Persistent BK papovavirus infection of transformed human fetal brain cells. I. Episomal viral DNA in cloned lines deficient in T-antigen expression.

After infection of permissive human fetal brain cells by BK human papovavirus (BKV), the vast majority of the cells were killed by the virus, but rare survivors were recovered after frequent medium changes. These surviving cells grew and formed visible colonies after 5 to 6 weeks and were thereafter established as permanent cell lines. These cells, designated as BK-HFB cells, were persistently infected and shed BKV. Morphologically, they were small polygonal cells and had transformed growth properties. Their plating efficiency on solid substrates or in semisolid medium was high, and they were tumorigenic in athymic nude mice. Cloning experiments in medium containing BKV antiserum revealed that BKV did not persist in the cultures in a simple carrier state. All cloned cell lines were initially T-antigen negative and virus-free. However, every clone began to release BKV and again became persistently infected within 3 weeks after removal of BKV antiserum. After rigorous antibody treatment, four of seven clones still released virus spontaneously upon removal of antiserum; three clones have remained virus-free and are apparently cured. Although these cloned cell lines are T- and V-antigen negative when grown in antiserum-containing medium, they retain "free" or episomal BKV genomes; integrated viral DNA was not detected in any of the clones. These free genomes are indistinguishable from prototype BKV DNA and are found in much larger amounts in virus-shedding cell lines.     

Biological and biochemical studies of African green monkey lymphotropic papovavirus.

The growth of African green monkey lymphotropic papovavirus (LPV) in human lymphoblastoid cell line BJA-B was found to be slow and inefficient due to the accumulation of defective particles. An analysis of molecularly cloned LPV DNAs showed that 3 of 19 clones had DNAs that were longer (5.1 kilobases) than the DNAs of the other clones. The 5.1-kilobase DNA was infectious for BJA-B cells, whereas the shorter (4.8-kilobase) molecules were defective. Unlike the wild-type virus, stocks of LPV made from cloned, infectious DNAs were homogeneous and had higher titers. Using stocks of nondefective LPV, we investigated other biological properties. LPV replication in another human B-lymphoblastoid cell line was observed. The virus did not cause tumors when it was inoculated into newborn hamsters. Serological surveys of human and nonhuman primate sera indicated that virtually all primates, including humans, show evidence of infection by viruses antigenically related to LPV.     

Sesquiterpenoids of Riccardia and Pallavicinia species

Riccardia species (Metzgeriales) contain various types of sesquiterpenes. R. jackii produces ent-selinane-, ent-aromadendrane-and ent-bicyclogermacrane-type sesquiterpenes together with (R)-cuparene and α-barbatene. Aneura pinguis (= Riccardia pinguis) is chemically quite different from R. multifida and R. jackii. The former produces a large amount of pinguisone. R. multifida contains 6-(3-methyl-2-butenyl)-indole and (+)-β-elemene as the major components. Pallavicinia longispina (Dilaenaceae; Metzgeriales) produces mainly spathulenol. The chiral properties of the sesquiterpenes isolated from R. jackii are quite similar to those of red algae, Laurencia species.     

Bibenzyls from Radula tokiensis and R. Japonica

A new bibenzyl having a dihydrooxepin ring was isolated from the acetone extract of the liverwort Radula tokiensis, together with the previously known 5 bibenzyls and 3 sesquiterpenes, trans-β-farnesene, cuparene and (Jcuparenol. Two known bibenzyls were isolated from R. japonica. The bibenzyl derivatives are significant chemosystematic markers of the Radulaceae.     

Phosphorylation of lens membrane: identification of the catalytic subunit of Na+, K+-ATPase

Receptor mediated endocytosis appears to depend on the action of a transglutaminase (TGase). Endocytosis can be induced in intact human RBC by the action of several classes of drugs. We tested the hypothesis that drugs acted by stimulating TGase activity. Of the endocytosis inducing drugs tested, neither primaquine nor vinblastine nor chlorpromazine enhanced TGase activity. We next tested the hypothesis that TGase activity was required for drug endocytosis in RBC by adding known TGase inhibitors. Paradoxically, m-Dansyl cadaverine, the most potent TGase inhibitor, produces endocytosis in human RBC. Therefore despite apparent striking morphologic similarities, drug induced endocytosis in RBC appears to proceed via different mechanisms from those involved in receptor mediated endocytosis in other cells.In the receptor-mediated endocytosis of some hormones and growth factors, it appears that the receptor-ligand complex forms clusters over clathrin coated pits which are then internalized as endocytic vacuoles. Both the clustering and internalization of ligands are inhibited by a variety of agents shown to inhibit transglutaminase (TGase) and it is therefore proposed that TGase participates in receptor-mediated endocytosis (1–3). Human erythrocytes undergo endocytosis when exposed to drugs like primaquine, chlorpromazine, and vinblastine (4), all of which are amphipathic cations (4). However, the mechanism of drug action is not known nor is it clear that this is a form of receptor-mediated endocytosis (4). Furthermore, clustering of receptors can occur in neonatal but not adult human RBC (5). TGase has been measured in human red cells (6) although its physiologic role is unknown. Like all TGases, it is calcium dependent (6,7), and primaquine induced red cell endocytosis is enhanced by Ca⁺⁺ addition (8). Therefore, we tested the hypothesis that TGase participates in drug induced endocytosis in intact human red cells.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

A new assay of X-prolyl dipeptidyl-aminopeptidase activity in human serum with glycylproline p-phenylazoanilide as substrate

Summary A new assay procedure for X-prolyl dipeptidyl-aminopeptidase activity in human serum was developed with glycylproline p-phenylazoanilide tosylate as substrate. p-Phenylazoaniline liberated by the enzyme reaction was measured photometrically at 493 nm after stopping the reaction with acid. This assay was simple and sensitive, and less than 50 l of human serum was required for the assay. Km value was 2.5 mm and the optimum pH was 8.7. After disc gel electrophoresis of human serum, the enzyme activity could be distinctly observed as a reddish band on the gel when the gel was incubated with this substrate.     

Two Phenolic Amides in the Seed Balls of Sugar Beet (Beta vulgaris L. var. saccharifera Alefeld)

Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Buty 1-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278, 390 (Soret band), 586 (a band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reduction. The absorbance ratios, A₃₉₀: A₂₁₈ and A₅₈₆ :A₃₉₀, were 0.32 and 0.31, respectively. A plot of the substrate (sulfite) and electron donor (ferredoxin) concentrations versus enzymatic (Fd-SiR) activity yielded sigmoidal curves, giving Hill coefficients («) of 2.3 (for sulfite) and 2.7 (for ferredoxin), respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion suggested that it was specific against isolated Fd-SiR. Using the antiserum, dot immunoblotting was performed to determine the immunological similarity of Fd-SiRs from Porphyra yezoensis, Spirulina platensis, Brassica chinensis and Spinacia oleracea. The tests revealed that the four forms of assimilatory Fd-SiR have antigenic determinants in common.     

Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 β-cell line

Bone homeostasis is maintained by a dynamic balance between bone resorption by osteoclasts and bone formation by osteoblasts. Since excessive osteoclast activity is implicated in pathological bone resorption, understanding the mechanism underlying osteoclast differentiation, function and survival is of both scientific and clinical importance. Osteoclasts are monocyte/macrophage lineage cells with a short life span that undergo rapid apoptosis, the rate of which critically determines the level of bone resorption in vivo. However, the molecular basis of rapid osteoclast apoptosis remains obscure. Here we report the role of a BH3-only protein, Noxa (encoded by the Pmaip1 gene), in bone homeostasis using Noxa-deficient mice. Among the Bcl-2 family members, Noxa was selectively induced during osteoclastogenesis. Mice lacking Noxa exhibit a severe osteoporotic phenotype due to an increased number of osteoclasts. Noxa deficiency did not have any effect on the number of osteoclast precursor cells or the expression of osteoclast-specific genes, but led to a prolonged survival of osteoclasts. Furthermore, adenovirus-mediated Noxa overexpression remarkably reduced bone loss in a model of inflammation-induced bone destruction. This study reveals Noxa to be a crucial regulator of osteoclast apoptosis, and may provide a molecular basis for a new therapeutic approach to bone diseases.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.