Enzymatic removal of bilirubin toxicity by bilirubin oxidase in vitro and excretion of degradation products in vivo

The toxic effects of the degradation products of bilirubin that were formed by reaction with bilirubin oxidase were investigated with the C 1300 mouse neuroblastoma cell line by examining the following parameters: growth inhibition, morphologic characteristics, membrane transport, DNA synthesis, and protein synthesis. The addition of bilirubin to the cells resulted in definite cytotoxic effects on all of these parameters in a dose-dependent fashion; the addition of bilirubin oxidase reversed the toxic effects on the C 1300 cells in vitro. Furthermore, we found that most of these enzymatic degradation products of bilirubin were excreted by the kidney into the urine in a few hours after intravenous injection of the degradation products; in contrast, no intact bilirubin was excreted. Thus, these findings suggest that hyperbilirubinemia in newborn infants (kernicterus) may be prevented by administering polyethylene glycol-conjugated bilirubin oxidase, with a longer plasma half-life which has been reported previously to oxidize bilirubin to its nontoxic components in the bloodstream.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies.

Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ration in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means +/- standard errors) 41 +/- 10 and 37 +/- 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.     

Enzymatic synthesis of alkyl xylobioside and xyloside from xylan and alcohol

Summary Alkyl -D-xylobioside and alkyl -D-xyloside were prepared by the one-pot reaction of xylan and a fatty alcohol, such as 1-octanol, 1-decanol, 2-octanol and 2-ethylhexanol using the cell-free culture filtrate of the xylan-assimilating strain, Aureobasidium pullulans KK415. Using this strain, a novel surfactant, alkyl -D-xylobioside, was produced as the main product when the alcohol and xylan was incubated at a temperature of 65 °C and pH 4.5.     

Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament-associated functions in cells in which it is differentially expressed, including neural cells and EBV-transformed B lymphocytes.     

Non-invasive detection of malignancy by identification of unusual CD44 gene activity in exfoliated cancer cells.

OBJECTIVE--To investigate non-invasive detection of cancer by testing for unusual CD44 gene activity in a clinical sample as an indicator of exfoliated tumour cells. DESIGN--Case-control study. SUBJECTS--44 unselected, consecutive patients with bladder cancer and 46 people with no evidence of neoplasia. MAIN OUTCOME MEASURE--Presence or absence of large CD44 gene products containing exon 6 derivatives in urine samples. RESULTS--Novel abnormalities in the pattern of expression of this gene, seen specifically in tumour tissue, led to cloning of a newly recognised coding region in it (exon 6). This was tested as a probe for detection of exfoliated malignant cells in naturally voided urine. CD44 gene products extracted from the urine and amplified with polymerase chain reaction contained predicted electrophoretic band of 735 base pairs in 40 of the 44 patients with bladder cancer (sensitivity 91%). Products from 38 of the 46 people with no evidence of neoplasia showed no such band (specificity 83%). CONCLUSIONS--Unusual activity of the CD44 locus in neoplasia and malignancy is confirmed, and techniques for the analysis of such activity can enable non-invasive investigation of patients for primary or recurrent bladder cancer or for other tumours that shed neoplastic cells into body fluids.     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.     

Comparison of production efficiency among field crops related to nitrogen nutrition and application

It has been generally considered that the low productivity of Leguminosae is caused by accumulation in the reproductive organs of a large amount of protein and lipid, since the biochemical costs of synthesizing these compounds is higher than that for carbohydrate. However, we report here on results which show that: the growth efficiencies (dry matter accumulated/ (dry matter accumulated + respiration)) of reproductive organs of Gramineae and Leguminosae were similar; the growth efficiency of rice in the vegetative stage was greater than that of soybean and field bean, regardless of nitrogen application rate; and when ¹⁴CO₂, ¹⁴C-sucrose or ¹⁴C-asparagine were introduced to the leaf at the maturation stage, respiratory loss of the introduced ¹⁴C was greater in soybean and field bean, especially in the light, than in rice. Thus, it is assumed that the low productivity in Leguminosae is caused by a larger respiratory loss under both dark and light condition in the shoot, and not in the reproductive organs.     

Reexamination of tetrodotoxin production by bacteria.

Vibrio alginolyticus has been reported as a good producer of tetrodotoxin (TTX), but the toxin extracted from this bacterium did not react to the monoclonal antibody against TTX. Surprisingly, chromatographic analyses detected high TTX peaks for polypeptone and yeast extracts used as medium materials, which were, as expected, all negative by the mouse bioassay. These results may require us to revise the bacterial production of TTX.     

Application of solvent extraction to ethanol fermentation

Summary The basic problems of applying solvent extraction to ethanol fermentation were investigated. The selection of solvents was based on the selectivity ratio, which was expressed as the ratio of the ethanol distribution coefficient to the water distribution coefficient. Solvents with high selectivity ratios of more than 50 were found mainly among the alcohols and esters. However, most of these solvents were toxic to ethanol-producing microorganisms. We tried to make a barrier to solvent molecules beneath the surface of gel beads immobilizing the cells as a protection against solvent toxicity. Porapack Q was found to be an effective barrier, and the ethanol production rate of immobilized cells protected with Porapack Q did not change event after the production of eight batches in medium saturated with sec-octanol, which was the most toxic solvent used in our experiments.