Differential expression of KAI1 metastasis suppressor protein in renal cell tumor histological subtypes.

PURPOSE: The similar appearance of renal tumor histological subtypes can complicate differential diagnoses. This problem is most notable for the chromophobe subtype of renal cell carcinoma, which can be histologically indistinguishable from oncocytoma with investigational molecular markers failing to provide reliable differentiation. KAI1 is a metastasis suppressor gene whose expression correlates inversely with the metastatic potential of most solid tumor cancer types. We tested the hypothesis that KAI1 is differentially expressed among renal tumor histological subtypes. MATERIALS AND METHODS: Immunohistochemical staining for KAI1 protein was performed in 152 nephrectomy specimens, including 48 clear cell, 35 papillary and 31 chromophobe renal cell carcinoma samples, 28 oncocytomas and 10 tumor-free kidneys. Staining was scored as none/minimal, low, moderate or high. KAI1 mRNA levels were compared by quantitative reverse transcriptase-polymerase chain reaction in an additional 22 chromophobe renal cell carcinoma and oncocytoma samples. RESULTS: In all 10 tumor-free kidneys KAI1 protein was detected exclusively in distal tubule cell membranes. Of the tumor specimens KAI1 protein was absent in all papillary renal cell carcinoma specimens. It was present in only 1 of 48 clear cell renal cell carcinomas (2%) and 2 of 28 oncocytomas (7%) but only at low levels. In contrast, 27 of 31 chromophobe renal cell carcinoma specimens (87%) expressed KAI1 protein, most at moderate or high levels. The diagnostic accuracy of KAI1 immunostaining for discerning chromophobe renal cell carcinoma from oncocytoma was 90% with similar results observed at the RNA level. CONCLUSIONS: KAI1 is an accurate biomarker for chromophobe renal cell carcinoma that may aid in the diagnostic differentiation of chromophobe renal cell carcinoma from oncocytoma. It remains to be determined whether KAI1 expression contributes to the low metastatic potential of chromophobe renal cell carcinoma.

West Nile virus infection in birds and mosquitoes, New York State, 2000.

West Nile (WN) virus was found throughout New York State in 2000, with the epicenter in New York City and surrounding counties. We tested 3,403 dead birds and 9,954 mosquito pools for WN virus during the transmission season. Sixty-three avian species, representing 30 families and 14 orders, tested positive for WN virus. The highest proportion of dead birds that tested positive for WN virus was in American Crows in the epicenter (67% positive, n=907). Eight mosquito species, representing four genera, were positive for WN virus. The minimum infection rate per 1,000 mosquitoes (MIR) was highest for Culex pipiens in the epicenter: 3.53 for the entire season and 7.49 for the peak week of August 13. Staten Island had the highest MIR (11.42 for Cx. pipiens), which was associated with the highest proportion of dead American Crows that tested positive for WN virus (92%, n=48) and the highest number of human cases (n=10).

Partial genetic characterization of West Nile virus strains, New York State, 2000.

We analyzed nucleotide sequences from the envelope gene of 11 West Nile (WN) virus strains collected in New York State during the 2000 transmission season to determine whether they differed genetically from each other and from the initial strain isolated in 1999. The complete envelope genes of these strains were amplified by reverse transcription-polymerase chain reaction. The resulting sequences were aligned, the genetic distances were computed, and a phylogenetic tree was constructed. Ten (0.7%) of 1,503 positions in the envelope gene were polymorphic in one or more sequences. The genetic distances were 0.003 or less. WN virus strains circulating in 2000 were homogeneous with respect to one another and to a strain isolated in 1999.

Fusion of docked membranes requires the armadillo repeat protein Vac8p.

The discovery of molecules required for membrane fusion has revealed a remarkably conserved mechanism that centers upon the formation of a complex of SNARE proteins. However, whether the SNARE proteins or other components catalyze the final steps of membrane fusion in vivo remains unclear. Understanding this last step depends on the identification of molecules that act late in the fusion process. Here we demonstrate that in Saccharomyces cerevisiae, Vac8p, a myristoylated and palmitoylated armadillo repeat protein, is required for homotypic vacuole fusion. Vac8p is palmitoylated during the fusion reaction, and the ability of Vac8p to be palmitoylated appears to be necessary for its function in fusion. Both in vivo and in vitro analyses show that Vac8p functions after both Rab-dependent vacuole docking and the formation of trans-SNARE pairs. We propose that Vac8p may bind the fusion machinery through its armadillo repeats and that palmitoylation brings this machinery to a specialized lipid domain that facilitates bilayer mixing.

High-throughput detection of West Nile virus RNA.

The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

Y chromosome sequence variation and the history of human populations.

Binary polymorphisms associated with the non-recombining region of the human Y chromosome (NRY) preserve the paternal genetic legacy of our species that has persisted to the present, permitting inference of human evolution, population affinity and demographic history. We used denaturing high-performance liquid chromatography (DHPLC; ref. 2) to identify 160 of the 166 bi-allelic and 1 tri-allelic site that formed a parsimonious genealogy of 116 haplotypes, several of which display distinct population affinities based on the analysis of 1062 globally representative individuals. A minority of contemporary East Africans and Khoisan represent the descendants of the most ancestral patrilineages of anatomically modern humans that left Africa between 35,000 and 89,000 years ago.

Molecular cloning and sequence analysis of the Danio rerio catalase gene.

Catalase is an antioxidant enzyme that plays a central role in the protection against oxidative stress through the metabolism of hydrogen peroxide. Catalase has been well studied in plants, bacteria, and mammals, but little work has been done in other vertebrate species. We have cloned the zebrafish (Danio rerio) catalase cDNA containing the complete coding region and analyzed expression by both reverse transcription polymerase chain reaction and western blot. The deduced amino acid sequence predicts a protein of 526 amino acids with both the primary DNA and amino acid sequences highly conserved among vertebrate species. The major protein-heme contact points in the catalase enzyme complex are also well conserved, although several amino acids associated with the second and third levels of the major substrate channel are not, suggesting potential differences in substrate access or specificity. The 3' flanking region of the cDNA contains a dinucleotide repeat near the termination codon consisting of a near perfect CA array that is polymorphic. The rat and mouse catalase genes also contain a CA repeat sequence in the 3' untranslated region, which, along with an adjacent 5' stem-loop structure, has previously been shown to be a site for mRNA protein binding (Clerch, 1995, Arch. Biochem. Biophys. 317 (1995) 267-274). A stem-loop structure is also predicted adjacent to the zebrafish CA repeat, suggesting a similar role in catalase gene regulation.

NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P.falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.

Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria.

NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria.

Human safety and immunogenicity of a canarypox-rabies glycoprotein recombinant vaccine: an alternative poxvirus vector system.

Avian poxvirus recombinants undergo abortive replication in nonavian cells, yet can achieve expression of extrinsic gene products. Canarypox-vectored vaccines have been innocuous and immunogenic in several mammalian species. ALVAC-RG, a canarypox recombinant expressing the rabies glycoprotein gene, was inoculated intramuscularly into adult volunteers on days 0, 28, and 180. Sequential cohorts received 10(3.5), 10(4.5), and 10(5.5) 50% tissue culture infective doses (TCID50); additional volunteers received the standard human diploid cell rabies vaccine (HDCV) on the same schedule. Reactogenicity of ALVAC-RG was minimal. The lowest dose of ALVAC-RG induced little antibody to rabies virus by ELISA or rapid fluorescent focus inhibition test (RFFIT), but 10(4.5) and 10(5.5) TCID50 doses elicited significant responses in both assays. All recipients of 10(4.5) and 10(5.5) TCID50 of ALVAC-RG attained RFFIT values above the presumed protective level. Canarypox-specific immune responses did not inhibit boosting of rabies-specific antibodies by the day 180 dose of ALVAC-RG. T cell proliferation in response to inactivated rabies virus in vitro was similar in HDCV and ALVAC-RG recipients after the first and second doses, although HDCV yielded superior results after the third dose. ALVAC-RG was safe in humans, induced functional antibody to rabies glycoprotein, elicited cellular responses to rabies virus, and could be used successfully for booster dosing at a 6 month interval.

Host-range restriction of vaccinia virus E3L-specific deletion mutants.

The vaccinia virus (VV) E3L gene product functions as a dsRNA binding protein that is involved in conferring an interferon-resistant phenotype upon the virus. Studies with a vaccinia virus (VV) E3L- deletion mutant (vP1080) have also demonstrated that the E3L gene product is critical for productive replication on certain cell substrates. While E3L was found to be nonessential for replication in chick embryo fibroblasts (CEFs), virus specifically deleted of E3L was found to be replication deficient in Vero, HeLa, and murine L929 cells. Further, the temporal block in replication appears to differ in these cell systems, as evidenced by the observed timing of protein synthesis inhibition. In Vero cells infected with the VV E3L- mutant, there was no detectable protein synthesis after 2 hr post-infection, whereas in L929 cells normal protein patterns were observed even at late times post-infection. Expression of a heterologous dsRNA binding protein, the reovirus sigma 3 protein, by the E3L- mutant virus restored near wild-type growth characteristics, suggesting the critical nature for regulating dsRNA levels in VV-infected cells.

Women in recovery. Their perceptions of treatment effectiveness.

Research with chemically dependent women over the past two decades indicates that women substance abusers have special characteristics and needs that warrant gender-sensitive drug-treatment approaches. While the potential benefit of such treatment seems clear, little empirical data is available on how women perceive the effectiveness of gender-sensitive specialized drug treatment. This article presents findings from an exploratory study of the present and past treatment experiences of 24 women in recovery. Results indicate that while some specialized services such as child care and women-only therapy groups are increasingly available, many drug-treatment programs fail to provide these services in a context which supports and promotes women. As a result, women in drug treatment continue to experience negative stereotyping and sexual harassment as their gender-specific needs remain ignored, silenced, or deemed pathological. Major gaps in drug treatment for women are discussed as are implications for the provision of effective gender-sensitive treatment.

Microsatellite-centromere mapping in the zebrafish (Danio rerio).

Ten (CA)n microsatellite simple sequence repeat (SSR) markers, 1, 2, 12, 14, 16, 18, 20, 22, 26, and 29, were used to show high chiasma interference and to determine centromere-marker map distances in the zebrafish (Danio rerio). Of these, SSR 12 exhibited no recombinant tetratypes among 175 half-tetrad embryos, placing this marker within 1 cM of the centromere of Linkage Group XVII. Fractions of heterozygous half-tetrads for the remaining nine markers ranged from 0.64 to 0.89. Of these, six recombinant fractions were more than 0.67 (P < 0.05), indicating strong chiasma interference during female meiosis in the zebrafish. Consistent with previous mapping data, SSRs 2 and 20 of Linkage Group VI were tightly linked. Half-tetrad analysis will allow the mapping of the remaining centromeres and may be useful in the mapping of new genes and mutations in the zebrafish.

The role of women's therapy groups in the treatment of chemical dependence.

The distinctive needs of chemically dependent women are contrasted with those of men and discussed in relationship to traditional mixed-gender therapy groups. A qualitative study of the contrasting experiences of women in mixed-gender and women-only treatment groups confirmed the hypothesis that their treatment needs are better met in the latter, and that these groups are an essential component of programs treating chemically dependent women.

Effect of herbal mixture student Rasayana on lipoxygenase activity and lipid peroxidation.

Brain cellular functions are affected by free radicals. Arachidonic acid and its 12-lipoxygenase metabolites have been proposed as important in enhancing long-term potentiation associated with learning. It has been reported that Student Rasayana (SR), an herbal mixture, improves brain functions. In this study we evaluated the antioxidant capacity of SR and its effect on lipoxygenase activity. Both alcoholic and aqueous extracts of SR inhibited enzymatic- and nonenzymatic-induced microsomal lipid peroxidation in a concentration-dependent manner. The agent concentrations (micrograms/mL) that produced 50% inhibition (IC50) of enzymatic- and nonenzymatic-induced microsomal lipid peroxidation, respectively, were 99.1 +/- 3.9 and 1992.0 +/- 122.7 for the aqueous extract, and 17.7 +/- 0.9 and 646.7 +/- 79.7 for the alcoholic extract. The aqueous extract inhibited soyabean lipoxygenase (SLP)-induced LDL oxidation in a concentration-dependent manner (IC50: 515.5 +/- 11.5), whereas the alcoholic extract enhanced SLP-induced LDL oxidation. Simultaneous addition of aqueous and alcoholic extracts inhibited SLP-induced LDL oxidation. The alcoholic extract (but not the aqueous extract) enhanced the ability of SLP to induce oxidation of linoleic acid. Rats fed 2% (w:w) SR showed inhibition of toluene-induced brain microsomal lipid peroxidation. These results suggest SR improves brain functions through scavenging free radicals as well as increasing the second messenger for long-term potentiation.

Gender differences in drug addiction and treatment: implications for social work intervention with substance-abusing women.

This article draws on current addiction research to describe the unique characteristics and treatment needs of chemically dependent women and how they differ from those of chemically dependent men. It explores similarities between women who are drug addicted and all women who experience gender-based oppression. The authors suggest that drug use is a coping strategy that some women adopt to manage this oppression. Finally, the article looks at traditional drug treatment programs, which have been designed to treat male addicts and fail to address the treatment needs of women. The authors offer an alternative treatment model designed to meet those needs. Parallels between characteristics of this alternative treatment model and social work practice are drawn, and opportunities and strategies for social workers to intervene with female addicts are identified.

Identity-by-descent and association mapping of a recessive gene for Hirschsprung disease on human chromosome 13q22.

Hirschsprung disease (HSCR) is a congenital disorder of unknown etiology characterized by the absence of enteric ganglia in the distal colon. We have ascertained a large, inbred, Mennonite kindred which demonstrates a high incidence of Hirschsprung disease (HSCR). Genealogical analysis of all kinship relationships identified a single common ancestral couple for all parents of affected offspring. Segregation analysis yielded a segregation ratio of 10.67% for males and 5.45% for females. We searched for locations of the gene(s) responsible for HSCR in this pedigree by genotyping three small multicase families and locating genomic regions demonstrating identity-by-descent followed by linkage disequilibrium analysis of 28 additional nuclear families. Based on this novel strategy, we report the mapping of a new locus for HSCR to chromosome 13q22. Nine microsatellite markers spanning 10 cM in this region were genotyped on thirty-one nuclear families. Significant nonrandom association was detected with alleles at markers D13S162, D13S160, D13S170, and AFM240zg9. In addition, our studies reveal preliminary evidence for a genetic modifier of HSCR in this kindred on chromosome 21q22.

In vitro and in vivo inhibition of microsomal lipid peroxidation by MA-631.

Excess free radicals are linked to many diseases, including aging, atherosclerosis, and cancer. Previously, we have shown that MA-631 (a complex herbal mixture) inhibits human low-density lipoprotein (LDL) oxidation and may play a role in prevention of atherosclerosis. In this study we further evaluated the in vivo and in vitro antioxidant activity of MA-631. Both the alcoholic and aqueous extracts of MA-631 inhibited enzymatic- and nonenzymatic-induced rat liver microsomal lipid peroxidation in a concentration-dependent manner. The thiobarbituric acid-reactive substances (TBARS) values (nmol malondialdehyde (MDA)/mg microsomal protein) were 1.43 +/- 0.18 for microsomes alone (baseline for enzymatic system), 19.63 +/- 2.50 for microsomes + reduced nicotinamide adenine dinucleotide phosphate (NADPH) (oxidation without inhibitor), 9.89 +/- 1.41 for heated microsomes (baseline for nonenzymatic system), and 27.15 +/- 0.08 for microsomes + ascorbate (oxidation without inhibitor). The concentrations (micrograms/2 ml) of MA-631 which produced 50% inhibition (IC50) of enzymatic- and non-enzymatic-induced lipid peroxidation were 15.2 +/- 2.0 and 17.0 +/- 2.6, respectively, for the aqueous extract, and 4.3 +/- 0.8 and 6.4 +/- 1.2, respectively, for the alcoholic extract. A 2% MA-631 (w:w) supplemented diet fed to rats for three weeks inhibited in vivo, toluene-induced microsomal lipid peroxidation in the brain, kidney, liver, and heart. These results imply that MA-631 may be useful in the prevention of free radical-linked diseases.