Effects of various alcoholic supplements on the growth rate of Clostridium acetobutylicum ATCC 824

Summary The inhibitory effect of various alkanols, benzyl alcohol and phenethyl alcohol on the growth rate of Clostridium acetobutylicum ATCC 824 was investigated. Inhibition of cell growth was studied by treating cultures with varied concentrations of alcohols. There was a threshold concentration above which growth inhibition occurred. The degree of inhibition was a linear function of the alcohol concentration used. The natural logarithm of the inhibition constant was shown to be: (1) a linear function of the chain length of the alkanols, (2) a linear function of the natural logarithm of the octanol/water partition coefficient for both aliphatic and cyclic alcohols.     

Kurloff cell proteoglycans: presence of two main size-populations of intracellular protease-resistant proteochondroitin sulphate. Effect of D-xyloside

This paper reports that the Kurloff cell sulphated and chondroitinase AC sensitive material previously described filtered on Sepharose CL4B columns as 2 main populations with Kav of 0.25 and 0.44. Its alkaline treatment resulted in the elution of 2 peaks with Kav of 0.52 and 0.78. Their reduction in size observed after alkaline treatment and the 6-fold increase in the (35S) sulphate incorporation after addition of 0.1 mM xyloside to the incubation medium indicate that these intracellular sulphated glycosaminoglycans exist in the form of proteoglycans. They were characterized by their resistance to degradation by pronase, papain or cathepsin D, as assessed by gel filtration chromatography on Sepharose CL6B or CL4B. After the glycosaminoglycans were digested with chondroitinase AC, thin-layer chromatography analysis indicated the presence of delta di-4S and delta di-6S in a ratio of 7:1. The presence of such protease-resistant proteochondroitin sulphate in intracytoplasmic granules of both Kurloff cells and other natural killer cell types is emphasized.     

Transport of CMP-N-glycoloylneuraminic acid into mouse liver Golgi vesicles

CMP-Neu5Gc has been shown to be transported into mouse liver Golgi vesicles by a specific carrier the characteristics of which were investigated in detail. In the system employed, CMP-Neu5Gc enters the Golgi vesicles within 2 min; transport was saturable with high concentrations of the sugar-nucleotide and was dependent on temperature. A kinetic analysis gave an apparent K_m of 1.3 μM and a maximal transport velocity of 335 pmol/mg protein per min. Almost identical values were obtained with CMP-Neu5Ac, under the same incubation conditions. Furthermore, the uptake of CMP-Neu5Gc was inhibited by CMP-Neu5Ac, a substrate analogue. Conversely, the uptake of CMP-Neu5Ac was inhibited by CMP-Neu5Gc to the same extent, leading to the conclusion that the transport of CMP-Neu5Ac and CMP-Neu5Gc is mediated by the same carrier molecule. This transport system for CMP-Neu5Gc involves both CMP and CMP-Neu5Gc since intravesicular CMP induced the entry of external CMP-Neu5Gc.     

Reconstituted skin in culture:a simple method with optimal differentiation

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.     

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cystein in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Cell kinetic disturbances induced by treatment of human diploid fibroblasts with 5-azacytidine indicate a major role for DNA methylation in the regulation of the chromosome cycle

Summary BrdU-Hoechst flow cytometry was used to investigate the effects of DNA hypomethylation, induced by treatment with 5-azacytidine (5AC), on cell proliferation. When human fibroblast-like cells derived from skin and amniotic fluid were exposed to 5AC during three successive cell cycles, their clone-forming ability was diminished after removal of the drug. Treated cells were rendered quiescent by culture with low serum in the absence of the drug. Upon serum stimulation, they showed a diminished fraction of proliferating cells, which exhibited a prolonged transit through the S and G2 phase of the cell cycle, and a permanent arrest within the G2 compartment. This pattern of disturbed cell proliferation may in part explain the changes in replication banding pattern reported in the literature. Cytogenetic analysis of 5AC-treated cells revealed numerous endomitoses and tetraploid metaphases indicating a disturbed chromosome cycle in association with these cell kinetic perturbations.     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237