A neutral proteinase catalyzing the removal of the C-terminal cross-linking region of type I collagen (telopeptidase) was extracted from rat uteri obtained 0-5 days postpartum. This enzyme showed lack of latency, in contrast to the collagenase which was activated by trypsin. Telopeptidase activity was highest at 0 and 1 day postpartum and decreased rapidly to the nulliparous level by 3 days. Collagenase activity increased slightly from 0 to 1 day postpartum and decreased rapidly there after. The elevated levels of these two enzymes were correlated with rapid loss of uterus collagen (77% by 3 days postpartum). The extracted telopeptidase had a neutral pH optimum. The major fraction isolated by gel-chromatography had an apparent molecular weight of approximately 15,000. It was inhibited by ethylenediamine-tetra-acetic acid and 1,10-phenanthroline but not by N-ethylmaleimide, nor by phenylmethanesulphonyl fluoride. It is therefore probably a metalloproteinase.
Three hybridomas which produce monoclonal antibodies (1D, 4C and 7B) against neurofilament protein were established by hybridizing mouse myeloma cells with spleen cells from BALB/c mice immunized with a human neurofrlament-rich fraction. The 1D antibody reacted with an epitope sharedby 200 K and 160 K neurofilament subunits. This epitope was sensitive to alkaline phosphatase treatment. The epitopes recognized by the 4C and 7B antibodies were, however, phosphataseresistant and localized in the 160K and 68 K subunits. All the three antibodies immunostained formalin-fixed, paraffin-embedded nervous tissue sections from humans, rats and mice. The 1D antibody stained axons as well as presynaptic terminals, but not perikarya. In contrast, both the axons and neuronal perikarya were immunostained with 4C and 7B antibodies. These observations suggest that there are isoforms of the neurofilament triplet in terms of the phosphorylation level, and heavily phosphorylated forms are distributed in axons distant from the cell body.
Isolated adult rat hepatocytes attached to flasks in the presence of frbronectin, spread, formed monolayer, and subsequently proliferated in Koga’s medium L under serum-free conditions. When a plasmin inhibitor, aprotinin (Trasylol) was omitted from the medium, hepatocytes tended to shrink after initial spreading, were detached from the substrata and died within 2 or 3 days. Examination of the hepatocyte-conditioned medium revealed the presence of plasmin-like protease activity. Since other plasmin inhibitors also prevented hepatocytes from shrinking and detachment, it is suggested that plasmin-like protease(s) is produced by hepatocytes and this causes detachment of hepatocytes. Thus, it is necessary to supplement a plasmin inhibitor such as aprotinin for successful cultivation of hepatocytes under serum-free conditions.
Secretory responses to ouabain and to lowering extracellular Na+ concentration have been studied in the isolated perfused pancreas of guinea pig. Ouabain (1-100 μM) produced dose-dependent secretory responses (pancreatic juice flow and protein output). The responses to 10 μM ouabain were completely abolished in Ca2+ deficient environment. The peak level of juice flow induced by 10 or 100 μM ouabain was partly reduced but the protein output remained almost unchanged after atropinization (2 μM). Lowering extracellular Na+ concentration from control (156 mM) to 25, 50, or 100 mM induced the secretory responses. The responses to 25 mM Na+ were nullified after the removal of extracellular Ca2+. The responses were reduced to about one half after atropinization (2 μM). The present results are compatible with the view that the major portion of the secretory responses to ouabain and to low Na+ environment may be mediated by the release of acetylcholine and other peptidergic transmitters (possibly VIP and PHI) from the intrinsic nerve endings distributed to the exocrine pancreas of guinea pig.
The microvascular architecture and the distribution of vascular resistance in the lungs of full term foetal and neonatal rabbits were studied by scanning electron microscopy of microvascular corrosion-casts and of specimens that had been perfusion-fixed by osmium. In the foetus, each potential air space was surrounded by its own capillary plexus; there were occasional, short anastamoses with adjacent plexuses. In the folded and compressed parenchymal tissue, two ‘lobule’ states were identified, one in which the vasculature was easy to perfuse, either with fixative or plastic, and another in which flow was inpeded. In the neonatal lung the parenchyma was inflated and the terminal air sacs rounded. The same capillary plexus arrangement was seen as in the foetus, but the perfusion of all the lobules was uniform. These findings suggest that the high pulmonary vascular resistance in the foetus is distributed unevenly. Such a system would allow maintenance of blood velocity sufficient to prevent sludging and stasis in the perfused, lower resistance areas, whilst total blood flow remained low. A cyclical pattern of vascular resistance and uniform lung development. The recruitment of the non-perfused areas at birth would rapidly drop pulmonary vascular resistance, and accommodate the vital increase in pulmonary blood flow at reduced perfusion pressures.
Localization of growth hormone-releasing hormone (GHRH) in normal human pancreas was examined immunohistochemically with four different anti-GHRH sera. Antisera raised aginst synthetic human GHRH(1-40)OH and GHRH(1-29)Gly4-Cys-NH2 gave no positive immunoreaction. Two other antisera (RG107 and #4676) raised against synthetic human GHRH(1-44)NH2 reveal GHRH-like immunoreactivity in pancreatic polypeptide (PP) cells, but the iminunoreactivity was abolished when RG107 or #4676 was absorbed by synthetic human PP(1-36)NH2- Among sixteen pancreatic endocrine tumors, only one tumor from an acromegalic patient contained many cells that reacted with all four anti-GHRH sera; this immunoreactivity disappeared by the addition of synthetic GHRH(1-44)NH2 but not of PP(1-36)NH2- The remaining fifteen tumors, which included eleven PP-positive tumors, did not show any immunoreactivity against the four anti-GHRH sera. The findings suggest that the anti-GHRH-44 sera (RG107 and #4676) contain not only an antibody for GHRH but also antibody that recognizes PP or an antibody that recognizes both GHRH and PP, and also that cells with genuine GHRH immunoreactivity are absent in normal human pancreas. In addition, it is suggested that the molecular structure of PP in normal pancreas differs from that in pancreatic endocrine tumors.
As one approach to examining the molecular functions of atrial natriuretic peptide (ANP), we examined the binding capacities of four human ovaries, one with a developing follicle of 7 mm (a) in diameter, two preovulatory follicles (small one of 12 mm (b) and large one of 18 mm (c) in diameter just before ovulation) and one (d) at menopause. The number of ANP binding sites of the ovary with a Graafian follicle (c) was far greater than those previously observed in various tissues. Four ovaries showed a drastic difference in concentrations of ANP binding sites. The binding capacity of membrane fractions from the ovary with a Graafian follicle just before ovulation (c) was 18 times greater than that of the ovary at menopause (d), 7.2 times that of the developing follicle (a), and 3.8 times that of the ovary with a preovulatory follicle (b). However, the dissociation constant (Kd) values showed no major difference at any stage among three ovaries except ovary at menopause. These findings suggest that ANP is involved in events related to the reproductive process.
1) We investigated the interactions between troponin C and troponin T, and between calmodulin and troponin T, in the presence of Ca2+ and Sr2+. Polyacrylamidegel electrophoresis under non-denaturing conditions and ultracentrifugation methods was used to examine the interactions. Calmodulin and skeletal troponin C bound to skeletal troponin T only in the presence of bivalent cations, whereas calmodulin and cardiac troponin C bound to cardiac troponin T in both the presence and absence of bivalent cations. 2) Skeletal troponin C bound to the skeletal troponin T-tropomyosin-actin complex only in the presence of bivalent cations, although cardiac troponin C hardly bound to the cardiac troponin T-tropomyosin-actin complex even in the presence of bivalent cations. 3) Calmodulin bound to the skeletal and cardiac troponin T-tropomyosin-actin complexes only in the presence of bivalent cations. 4) Trifluoperazine, a calmodulin antagonist, inhibited the bivalentcation-dependent interaction between calmodulin and skeletal troponin T. 5) The results suggest that the conformation of cardiac troponin T coupled with the tropomyosin-actin complex differs considerably from that of troponin T alone, and that the interaction between troponin C and troponin T does not play a major role in the troponin regulation of muscle contraction. The bivalent-cation-induced exposure of the hydrophobic region may enable calmodulin to bind to skeletal troponin T.
The effects of bromodeoxyuridine (BrdU), an analogue of thymidine, on the DNA synthesizing cells of the rat fetal brain were examined at the stage of neuroblast formation. BrdU in the cell nuclei was clearly detected by avidin-biotin complex peroxidase method using an anti-BrdU monoclonal antibody in paraformaldehydefixed, paraffin-embedded tissue sections 27 min after the intravenous administration of BrdU. The sufficient dose of BrdU required for immunohistochemistry was 20 mg/kg of body weight. By this method, the elevator movement of matrix cell nuclei and the migration profiles of the neuroblasts in the migrating zone and cortical plate were demonstrated. The labeling indices of the matrix cells in the cerebral neopallium during the neuroblast formation gave essentially equal or even higher values than those previously reported by tritiated thymidine autoradiography. BrdU incorporated at the embryonic stage was also detectable in 3-month-old rats. Rats that received 20-60 mg/kg BrdU on the embryonic day 16 revealed no significant abnormalities in behaviors and morphology. The results suggest that the BrdU immunohistochemical method is a useful tool in cell kinetic studies ofthe developing nervous system in vivo.
We examined the interaction between a protein-bound polysaccharide (PSK) and rabbit skeletal muscle myosin. PSK inhibited the binding of tubulin to myosin in a concentration-dependent manner, whereas it had small effect on actin-myosin interaction as shown by co-sedimentation methods. Native and PSK-bound myosins were subjected to limited proteolysis with trypsin or chymotrypsin. The digestion patterns were similar between native and PSK-bound myosins. However, PSK binding delayed the digestion of 200 and 130 kDa fragments of heavy chain; also DTNB light chain was not digested by chymotrypsin in the presence of PSK. Electron microscopic observations indicated that myosin filaments in the presence of PSK appeared to be linked to each other and were aligned roughly parallel to the long axis.
1,2,3,4-Tetrahydroisoquinoline (TIQ) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) were detected in two regions (frontal lobe and caudate nucleus) of eight human brains, four from the control and four from patients with parkinsonism. The TIQ content did not significantly differ between control and parkinsonian brains in both regions. In contrast, the 1MeTIQ content was markedly reduced in the parkinsonian brain, particularly in the frontal lobe, suggesting an important role of this amine in parkinsonism.
Chemical synthesis of pancreastatin fragments (33-49), (39-49), (42-49) and (45-49) was carried out using a solid-phase synthesizer, and antisera were characterized. The synthetic pancreastatin (33-49) coupled with keyhole limpet hemocyanine (KLH) was used as immunogen and antibodies were produced in New Zealand white rabbits. One of the antisera, T-2602, which showed the highest titer, recognized the C-terminal region of pancreastatin and cross-reacted equally with both pancreastatin (33-49) and native pancreastatin, but not with other known peptide hormones. Gel-filtration analysis of aliquots of tissue homogenates revealed the existence of four major components of pancreastatin immunoreactivity which corresponded to the elution position for pancreastatin precursor (void volume), native pancreastatin, pancreastatin (33-49), and related small peptide fragment in porcine pancreas. Analysis of tissue homogenates from the porcine adrenal gland revealed a predominant large-molecular form and a small amount of native pancreastatin. The immunohistochemical study using the antiserum T-2602 showed widespread immunoreactivity in amine/peptide producing endocrine cells. Not only pancreatic islet cells but also gut endocrine cells and adrenal chromaffin cells showed intense immunoreactivity in their secretory granules. As this immunoreactivity precisely coincided with that for chromogranin A, it is suggested that pancreastatin is a peptide closely correlated with chromogranin A.