Viability tests of cryopreserved endocrine pancreatic cells

Collagenase-isolated islets, which had been cultured for 1 week, were frozen at two different cooling rates. Islets frozen at 5 degrees C/min behaved from a functional point of view very similarly to that of nonfrozen, cultured control islets, except for a reduced maximal insulin secretory capacity and a reduced insulin content. Slowly frozen islets (0.5 degrees C/min), however, displayed reduced rates of both proinsulin biosynthesis and glucose oxidation. It is concluded that isolated islets can be cryopreserved with great success and that the methods of choice for viability tests are those characterizing the dynamics of insulin secretory capacity of the cryopreserved islets.     

Structural and biochemical aspects of the effects of various media used for isolation and fractionation of mouse liver nuclei

We have carried out an electronmicroscopic study of replicas of the underside of culture cells. The techniques developed for the preparation of such replicas are described in detail. We find that the cells do not rest on the surface of the culture dish itself, but rather are attached to a protein film adsorbed from the culture medium. The underside of cells is usually rather smooth looking. It is difficult to clearly define the site of attachment of cells to their substrate. The features interpreted as attachment sites are often bored and diffuse. Heavy bundles of micro-filaments can be recognized in the sole plate of BHK cells. These bundles are usually parallel to the long axes of the cells, and often terminate in cell projections. They are only rarely encountered in the sole plate of transformed BHK cells.     

Distribution and shifts of ingested marker particles in residual bodies and other lysosomes. Studies on in vitro cultivated human glia cells in phase II and 3

Human glia cells approaching phase III in vitro were exposed to thorium dioxide particles. The intracellular distribution of the markers was studied by electronmicroscopy after intervals ranging between 15 min and 3 days. In a second experiment glia cells in phase II were labelled and kept at confluence for time periods varying between 1 day and 22 weeks before they were studied ultrastructurally.The findings showed that secondary lysosomes form part of a vacuome which, by fusion and fission, allows material within membrane-limited vacuoles to spread in the system, although it is in any moment discontinuous. The results further indicated that residual bodies form an integrated part of this vacuome and probably regularly receive lytic enzymes by fusion with other types of lysosomes. The rate of extrusion of residual bodies or exocytosis of indigestible material was found to be low, if it occurred at all in the cell strains examined.     

The isolation and properties of mitochondria from rat pancreas

A technique for the isolation of functional rat pancreatic mitochondria is described. The resultant mitochondrial preparations contained oligomycin-insensitive Mg²⁺-ATPase activity and coupled respiration could only be demonstrated in the absence of Mg²⁺ and in the presence of EDTA.     

GABA and endocrine regulation—Relation to neurologic-psychiatric disorders

Data obtained with experimental animals and with humans concerning the involvement of GABA-ergic systems in the control of hormonal secretion have been discussed and analyzed. The available evidence indicates that GABA-ergic systems might modulate the release of several hypothalamic-hypophyseal hormones that are involved in behavioral regulation, either via their endocrine actions or via their direct actions on the brain. Further studies along this line might lead to the development of GABA-ergic drugs that will be useful for treating certain hormonal or neuropsychiatric disorders.     

Changes in inducibility of ornithine decarboxylase activity in differentiating human neuroblastoma cells

Human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), or a combination of these two substances. The phenotypical changes induced by these substances differed, but one effect of both was an inhibition of the cell growth. Addition of TPA or RA to non-treated cells had no effect on the activation of ornithine decarboxylase (ODC, EC 4.1.1.17.), while a change to fresh medium stimulated the ODC to maximum activity after 4-6 h. The activity was not altered by the presence of RA in the fresh medium, but TPA partially inhibited the medium-stimulated ODC activity. Cells treated for 4 or 8 days with TPA or a combination of TPA and RA had a low ODC activity which could not be induced by fresh medium. However, RA-treated (and thus growth-inhibited) cells still responded to a change of medium by exhibiting an ODC activity of the same magnitude and duration as in medium-stimulated control cells. The results seem to suggest that the growth inhibition induced by TPA and RA, respectively, is mediated by different mechanisms.     

Mammary serum factor initiates cell growth in 3T3 cell variants which are non-responsive to EGF

The structural organization of the histone-containing spermatozoa of the goldfish C. auratus is studied. Chemical fractionation shows that the five standard histones are present. Histone H1 subfractions are also similar to those found in other organisms. This is the first organism studied in which no peculiar basic protein is found in spermatozoa, since in all the organisms described up to now, unique protein components are always present, either alone or accompanied by somatic-like histones. Ultrastructural studies show that this chromatin is organized as a bundle of fibers of about 25 nm diameter, which upon spreading give the typical ‘beads-on-a-string’ appearance. Nuclease digestion demonstrates a repeat length of 205 base pairs (bp), slightly longer than in somatic tissues, but shorter than in echinoderm spermatozoa.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Modulation of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithelial cell line

Our recent studies have shown that chick embryo epiphyseal cartilage synthesizes three distinct species of proteoglycan (PG-H, PG-Lb, and PG-Lt) which are analogous in having glycosaminoglycan side chains of the chondroitin (dermatan) sulfate type but different from one another in regard to the structure of core protein. In the present report, the expression of PG-H and PG-Lb has been studied in developing chick hind limbs (stages 19-33), using antibodies specific for these substances in indirect immunofluorescence. At the onset of cartilage morphogenesis (stage 24), PG-H became recognizable in the cartilage primordia, whereas a parallel section stained for PG-Lb showed no reaction. The first evidence of PG-Lb appearance was seen in a stage 28 cartilage (e.g., tibia) in which the cells in the middiaphysis became elongated in a direction perpendicular to the long axis of the cartilage. The PG-Lb fluorescence was confined to the zone of these flattened, disc-like cells, whereas the fluorescence for PG-H was uniformly distributed throughout the cartilage. With further development of cartilage (stage 29 approximately), the zone of flattened cells spread proximally and distally, and simultaneously large hypertrophied cells appeared at the diaphyseal region. During these zonal changes of cell morphology, the PG-Lb fluorescence remained restricted to the zone of flattened cells. Parallel sections stained for PG-H, in contrast, showed an evenly distributed pattern of the PG-H fluorescence throughout the cartilage. The results indicate that the appearance of PG-Lb is closely associated with the zonal changes of cell shape and orientation along the proximal-distal axis of the developing limb cartilage, and further suggest that the flattened chondrocytes in this particular zone have undergone additional changes in gene expression to form an extracellular matrix of still another chemical property.     

Induction of fructose 1,6-bisphosphatase and glucose 6-phosphatase in fetal mouse liver

Fructose 1,6-bisphosphatase and glucose 6-phosphatase were induced in organ cultures of liver tissues from 15- and 19-day-old fetal mice, using a culture method that allowed the tissues to be maintained for 7 days in the absence of serum. In cultures from 15-day-old fetal liver, both enzyme activities increased significantly per milligram of DNA after a lag period of 1 to 3 days. In cultures from 19-day-old fetal liver only glucose 6-phosphatase increased in the absence of inducer. N6,O2'-Dibutyryladenosine 3',5'-monophosphate enhanced the rate of increase in fructose 1,6-bisphosphatase and glucose 6-phosphatase activities. The minimum effective concentration of the cyclic nucleotide was approximately 10(-6) M. Dexamethazone inhibited the increase in fructose 1,6-bisphosphatase during culture for 7 days. Glucose 6-phosphatase activity was enhanced by dexamethazone in cultures from 19-day-old fetal liver, but was without effect on glucose 6-phosphatase in cultures from 15-day-old fetal liver. The minimum inhibitory concentration of dexamethazone was less than 10(-8) M. The results suggest a complicated effect of the cyclic nucleotide on the two enzymes in fetal mouse liver as well as different mechanisms of action of dexamethazone on the induction of two enzymes.     

Characterization of microsomal glycosyl-transferases of rat pancreas and influence of diets

Four rat pancreatic microsomal glycosyl-transferases (fucosyl-, galactosyl-, mannosyl- and N-acetylglucosaminyl-transferases) are studied and characterized for their optimal conditions and their relation with interfering reaction (glycosyl-nucleotide pyrophosphatases, osidases and proteinases). Dietary treatments of the rats induce modification: for all the transferase activities, the highest levels are found in a high-starch diet and the lowest one in a high-fat diet. The activities found in the standard diet are at the level of the high-starch or of the high-fat diet depending on the enzyme studied. The observed modifications are not explained by alterations in physico-chemical parameters of the enzymes or by intervention of glycosyl-nucleotide pyrophosphatases, osidases or proteolytic enzymes. The modifications observed for the mannosyl-transferase are predominantly found in a lipid fraction extracted by chloroform-methanol ($\text{2}\text{1}$).     

Cyclic nucleotide phosphodiesterase and protein activator in fetal rabbit tissues.

Cyclic nucleotide phosphodiesterase activity (EC 3.1.4.17) was studied in fetal and newborn rabbit brain, heart, liver, kidney, and lung. Kinetic analysis of phosphodiesterase activity from homogenates of organs from the 25-day embryo suggested the presence of a high K_m and a low K_m activity for both cyclic AMP and cyclic GMP hydrolysis. The addition of 1 μm cyclic GMP to the assay stimulated the hydrolysis of cyclic AMP by whole homogenates of liver, brain, lung, and kidney, but not heart, at all of the ages studied. The addition of micromolar levels of calcium ion stimulated cyclic GMP hydrolysis by homogenates of fetal brain, heart, and kidney, with or without added protein activator. Cyclic GMP phosphodiesterase activity was not stimulated by the addition of calcium ion in homogenates of early fetal rabbit liver and lung, but stimulation was detected in the late embryo and newborn. The presence of the heat-stable protein activator was demonstrated in brain, heart, kidney, liver, and lung tissue at all of the fetal ages studied, and in the newborn rabbit. DEAE-cellulose chromatography demonstrated the presence of three separable enzymes in brain and liver at 15 days, heart at 19 days, and lung and kidney at 25 days of gestation, with no changes in the kinetic properties of the isolated enzymes during development. These experiments suggest that all of the organs studied have the mature array of phosphodiesterases early in development, but an enzyme from liver and lung becomes sensitive to regulatory control by calcium only late in gestation.     

The complexing of yeast transfer-RNATyr by tyrosyl-transfer-RNA synthetase of hog pancreas.

Using methyl-tryoctyl-ammonium chloride (which is soluble in cyclohexane and insoluble in water) it is possible to transport α-chymotrypsin in 20% yield from a water solution to a supernatant cyclohexane solution. The spectroscopic properties of the protein in the aprotic phase are investigated. On the basis of these spectroscopic data, it is argued that under certain conditions no extensive denaturation of the protein takes place in cyclohexane in the presence of the ammonium salt. The possible reason for this unexpected finding and its implications, are discussed.     

Molecular interactions in human T-cell-mediated cytotoxicity to Epstein-Barr virus. I. Blocking of effector cell function by monoclonal antibody OKT3

The ability of different anti-human T-cell lymphocyte monoclonal antibodies to inhibit the effector function of the cytotoxic T-cell response against autologous Epstein-Barr virus (EBV)-infected B-cell targets has been tested. It was found that monoclonal antibody, OKT3, which reacts with most human T cells, blocks the effector cell function in the absence of complement, an effect that was dose dependent. When monoclonal antibody OKT3 was tested at a concentration of 1 μg/ml, inhibition of cytotoxicity ranged between 50 and 80%. The F(ab′)₂ fragment of OKT3 inhibited as well as the intact IgG molecule, indicating that the Fc portion of the antibody is not necessary for the cytotoxicity blocking. The Fab fragment of OKT3 had lower blocking activity per microgram of protein tested. Antibodies SC1, OKT11 (anti-pan T cell), OKT8 (anti-cytotoxic/suppressor subset), and L368 (anti-HLA) did not have any discernible blocking effects. However, antibodies SC1, OKT8, and L368 could abrogate the cytotoxic activity in the presence of complement. Blocking by OKT3 was not due to its being present on the cell surface in higher concentrations than the other monoclonal antibodies since cytofluorographic analysis demonstrated that the amount of OKT8 or L368 antibodies bound on the cells was greater than OKT3. In addition, blocking was not due to antigenic modulation since incubation with antibody OKT3-F(ab′)₂ was not associated with a significant decrease in the amount of its reactive antigen. Under the conditions tested OKT3 did not affect cell viability or cause agglutination.     

Structure and flanking regions of soybean seed protein genes

We have characterized the structure and flanking region of genes representing two, coordinately expressed, soybean seed protein gene families. One family directs the synthesis of the major storage protean glycinin; the other encodes a 15.5 kd polypeptide of unknown function. DNA blot hybridization experiments showed approximately three, nonallelic genes in the glycinin family and two in the 15 kd protein family, and showed that these families are not selectively amplified or rearranged during embryogeny. R-loop and S1 nuclease mapping studies demonstrated no detectable introns in the 15 kd protein genes but at least one and possibly two in the glycinin genes. No interfamily clustering of these genes occurs within a 10-15 kb chromosomal domain. Nor are they contiguous to other genes expressed at moderate levels during embryogenesis. Each of them, however, is contiguous to a gene expressed at another developmental period in the leaf. These leaf genes encode rare class messages which constitute only 1 X 10(-5%) of the leaf mRNA, or about one molecule per cell. R-loop analysis of two leaf genes showed that one contains no detectable introns while the other possesses at least three. DNA gel blot studies showed that only one of the seed protein genomic clones contains an interspersed repetitive DNA element. Pairwise cross-hybridization studies did not detect any flanking sequences shared by the 15 kd protein, glycinin and leaf genes.     

Behavioural and endocrine responses of female mice to synthetic analogues of volatile compounds in male urine

The urine of intact, adult male mice elicits more investigatory sniffing from female mice than does the urine of castrated males. When either of two androgen-dependent urinary compounds, 2-sec-butyl dihydrothiazole or dehydro-exo-brevicomin are added to castrate urine, its relative attractiveness remains the same. When both compounds are added to castrate urine, however, its activity is enhanced and the castrate urine becomes as attractive to females as whole, intact male urine. Females exposed to the reconstituted ‘normal’ urine for 3 min per day, displayed more frequent oestrus cycles. The two synthetic compounds are synergistic in the context of castrate urine, producing an olfactory message that behaviourally and physiologically mimics the activity of the normal biological signal.     

Differences in iodinated peptides and thyroid hormone formation after chemical and thyroid peroxidase-catalyzed iodination of human thyroglobulin

The distribution of iodine among the polypeptides of human goiter thyroglobulin (Tg) was examined. Tg was iodinated in vitro with ¹³¹I to levels of 2 to 84 gram atoms (g.a.)/mol using thyroid peroxidase (TPO) or a chemical iodination system. The samples were reduced, alkylated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two low-molecular-weight peptides appeared preferentially in radioautograms of the sodium dodecyl sulfate (SDS) gels of TPO-iodinated samples. Iodination of these peptides increased sharply in the TPO-treated Tg as the level of total iodine/ molecule rose. Radioiodine was incorporated into these same gel regions in the chemically treated Tg, but only after much higher levels of total iodination were reached. Differences in iodoamino acid distribution were also noted between the chemically and enzymatically iodinated thyroglobulins. In the chemically iodinated samples, little thyroxine (T₄) was synthesized, even at high iodine levels. In the TPO-treated samples only small amounts of T₄ were seen below 14 g.a. total I/mol, while at or above that level of iodination T₄ formation increased sharply. To examine the coupling process, Tg was chemically iodinated, excess I^? removed, and the samples treated with TPO and a H₂O₂-generating system in the absence of iodide. Radioautograms obtained from SDS-polyacrylamide gels of reduced and alkylated protein from such coupling assays showed an increase in the level of iodine in the low-molecular-weight peptides after TPO treatment. Thyroxine production also increased with TPO treatment. The addition of free DIT (a known coupling enhancer) to the [¹³¹I]Tg/TPO incubation increased both the production of T₄ and the amount of iodine in the smaller polypeptides. Two-dimensional maps prepared from CNBr-digested TG showed differences between the coupled and uncoupled samples. Our observations confirm the importance of the lowmolecular-weight peptides derived from Tg in thyroid hormone synthesis. At total iodine levels above 14 g.a./mol Tg in enzymatically treated samples there is selective incorporation of iodine into both the low-molecular-weight polypeptides and into thyroid hormone.     

Elevated intramitochondrial adenine nucleotides and mitochondrial function

Several groups of investigators have shown that treatment of rats with glucagon produces an increase in the adenine nucleotide content of hepatic mitochondria. It has been suggested that this enlarged pool of exchangeable nucleotides may be responsible for several of glucagon's stimulatory effects on mitochondrial functions by accelerating the transport of adenine nucleotides across the inner mitochondrial membrane. This hypothesis was tested by loading rat liver mitochondria in vitro with adenine nucleotides to supranormal levels. This procedure did result in stimulation of several metabolic and bioenergetic functions including pyruvate carboxylation, uncoupler-dependent ATPase, and succinic dehydrogenase activity but not formation of citrulline. However, a sham loading that did not increase the nucleotide content of the mitochondria was essentially as effective as the loading procedure in stimulating those functions assayed. Mitochondria, loaded in vitro with supranormal levels of adenine nucleotides, were shown to have an enlarged pool of exchangeable nucleotides. This exchange was atractyloside sensitive, but the rate of exchange was only slightly increased as a consequence of enlargement of the pool. Similarly, mitochondria isolated from glucagon-treated rats showed no increase in the rate of exchange, although the exchangeable pool was increased. There was no correlation between the rate of nucleotide exchange and the rate of the uncoupler-dependent ATPase.     

Lack of circadian rhythmicity of rat fetal intestinal enzymes as compared to the dams

The 24-hr activity patterns of intestinal maltase, lactase, leucylnaphthylamine hydrolyzing activity, γ-glutamyltransferase, and alkaline phosphatase were determined in pregnant rats maintained on a 12-12 light-dark cycle, with feeding during the dark period (1800-0600 hr, EST). The activities of these enzymes plus those of lysosomal maltase and lactase were followed during the same time period in 19- to 20-day-old fetuses. The activity patterns in the dams followed circadian rhythms, with peak activities occurring during the feeding-dark period. These rhythms are similar to the feeding schedule-cued rhythms observed in male rats and, therefore, are assumed to be feeding schedule cued also. In the fetuses, which obtained nutrients through the placenta, the activities increased in a somewhat nonlinear manner throughout the entire 24-hr period, but did not display a defined rhythm. It is concluded that endogenous intestinal enzyme rhythms do not exist in utero, and that oral and/or intermittent feeding is necessary for these rhythms to occur.