Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose

To evaluate the influence of cell density on the activity of fibroblast prolidase (EC 3.4.13.9), we determined this activity in sparse and dense cultures. We also investigated, the effects of different concentrations of β-d(?) fructose and l(+) ascorbate, which both increased cell density at confluency. For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5-fold and prolidase specific activity, 1.8-fold. For ascorbate, a broad optimum concentration was found (range 0.01 – 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4-fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27–31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density.     

Interaction of Neuropeptides and Cultured Glial (Müller) Cells of the Chick Retina: Elevation of Intracellular Cyclic AMP by Vasoactive Intestinal Peptide and Glucagon

Vasoactive intestinal peptide (VIP) and, to a lesser extent, glucagon were found to increase intracellular cyclic AMP rapidly in cultured glial (Müller) cells of the chick embryo retina. Although VIP elicited higher cyclic AMP accumulation than glucagon at each concentration tested, the half-maximal concentrations were similar, i.e., 6 X 10(-8) M for VIP and 8 X 10(-8) M for glucagon. Secretin had a minimal effect on cyclic AMP accumulation even at a very high (5 X 10(-6) M) concentration. Several other peptide and nonpeptide putative agonists also had little effect on cyclic AMP accumulation. The cultured Müller cell may thus be a useful model for examining VIP and glucagon effects on glial elements of the CNS.     

Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes,and in leukemic cells

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained.At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP.At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.Abbreviations dCMP deaminase deoxycytidylate deaminase - PHA Phytohemagglutinin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - AMOL acute monocytic leukemia - WBC white blood cells     

A new procedure for rapidly scoring acrosome reactions of human sperm

Because the acrosome of human sperm is too small to be directly visualized by phase-contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome-reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein-conjugated Ricinus communis agglutinin-60 (FITC-RCA); we show that in sperm without acrosomal caps, FITC-RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC-RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC-RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC-TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome-reacted human sperm.     

Identification of up-regulated genes in human uterine leiomyoma by suppression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.     

Culture of human corpus cavernosum endothelium

Summary A method for culturing endothelial cells (HCC-EC) from surgical specimens of human corpus cavernosum has been developed. The approach involves selective endothelial outgrowth from explants and may be generally applicable to tissue whose endothelium is not amenable to isolation by routine mechanical or enzymatic methods. The tissue is minced into pieces which are placed onto gelatin-or fibronectin-coated tissue culture plastic, and grown in medium suitable for microvascular endothelial cell growth (Carson and Haudenschild, In Vitro 22:344–354, 1986). By Days 5 to 7 EC colonies are found. Within a day or two after the appearance of the EC colonies, a non-EC cell type appears and, if undisturbed, quickly overgrows the EC. An exploitable temporal separation between the emergence of EC and non-EC is obtained when both conditioned medium (from bovine aortic endothelium) and retinal extract are present during the outgrowth period. Explants are removed by pipetting at the first sign of the emergence of the non-EC cell type. Once isolated, HCC-EC do not require conditioned medium but do require either retinal extract or acidic fibroblast growth factor for survival and growth. Approximately 60% of the first passage cultures are at least 80% EC as judged by DiI-Ac-LDL labeling. One corpus (0.3×0.3×0.5 cm) usually produces 120 cm² of primary culture within 2 wk. These EC form contact-inhibited monolayers and stain positively for Factor VIII. They have a doubling time at 6th passage of 48 h and a plateau density of 5 to 7×10⁴ cells/cm². The availability of such cultures should facilitate the study of endothelium-mediated responses which play an important role in the erectile function of human penile corpus cavernosum. Supported by NIH R01 HL23567-09, DK-39080-01, DK40025-01, DK40487-01.     

Isolation of inhibin like peptides from human placenta

Two moieties of inhibin could be obtained by chromatography of partially purified preparations of inhibin from human placenta on Sephadex G-100, G-25 and ion exchange chromatography on diethylaminoethyl Sephadex A-50. The higher molecular weight moiety (14,000) designated as HPI-H appears to be similar to inhibin from human seminal plasma. While the lower molecular weight moiety (1500) designated as HPI-L appears to be similar to that of sheep testicular inhibin. The preparations from both human term placenta and human seminal plasma inhibited the binding of [¹²⁵I] human follicle stimulating hormone to rat testicular receptors. This effect of inhibins could be neutralized by antisera raised against corresponding polypeptide. Further these antibodies could neutralize endogenous inhibin resulting in 2 to 3 fold increase in serum follicle stimulating harmone levels, which could then be reversed by exogenous administration of the isolated inhibin preparations.