Biosynthesis of various types of collagen by human hepatoma cells in vitro

A cloned human hepatoma cell line (HH2-1) produced and formed collagen fibers in vitro. The relative rate of collagen synthesis by the cells was increased with an enhancement of the cell density. An analysis of the components of the collagen using sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the cells synthesized interstitial collagen, types I and III, and other collagenous proteins. Thus, human hepatoma cells may play an important role in the formation of stromal collagen in the tumor.     

Evidence of intracellular and trans-acting differentiation-inducing activity in human promyelocytic leukemia HL-60 cells: its possible involvement in process of cell differentiation from a commitment step to a phenotype-expression step

We previously reported that human promyelocytic leukemia HL-60 cells, when treated with various inducers in magnesium-deficient medium, became committed to differentiate but did not express the differentiation-related phenotypes (Okazaki et al., J. Cell. Physiol., 131:50-57, 1987). In the present study we demonstrated the existence of an intracellular differentiation-inducing activity (int-DIA) in differentiation-committed phenotype-nonexpressing HL-60 cells by using cybrid formation between untreated HL-60 cells and cytoplasts from HL-60 cells treated in magnesium-deficient medium with 100 nM 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell extracts from similarly treated HL-60 cells also showed int-DIA, which when added (10 mg total protein/ml) to culture of untreated HL-60 cells, could increase the percentages of nitroblue tetrazolium (NBT)- and nonspecific esterase (NSE)-positive cells from 1% to 53%, and from 0 to 32%, respectively. They also induced differentiation of human monoblastic leukemia U-937 cells and of human myeloblastic leukemia KG-1 cells but not of erythroleukemia K-562 cells. These results suggested that the int-DIA had a common effect on differentiation induction in several human myeloid cell lines and may be involved in inducing cells to proceed from a commitment to a phenotype-expression step during human myeloid cell differentiation.     

Specific cytolysis of fresh tumor cells by an autologous killer T cell line derived from an adult T cell leukemia/lymphoma patient

Cytotoxic T cells (Tc) derived from one patient with adult T cell leukemia/lymphoma (ATL) killed fresh autologous lymphoma cells in vitro. The Tc were induced from peripheral blood leukocytes (PBL) of this patient during remission by multiple in vitro stimulations with an autologous ATLV-bearing cell line (ILT) that was previously established by cloning of PBL in the presence of interleukin 2 (IL 2). PBL from eight other ATL patients were stimulated in the same manner, and responder cells from a patient in remission also showed cytotoxicity specific for ATL virus (ATLV)-bearing cells. Fresh lymphoma cells were obtained in relapse and were used as target cells for the autologous Tc induced. They became susceptible to the Tc within 4 hr of in vitro incubation, and their susceptibility increased with incubation time for at least 12 hr. ATLV antigens on the cell surface of these lymphoma cells, however, were not detected by radioimmunoassay during these incubation periods, but were detectable after 16 hr of incubation. In addition, cytotoxicity against lymphoma cells was completely inhibited by autologous ILT cells used as "cold" target competitor cells. These findings indicate that the target antigen of the Tc was expressed on both autologous ILT cells and lymphoma cells, and it may be different from ATLV antigens detected by serologic methods. In addition, the data suggested allogeneic restriction of the Tc in that the preferentially killed allogeneic ATLV-bearing cells share several HLA antigens.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.     

Taxonomic Characteristics of a Thermophilic Acidophilic Strain of Bacillus Producing Thermophilic Acidophilic Amylase and Thermostable Xylanase

Taxonomic characteristics of a strain of thermophilic acidophilic bacillus, Bacillus sp. 11-1S, which had the ability to produce thermophilic acidophilic amylase and thermostable xylanase were examined. Cells of the organism were aerobic, heterotrophic, Gram-positive, spore-forming rods. It grew at temperatures between 45 and 70°C (optimum 65°C) in media of pHs ranging from 2.0 to 5.0 (optimum 3.5 ~ 4.0). Physiological and biochemical characteristics were identical with those of Bacillus acidocaldarius, and % GC of DNA (59%) was close to that of the latter (61 ~ 62%). From these results it was concluded that the organism belongs to B. acidocaldarius Darland and Brock.     

Control of pyrimidine biosynthesis in human lymphocytes. Inhibitory effect of guanine and guanosine on induction of enzymes for pyrimidine biosynthesis de novo in phytohemagglutinin-stimulated lymphocytes.

Human peripheral lymphocytes were incubated with Phaseolus vulgaris phytohemagglutinin. The induction of glutamine-utilizing carbamyl phosphate synthetase (EC 2.7.2.5) and aspartate transcarbamylase (EC 2.1.3.2) for pyrimidine biosynthesis de novo and the induction of uridine kinase were observed as described previously (Ito, K., and Uchino, H. (1971) J. Biol. Chem. 246, 4060-4065; Ito, K., and Uchino, H. (1973) J. Biol. Chem. 248, 389-392; Lucas, Z.J. (1967) Science 156, 1237-1240). By the addition of 1 mM guanine to the culture, the induction of the former two enzymes was inhibited, while that of uridine kinase was not, and even accelerated. An increase in the rate of [14C] bicarbonate incorporation into the acid-soluble uridine nucleotides via the de novo pathway for pyrimidine biosynthesis after phytohemagglutinin stimulation was inhibited by guanine, the incorporation rate being almost at the level of the control culture without phytohemagglutinin. Guanosine had a similar effect on pyrimidine biosynthesis. The induction of the three enzymes mentioned above was completely inhibited by adenine (1 mM). Guanine and guanosine seem to have a unique inhibitory effect on the induction of glutamine-utilizing carbamyl phosphate synthetase and aspartate transcarbamylase.     

Lipopolysaccharide Disrupts the Milk-Blood Barrier by Modulating Claudins in Mammary Alveolar Tight Junctions

Mastitis, inflammation of the mammary gland, is the most costly common disease in the dairy industry, and is caused by mammary pathogenic bacteria, including Escherichia coli. The bacteria invade the mammary alveolar lumen and disrupt the blood-milk barrier. In normal mammary gland, alveolar epithelial tight junctions (TJs) contribute the blood-milk barrier of alveolar epithelium by blocking the leakage of milk components from the luminal side into the blood serum. In this study, we focused on claudin subtypes that participate in the alveolar epithelial TJs, because the composition of claudins is an important factor that affects TJ permeability. In normal mouse lactating mammary glands, alveolar TJs consist of claudin-3 without claudin-1, -4, and -7. In lipopolysaccharide (LPS)-induced mastitis, alveolar TJs showed 2-staged compositional changes in claudins. First, a qualitative change in claudin-3, presumably caused by phosphorylation and participation of claudin-7 in alveolar TJs, was recognized in parallel with the leakage of fluorescein isothiocyanate-conjugated albumin (FITC-albumin) via the alveolar epithelium. Second, claudin-4 participated in alveolar TJs with claudin-3 and claudin-7 12 h after LPS injection. The partial localization of claudin-1 was also observed by immunostaining. Coinciding with the second change of alveolar TJs, the severe disruption of the blood-milk barrier was recognized by ectopic localization of β-casein and much leakage of FITC-albumin. Furthermore, the localization of toll-like receptor 4 (TLR4) on the luminal side and NFκB activation by LPS was observed in the alveolar epithelial cells. We suggest that the weakening and disruption of the blood-milk barrier are caused by compositional changes of claudins in alveolar epithelial TJs through LPS/TLR4 signaling.     

Heme-binding properties of heme detoxification protein from Plasmodium falciparum

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.     

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori

Dietary carotenoids are absorbed in the intestine and delivered to various tissues by circulating lipoproteins; however, the mechanism underlying selective delivery of different carotenoid species to individual tissues remains elusive. The products of the Yellow cocoon (C) gene and the Flesh (F) gene of the silkworm Bombyx mori determine the selectivity for transport of lutein and β-carotene, respectively, to the silk gland. We previously showed that the C gene encodes Cameo2, a CD36 family member, which is thought to function as a transmembrane lipoprotein receptor. Here, we elucidated the molecular identity of the F gene product by positional cloning, as SCRB15, a paralog of Cameo2 with 26% amino acid identity. In the F mutant, SCRB15 mRNA structure was severely disrupted, due to a 1.4 kb genomic insertion in a coding exon. Transgenic expression of SCRB15 in the middle silk gland using the binary GAL4-UAS expression system enhanced selective β-carotene uptake by the middle silk gland, while transgenic expression of Cameo2 enhanced selective lutein uptake under the same GAL4 driver. Our findings indicate that divergence of genes in the CD36 family determines the selectivity of carotenoid species uptake by silk gland tissue and that CD36-homologous proteins can discriminate among carotenoid species.