Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione

A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion-exchange chromatography with a FPLC system and reversed-phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N-terminal amino group was not blocked, peptide sequencing with common techniques was not possible. Comparison of the isolated peptide with the well-known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identity of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10-100 nM, a so far unknown property of this well-known peptide.     

Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells. Evidence for increased degradation of catalytic subunit in the presence of cAMP

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.     

Hormonal asynchrony and embryonic development

Luteinizing hormone (LH), progesterone and estradiol profiles in peripheral blood serum were compared among parous and nonparous females with normal, abnormal or no embryonic development. Hormonal profiles between parous and nonparous females of the same embryonic status did not differ and the data were combined. Estrous cycle length was longer (P<.05) in parous (22.3±.4 days) than nonparous females (21.0±.4 days). Females with normal developing embryos had a higher serum progesterone concentration at Days 3 and 6 and a lower ratio of estradiol to progesterone than did females with abnormal embryonic development. Females with a normal embryo had higher (P<.05) preovulatory LH peaks than females with abnormal development or no recovery of an oocyte or embryo (34.3±4.7, 11.8±6.8 and 13.3±2.5 ng/ml, respectively). The interval from onset of estrus to LH peak was 8.9±2.1, 13.7±3.7 and 13.5±6.2 hr for females with normal, abnormal or no recovery of an embryo. The lower LH peak, the longer interval from onset of estrus to LH peak, and lower progesterone concentration in peripheral blood serum in females with abnormal embryos or no recovery indicated that these females had a hormonal asynchrony. The hormonal asynchrony may produce an undesirable uterine environment for male and female gametes or embryos which resulted in fertilization failure or embryonic death. In the second experiment, more transferable embryos were obtained when superovulated females received prostaglandin F_2α (PGF_2α) intravenously rather than intramuscularly. Administering PGF₂α intravenously rather than intramuscularly may have caused the demise of the corpus luteum sooner and thereby produced a more normal uterine environment which allowed more embryos to develop normally.     

Structure of the rat prolactin gene

The organization and sequence of the rat preprolactin gene has been investigated. Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon. Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments. The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments. The sequence of more than 1800 nucleotides of the cloned DNA has been determined. The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin. These coding regions are separated by an intervening sequence of 597 nucleotides. At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin. Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Transit times through the cycle phases of jejunal crypt cells of the mouse. Analysis in terms of the mean values and the variances.

Mean transit times as well as variances of the transit times through the individual phases of the cell cycle have been determined for the crypt epithelial cells of the jejunum of the mouse. To achieve this the fraction of labelled mitoses (FLM) technique has been modified by double labelling with [3H] and [14C]thymidine. Mice were given a first injection of [3H]thymidine, and 2 hr later a second injection of [14C]thymidine. This produces a narrow subpopulation of purely 3H-labelled cells at the beginning of G2-phase and a corresponding subpopulation of purely 14C-labelled cells at the beginning of the S-phase. When these two subpopulations progress through the cell cycle, one obtains FLM waves of purely 3H- and purely 14C-labelled mitoses. These waves have considerably better resolution than the conventional FLM-curves. From the temporal positions of the observed maxima the mean transit times of the cells through the individual phases of the cycle can be determined. Moreover one obtains from the width of the individual waves the variances of the transit times through the individual phases. It has been found, that the variances of the transit times through successive phases are additive. This indicates that the transit times of cells through successive phases are independently distributed. This statistical independence is an implicit assumption in most of the models applied to the analysis of FLM curves, however there had previously been no experimental support of this assumption. A further result is, that the variance of the transit time through any phase of the cycle is proportional to the mean transit time. This implies that the progress of the crypt epithelial cells is subject to an equal degree of randomness in the various phases of the cycle.     

Seasonal fluctuation of zooplankton populations in lower Delaware Bay

Quantitative zooplankton samples were obtained monthly or bi-monthly 15 times from June 1974 to May 1975 at three stations in lower Delaware Bay. Two 12-hour cruises were also conducted at one of the stations.Arthropods dominated the samples in terms of number of species and number of individuals. The number of zooplankton from surface samples ranged from 58/m³ in August to 21,092/ m³ in June, while bottom samples varied from 259/m³ in August to 30,395/ m³ in October. In general, larger concentrations of individuals were found in bottom samples.Only on three occasions did meroplankton exceed the holoplankton, and these occurred at the shallow water stations. Meroplankton comprised a larger percentage of the bottom samples than surface samples. Zoeae of Neopanope sayi and Uca sp. contributed mainly to the large proportion of meroplankton in July 1974, veligers of Mytilus edulis in January 1975, and nauplii of Balanus sp. in May 1975.Copepods were the largest component of the population throughout most of the year. At all stations and depths, Arctica tonsa dominated most of the summer samples. In the spring of 1975, A. tonsa was replaced by Centropages hamatus, Temora longicornis, and Pseudocalanus minutus.During the 12-hour cruises there were higher numbers of individuals in the bottom waters in the day with migration to surface waters in the afternoon and evening. Based on cluster analysis, five time-related assemblages were discerned: June, July–August, September–November, December, January–May. Comparison of Delaware Bay zooplankton with other estuarine systems indicated that the densities obtained locally were most similar to those reported in the York River, Virginia.     

The immune response of allophenic mice to the synthetic polymer GLΦ

The immune response of allophenic mice of type C57BL/6(A × SJL) F₁ to GL administered in complete Freund's adjuvant was tested. Control mice of the three strains C57BL/6, A, and SJL are all nonresponders to this antigen. However, the F₁ generations of C57BL/6 × A, C57BL/6 × SJL, and A × SJL were all responders to the antigen, so that the complementarity of at least two genes is confirmed. The allophenic mice showed no further complementation beyond the F₁ generation, a result which may argue against the possibility that more than two genes control the response to GL in these mouse strains. Characterization of the allophenic mice over several months showed that they exhibit chimeric drift, both in their coat color and in peripheral white blood cell population. There is no apparent correlation of coat color to the lymphocyte composition of the mice at any one time. The mice are true chimeras, since killing of the two populations of white blood cells with two different anti-H-2 sera produced a 100 percent killing. The immune response of individual allophenic mice to GL showed a good correlation to the number of A × SJL lympho-cytes in the animal.Abbreviations used in this paper are GL an amino acid polymer of 57 %l-glutamic acid, 38%l-lysine, and 5%l-phenylalanine - GLT¹⁵ an amino acid polymer ofl-glutamic acid,l-lysine, and 15 %l-tyrosine - (T,G)-A-L an amino acid polymer having a polylysine backbone with side chains of polyd-l-alanine, terminating in short sequences of tyrosine and glutamic acid - GAT₁₀ an amino acid polymer of 60%l-glutamic acid, 30%l-alanine, and 10%l-tyrosine - GLA₅ an amino acid polymer of 57%l-glutamic acid, 38%l-lysine, and 5%l-alanine - DNP 2,4 dinitrophenyl - BGG bovine gamma globulin - FCS fetal calf serum - PWBC peripheral white blood cell - SWBC spleen white blood cell - T cell thymus-derived lymphocyte - B cell bone marrow-derived lymphocyte     

Cell-free synthesis of a large translation product of prolactin messenger RNA.

RNA prepared from rat anterior pituitaries or from prolactin-secreting pituitary tumors has been shown to direct the synthesis of a large form of prolactin in a cell-free system derived from wheat germ. Immunoprecipitation of cell-free reactions demonstrated the synthesis of a product which was recognized by a specific antiprolactin antisera. Analysis of the immunoprecipitate on sodium dodecyl sulfate containing polyacrylamide gels suggested that the cell-free product has a molecular weight of approximately 28,000 compared to 22,500 for prolactin. RNA prepared by completely different techniques from rat pituitary and a pituitary tumor resulted in identical large translation products. Translation of tumor RNA in a cell-free system from Krebs ascites cells also resulted in a similar large product. The identity of the cell-free product as prolactin was confirmed by comparing peptides derived from the cell-free product and prolactin. The results of these studies suggest that prolactin messenger RNA directs the cell-free synthesis of a product which contains the amino acid sequence of prolactin but which has an addition at one or both ends of the molecule.