JAR human placental choriocarcinoma cells actively synthesize,take up and release polyamines

Uptake of polyamines has been investigated extensively in many cells, but not in placenta, where the polyamine– polyamine oxidase system is supposed to have an immunoregulatory function in pregnancy. Due to the importance of the transfer in this tissue, we have started this study. JAR human placental choriocarcinoma cells in monolayer at confluency were used as a model for measuring the key enzymes of polyamine synthesis and interconversion, rate of uptake and efflux, and the polyamine content. Polyamines were taken up by JAR cells and released by an independent mechanism. Ornithine decarboxylase and spermidine acetyltransferase activities and the rate of transport in and out of the cell were much higher than in other cells, such as L1210 cells. However the systems used for uptake and release appear in many respects to be similar to those observed in L1210 cells, but different from others. The uptake appears to be regulated by an inhibitory protein. Moreover, protein kinase C appears to be involved in the process. The efflux also is regulated as in L1210 cells, through control of H⁺ and Ca²⁺ concentration. In conclusion, this study shows that, in JAR cells, ornithine decarboxylase and spermidine acetyltransferase activities were much higher than in other cells, and so was the rate of transport in and out of the cells. As a result, a much higher polyamine content was observed.     

Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Streptococcus faecium mutants that are temperature sensitive for cell growth and show alterations in penicillin-binding proteins.

The penicillin-binding proteins (PBPs) of 209 cell division (or growth) temperature-sensitive mutants of Streptococcus faecium were analyzed in this study. A total of nine strains showed either constitutive or temperature-sensitive conditional damage in the PBPs. Analysis of these nine strains yielded the following results: one carried a PBP 1 constitutively showing a lower molecular weight; one constitutively lacked PBP 2; two lacked PBP 3 at 42 degrees C, but not at 30 degrees C; one was normal at 30 degrees C but at 42 degrees C lacked PBP 3 and overproduced PBP 5; two were normal at 42 degrees C and lacked PBP 5 at 30 degrees C; one constitutively lacked PBP 5; and one carried a PBP 6 constitutively split in two bands. The mutant lacking PBP 3 and overproducing PBP 5 continued to grow at 42 degrees C for 150 min and then lysed. Revertants selected for growth capability at 42 degrees C from the mutants altered in PBPs 5 and 6 maintained the same PBP alterations, while those isolated from the strains with altered PBP 1 or lacking PBP 2 or PBP 3 showed a normal PBP pattern. Penicillin-resistant derivatives were isolated at 30 degrees C from the mutants lacking PBP 2 and from that lacking PBP 3. All these derivatives continued to show the same PBP damage as the parents, but overproduced PBP 5 and grew at 42 degrees C. These findings indicate that high-molecular-weight, but not low-molecular-weight, PBPs are essential for cell growth in S. faecium. This is in complete agreement with previous findings obtained with a different experimental system. On the basis of both previous and present data it is suggested that PBPs 1, 2, and 3 appear necessary for cell growth at optimal temperature (and at maximal rate), but not for cell growth at a submaximal one (or at a reduced rate), and an overproduced PBP 5 is capable of taking over the function of PBPs 1, 2, and 3.     

Modulation of the cell-mediated immune function by interferon α, β or γ can partially reverse the immunosuppression induced by human T-cell leukemia virus I in human cord blood cultures

Summary Infection with human T-cell leukemia virus type I (HTLV-I) is associated in vitro and in vivo with a remarkable depression of cell-mediated immune functions. In the present report it is shown that early events following virus-induced suppression of the cell-mediated immune response of freshly isolated cord blood mononuclear cells (CBL) infected with HTLV-I can be partially counteracted by treatment with interferons , or (IFN). All three types of IFN exerted a protective effect on CBL cultures exposed to the virus. This resulted in: (a) a reduced number of virus-positive cells until 4 weeks of culture; (b) delay in the clonal expansion of infected cells (IFN and ); (c) increased natural killer cell activity of CBL, 1 week post-infection (p.i.), mediated by IFN; (d) increase of allospecific recognition of infecting and priming HTLV-I donor MT-2 cells by CBL in a cytotoxic-T-lymphocyte-like response, mediated by IFN and particularly by IFN; (e) phenotype distribution of CBL subpopulations, tested 4 days p.i., more similar to that of non-infected CBL cultures.In contrast, the overall CBL proliferation, that is profoundly depressed during the first week p.i., was not restored by IFN treatments, suggesting that boosting of the cell-mediated killing induced by IFN might involve the maturation of undifferentiated precursor cells rather than stimulation of their proliferation. The improvement of the efficiency of the antiviral immune response induced by treatment with IFN is likely to contribute to the clearance of virus-positive cells during the early phase of infection. This would provide experimental evidence to support an immunopharmacological approach contributing to the conversion of HTLV-I carriers from positive to negative.     

Survey of allergic respiratory symptoms in grain terminal workers in the Port of Genoa

Summary Fifty workers from two grain elevator terminals were examined to evaluate the prevalence of respiratory symptoms and their relationship with grain dust exposure.Fifty per cent of subjects complained of respiratory symptoms (rhinitis, asthma, chronic cough and dyspnea on exertion). In 34% of the workers the ventilatory function revealed some abnormalities (slight or moderate obstruction).Twenty-two (44%) showed a positive cutaneous reaction to one or more of the allergens tested, mostly towardsDermatophagoides and storage mites. The self-measurement of PEF over 14 days in 27 workers, showed significant differences between symptomatic and asymptomatic subjects. In particular, the results obtained from the cutaneous reactions could suggest a bronchial hypereactivity, worsened by the working exposure to dusts. The monitoring of PEF appears to be a simple and useful method for investigating the relationship between respiratory symptoms and the working environment.     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.     

Calcium-activated, phospholipid-dependent protein kinase activity and protein phosphorylation in HL60 cells induced to differentiate by retinoic acid

Treatment of human promyelocytic (HL60) cells with retinoic acid for at least 48 h causes differentiation to more mature myeloid forms. Prior to commitment of cells to the myeloid pathway there is a marked increase in cytosolic calcium-activated, phospholipid-dependent protein kinase activity. This increase does not result from an intracellular redistribution of the enzyme. Concomitant with the increased enzyme activity there is enhanced phospholipid-dependent phosphorylation of proteins of 29, 49, 52, 58, 68, 69, 120, 170, 200 and 245 kDa.     

Partial purification and biochemical characterization of a T cell suppressor factor produced by human glioblastoma cells

The human glioblastoma cell line 308 constitutively secretes a soluble factor with biologic and biochemical characteristics of human monocyte-derived interleukin 1 (IL 1). The 308 cells also produce a 97,000 m.w. factor that inhibits the effects of IL 1 and interleukin 2 (IL 2) on T lymphocytes. By using sequential chromatography on Blue Affigel, hydroxyapatite, and Ultrogel AcA54, the inhibitory factor, termed glioblastoma-derived T cell suppressor factor (G-TsF), was separated from IL 1 and purified 2000-fold with respect to the protein present in the crude 308 cell supernatant. This G-TsF preparation was sensitive to tryptic proteolysis, showed a peak of pI 4.6 on isoelectric focusing, and when labeled with 125I, revealed six protein bands in the range of 30 to 100 kdaltons on SDS gel.     

Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure

The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin.     

Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.