HG cell migration. Accordingly, mitoses of the halibut HG cells have generally ceased prior to morphogenesis, and cytodifferentiation is already quite advanced when cell migration starts.

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.

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.

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.

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.

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.

Electrolytic lesions in the lateral hypothalamus (LH) seriously affect ingestive behavior and sensorimotor functions in the rat. We here report that bilateral infusions of the neurotoxin, ibotenic acid (IBO) in the LH yield a decrease in body weight, but not to the same extent as electrolytic lesions. The sensorimotor impairments were most severe after electrolytic lesions. When tested in a residential maze on days 5–7 and 18–20 after surgery, both lesioned groups showed no lack of motivation to seek food and water. Histological examination of the LH following IBO exposure revealed extensive degeneration of neuronal cell bodies with little evidence of non-specific damage. Biochemical analysis of the rostral forebrain content of norepinephrine (NE) and serotonin (5-HT) revealed that the fibers passing through the LH remained largely intact in the IBO treated rats. The results suggest that the observed aphagia and adipsia is not due to a lack of motivation, but rather reflects changes in the process which operate to initiate eating and drinking. Furthermore, selective neuronal degeneration induced the same behavioral changes as the electrolytic ones, though not to the same extent.

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...

SUMMARY A quantitative method was developed for measuring cell agglutination by the plant lectin, concanavalin A. The method, based on an assay for intercellular adhesion, determines the concanavalin A-stimulated rate of attachment of single cells to a cell layer. The rate depended on the number of single cells and the concentration of concanavalin A. Experiments on concanavalin A agglutinability of mouse BALB/c 3T3 cells agreed qualitatively with observations obtained with a visual method presently in use. For example, the concanavalin A-stimulated rate was low when EDTA was used to prepare the single cells, high when trypsin-dispersed cells were employed, and highest when the latter cells were assayed with trypsin-treated cell layers. The latter result was not explicable in terms of concanavalin A binding to the cell layer, since the same quantities of labeled lectin attached to untreated and to trypsin-treated cell layers. Concanavalin A attached to an untreated cell layer in such a manner that it retained high binding activity toward surface receptors on trypsin-treated single cells. Above a threshold density of concanavalin A bound to the cell layer, the stimulation of single cell attachment to the layer was directly proportional to the amount of concanavalin A bound to the layer. The concanavalin A-stimulated rate of attachment was specifically inhibited by methyl a-D-mannopyranoside. However, once single cells were attached to the cell layer in the presence of concanavalin A, they were not released by methyl a-mannoside. Agglutination is considered to be more complex than the simple binding of cells together through lectin bridges.

The origin, morphogenesis, and biochemical differentiation of the dorsal and ventral pancreas of the rat embryo have been investigated in order to ascertain the similarities and dissimilarities between the two lobes. We have utilized a culture system in which the primitive gut gives rise to a number of differentiated organs, including the dorsal and ventral pancreas. The two pancreases do not undergo fusion in these cultures, thus allowing independent analyses of the two lobes for comparison with in vivo results. The dorsal pancreas first appeared at the 23–25 somite stage while the ventral pancreas appeared approximately 12 hr later at the 29–30 somite stage. Guts from embryos as young as 12 somites were capable of developing both pancreases in vitro. In spite of the 12 hr difference between the times of their appearance, the dorsal and ventral pancreases exhibited identical patterns of morphological and biochemical differentiation. The two lobes contained the same exocrine enzymes and hormones, at similar levels, differing only in their glucagon content, the dorsal pancreas possessing a fivefold higher glucagon specific activity. The implications of these results are discussed.

The patterns of protein synthesis during the in vitro development of the embryonic rat pancreas have been studied. Electrophoresis in discontinuous acidic and basic urea polyacrylamide gels was used to resolve the exocrine pancreatic proteins. This procedure is shown to give specific profiles of the proteins from different tissues, and may thus be of general usefulness. The pancreas-specific proteins were identified on the discontinuous gels by co-migration with the purified proteins, by enzymatic activity, and by reaction with specific antibodies against the rat exocrine proteins. The rates of synthesis of the specific pancreatic proteins were estimated from the incorporation of l-[3H]leucine into these proteins resolved on the discontinuous urea polyacrylamide gels. The specific radioactivity of the cellular leucine pool was calculated indirectly from leucine pool saturation experiments and from the rate of the protein turn-over. The calculated value was verified by several means including the prediction of the accumulation curves for total and specific proteins in embryonic rat pancreases grown in vitro. The results show that while the rate of total protein synthesis during this period only increases 25% on a cell basis, the synthetic rates for amylase and chymotrypsinogen increase at least 100-fold. The increased synthetic rates account for the observed accumulation of the specific proteins during the secondary developmental transition. Thus, the mechanism of pancreatic cytodifferentiation involves the selective regulation of the synthesis of cell-specific proteins.

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.

Forced immobilisation or restraint stress is one of the many available techniques for the induction of the so-called experimental stress ulcer in rats. The etiology of these gastric erosions is undoubtedly complex, involving simultaneously a number of central and peripheral factors [1]. At the peripheral level, it is common to consider the so-called aggressive factors (gastric motility and pH), defensive factors (the integrity of the buffering glyco-protein barrier), and the balance between these.

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.

1. A proteinase has been isolated from the ovarian fluid of the lumpsucker (Cyclopterus lumpus). 2. The enzyme was purified essentially to homogeneity by a one step purification procedure using anion-exchange chromatography. 3. The mol. wt of the denatured enzyme is approximately 20,000 as judged by SDS-polyacrylamide gel electrophoresis. 4. The enzyme is inhibited by serine-proteinase inhibitors and acts in the manner of a trypsin-type proteinase both with respect to specific peptide substrates and enzyme inhibitors. 5. The lumpsucker proteinase exhibits low general proteolytic activity but acts effectively on the specific chromogenic peptide substrates.

Publisher Summary The assays described in this chapter are kinetic and are aimed at measuring the first detectable events that occur in a population of single cells. Adhesion is defined as the rate of attachment or of adsorption of single ceils to other surfaces. Three types of adhesion can be measured: Nonspecific cell-substrate adhesion, or more simply, nonspecific adhesion (Reaction c above) is defined as the rate at which single cells attach to a solid substrate such as glass, tissue culture plastic, collagen. Intercellular adhesion is the rate at which cells attach to cells. This is divided into specific intercellular adhesion, the rate at which cells attach to homologous cells, and nonspecific intercellular adhesion, the rate at which cells attach to heterologous cells.