Basement membrane (type IV) collagen was isolated from limited pepsin digests of rabbit lens capsules by differential salt fractionation and DEAE-cellulose chromatography. The sheep antibodies raised against this type IV collagen reacted with the vascular basement membrane, tubular basement membrane and glomerulus of the rabbit, rat, guinea pig and human kidney. In these glomeruli, a strongly positive immunofluorescent reaction was observed only in the mesangium and Bowman’s capsule basement membrane; in contrast, the reaction was weak in the glomerular capillary basement membrane (GBM). However, tissue sections digested with microbial protease (Streptomyces griseus) showed a strongly positive immunofluorescent reaction in the GBM. Digestion with trypsin or heparitinase (Flavobacrerium heparium) failed to enhance immunofluorescent staining of the GBM. The results suggest that type IV collagen in the GBM is covered with a protein or proteins sensitive to microbial protease, in contrast to type IV collagen in other basement membranes.

Experiments were performed to investigate the mechanism underlying the inhibitory effect of Ca ions on the Ba-dependent electrical and mechanical activity in the myocardium. In isolated strips of guinea-pig ventricular muscle perfused with Ca-free, K-rich (27 mM) depolarizing solution, addition of 0.6-1 mM Ba to the bath induced the Badependent slow action potentials. These action potentials were blocked by Co ions in concentrations much lower than those required to block the Ca-dependent action potentials. Development of the Ba-dependent action potentials was accompanied by a rise in resting tension (contracture). Addition of low concentrations (0.05-0.2 mM) of Ca to the depolarizing solution inhibited the action potentials induced by Ba and the contracture was suppressed. Further increase in Ca concentration enhanced the action potentials and these had a property characteristic of the Ca-dependent potential. The effect of Ca was accompanied by little change in the membrane resistance, determined using the sucrose gap method. The results suggest that Ca ions inhibit the Ba current through the slow channel and hence exert a relaxing effect on the Ba-induced contracture.

The gastrointestinal tract of three rodents (the mouse, hamster and guinea pig) was investigated immunocytochemically using antisera raised against porcine polypeptide YY (PYY), bovine pancreatic polypeptide (BPP) and the C-terminus (sequence 24-36) of BPP. PYY-Immunoreactive cells were found in the alimentary tract of the three species studied. Neither BPP- nor BPP C-terminus-immunoreactive cells were detected in any parts of the digestive tract of these species. PYY-Immunoreactive cells were observed in the colon and caecum of the three species investigated and in the rectum of the mouse and guinea pig. These cells were seen also in the pylorus, duodenum and ileum of the mouse. PYY-Immunoreactive cells in the three rodents studied were of the open type, i.e. they extended from the basal lamina to the gut lumen. In the mouse colon these cells were found to extend a cytoplasmic process from the basal part towards the neighbouring cells.

Tumor promoter, 12-o-tetradecanoylphorbol-13-acetate (TPA) induced the appearance of larger extrachromosomal circular DNA molecules (more than 1 μm in contour length) in mouse B16 cells, but no such effect in rat L6 cells. This may be due to the presence of a TPA-inactivating enzyme in the rat skin. Teleocidin induced an increase in the number of smaller circular DNAs (less than 1 μm in contour length) in the mouse cells, and the appearance of larger circular DNA molecules in the rat cells. The persistent effect of TPA on the appearance of circular DNAs was shown in interspecific mouse-rat reconstituted cells, in which the phenotype of unique cell morphology is reversibly changed to that of parent mouse by TPA. The persistence of induction ofcircular DNAs may be related to the irreversibility of tumor promotion at the first stage.

Plasma thyroid hormone concentrations were evaluated in three experiments after treatment of male and female Syrian hamsters with two indoles, 6-chloromelatonin and melatonin. In experiment 1, female hamsters given daily afternoon subcutaneous injections of 2.5, 15 or 25 μg of either 6-chloromelatonin or melatonin had the same doserelated depression of circulating levels of thyroxine (T₄) and free T₄ index compared to the saline-injected control animals. No effect of either indole was observed on T₃ levels or on the free T₃ index. Daily afternoon injections of melatonin reduced T₄ titers and the free T₄ index in male hamsters; these effects were prevented if the melatonin-injected hamsters were concurrently receiving a biweekly subcutaneous implant of 1 mg of 6-chloromelatonin or 1 mg of melatonin (experiment 2). Injections of 6-chloromelatonin caused a significant reduction in free T₄ index but not in plasma T₄ titers. The effect on free T₄ index in these 6-chloromelation-treated animals was reversed by biweekly implantation of a 1 mg melatonin pellet (experiment 3). In conclusion, daily afternoon injections of 6-chloromelatonin are as effective as melatonin in depressing circulating concentrations of T₄ and the free T₄ melatonin in female hamsters; in male animals, melatonin injections were somewhat more effective than 6-chloromelatonin in this regard. However, either melatonin or 6-chloromelatonin implants are capable of reversing the T₄ suppression induced by melatonin injections. Thus, 6-chloromelatonin acts as a melatonin agonist and may be of further use in determining the role of melatonin in future physiological studies.

Distribution of calpain I and calpain II was studied in various rat tissues by peroxidase anti-peroxidase method. 1) The distribution of calpain I showed a pattern very similar to that of calpain II. 2) Almost exclusively intracellular distribution of both types of calpain was noted. Extracellular space such as connective tissues was essentially devoid of calpains. 3) Both calpains I and II were distributed mainly in parenchymal or epithelial cells of various tissues; cells of mesenchymal origin showed no or much less significant staining with the antibodies. 4) Cell-type specific distribution of calpains was noted in certain tissues. In liver, calpains were distributed in the parenchymal cells preferentially in the pericentral zone of hepatic lobules. In kidney, calpains were distributed in epithelia of proximal and distal tubules, loops of Henle and collecting ducts; glomeruli were essentially immuno-negative. In submandibular gland, calpains were densely distributed in epithelia of the mucous gland and ducts, whereas epithelia of the serous gland were completely immuno-negative. In the skeletal muscle, the submembraneous portion was selectively stained.

In the presence of Ca²⁺ and ionophore A23187, axopodial retraction was induced, suggesting that the intracellular Ca²⁺ is involved in the axopodial retraction. Localization of intracellular Ca²⁺ was examined during the axopodial contraction induced by adding food organisms to heliozoan cell, by calcium pyroantimonate cytochemistry and energy dispersive X-ray microanalysis. Calcium antimonate deposits were detected inside many vesicles and in the cell-coated layer in both the axopodium and cell body. In axopodia stimulated for 20 sec by giving flagellates, a number of small electron-dense deposits were seen in association with the axonemal microtubules. In cells stimulated for 5 min, deposits in vesicles and in the middle layer of the cell coat were no longer seen. Results of energy dispersive X-ray microanalyses and disappearance of dense deposits after EGTA treatment indicate that the electron-dense deposits contain Ca²⁺ ions. The results suggest that the axopodial contraction is caused by Ca²⁺ ions released from the cellcoated layer or from the vesicles surrounding the axonemal microtubules.

The structure of parotid serous cells of the musk shrew was examined by transmission electron microscopy with special reference to intercellular canaliculi. Parotid serous cells possessed an intricate system of intercellular canaliculi associated with well-developed basolateral interdigitations. These two structures were demarcated by leaky limiting junctions of extreme luminal-basal shallowness. Mitochondria often accompanied interdigitations. The intercellular canaliculi together with the basolateral interdigitations are considered to favour the secretion of primary saliva by the model of Silva et al. (28) and of Poulsen et al. (21).

The gastrointestinal tract of two bony fish species, the daddy sculpin (Cottus scorpius) and the cod (Gadus callarias), was investigated immunocytochemically for the occurrence of polypeptide YY (PYY) and pancreatic polypeptide (PP). In C. scorpius, PYY-immunoreactive cells were found in the cardiac portion of the stomach. Beaded PYY-immunoreactive nerve fibers were located in the muscle layers of the stomach as well as under the mucosal basement membrane of the pyloric portion of the stomach. In G. callarias, PYY-immunoreactivity was not detected in any part ofthe gut. In C. scorpius, PP-immunoreactive cells were observed in the gastric caeca and duodenum. In G. callarias, PP-immunoreactive cells were distributed widely in the stomach, gastric caeca, duodenum, ileum and colon. All the endocrine cells observed here were of the open-type.

A technique for electrical field-induced cell fusion was modified to obtain a large amount of giant insulin-producing cells from normal pancreatic β-cells as well as from the clonal cell line RINm5F. The fusion was established by one or several pulses of 1.50-2.75 kV with a time constant of 175-550 μsec. The resulting electrical field strength corresponded to 2.5 - 10⁵ V/m. After fusion the giant cells (diameter ≥20 μm) comprised as much as 60% of the cells excluding trypan blue. Except for being polynucleated, the giant cells displayed similar cytological characteristics as their normal-sized counterparts both with regard to appearance and distribution of insulin-containing secretory granules and other intracellular organelles. The fused RINm5F cells survived under tissue culture conditions, but when cultured for more than one week, division of the giant cells resulted in normal-sized cells.