Specificity of blebbistatin, an inhibitor of myosin II

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC₅₀ values ranging from 0.5 to 5 μM. Interestingly, smooth muscle which is highly homologous to vertebrate nonmuscle myosin is only poorly inhibited (IC₅₀=80 μM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X. This revised version was published online in July 2006 with corrections to the Cover Date.     

Emphysematous lung destruction by cigarette smoke. The effects of latent adenoviral infection on the lung inflammatory response.

This study was designed to test the hypothesis that cigarette smoke-induced inflammation and emphysema are amplified by the presence of latent adenoviral (Ad) infection, and to determine whether this emphysematous process can be reversed by all-trans-retinoic acid (RA) treatment. The results confirm that in guinea pigs, chronic cigarette-smoke exposure caused lesions similar to human centrilobular emphysema. They also show that latent Ad infection combined with cigarette-smoke exposure caused an excess increase in lung volume (P < 0.001), air-space volume (P < 0.001), and lung weight (P < 0.01), and further decrease in surface-to-volume ratio (P < 0.001) compared with smoke exposure alone. RA treatment failed to reverse these emphysematous changes. Analysis of inflammatory response in parenchymal and airway tissue showed that smoking caused an increase of polymorphonuclear leukocytes (PMNs) (P < 0.0002), macrophages (P < 0.001), and CD4 cells (P < 0.0009), and that latent Ad infection independently increased PMNs (P < 0.001), macrophages (P = 0.003), and CD8 cells (P < 0.001). We conclude that latent Ad infection amplifies the emphysematous lung destruction and increases the inflammatory response produced by cigarette-smoke exposure. In this study, the increase in CD4 was associated with cigarette smoke and the increase in CD8 cells with latent Ad infection.     

Multiple transformation phenotypes among revertants of temperature-sensitive mutants in the type 5 adenovirus DNA-binding protein

Seven independently isolated revertants of temperature-sensitive mutants in the 72K protein of Ad5 were tested for the ability to transform rat cells under a variety of conditions. Using the continuous cell line designated CREF, at 36° the range of transformation phenotypes of the different revertants included a frequency characteristic of WT and also the elevated frequency associated with the parental temperature-sensitive alleles. Transformation frequency did not correlate with other phenotypes, such as plaque size or plaquing efficiency on HeLa cells at 38.5°–39°. Therefore, although it is likely that the 72K protein modulates transformation, it is possible to dissociate genetically this regulatory function of the protein from its replicative function in permissive infection.     

A novel ELISpot method for adherent cells

The purpose of this study was to develop an enzyme-linked immunospot assay (ELISpot assay) that can be used with human adherent cells. While standard enzyme-linked immunosorbent assays (ELISAs) are available and widely used and ELISpot assays are used for nonadherent lymphocytes, no ELISpot assay has been developed for adherent cells. We used primary human fibroblasts from four different tissues (myometrium, lung, gingiva, and orbit), either unstimulated or interleukin (IL)-1beta-activated, to evaluate an ELISpot assay. Antibody pairs for IL-6 and IL-8 were used and results were compared to a standard ELISA. We found that we could reliably detect IL-6 and IL-8 spots with as few as 10 fibroblasts. Optimal cell numbers were 50 cells per well incubated for 8 h, although spots appeared as early as 2 h after incubation. Spots were absent when cells, primary, or secondary anti-cytokine antibodies were omitted from the protocol. Spot number and size can be ascertained using current automated ELISpot reader technology. The frequency of IL-6 and IL-8-producing human fibroblasts could also be determined. For example, 60% of the lung fibroblasts express IL-6, but IL-8 can be detected from only 40% of the cells. Approximately 80% of the human orbital fibroblasts make IL-6, whereas approximately 50% generate IL-8 following IL-1beta stimulation. These new findings show that fibroblasts from different human tissues display different frequencies of cytokine production and this further supports the concept of fibroblast diversity. The sensitivity of this new ELISpot assay is adequate for cytokine detection in just a few cells, unlike the standard ELISA. It should permit ascertaining the frequency of fibroblasts and other adherent cells that produce cytokines and, if desired, can be used in tandem with a standard ELISA to determine total cytokine produced. Moreover, the assay is suitable for normal human adherent cells that are often short-lived and difficult to cultivate.     

High level expression of apoptosis inhibitor in hepatoma cell line expressing Hepatitis B virus

The serious result of hepatitis B (HBV) virus infection is development of hepatocellular carcinoma (HCC). However, the reason of development of HCC in HBV infected patients is still unclear. Recently, the suppression of cell apoptosis is found to relate with the development of cell carcinogenesis, therefore, the expression of apoptosis inhibitor in the virus related cancer line such as hepatoma cell line HepG2.215 was investigated. There are at least six Human apoptosis inhibitors (IAP) have been identified now. They are cIAP1, cIAP2, XIAP, NAPI, survivin and pIAP. Using gene-assay technology, we have recently compared the expression of IAPs in the HepG2.215 cells that persistently expresses Hepatitis B virus by integrated HBV genome with its parent cell line HepG2. The results suggest that there was obviously increase of cIAP2 and cIAP1 in the HepG2.215 cells versus HepG2 cells. Those observations imply a possibility of long time HBV infection could induce the over-expressing apoptosis inhibitors, furthermore, causing the liver cancer. The high expression of cIAP1 and cIAP2 in HBV expressing cells was confirmed by RT-PCR and Northern blot analysis. However, we did not find the change of NIAP and suvivin in HepG2.215 cells. In contrast, the expression of XIAP was down in the HepG2.215 cells comparing with HepG2 cells. How HBV triggers the over-expression of apoptosis inhibitor is unclear. Transient transfection of HepG2 cells with the plasmids expressing different HBV proteins such as S, M, L, X and core proteins did not give a decisive conclusion. Further study is going on now.     

Mutagenicity of bisphenol A (4,4'-isopropylidenediphenol) in vitro: effects of nitrosylation

Bisphenol A (4,4'-isopropylidenediphenol) is a common component of polycarbonate plastics and epoxy resins. Since bisphenol A-containing plastics and resins have found uses in food-contact items, its potential migration into foodstuffs and possible health consequences have been the focus of many recent studies. However, the potential mutagenic activation of bisphenol A by nitrosylation has received little attention. Incubation of bisphenol A with sodium nitrite under acidic conditions produced a yellow-brown product. When nitrosylated bisphenol A was tested in the Ames Salmonella/microsome assay at 100 ng to 1 mg/plate, dose-dependent increases in mutagenicity were found in both TA98 and TA100 Salmonella strains. These results indicated the presence of a direct-acting mutagenic activity causing both frameshift and base pair mutations, respectively. When compared to colony formation in untreated controls, the addition of rat liver S9 for metabolic activation had little influence on revertant colony formation. Unreacted bisphenol A dissolved in DMSO, acidic buffer, or inactivated nitrosylation solution showed negligible mutagenicity. When the nature of the mutagenic changes was examined using the Ames II trade mark Assay, a variety of base pair changes was found including T:A to A:T - S9, G:C to A:T +/- S9,C:G to A:T +/- S9 and C:G to G:C +/- S9. Bisphenol A also induced frameshift mutations at G:C sites. In addition, the presence of electrophiles was shown by the production of an intensely coloured orange-red product upon incubation of nitrosylated bisphenol A with the nucleophile 4-(4'-nitrobenzyl)pyridine. These findings suggest that migration of bisphenol A into nitrite containing foodstuffs, or its ingestion in the presence of nitrite, could lead to the formation of mutagenic compounds.     

The primate Harderian gland: Does it really exist?

The Harderian gland, an anterior orbital structure, is either absent or vestigial in primates. This is based upon gross anatomical observations of scattered adult specimens. Though largely absent in the adult human, it is present in the fetal and neonatal stages. Thus, histological examination of the orbital region of neonatal material was undertaken in other primates. The orbital region of neonatal specimens of 12 species of strepsirrhines (Lemuriformes and Lorisiformes), and haplorhine (tarsiers and callitrichids) was examined. The Harderian gland is ensconced in either periorbital fat or connective tissue and thus was not readily identifiable gross anatomically. Thus, it may have been missed in the anatomical studies. Tarsal glands are present in all neonatal primate eyelids. The relative size of the neonatal primate Harderian gland can be subdivided into five separate categories, ranging from large to absent (tarsiers), with no apparent phylogenetic trends. Thus, the Harderian gland is present in numerous primates at birth, quite possibly all strepsirrhines. The positive findings on callitrichids question whether any anthropoids lack the Harderian gland postnatally. The enigmatic tarsier appears to possess another apomorphic trait in lacking a Harderian gland. Further study is required to determine the role of this gland and its relationship with the tarsal glands.     

Alcoholism and drug abuse in three groups — bipolar I, unipolars and their acquaintances

Objective: Previous work has shown that manic-depressive illness and alcohol abuse are linked. This study further explores the relationship of alcohol and drug abuse in bipolar I patients and unipolar depressives and a comparison group obtained through the acquaintance method. Method: Diagnosis was accomplished according to Research Diagnostic Criteria (RDC): controls=469; bipolars=277; unipolar depressives=678. Systematic data were gathered using the SADS on lifetime and current drug abuse and alcoholism. Both patients and comparison subjects were then followed prospectively for 10 years. First degree family members were interviewed using the RDC family history method. Results: The group of bipolar patients and the group of unipolar patients had higher rates of drug and alcohol abuse than the comparison group when primary and secondary affective disorder patients were combined. However, primary unipolar patients did not have higher rates of alcohol or drug abuse than the comparison group. In contrast, primary bipolar patients had higher rates of alcoholism, stimulant abuse, and ever having abused a drug than the primary unipolar group and the control group. In an evaluation of the bipolar patients, drug abusers were significantly younger at intake and had a significantly younger age of onset of bipolar disorder. There was a significant increase in family history of mania or schizoaffective mania in the drug-abusing bipolar patients as compared to the non-abusing bipolar patients. Limitation: As in all adult samples of patients with affective illness, the chronology of alcohol and substance problems vis-à-vis the onset of illness was determined retrospectively. Conclusions: (1) Alcoholism and drug abuse are more frequent in bipolar than unipolar patients. (2) The drug abuse of bipolar patients tends toward the abuse of stimulant drugs. (3) In a bipolar patient, familial diathesis for mania is significantly associated with the abuse of alcohol and drugs. (4) More provocatively, these findings suggest the hypothesis of a common familial-genetic diathesis for a subtype of bipolar I, alcohol and stimulant abuse. Clinical implications: The present analyses, coupled with two previous ones from the CDS, suggest that drug abuse may precipitate an earlier onset of bipolar I disorder in those who already have a familial predisposition for mania. Furthermore, in dually diagnosed patients with manic-depressive and alcohol/stimulant abuse history, mood stabilization of the bipolar disorder represents a rational approach to control concurrent alcohol and drug problems, and should be studied in systematic controlled trials.