Constitutive expression of the nef gene suppresses human immunodeficiency virus type 1 (HIV-1) replication in monocytic cell lines.

In order to study the effect of nef gene expression on viral replication in monocytic cells, we established monocytic (U937 and THP-1) cell transfectants constitutively expressing the human immunodeficiency virus type 1 nef gene. We constructed a plasmid expressing the nef gene derived from an infectious clone, NL432, under the control of SR alpha promoter which can drive a high level of gene expression. We found suppressed viral replication in nef-expressing monocytic cells, although a negative effect of nef was observed, with some variation depending on the virus strain and the cell. We also observed that the expression of the surface CD4 molecule is inversely related to the expression of the nef gene, especially in the U937 transfectants. These results indicate that the suppression of viral replication and the down-modulation of CD4 molecule by nef gene expression occur in monocytic cell lines as in T cell lines.     

Activities of creatine kinase isoenzymes in single skeletal muscle fibres of trained and untrained rats

Biochemical changes in the creatine kinase isoenzyme compositions in single muscle fibres of different types in rats were induced by endurance running training. Single muscle fibres were dissected from the soleus and extensor digitorum longus muscles of Wistarstrain male rats trained on a motor-driven treadmill for 16 weeks. Each fibre was typed histochemically (SO, slow-twitch oxidative; FOG, fast-twitch oxidative glycolytic; FG, fast-twitch glycolytic), and the activities of total creatine kinase and its four isoenzymes (CK-MM, -MB,-BB, and mitochondrial creatine kinase) were measured. The endurance training did not affect the total creatine kinase activity, but resulted in significantly increased activities of CK-MB and CK-BB in SO and FOG fibres, and the mitochondrial enzyme activity in FOG and FG fibres. Endurance training induced biochemical changes in the isoenzyme compositions, specifically in FOG fibres. These results suggest that changes in creatine kinase isoenzymes with endurance training reflect changes in the energy metabolism in the different muscle fibres, supporting the hypothesis that the different isoenzymes play different roles in energy transduction.     

Mediation of nitrous oxide analgesia in mice by spinal and supraspinal kappa-opioid receptors

Exposure to nitrous oxide produced concentration-dependent analgesia in the mouse abdominal constriction test. Intracerebroventricular or intrathecal pretreatment with naltrexone or nor-binaltorphimine significantly reduced nitrous oxide analgesia. However, similar pretreatment with beta-funaltrexamine had no appreciable effect. These findings suggest that nitrous oxide analgesia involves spinal and supraspinal kappa-opioid receptors.     

Phenotypic and Genotypic Lineage Switch of a Lymphoma with Shared Chromosome Translocation and T-Cell Receptor γ Gene Rearrangement

A case of non-Hodgkin's lymphoma showed a phenotypic and genotypic cell lineage switch twice during nine years of his clinical history; first, T-cell type, pleomorphic small cell lymphoma developed, followed by B-cell type, diffuse centroblastic/centrocytic lymphoma, and finally T-zone lymphoma without follicles again developed, from which AST-1 cultured cell line was established. Karyotype analysis demonstrated a shared abnormal chromosome, der(1)t(1;?)(p36;?), among the first relapsed B-cell tumor, the second relapsed T-cell tumor and AST-1 cell line. Furthermore, T-cell receptor (TCR) γ gene rearrangement bands of the same size were observed in the first relapsed B-cell tumor and the second relapsed T-cell tumor as well as AST-1 cell line. These results suggested that both relapsed tumors of different cell lineages are derived from a common malignant clone, presumably a committed lymphoid stem cell. A unique translocation, t(2;14)(q37;q11.2), which may involve TCR δ/α gene complex, was observed in the second relapsed tumor and AST-1 cells. To attempt to isolate the breakpoint of this translocation, the configuration of TCR δ/α gene complex was studied. The result showed that two rearrangements of TCR α gene detected with J_α probes were the products of the normal TCR rearrangement process, and were not involved in the translocation at this region. This patient, together with the AST-1 cell line, provided us a unique opportunity to study the development and clonal evolution of malignant lymphoma.     

Differential protective effects of varying degrees of hypoxia on the cytotoxicities of etoposide and bleomycin

Summary Oxygen is thought to be involved both directly and indirectly in the mechanisms of action of several anticancer agents. We studied the effects of various oxygen concentrations on the cytotoxicities of the following drugs: bleomycin (BLM), etoposide (VP-16), doxorubicin (DOX), and mitomycin C (MMC). Human sarcoma cells, MESSA, were exposed to drug for 1 h at one of several oxygen concentrations: less than 1%, 2.5%, 5%, 21%, and 95%. Cytotoxicity was assessed by cellular incorporation of ³H-thymidine into DNA 5 days after drug exposure. Control experiments varying oxygen concentration without drugs demonstrated toxicity only at the highest concentration (95%). Three different responses of drug sensitivity to varying oxygen tensions were observed. BLM, which has been shown to utilize oxygen as a substrate in generating free radicals and producing DNA scission, demonstrated a progressive increase in cytotoxicity over the entire range of increasing oxygen concentrations. This is consistent with the model of a BLM-cation-oxygen complex and catalytic reduction of oxygen. VP-16, which also produces DNA strand breakage but by interaction with topoisomerase II, exhibited a threshold response. VP-16 toxicity was ameliorated by anoxic conditions (less than 1% O₂), but not by oxygen concentrations of 2.5%–95%. The reason for this protective effect of anoxia with VP-16 is not clear. In contrast, acute anoxia had no effect on the cytotoxicities of DOX and MMC. We conclude that acute hypoxia protects cells from both BLM and VP-16 but that the nature of that protection is different. VP-16 toxicitiy is blunted only by severe anoxia, wheaeas BLM exhibits a dose response effect over the entire range of oxygen concentrations.Supported by NIH grant CA-27478 from the US Department of Health and Human Services, and by the American Lung Association. Dr. Sikic is a recipient of a Faculty Development Award in Clinical Pharmacology from the Pharmaceutical Manufacturer's Association Foundation.     

ERBIN associates with p0071, an armadillo protein,at cell-cell junctions of epithelial cells

BACKGROUND: ERBIN, an ErbB2 receptor-interacting protein, belongs to a recently described family of proteins termed the LAP [leucine-rich repeats and PSD-95/dLg-A/ZO-1 (PDZ) domains] family which has essential roles in establishment of cell polarity. RESULTS: To identify new ERBIN-binding proteins, we screened a yeast two-hybrid library, using the carboxyl-terminal fragment of ERBIN containing PDZ domain as the bait, and we isolated p0071 (also called plakophilin-4) as an ERBIN-interacting protein. p0071 is a member of the p120 catenin family, which are defined as proteins with 10 armadillo repeats, and localizes along the cell-cell border. The ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence. Endogenous ERBIN was co-immunoprecipitated with p0071. In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localized largely with beta-catenin and partly with desmoplakin along the lateral plasma membrane domain. At these cell-cell contact regions, ERBIN co-localizes with p0071. Over-expression of the dominant active forms of Cdc42, Rac1 or RhoA, Rho family small GTPases, resulted in a marked accumulation of ERBIN at the cell-cell contacts of MDCK and HeLa cells. CONCLUSION: These results show that ERBIN interacts in vivo with p0071 and that it may be involved in the organization of adherens junctions and the desmosomes of epithelia. In addition, we demonstrated that the subcellular localization of ERBIN might be regulated by Rho family small GTPases.     

Positioning of actin filaments and tension generation in skinned muscle fibres released after stretch beyond overlap of the actin and myosin filaments

Summary Skinned fibres from frog semitendinosus muscle were stretched in relaxing solution from a sarcomere length of 2.5 m to greater sarcomere lengths, and then shortened back to the original length. Fibres could be stretched up to sarcomere lengths of 3.3 m, and reshortened fully. If the original stretch was to a sarcomere length greater than 3.3 m, the extent of recovery was dependent on the magnitude of the stretch and the number of times the stretch/shorten cycle was repeated. When the original stretch was to sarcomere lengths beyond overlap of the thick and thin filaments, the thin filaments did not re-enter the thick filament array but buckled at the A-I junction. If these fibres were subsequently activated and contracted, the thin filaments re-entered the thick filament array, taking up approximately their former positions, and allowing reduced development of isometric tension.Finally, the present observations support the view that calcium-induced interactions of actin and myosin filaments in the presence of ATP play an important role in the organization of myofibrillar structure during differentiation (compare Hayashiet al. 1977; Shimada & Obinata, 1977).     

Determination of metabolic profiles on single muscle fibres of different types

Summary Single muscle fibres separated from extensor digitorum longus (EDL) as well as soleus (SOL) in the Wistar strain male rat in relaxing solution were typed histochemically, then glycolytic and oxidative enzyme activities were determined on the same fibres. Glycolytic enzyme lactate dehydrogenase (LDH), phosphofructokinase (PFK), pyruvate kinase (PK) and creatine kinase (CK) showed highest activities in fast-twitch glycolytic (FG), lower in fast-twitch oxidative glycolytic (FOG) and lowest in slow-twitch oxidative (SO) fibres. Also oxidative enzyme succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) showed highest activities in SO, lower in FOG and lowest in FG fibres. The activities of LDH, PFK, PK and CK in FOG fibres separated from EDL showed higher activity compared to those separated from SOL, whereas the opposite result was obtained for the activities of SDH and MDH. Enzyme activities in a single muscle fibre type were not distinguishable from those of another type, and the activity profiles overlapped over a considerable range. The correlations among the separate enzyme activities of CK, LDH and MDH obtained from the same single fibre overlapped over a considerable range.     

Effects of 2,3-butanedione monoxime on contraction of frog skeletal muscles: An X-ray diffraction study

Summary We studied the effects of BDM (2,3-butanedione monoxime) on the tetanic contraction of frog skeletal muscles using an X-ray diffraction technique. BDM significantly increased the resting equatorial intensity ratio (I _1,0/I _1,1). In sartorius muscle, 3mm BDM suppressed tetanic tension by 40–70% whereas the equatorial intensity ratio, which is 2.6 at rest, decreased to 0.75 during tetanus, close to the value in normal contraction (about 0.50). BDM (3mm) reduced the intensity increase of the 5.1-nm layer-line to 41%, that of the 5.9-nm layer-line to 24%, and the intensity decrease of the second myosin meridional reflection (at 1/21.5 nm^–1) at 81%. In overstretched semitendinosus muscle, 3mm BDM did not significantly reduce the intensity increase of the second actin layer-line during activation, suggesting that enough calcium is released to activate the regulatory system and the regulatory proteins are intact. These results indicate that BDM suppresses tetanic tension by mainly inhibiting actin-myosin interaction. It has a smaller effect on the equatorial reflections and myosin layer-lines than on the actin layer-lines, suggesting that BDM-influenced myosin heads may bind to actin without following the symmetry of the actin helix.