Localization of a C-terminal region of lambda2 protein in reovirus cores

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.     

Antimycobacterial matricaria esters and lactones from Astereae species

Six matricaria esters (MEs) and two matricaria lactones (MLs), isolated from members of the tribe Astereae (Asteraceae), were tested against Mycobacterium tuberculosis and M. avium, using a radiorespirometric bioassay. (2Z,8Z)-ME and (2E-8Z)-ME gave minimum inhibitory concentrations (MICs) of 50 micrograms ml-1 against M. tuberculosis and respective MICs of 25 and 50 micrograms ml-1 against M. avium. The (4Z,8Z)-ML, (2Z)-8-dehydro-ME and (2Z,8Z)-10-angeloyloxy-(2Z,8Z)-ME showed respective MICs of 12.5, 25, 25 micrograms ml-1 against M. tuberculosis and MICs of 50, 25, 25 micrograms ml-1 against M. avium, respectively. The MICs of (2Z,8Z)-10-tigloyloxy-ME and (2E,8Z)-10-angeloyloxy-ME and (4E,8Z)-ML ranged from 50 to > 100 micrograms ml-1 against both pathogenic mycobacteria.     

Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryoelectron microscopy and X-ray crystallography of virus-Fab complexes

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the beta-B-beta-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.     

Rapid stress-induced elevations in corticotropin-releasing hormone mRNA in rat central amygdala nucleus and hypothalamic paraventricular nucleus: an in situ hybridization analysis

High densities of nerve cells containing corticotropin-releasing hormone (CRH) are located in the central nucleus of the amygdala (CeA) and paraventricular nucleus (PVN) of the hypothalamus. These brain regions play an important role in activating autonomic, behavioral, and endocrine responses to stress. This study was conducted to provide needed information concerning the acute effects of stress on CeA and PVN CRH mRNA expression. Rats were exposed to restraint stress for 1 h and brains collected after a 1-h post-stress interval. CRH mRNA expression occurring in the CeA and PVN was examined using in situ hybridization techniques. Densitometric analysis revealed that acute restraint stress produced significant increases in CRH mRNA levels in the PVN and in the rostral CeA region. In addition, the area in the rostral CeA encompassing high CRH mRNA signals increased significantly after stress. Results provide clear evidence that CRH neurons in the CeA and PVN exhibit rapid increases in CRH mRNA expression after exposure to stress.     

Virus- and interferon-induced loss of inhibitory M2 muscarinic receptor function and gene expression in cultured airway parasympathetic neurons

We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis.     

Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.     

Omental transfer for salvage of the moribund lower extremity

Use of omental flaps is well documented in soft tissue reconstruction of the head and neck, chest wall, and abdomen. Three cases of omental transfer for soft tissue reconstruction of the lower extremities are presented. In two patients, free vascularized omental flaps were used to cover deep soft tissue defects over the lower leg and in one patient, a pedicle flap was used to cover a deep groin defect extending into the hip joint. In all patients, use of an omental graft allowed revascularization and subsequent wound healing with good cosmetic results.     

Inhibitory effects of hydroxystilbenes on cyclooxygenase from sheep seminal vesicles

Therapy-related acute myeloid leukemias with balanced translocations affecting the 11q23 chromosome region are one of the most serious complications of treatments with topoisomerase II inhibitor drugs as epipodophillotoxins and anthracyclines. 1,2-5 These cases are usually associated with short interval time from previous chemotherapies, absence of myeloid dysplastic phase, hyperleukocytosis and young age. We and others have recently identified and cloned the ALL1 gene at 11q23 band (also named MLL, HRX. Hrxt) which is consistently altered in t-AML following therapies with topo II targeting drugs. However, there are few reports of cases of t-AML, clinically and biologically similar to the subtype of leukemias secondary to exposure to topo II inhibitors drugs but without the involvement of the ALL1 gene. These observations suggest that genes other than ALL1 which are etiopathogenetically relevant for hematological neoplasias are located in this cytogenetic region.     

Control of Leishmania major by a monoclonal alpha beta T cell repertoire

Little is known regarding the diversity of the host T cell response that is required to maintain immunologic control of microbial pathogens. Leishmania major persist as obligate intracellular parasites within macrophages of the mammalian host. Immunity is dependent upon activation of MHC class II-restricted T cells to an effector state capable of restricting growth and dissemination of the organisms. We generated alpha-beta Leishmania-specific (ABLE) TCR transgenic mice with MHC class II-restricted T cells that recognized an immunodominant Leishmania Ag designated LACK. Naive T cells from ABLE mice proliferated in vitro after incubation with recombinant LACK or with Leishmania-parasitized macrophages and in vivo after injection into infected mice. Infected ABLE mice controlled Leishmania infection almost as well as wild-type mice despite a drastic reduction in the T cell repertoire. ABLE mice were crossed to mice with disruption of the TCR constant region alpha gene to create animals with a single alpha beta T cell repertoire. Although mice deficient in all alpha beta T cells (TCR-C alpha 0 mice) failed to control L. major, mice with a monoclonal alpha beta T cell repertoire (ABLE TCR-C alpha 0 mice) displayed substantial control. The immune system is capable of remarkable efficiency even when constrained to recognition of a single epitope from a complex organism.     

The isolated choroid plexus cyst

A 48-year-old asymptomatic male hepatitis B virus carrier presented with a 2-day history of fever, chills, right upper quadrant abdominal pain, and jaundice. Shock was detected on admission. Emergency abdominal computed tomography (CT) scanning without contrast enhancement showed the features of acute pancreatitis. Hemobilia, edematous pancreatitis, cholestasis and cholecystitis were found on exploratory laparotomy. Neither stone nor active bleeding were detected on intraoperative choledochoscopic examination. Postoperative T-tube cholangiography one month later revealed non-opacification of the left intrahepatic duct. The patient's abdominal pain and hemobilia recurred. Celiac angiography and CT scanning with contrast showed two hepatocellular carcinomas (HCC) in the left lobe of the liver. This is the first case report in the English literature of HCC presenting as jaundice, hemobilia, and acute pancreatitis.