Low oxygen levels (hypoxia) play a role in clinical conditions such as stroke, chronic ischemia, and cancer. To better understand these diseases, it is crucial to study the responses of vertebrates to hypoxia. Among vertebrates, some teleosts have developed the ability to adapt to extremely low oxygen levels. We have studied long-term adaptive responses to hypoxia in adult zebrafish. We used zebrafish that survived severe hypoxic conditions for 3 wk and showed adaptive behavioral and phenotypic changes. We used cDNA microarrays to investigate hypoxia-induced changes in expression of 15,532 genes in the respiratory organs (the gills). We have identified 367 differentially expressed genes of which 117 showed hypoxia-induced and 250 hypoxia-reduced expressions. Metabolic depression was indicated by repression of genes in the TCA cycle in the electron transport chain and of genes involved in protein biosynthesis. We observed enhanced expression of the monocarboxylate transporter and of the oxygen transporter myoglobin. The hypoxia-induced group further included the genes for Niemann-Pick C disease and for Wolman disease [lysosomal acid lipase (LAL)]. Both diseases lead to a similar intra- and extracellular accumulation of cholesterol and glycolipids. The Niemann-Pick C protein binds to cholesterol from internal lysosomal membranes and is involved in cholesterol trafficking. LAL is responsible for lysosomal cholesterol degradation. Our data suggest a novel adaptive mechanism to hypoxia, the induction of genes for lysosomal lipid trafficking and degradation. Studying physiological responses to hypoxia in species tolerant for extremely low oxygen levels can help identify novel regulatory genes, which may have important clinical implications.

JNK proteins are ubiquitously expressed, evolutionarily conserved MAP kinases that are involved in stress responses. Recently, it was shown that the JNK cascade in Xenopus oocytes exhibits sustained, all-or-none responses to graded, transient stimuli. Here, we have examined the character of the JNK cascade's response in mammalian cells. The steady-state responses of JNK to sorbitol and anisomycin were found to be highly ultrasensitive in HeLa cells, HEK 293 cells, and Jurkat T cells. The JNK responses were also reversible, not sustained, as was the case in oocytes. Jurkat cells activated their JNK in response to phorbol myristate acetate (PMA), and the response of the entire population of Jurkat cells was graded. However, analysis of subpopulations of the PMA-treated Jurkat cells revealed that the steady-state responses of both JNK and CD69, a T cell surface activation marker, were essentially all-or-none in character. These studies show that the JNK cascade commonly exhibits switch-like responses to a variety of stimuli.

3Varicella-zoster virus (VZV) is an alphaherpesvirus with a genome of ϳ125,000 bp encoding at least 70 unique open reading frames (ORFs) (3,9,10,15). Primary VZV infection is associated with a cell-associated viremia and a diffuse cutaneous rash recognized as varicella, or chickenpox. VZV appears to cause viremia by infecting lymphocytes. VZV establishes latency in sensory ganglia and causes herpes zoster upon reactivation. VZV is sustained in the human population, which is its only natural reservoir, primarily by direct contact with infectious virus in varicella or zoster skin lesions or in respiratory secretions. Investigating the molecular mechanisms of VZV pathogenesis has been difficult because of VZV's restricted infectivity for nonhuman species in vivo and the highly cellassociated nature of VZV replication in vitro. These obstacles have been addressed by the development of the SCIDhu mouse model to examine VZV pathogenesis and immunobiology in vivo (5-7, 19, 30-34, 37, 40-42) and by the making of genetically altered VZV recombinants from VZV cosmids (11,20,28). Cosmid mutagenesis can be useful to define essential regions of the VZV genome if infectious virus cannot be recovered without genetic complementation. When genetic changes are not lethal, VZV mutants can be evaluated in vitro and in human T-cell and skin xenografts in SCID mice to determine how particular VZV gene products contribute to virulence in differentiated human cells within their unique tissue microenvironments in vivo. The purpose of this review is to highlight new insights about VZV pathogenesis and immunobiology that have emerged from analyses of VZV infection in the SCIDhu mouse model and from examination of the differential effects of mutations targeting ORF47, which encodes a viral kinase/tegument protein, on VZV tropism for skin and T cells.Clinical observations indicate that primary VZV infection begins with respiratory mucosal inoculation and that the characteristic chickenpox rash develops after an incubation period of 10 to 21 days (3, 9). In the absence of experimental data, early events in VZV pathogenesis have been compared to mousepox (16). According to this model, VZV is presumed to infect mononuclear cells in regional lymph nodes, causing a primary viremia that carries the virus to reticuloendothelial organs, such as the liver, for a phase of viral amplification, which is followed by a secondary viremia in the late incubation period that results in VZV transport to skin. Instead, our recent experiments with the SCIDhu mouse model support the concept that infected T cells have the potential to mediate VZV transfer to skin immediately after entering the circulation during primary viremia and suggest that the prolonged interval between exposure and the appearance of varicella skin lesions reflects the time required for VZV to overcome previously unrecognized but potent innate immune barriers, especially alpha interferon (IFN-␣) production, mounted directly by epidermal cells in vivo (27). Experiments analyzing VZV recombin...

The 12 histidine and four cysteine residues of the Fur repressor of Escherichia coli were changed, respectively, to leucine and serine by site-directed mutagenesis of the fur gene. The affects of these mutations were measured in vivo by ligation of the mutated genes to a wild-type fur promoter followed by measurement of the ability of these plasmids to regulate expression of a lacZ fusion in the aerobactin operon. In vitro affects were assayed by insertion of the mutated genes in the expression vector pMON2064 attended by isolation of the altered Fur proteins and appraisal of their capacity to bind to operator DNA. The results suggest that cysteine residues at positions 92 and 95 are important for the activity of the Fur protein.

To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.Varicella-zoster virus (VZV) is a ubiquitous human alphaherpesvirus that causes varicella (chicken pox), establishes latency in sensory ganglia, and can reactivate to cause herpes zoster. The pathogenesis of primary VZV infection involves inoculation of respiratory mucosa, initiation of a cell-associated viremia, and the appearance of widely distributed vesicular skin lesions (1, 4). T lymphocytes appear to be a major target cell for VZV viremia, and these migrating cells have the capacity to transport the virus to epidermal and dermal cells (15,19). VZV skin lesions contain high titers of infectious virus, which is transmissible to other susceptible individuals in the population.VZV ORF47 encodes the ORF47 protein, which has the characteristic amino acid sequence and functions of a serine/ threonine protein kinase and has homologies to herpes simplex virus (HSV) U L 13 and cellular casein kinase II (CKII) (5,6,12,27,34). The ORF47 protein is dispensable for VZV replication in vitro, as shown in studies of ROka47S, a recombinant virus described by Heineman and Cohen, in which ORF47 transcription was blocked by a stop codon mutation (8). In contrast, the ORF47 protein is essential for VZV infection of differentiated human T-cell and skin xenografts in the SCID-hu model of ...