Internalization of the vasoactive intestinal peptide (VIP) in a human adenocarcinoma cell line (HT29)

The time course of internalization of radioiodinated vasoactive intestinal peptide (VIP) in HT29 cells was obtained using the technique of acetic acid removal of cell-surface-bound peptide. Even after 10 min incubation at 37 degrees C, 125I-VIP, initially bound on the HT29 cell surface, was compartmentalized within the cells. During the same time, degraded radioactive material was released by cells in the incubation medium. Localization of internalized 125I-VIP was investigated using two different subcellular fractionation techniques. 10 min after the onset of internalization, 125I-VIP labelling was found in intermediate structures and 10 min later the bulk of the radioactivity was detected in a low-density fraction containing very large lysosomes with a multivesicular aspect. The lysosomotropic agent NH4Cl appeared to inhibit 125I-VIP internalization, degradation and appearance of radiolabelled peptide in the large lysosomes in a time-dependent manner. Moreover, the effect of NH4Cl resulted in an accumulation of radioactive material in fractions containing microsomal structures. On the other hand, bacitracin, together with methylamine, highly enhanced 125I-VIP labelling in a membrane fraction, suggesting that these agents possibly act on a cell surface component of HT29 cells. These results support the conclusion that in HT29 cells, prelysosomal structures and large secondary lysosomes are probably part of the intracellular pathway of internalized VIP.     

The antenna system of Rhodospirillum rubrum. Detection of macromolecular constituents not stainable by Coomassie brilliant blue in solubilized preparations of the B880 complex

A solubilized preparation of the major Rhodospirillum rubrum antenna complex (B880) was obtained by a described procedure and its polypeptide composition was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only two polypeptides of molecular weights close to 7000 were detected after staining the gels with Coomassie brilliant blue. However, several other constituents could be visualized by silver staining or by an immunochemical method. When the preparation was chromatographed on Sephacryl, some of the resulting fractions exhibited the characteristic B880 absorption spectrum and contained only the two proteins that were detectable with Coomassie brilliant blue. In those fractions the A ₂₈₀/A ₈₈₀ratio was 0.4, which indicated a significant improvement of the bacteriochlorophyll to protein ratio over the unchromatographed preparation (A ₂₈₀/A ₈₈₀=0.7). Other chromatography fractions lacked bacteriochlorophyll and contained a carotenoid which seemed to be bound to protein. The macromolecular constituents present in these latter fractions differed from those associated to the purified B880 complex in their electrophoretic moblities and/or in their staining properties. That suggested the possible existence of a carotenoprotein that did not result from the B880 complex upon loss of bacteriochlorophyll.     

Short term regulation by lysine of ornithine decarboxylase activity in Dictyostelium discoideum

A transitory increase in ornithine decarboxylase activity has been observed soon after food removal from Dictyostelium discoideum amoeba. This increase can be prevented by supplementation of the differentiation buffer with the 11 amino acids known for their ability to retard the development of this slime mold. Lysine can replace the amino acid mixture with an apparent inhibition constant of 50 micromolar. This inhibition by lysine, which was only observed in vivo, took place within 5 min and was readily reversed upon lysine removal.     

Locus determining the human sperm-specific lactate dehydrogenase,LDHC, is syntenic with LDHC

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldhl, Ldh2, and Ldh3, respectively.     

Some observations on the urea-degrading enzyme of the diatom Cyclotella cryptica and the role of nickel in its production

The urea-degrading enzyme of Cyclotella cryptica was testedin crude cell-free extracts for effects from chemical reagentsknown to distinguish between urease and ATP:urea amidolyase.Inhibition of the enzyme by hydroxyurea and its indifferenceto added ATP, Mg²⁺ or K⁺ avidin or biotin clearly characterizedthe enzyme as urease (EC 3.5.1.5 [EC] ). The Cyclotella urease wasunaffected by thiourea addition, as was also the growth of thediatom in the presence of this substrate analogue. Indirectevidence was obtained from growth studies of the diatom andcorresponding urease production showing that the enzyme: (i)contains Ni²⁺ tightly bound to an apoprotein; (ii) is producedconstitutively even from growth on nitrate and does not requireextracellular urea for its synthesis, although quantitativelythe activity is greatest from growth on urea. It is concludedthat Cyclotella urease is a Ni²⁺ constitutive enzyme similarin many respects to those previously reported from Phaeodactylumtricornutwn and Tetraselmis maculata.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.