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Background: Patients with advanced neuroblastoma have a poor prognosis. The antiapoptotic protein Akt has been implicated as a possible mediator of the resistance of human neuroblastoma cells to apoptosis; the proapoptotic protein Bid, is inhibited by activated Akt. Neuroblastoma has demonstrated responsiveness to immunotherapeutic approaches in preclinical studies, prompting investigation of new therapeutic strategies based on potentiation of the host immune response, including the use of systemic cytokines. Methods: We examined the antitumor efficacy and mechanisms of action of the central immunoregulatory cytokine interleukin-12 (IL-12) in mice bearing established orthotopic neuroblastoma tumors derived from murine TBJ and Neuro-2a cells. Cohorts of mice (10 mice/group) bearing established orthotopic neuroblastoma tumors were injected intraperitoneally with IL-12 or vehicle and monitored for survival. IL-12-induced apoptosis within the tumor microenvironment was investigated using ribonuclease protection assays, nuclear staining, and electron microscopy. Protein expression was determined via Western blot analysis and enzyme-linked immunosorbent assays. Confocal microscopy was used to examine the distribution of overexpressed Bid-enhanced green fluorescent protein fusion protein (Bid-EGFP) in TBJ cells. All statistical tests were two-sided. Results: IL-12 induced complete tumor regression and long-term survival of 8 (80%) of 10 mice hearing established neuroblastoma tumors compared with 1 (10%) of 10 control mice (P = .0055) and profound tumor cell apoptosis in vivo despite the fact that TBJ and Neuro-2a cells were resistant to receptor-mediated apoptosis in vitro. These cells expressed high levels of phosphorylated Akt, a key prosurvival molecule, and Akt inhibitors sensitized neuroblastoma cells to apoptosis mediated by IL-12-inducible cytokines including tumor necrosis factor-[alpha] and interferon-[gamma] in vitro. IL-12 increased the expression of proapoptotic genes and decreased Akt phosphorylation within established TBJ tumors in conjunction with activation and subcellular translocation of Bid. Conclusions: Our results suggest that IL-12 overcomes a potentially critical mechanism of tumor self-defense in vivo by inhibiting Akt activity and imply that IL-12 may possess unique therapeutic activity against tumors that express high levels of activated Akt.

Background: In the autumn of 1992, a novel form of chronic, active hepatitis of unknown etiology was discovered in mice at the National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC), Frederick, Md. A high incidence of hepatocellular tumors occurred in affected animals. The disease entity was originally identified in A/JCr mice that were untreated controls in a long-term toxicologic study. Purpose: Our original purpose was to determine the origin and etiology of the chronic hepatitis and to quantify its association with hepatocellular tumors in mice of low liver tumor incidence strains. After a helical microorganism was discovered in hepatic parenchyma of diseased mice, we undertook characterization of the organism and investigation of its relationship to the disease process. Methods: Hepatic histopathology of many strains of mice and rats, as well as guinea pigs and Syrian hamsters, in our research and animal production facilities was reviewed. Steiner's modification of the Warthin-Starry stain and transmission electron microscopy were used to identify bacteria in the liver. We transmitted the hepatitis with liver suspensions from affected mice and by inoculation with bacterial cultures. Bacteria were cultivated on blood agar plates maintained under anaerobic or microaerophilic conditions and characterized morphologically, biochemically, and by 16S rRNA sequence. Results: We report here the isolation of a new species of Helicobacter (provisionally designated Helicobacter hepaticus sp. nov.) that selectively and persistently colonizes the hepatic bile canaliculi of mice (and possibly the intrahepatic biliary system and large bowel), causing a morphologically distinctive pattern of chronic, active hepatitis and associated with a high incidence of hepatocellular neoplasms in infected animals. Conclusions: The novel Helicobacter is a likely candidate for the etiology of hepatocellular tumors in our mice. The Helicobacter-associated chronic active hepatitis represents a new model to study mechanisms of carcinogenesis by this genus of bacteria. Implications: Adenocarcinoma of the stomach, the second most prevalent of all human malignancies worldwide, is associated with infection at an early age with Helicobacter pylori. Infection leads to several distinctive forms of gastritis, including chronic atrophic gastritis, which is a precursor of adenocarcinoma. H. hepaticus infection in mice constitutes the only other parallel association between a persistent bacterial infection and tumor development known to exist naturally. Study of the H. hepaticus syndrome of chronic active hepatitis and liver tumors in mice may yield insights into the role of H. pylori in human stomach cancer and gastric lymphoma. [J Natl Cancer Inst 86:1222-1227, 1994]

ABSTRACTPolo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. Studies have shown that Plk4 undergoes dynamic relocalization from a ring-like pattern around a centriole to a dot-like morphology at the procentriole assembly site and this event is central for inducing centriole biogenesis. However, the detailed mechanisms underlying Plk4’s capacity to drive its symmetry-breaking ring-to-dot relocalization remain largely unknown. Here, we showed that Plk4 self-initiates this process in an autophosphorylation–dependent manner and that STIL, its downstream target, is not required for this event. Time-dependent analyses with mEOS-fused photoconvertible Plk4 revealed that a portion of ring-state Plk4 acquires a capacity, presumably through autophosphorylation, to linger around a centriole, ultimately generating a dot-state morphology. Interestingly, Plk4 WT, but not its catalytically inactive mutant, showed the ability to form a nanoscale spherical assembly in the cytosol of human cells or heterologous E. coli, demonstrating its autophosphorylation-dependent self-organizing capacity. At the biochemical level, Plk4 – unlike its N-terminal βTrCP degron motif – robustly autophosphorylated the PC3 SSTT motif within its C-terminal cryptic polo-box, an event critical for inducing its physical clustering. Additional in vivoexperiments showed that although STIL was not required for Plk4’s initial ring-to-dot conversion, coexpressed STIL greatly enhanced Plk4’s ability to generate a spherical condensate and recruit Sas6, a major component of the centriolar cartwheel structure. We propose that Plk4’s autophosphorylation-induced clustering is sufficient to induce its ring-to-dot localization conversion and that subsequently recruited STIL potentiates this process to generate a procentriole assembly body critical for Plk4-dependent centriole biogenesis.

Mutations in CEP290, a transition zone protein in primary cilia, cause diverse ciliopathies, including Leber congenital amaurosis (LCA) and Joubert-syndrome and related disorders (JSRD). We examined cilia biogenesis and function in cells derived from CEP290-LCA and CEP290-JSRD patients. CEP290 protein was reduced in LCA fibroblasts with no detectable impact on cilia; however, optic cups derived from induced pluripotent stem cells (iPSCs) of CEP290-LCA patients displayed less developed photoreceptor cilia. Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia and decreased ciliogenesis. We observed selectively reduced localization of ADCY3 and ARL13B. Notably, Hedgehog signaling was augmented in CEP290-JSRD because of enhanced ciliary transport of Smoothened and GPR161. These results demonstrate a direct correlation between the extent of ciliogenesis defects in fibroblasts and photoreceptors with phenotypic severity in JSRD and LCA, respectively, and strengthen the role of CEP290 as a selective ciliary gatekeeper for transport of signaling molecules in and out of the cilium.

Despite successfully suppressed viremia by treatment, patients with high levels of biomarkers of coagulationinflammation are at an increased risk of developing non-AIDS defining serious illnesses such as cardiovascular diseases. Thus, there is a relationship between persistent immune activation and coagulationinflammation, although the mechanisms are poorly understood. Platelets play an important role in this process. Although interactions between platelets and elements of the innate immune system, such as monocytes, are well described, little is known about the interaction between platelets and the adaptive immune system.

The inflammatory microenvironment promotes skin tumorigenesis. However, the mechanisms by which cells protect themselves from inflammatory signals are unknown. Downregulation of IKKα promotes skin tumor progression from papillomas to squamous cell carcinomas, which is frequently accompanied by genomic instability, including aneuploid chromosomes and extra centrosomes. In this study, we found that IKKα promoted oligomerization of nucleophosmin (NPM), a negative centrosome duplication regulator, which further enhanced NPM and centrosome association, inhibited centrosome amplification, and maintained genome integrity. Levels of NPM hexamers and IKKα were conversely associated with skin tumor progression. Importantly, proinflammatory cytokine-induced IKKα activation promoted the formation of NPM oligomers and reduced centrosome numbers in mouse and human cells, whereas kinase-dead IKKα blocked this connection. Therefore, our findings suggest a mechanism in which an IKKα-NPM axis may use inflammatory signals to suppress centrosome amplification, promote genomic integrity, and prevent tumor progression.

GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

Primary cilia are microtubule-based solitary sensing structures on the cell surface that play crucial roles in cell signaling and development. Abnormal ciliary function leads to various human genetic disorders, collectively known as ciliopathies. Outer dense fiber protein 2 (Odf2) was initially isolated as a major component of sperm-tail fibers. Subsequent studies have demonstrated the existence of many splicing variants of Odf2, including Cenexin1 (Odf2 isoform 9), which bears an unusual C-terminal extension. Strikingly, Odf2 localizes along the axoneme of primary cilia, whereas Cenexin1 localizes to basal bodies in cultured mammalian cells. Whether Odf2 and Cenexin1 contribute to primary cilia assembly by carrying out either concerted or distinct functions is unknown. By taking advantage of odf2−/−cells lacking endogenous Odf2 and Cenexin1, but exogenously expressing one or both of these proteins, we showed that Cenexin1, but not Odf2, was necessary and sufficient to induce ciliogenesis. Furthermore, the Cenexin1-dependent primary cilia assembly pathway appeared to function independently of Odf2. Consistently, Cenexin1, but not Odf2, interacted with GTP-loaded Rab8a, localized to the distal/subdistal appendages of basal bodies, and facilitated the recruitment of Chibby, a centriolar component that is important for proper ciliogenesis. Taken together, our results suggest that Cenexin1 plays a critical role in ciliogenesis through its C-terminal extension that confers a unique ability to mediate primary cilia assembly. The presence of multiple splicing variants hints that the function of Odf2 is diversified in such a way that each variant has a distinct role in the complex cellular and developmental processes.

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H2O2but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O2-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.

The role of ArfGAP1 in COPI vesicle biogenesis has been controversial. In work using isolated Golgi membranes, ArfGAP1 was found to promote COPI vesicle formation. In contrast, in studies using large unilamellar vesicles (LUVs) as model membranes, ArfGAP1 functioned as an uncoating factor inhibiting COPI vesicle formation. We set out to discriminate between these models. First, we reexamined the effect of ArfGAP1 on LUVs. We found that ArfGAP1 increased the efficiency of coatomer-induced deformation of LUVs. Second, ArfGAP1 and peptides from cargo facilitated self-assembly of coatomer into spherical structures in the absence of membranes, reminiscent of clathrin self-assembly. Third, in vivo, ArfGAP1 overexpression induced the accumulation of vesicles and allowed normal trafficking of a COPI cargo. Taken together, these data support the model in which ArfGAP1 promotes COPI vesicle formation and membrane traffic and identify a function for ArfGAP1 in the assembly of coatomer into COPI.

Ceramidases catalyze the conversion of ceramide to sphingosine. They are acylaminohydrolases that catalyze the deacylation of the amide-linked saturated fatty acid from ceramide to generate sphingosine. They also catalyze the reverse reaction of ceramide biosynthesis using sphingosine and fatty acid. In mammals, different proteins catalyze these reactions while individually exhibiting optimal activity over a narrow pH range and have been accordingly called acid, neutral, and alkaline ceramidases. Several genes encode for variants of alkaline ceramidase in mammals. Brainwashing (Bwa) is the only putative alkaline ceramidase homologue present in DROSOPHILA: In this study we have demonstrated that BWA does not exhibit ceramidase activity and that bwa null mutants display no loss of ceramidase activity. Instead, the neutral ceramidase gene CDase encodes the protein that is responsible for all measurable ceramidase activity in DROSOPHILA: Our studies show strong genetic interaction of Bwa with CDase and the Drosophila ceramide kinase gene (DCERK). We show that, although BWA is unlikely to be a ceramidase, it is a regulator of sphingolipid flux in DROSOPHILA: Bwa exhibits strong genetic interaction with other genes coding for ceramide-metabolizing enzymes. This interaction might partly explain its original identification as a ceramidase.

Mammalian antimicrobial peptides, including β‐defensins, represent an ancient arm of innate immunity designed to directly neutralize invading microbes. Previously, we demonstrated that murine β‐defensin 2 (mDF2β) also acted as an endogenous ligand for TLR‐4‐activating maturation of dendritic cells (DCs). Herein, we report that this TLR‐4 –dependent activation leads to induction of an atypical cell death that is unexpectedly exaggerated by the inhibition of caspases. Experiments using APCs with nonfunctional TNF‐α or its receptors suggest that this is a NF‐κB‐ and TNF‐α‐dependent process that does not require TNFR1. We demonstrate that mDF2β triggers a TNFR2‐mediated signaling cascade of “self‐destruction” through up‐regulation of membrane‐bound TNF‐α and TNFR2. This appears not to be an isolated phenomenon, as human synthetic β‐defenisn 3 was also able to activate and kill DCs. We propose that β‐defenins may play an important immunoregulatory role as controllers of the natural process of elimination of activated APCs.

Abstract: Hearts from 1-yr-old Erythrocebus patas monkeys were examined after in utero and 6-wk-postbirth exposure to antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs). Protocols were modeled on those given to human immunodeficiency virus (HIV)-1-infected pregnant women. NRTIs were administered daily to the dams for the last 20% or 50% of gestation, and to the infants for 6 wk after birth. Exposures included: no drug (n=4); Zidovudine, 3′-azido-3′-deoxythymidine (AZT; n=4); AZT/Lamivudine, (−)-β-l-2′, 3′-Dideoxy-3′-thiacytidine (Epivir, 3TC) (n=4); AZT/Didanosine (Videx, ddl) (n=4); and Stavudine (Zerit, d4T)/3TC (n=4). Echocardiograms and clinical chemistry showed no drug-related changes, but the d4T/3TC-exposed fetuses at 6 and 12 mo had increased white cell counts (p<0.05). At 1 yr of age, oxidative phosphorylation (OXPHOS) enzyme activities were similar in heart mitochondria from all groups. Mitochondrial pathology, that included clones of damaged mitochondria (p<0.05), was found in hearts of all 1-yr drug-exposed infants. Levels of mtDNA were elevated (p<0.05) in hearts of all NRTI-exposed monkeys in the following order: control < d4T/3TC

Abstract: Cardiac toxicity has been associated with HIV infection and exposure to nucleoside reverse transcriptase inhibitors (NRTIs), but the role of the latter in the development of cardiac disease of HIV-infected patients is uncertain. To investigate the cardiotoxicity of transplacentally administered zidovudine (AZT) or AZT plus lamivudine (3TC) in the absence of HIV infection, we evaluated several biomarkers of cardiac mitochondrial structure and cardiac structure and function in a B6C3F1 mouse model. In utero exposure to AZT-3TC resulted in ultrastructural pathology, loss of mitochondria, and altered echocardiographic measurements in newborn mice. Cardiac pathology and dysfunction persisted into the adult life of female mice exposed in utero to AZT, as evidenced by significant dose-dependent heart enlargement, clusters of atypical mitochondria and myofibril alterations, significantly increased cytochrome c oxidase activity, and significantly higher numbers of mutations in mitochondrial tRNA genes compared with unexposed controls at 18 to 24 mo of age. These data led to the hypothesis that the long-term pathology of perinatal exposure to these NRTIs is related to persistent mitochondrial DNA mutations in cardiac tissue; that is, the primary damage during drug treatment is mutational (as opposed to affecting polymerase γ and/or other mitochondrial elements) and leads over time to delayed, progressive cardiotoxicity.

This study was designed to investigate fetal mitochondrial toxicity in Erythrocebus patas monkeys exposed in utero to zidovudine (AZT) and lamivudine (3TC), and taken at term. Pregnant patas monkeys were given a daily dose of 40 mg AZT (86% of the human daily dose, based on body weight), for the last 10 weeks (50%) of gestation, and a daily dose of 24 mg 3TC (84% of the human daily dose, based on body weight) for the last 4 weeks of gestation. At term, AZT was found to be incorporated into fetal mitochondrial DNA from skeletal muscle, liver, kidney, and placenta. By transmission electron microscopy (EM) drug-exposed fetal cardiac and skeletal muscle cells showed mitochondrial membrane compromise, mitochondrial proliferation, and damaged sarcomeres, while mitochondria in brain cerebrum and cerebellum were morphologically normal. Substantial depletion of oxidative phosphorylation (OXPHOS) Complex I specific activities was observed in heart (87% reduction in mean, p = 0.02) and skeletal muscle (98% reduction in mean, p = 0.002) from drug-exposed fetuses, compared to unexposed fetuses. In addition Complex IV activity was highly depleted (85% reduction in mean, p = 0.004) in skeletal muscle from the drug-exposed fetuses (p = 0.004). Brain cerebrum and cerebellum showed no statistically significant OXPHOS changes with drug exposure. Mitochondrial DNA quantity was substantially depleted (>50%) in heart, skeletal muscle, cerebellum, and cerebrum from drugexposed fetuses compared to unexposed controls. Overall, the data indicate that significant mitochondrial damage was observed at birth in monkey fetuses exposed in utero to AZT plus 3TC in a human-equivalent dosing protocol.

ABSTRACTA single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal (“?”) in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and ?- particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed.

ABSTRACTWe previously reported that if murine leukemia virus particles are produced in the presence of the mild oxidizing agent disulfide-substituted benzamide-2, they fail to undergo the normal process of virus maturation. We now show that treatment of these immature particles with a reducing agent (dithiothreitol) induces their maturation in vitro, as evidenced by proteolytic cleavage of Gag, Gag-Pol, and Env proteins and by their morphology. The identification of partial cleavage products in these particles suggests the sequence with which the cleavages occur under these conditions. This may be a useful experimental system for further analysis of retroviral maturation under controlled conditions in vitro.

Development and differentiation of the mammary gland occurs by means of critical stromal-epithelial interactions. Although many studies have attempted to understand these complex interactions, it has been difficult to demonstrate the essential role of adipose tissue in the development and function of the mammary gland. By using the A-ZIP/F-1 transgenic mice lacking in white adipose tissue (WAT), we have studied the role of adipocytes in mammary gland development and differentiation. In the absence of WAT, rudimentary mammary anlagen form but are unable to grow and branch normally, resulting in a few, short, severely distended ducts. However, during pregnancy, a tremendous amount of epithelial cell division and alveolar cell formation occurs even in the absence of adipocytes, illustrating that adipose tissue is not required for mammary gland differentiation. Mammary gland transplantation revealed that epithelial cells from these transgenic mice possess the potential for normal growth and differentiation when placed into a normal stromal environment. These experiments clearly demonstrate that the absence of adipocytes in the mammary gland results in disruption of stromal-epithelial interactions that prevent normal mammary gland development. The rudimentary epithelial anlage, however, contain mammary stem cells, which are fully capable of alveolar differentiation. Published 2002 Wiley-Liss, Inc.

ABSTRACTSome retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9Gagproteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.

T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is down-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21–23. Theclaudin-18gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for transactivation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18transcript has an alternative 12-bp insertion derived from the 5′ end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach.

ABSTRACTAlthough the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3' end of the HIV-1 gag-poltransframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6Gagprotein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNAPR2. A disrupted translation of gag-polmRNA induced at the PNAPR2-annealing site resulted in a decreased synthesis of Pr160Gag-Polpolyprotein, hence the viral protease, a predominant expression of Pr55Gagdevoid of a fully functional p6Gagprotein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNAPR2abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-poltransframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.

3'-azido-3'-deoxythymidine (AZT) is given to pregnant women positive for the human immunodeficiency virus type 1 (HIV-1) to reduce maternal–fetal viral transmission. To explore fetal mitochondrial consequences of this exposure, pregnant Erythrocebus patas monkeys were given daily doses of 1.5 mg (21% of the human daily dose) and 6.0 mg (86% of the human daily dose) of AZT/kg body weight (bw), for the second half of gestation. At term, electron microscopy of fetal cardiac and skeletal muscle showed abnormal and disrupted sarcomeres with myofibrillar loss. Some abnormally shaped mitochondria with disrupted cristae were observed in skeletal muscle myocytes. Oxidative phosphorylation (OXPHOS) enzyme assays showed dose-dependent alterations. At the human-equivalent dose of AZT (6 mg of AZT/kg bw), there was an ~85% decrease in the specific activity of NADH dehydrogenase (complex I) and three- to sixfold increases in specific activities of succinate dehydrogenase (complex II) and cytochrome-c oxidase (complex IV). Furthermore, a dose-dependent depletion of mitochondrial DNA levels was observed in both tissues. The data demonstrate that transplacental AZT exposure causes cardiac and skeletal muscle mitochondrial myopathy in the patas monkey fetus.

An automatic monitor has been developed for measuring formaldehyde in air using a sensitive tape for formaldehyde. It is based on the color change of the tape on reaction with formaldehyde. The porous cellulose tape, containing silica gel as an absorbent and impregnated with the processing solution containing hydroxylamine sulfate, Methyl Yellow (pH indicator; pH 2.9-4.0, red-yellow), glycerin and methanol, was found to be a highly sensitive means of detecting formaldehyde and maintains a stable sensitivity. When the tape was exposed to a sample of air containing formaldehyde, the color of the tape changed from yellow to red. The degree of color change was proportional to the concentration of formaldehyde at a constant sampling time and flow rate, and it could be recorded by measuring the intensity of reflected light (555 nm). The tape could be used to detect down to 0.08 ppm (World Health Organization standard) of formaldehyde with a sampling time of 30 min and a flow rate of 100 mL min–1. Reproducibility tests showed that the relative standard deviation of response (n=10) was 3.8% for 0.1 ppm formaldehyde. The monitor is simple, specific, capable of unattended operation and is recommended for both laboratory and field operation.

Insect cell expression of the HIV-1 Gag precursor protein by recombinant baculoviruses results in the assembly and budding of noninfectious virus-like particles (VLPs). The VLPs resemble immature virus in ultrastructural morphology and can be purified by conventional retroviral techniques. The virus-like appearance of the particles suggested that they could be used to package additional peptides. The retroviral frameshift mechanism was used to translate thepolgene products by expressing additional genetic information as chimeric Gag–Pol fusion proteins. Sequences encoding the carboxyl 65% of the HIV-1 surface glycoprotein (gp120, SU) were inserted into the Gag–Pol reading frame immediately downstream of the Gag stop codon. The assembly and budding of large quantities of Gag and chimeric Gag–SU VLPs were observed by standard transmission electron microscopy. The presence of gp120 epitopes in the Gag–SU VLPs was confirmed by immunoelectron microscopy and Western blot analysis using monoclonal anti-gp120 antibodies. Mice inoculated with the Gag–SU pseudovirions developed cytotoxic lymphocyte responses to both HIV-1 Gag and Env epitopes yet humoral immune responses only to Gag epitopes. The chimeric Gag–SU particles may have applications as vaccines or immunotherapeutic treatments for HIV-1 infection. In addition, the frameshift mechanism can be applied to the packaging of other viral or cellular proteins.

A galvanic sensor for monitoring nitrogen dioxide was developed by using a poly(ethylene oxide) complex of silver trifluoromethanesulphonate electrolyte. The sensor, which is expressed as Au/P(EO)4.5 AgCF3SO3/Ag, is a small disk (i.d. 13 mm). The polymeric electrolyte film was made by casting the mixture of acetonitrile solutions of both P(EO) (MW 6×105) and AgCF3SO3. The working electrode was made by sputtering of gold in argon. The thicknesses of the desposited gold, polymeric electrolyte film and silver are 25 nm, 30 µm and 1 mm, respectively. When the sample gas containing nitrogen dioxide impinges at 20 ml min-1 on the gold cathode, the current flowing in the external circuit was linearly related to the concentration of nitrogen dioxide from 20 ppb to about 10 ppm. The current efficiency of the cell was 0.051%. The cell's response time was about 2 min for 0.5 ppm of nitrogen dioxide.

cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized. BIV expresses two distincttatmRNAs composed of three exons that are derived by alternative splicing. The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids. The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3. Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses. BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria. BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells. Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells. However, an intact BIVtatgene is required for viral replication in both Cf2Th and EREp cells. Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain. BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells. In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types. In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants. The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.

Hydrogen fluoride in a standard or sample gas stream at 200 ml min−1permeates through a teflon membrane (0.8 μm pore size, 0.08 mm thick) into an absorption solution (citrate/acetate buffer at pH 5.4) flowing at 30 ml min−1. The fluoride produced is measured with the fluoride-selective electrode. The response time is about 12 min. The absorption efficiency of hydrogen fluoride is about 70% between 6.5 and 0.25 ppm by volume (5.2 and 0.2 mg m−3). In this range, the Nernst equation is valid with a relative standard deviation of less than 1.8%. The lower determination limit for hydrogen fluoride is 0.1 ppm (0.08 mg m−3).

Large granular lymphocytes (LGL) comprise a natural defense system in the liver and exert an inhibitory effect on tumor cell metastasis. In order to demonstrate the maturation of LGL in the liver from the morphological aspect, we evaluated electron-microscopically the frequency of 0.2 µm vesicles (rod-cored and “empty” vesicles) and dense granules in LGL from the liver, spleen, and peripheral blood of the rat. Both of these cell organelles are characteristic to LGL and may relate to natural killer-mediated cytolysis. On the average, there were 12.7 of the 0.2 µm vesicles and 4.3 rod-cored vesicles (RCV) per cell section in the liver, 6.6 0.2 µm vesicles and 1.6 RCV in the spleen, and 8.6 0.2 µm vesicles and 0.9 RCV in the peripheral blood. The number of 0.2 µm vesicles per cell section ranged from 0 to 19 with the exception of a few higher instances. Therefore, LGL were divided into vesicle-rich(>9 0.2 µm vesicles per cell section) and vesicle-poor (<8 per cell section) populations. Hepatic LGL consisted mainly of a vesicle-rich population while splenic LGL consisted mainly of a vesicle-poor population, and peripheral blood contained equal proportions of both populations. In addition to diversity with regard to the number of 0.2 µm vesicles, LGL obtained from various organs also displayed heterogeneity in the number and size of dense granules. Since the number of dense granules per cell section usually ranged from 1 to 13, LGL were diveded into 2 populations, i.e., LGL with many (>7 per cell section) granules and those with a few(<6 per cell section) granules. Specifically, splenic LGL had a few small (average diameter, less than 400 nm) dense granules, while sections of LGL from the liver and peripheral blood displayed many small dense granules and a few large (>400 nm) ones, respectively, in addition to the populations seen in the spleen. Thus, the present study has demonstrateda difference in the distribution of 0.2 µm vesicles in LGL based on the tissue of origin. The present study has revealed the difference in the distribution of 0.2 µm vesicles of LGL by tissue and indicated that immature LGL are predominant in the spleen, while hepatic LGL are generally more mature as defined by the number of vesicles. These data suggest that the microenvironment of the liver may contribute to the increased expression of these vesicles in LGL.

Expression of human immunodeficiency virus (HIV) Gag precursor protein (Pr55) by recombinant baculoviruses in insect cells results in the assembly and budding of Pr55 as virus-like particles, or Gag pseudovirions. The ultrastructural morphology, size, and sucrose sedimentation rate of Gag pseudovirions are indistinguishable from immature lentivirus particles produced by HIV-infected human cells. Recombinant baculoviruses were engineered to express individually Pr55 and HIV Env glycoprotein precursor (gp160). These recombinant baculoviruses were used to co-infect insect cells to produce chimeric HIV Gag pseudovirions containing gp160 in experiments to develop methodologies for producing complex noninfectious particulate vaccines for HIV. Coexpression of HIV Pr55 and gp160 resulted in the apparent incorporation of gp160 into Gag pseudovirions as determined by immunoblotting with envelope-specific monoclonal antibodies. Furthermore, results from indirect immunogold electron microscopy using monoclonal antibodies to HIV gp120, a component of the Env glycoprotein precursor, suggested that HIV gp160 was specifically incorporated during the budding process into the outer surface of chimeric Gag pseudovirions. Parallel labeling experiments to localize gp120 and Pr55 epitopes on HIV-infected H9 lymphocytes provided results similar to those obtained with chimeric Gag pseudovirions producing recombinant baculovirus-infected insect cells. Parameters influencing immunoelectron microscopy results in cell-surface and postembedding labeling experiments are discussed.

A flow-injection analysis of sulphate using a reaction column packed with barium chloranilate (BaCh) is described. The chloranilate ion released by the reaction is monitored at the isosbestic point (310 nm) of chloranilate. An aqueous solution of 2-propanol (60%) is used as the carrier solution at a flow-rate of 1 ml . min-1. The linear dynamic range of the detection system is approximately 2.5×10-6~1.0×10-3 mol/l per 10 µl sample injection. The time required for a determination is only 20 s per sample injection. The method has also been applied to the amplified determination of phosphate by reaction with molybdate resulting from the decomposition of molybdophosphate (H3PO4.MoO3) extracted.

Some retroviruses, including HIV-1, regulate the relative amounts of gag and pol gene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which the gag and pol genes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fused gag-pol gene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-type gag-pol gene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-type gag-pol gene was expressed, whereas no particles were detected in cells that expressed the fused gag-pol gene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented. Copyright 1993, 1999 Academic Press

Concentrations of the potent, HIV(human immunodeficiency virus) inactivating protein, cyanovirin-N (CV-N), which completely inhibit HIV-1 infectivity, do not block the binding of soluble CD4-receptor (sCD4) to HIV-1 lysates nor the attachment of intact HIV-1 virions to several target T-cell lines. Furthermore, in contrast to the known disassociative effects of sCD4 on viral envelope glycoproteins, treatment of HIVRFwith high concentrations of CV-N results in complete viral inactivation but without apparent shedding of gp120 or other ultrastructural changes. These results are consistent with the view that the virucidal effects of CV-N result from interference with step(s) in the fusion process subsequent to the initial binding of the virus to target cells.

We have defined a population of CD3‐, CD56+small lymphocytes (SLs) that exhibit the same phenotype and lytic capacity as natural killer (NK) cells. NK cells characteristically express the surface markers CD16 and CD56, mediate non–major histocompatibility complex (MHC)–restricted lysis, and have been equated with CD3‐large granular lymphocytes (LGLs). In the present study we extended the observation that CD3‐, CD56+SLs can mediate NK‐ and antibody‐dependent cellular cytotoxicity activity by studying the activation signals and lytic mechanisms that might be utilized by CD3‐, CD56+SLs in comparison to CD3‐CD56+LGLs. Our results show that CD3‐SLs, similar to CD3‐LGLs, exhibited activated killing in response to interleukin‐2 (IL‐2). In addition, after IL‐2 activation, the CD3‐SLs exhibited morphologic changes, including increases in size and granularity, and both morphologically and phenotypically became virtually indistinguishable from CD3‐LGLs. Similar to CD3‐LGLs, CD3‐SLs could be directly activated by IL‐2 alone to secrete significant quantities of interferon‐γ (IFN‐γ) and to express IL‐2 receptor (IL‐2R) p55. Examination of serine esterases and pore‐forming protein (PFP) demonstrated that these cells exhibited a cytoplasmic distribution of perforin, which, unlike that of CD3‐LGLs, was not associated with dense cytoplasmic azurophilic granules. Serine esterase levels were similar. However, after IL‐2 activation PFP was concentrated in dense cytoplasmic granules, similar or identical to the situation in CD3‐, CD56+LGLs. These CD3‐, CD56+subsets appear to represent a continuum of activated cells that might represent various states of maturation of NK cells.

In the present study, we have examined the exocytosis model for cellular lysis. Using monoclonal antibodies reactive with human pore‐forming protein (PFP), we examined the localization of PFP at the interaction site of natural killer (NK) cells and the NK tumor targets K562 and Molt‐4 as well as during antibody‐ dependent cellular cytotoxicity. Following the interaction of effector‐target cell contact, an increased frequency of PFP was detected on the effector surface, in the micro‐ environment, and on the target surface of the interaction site. This temporal deposition of PFP was paralleled by loss of target cell integrity and release of chromium. In addition, selective deposition of PFP was seen at the interaction site of the target cell compared to other target cell regions. Collectively, these results are consistent with the exocytosis model and further support the hypothesis that PFP is one of the secreted moieties involved in NK cellular cytotoxicity

Characterization of human foamy virus (HFV) gag-encoded precursors and the search for a Gag-Pol polyprotein and mature proteins derived from proteolytic processing were carried out in HFV-infected cells and with purified preassembled cores and extracellular virus by Western blotting and radioimmunoprecipitation using antisera against synthetic peptides corresponding to putative Gag and protease proteins. Precursor proteins, Pr78gag/74gagand Pr135pol, were found in the nucleus of epithelial and fibroblast cells 3–4 days after HFV infection. Kinetic analysis of HFV Pr78gagand Pr74gagindicated that Pr78gagis a precursor to Pr74gag. South-Western blot analysis indicated that Pr78gagand Pr74gaghave properties associated with nucleic acid binding protein although they lack the typical zinc-finger motifs found in retroviral nucleocapsid proteins. Western blot analyses of preassembled HFV cores isolated from the cytoplasm of infected cells and purified by sucrose gradient centrifugation demonstrated the presence of Pr78gag/74gagand Pr135pol, but no proteolytically processed Gag proteins were observed. The majority of extracellular HFV particles were found to have pentagon-shaped cores, as observed intracellularly, and are believed to be the immature extracellular form of the virus. The highest concentration of extracellular particles, estimated by EM, Western blot, and reverse transcriptase assays were found in sucrose gradient fractions having a density of 1.21–1.24 g/cm3. Western blot analysis revealed that Pr78gag/74gagand Pr135polwere the major viral proteins associated with these extracellular particles, as only small amounts of putative proteolytically cleaved capsid (p32) were observed. Our results support the notion that Pol is translated independent of Gag in HFV-infected cells.

The waste water sample is pumped at the flow rate of 4 cm3min-1to the mixing joint, where a reaction solution (5×10-3M bismuth nitrate-containing sodium acetate buffer at pH 5.0) is mixed at the rate of 1 cm3min-1. Then the mixture containing hydrogen cyanide is fed into a gas-liquid separator. The hydrogen cyanide permeates through a Teflon membrane (0.80 μm pore size, 0.08 mm thick) into an absorption solution (0.02% sodium hydroxide) flowing at 24 cm3h-1. The cyanide ion produced is measured with the cyanide selective electrode. The response time is about 1.3 min at a concentration of 1 ×10-4M cyanide. The Nernst equation is valid with a relative standard deviation of less than 2.8% in the range between 1 ×10-3and 1 ×10-6M. The lower detection limit is 1 ×10-7M (2.6 CN-ppb). This method can detect free cyanide ion and indirectly metal cyano complexes whose stability constants are below 1017.

The tumor suppressor folliculin (FLCN) suppresses nuclear translocation of TFE3, a master transcription factor for lysosomal biogenesis, via regulation of amino-acid-sensing Rag GTPases. However, the importance of this lysosomal regulation in mammalian physiology remains unclear. Following hematopoietic-lineage-specific Flcndeletion in mice, we found expansion of vacuolated phagocytes that accumulate glycogen in their cytoplasm, phenotypes reminiscent of lysosomal storage disorder (LSD). We report that TFE3 acts in a feedback loop to transcriptionally activate FLCN expression, and FLCN loss disrupts this loop, augmenting TFE3 activity. Tfe3deletion in Flcnknockout mice reduces the number of phagocytes and ameliorates LSD-like phenotypes. We further reveal that TFE3 stimulates glycogenesis by promoting the expression of glycogenesis genes, including Gys1and Gyg, upon loss of Flcn. Taken together, we propose that the FLCN-TFE3 feedback loop acts as a rheostat to control lysosome activity and prevents excessive glycogenesis and LSD-like phagocyte activation.

We have already developed a HCHO monitoring system which is called FP-30. In this experiment, we have developed a NO2detection tablet which can be used by the monitoring system. The detection tablet for the NO2was constructed with the sensing paper: porous cellulose paper that contains silica gel as an adsorbent, N-1-naphthylethylenediamine dihydrochloride (NED), and glycerin. The NO2in sample gas was blown over and adsorbed on the surface of the sensing paper. Then the NO2reacted with NED, producing a yellow compound. The coloring reaction took place on the surface of the sensing paper. The degree of color change of paper from white to yellow was monitored as a function of the intensity of the reflected light (λ= 475 nm) of an LED. The detection limit was 0.01 ppm when the sampling time was 30 min, and the flow rate of sample gas was 250 ml/min. This sensing paper process was not interfered with by acetaldehyde, acetone, alcohols, hydrocarbons, carbon monoxide or carbon dioxide. The NO2concentrations in the rooms of a house or school were monitored using this monitoring system and the standard chemiluminescence method. The concentrations of NO2monitored by both methods were within 18% of the average. This highly sensitive, selective, and handy NO2gas monitoring system will be widely applicable and convenient for users who are not specialists in this field.

A compound that reduces the prevalence of perinucleolar compartment in cancer cells inhibits metastasis in vivo.

An amplification method is described for the determination of selenious acid with bismuthiol II. A 9.4 fold amplification is achieved.