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The purpose of this study was to evaluate associations between germline epidermal growth factor receptor (EGFR) variants involved in transcriptional regulation and overall survival in white patients with non-small-cell lung cancer (NSCLC) treated with the EGFR tyrosine kinase inhibitor, gefitinib. Of 175 consecutive patients treated with oral gefitinib (250 mg/day), 170 (median age: 67 years; 72% men) were evaluable for genotyping and survival. Fifty-five patients (33%) had stable disease and 17 (10%) had an objective response. The most common of four haplotypes was G-C (EGFR*1) at the EGFR -216G>T and -191C>A loci (frequency, 0.45). After adjusting for performance status, previous platinum-containing chemotherapy and occurrence of skin rash or diarrhea during the first treatment cycle in patients with performance status 0 or 1 (N=139), the absence of EGFR*1 was associated with significantly better survival (hazard ratio: 0.54; 95% confidence interval: 0.32-0.91; P=0.015). The results may help identify patients with NSCLC who can benefit from gefitinib treatment.

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Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2-5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright's fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6-WNT10A and COL4A3-COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.

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BACKGROUND: Recent work suggests that multiple genes and several environmental risk factors influence risk for non-syndromic oral clefts, one of the most common birth defects in humans. Advances in high-throughput genotyping technology now make it possible to test multiple markers in many candidate genes simultaneously. METHODS: We present findings from family based association tests of single nucleotide polymorphism (SNP) markers in 64 candidate genes genotyped using the BeadArray approach in 58 case-parent trios from Maryland (USA) to illustrate how multiple markers in multiple genes can be analysed. To assess whether these genes were expressed in human craniofacial structures relevant to palate and lip development, we also analysed data from the Craniofacial and Oral Gene Expression Network (COGENE) consortium, and searched public databases for expression profiles of these genes. RESULTS: Thirteen candidate genes showed significant evidence of linkage in the presence of disequilibrium, and ten of these were found to be expressed in relevant embryonic tissues: SP100, MLPH, HDAC4, LEF1, C6orf105, CD44, ALX4, ZNF202, CRHR1, and MAPT. Three other genes showing statistical evidence (ADH1C, SCN3B, and IMP5) were not expressed in the embryonic tissues examined here. CONCLUSIONS: This approach demonstrates how statistical evidence on large numbers of SNP markers typed in case-parent trios can be combined with expression data to identify candidate genes for complex disorders. Many of the genes reported here have not been previously studied as candidates for oral clefts and warrant further investigation.

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Proinflammatory and immunoregulatory products from C3 play a major role in phagocytosis, respiratory burst, and airways inflammation. C3 is critical in adaptive immunity; studies in mice deficient in C3 demonstrate that features of asthma are significantly attenuated in the absence of C3. To test the hypothesis that the C3 gene on chromosome 19p13.3-p13.2 contains variants associated with asthma and related phenotypes, we genotyped 25 single nucleotide polymorphism (SNP) markers distributed at intervals of approximately 1.9 kb within the C3 gene in 852 African Caribbean subjects from 125 nuclear and extended pedigrees. We used the multiallelic test in the family-based association test program to examine sliding windows comprised of 2-6 SNPs. A five-SNP window between markers rs10402876 and rs366510 provided strongest evidence for linkage in the presence of linkage disequilibrium for asthma, high log[total IgE], and high log[IL-13]/[log[IFN-gamma] in terms of global P-values (P = 0.00027, 0.00013, and 0.003, respectively). A three-SNP haplotype GGC for the first three of these markers showed best overall significance for the three phenotypes (P = 0.003, 0.007, 0.018, respectively) considering haplotype-specific tests. Taken together, these results implicate the C3 gene as a priority candidate controlling risk for asthma and allergic disease in this population of African descent.

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Analysis of haplotypes based on multiple single-nucleotide polymorphisms (SNP) is becoming common for both candidate gene and fine-mapping studies. Before embarking on studies of haplotypes from genetically distinct populations, however, it is important to consider variation both in linkage disequilibrium (LD) and in haplotype frequencies within and across populations, as both vary. Such diversity will influence the choice of "tagging" SNPs for candidate gene or whole-genome association studies because some markers will not be polymorphic in all samples and some haplotypes will be poorly represented or completely absent. Here we analyze 11 genes, originally chosen as candidate genes for oral clefts, where multiple markers were genotyped on individuals from four populations. Estimated haplotype frequencies, measures of pairwise LD, and genetic diversity were computed for 135 European-Americans, 57 Chinese-Singaporeans, 45 Malay-Singaporeans, and 46 Indian-Singaporeans. Patterns of pairwise LD were compared across these four populations and haplotype frequencies were used to assess genetic variation. Although these populations are fairly similar in allele frequencies and overall patterns of LD, both haplotype frequencies and genetic diversity varied significantly across populations. Such haplotype diversity has implications for designing studies of association involving samples from genetically distinct populations.

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N-t-butyl hydroxylamine (NtBHA) delays senescence-dependent changes in human lung fibroblasts (IMR90) (Atamna et al., J. Biol. Chem. 275, 6741-6748). The current study examines the effect of NtBHA on mitochondria in old and young rats and human primary fibroblasts (IMR90). In NtBHA-treated rats, the age-dependent decline in food consumption and ambulatory activity was reversed without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding NtBHA. These findings suggest that NtBHA improved mitochondrial function in vivo. The age-dependent increase in proteins with thiol-mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats; no significant effect was observed in young rats. In IMR90 cells, NtBHA delayed senescence-associated changes in mitochondria and cellular senescence induced by maintaining the cells under suboptimal levels of growth factors. Proteasomal activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates in cells 10- to 15-fold the extracellular concentration and is maintained by mitochondrial NADH. NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radical-induced toxicity to mitochondria.

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CONTEXT: The continued release of human immunodeficiency virus type 1 (HIV-1) into plasma at very low levels during highly active antiretroviral therapy (HAART) can be detected using specialized techniques, but the nature and significance of this low-level viremia, especially as related to acquisition of drug resistance mutations, are unclear. OBJECTIVE: To determine genetic resistance profiles of low-level plasma HIV-1 in patients with prolonged viral suppression (<50 copies/mL of plasma HIV-1 RNA) while receiving HAART. DESIGN AND SETTING: Cross-sectional study conducted at a US academic hospital from November 1999 to February 2001 using a novel method for amplification of low levels of viral genomes in plasma. PATIENTS: Eighteen HIV-1-infected patients (7 children and 11 adults), enrolled in a longitudinal study of HIV-1 reservoirs, who had suppression of viral replication while receiving protease inhibitor-containing combination therapy. Two patients (1 adult and 1 child) with less optimal suppression of viral replication were included to assess virus predominating when plasma HIV-1 RNA levels are low but detectable (<1000 copies/mL). Follow-up analyses were conducted in 3 patients. MAIN OUTCOME MEASURE: Detection of drug resistance mutations in clones amplified from low-level plasma virus. RESULTS: Viral sequences were amplified from 8 of the 18 patients with simultaneous plasma HIV-1 measurements of less than 50 copies/mL and from 2 patients with 231 and 50 copies/mL. Clones from 3 treatment-naive patients with less than 50 copies/mL of plasma HIV-1 RNA showed continued release, for as long as 42 months, of wild-type drug-sensitive virus. The 7 patients with prior nonsuppressive therapy, with viral loads below 50 copies/mL and during "blips" to 231 and 64 copies/mL, had only resistance mutations consistent with pre-HAART therapy (although reverse transcriptase inhibitor mutations may have continued to occur). New HAART-related mutations were seen in a control patient with prior viral load levels of about 400 to 1000 copies/mL. For phylogenetic analysis, sequences were available for both resting CD4(+) T cells and plasma HIV for 7 of 10 patients and showed patient-specific clustering of sequences and a close relationship between virus in the plasma and the latent reservoir. CONCLUSIONS: Based on the samples that could be amplified, low-level viremia in children and adults receiving HAART with prolonged suppression of viremia to less than 50 copies/mL of HIV-1 RNA may result primarily from archival, pre-HAART virus, reflecting earlier treatment conditions, and does not appear to require development of new, HAART-selected mutations reflecting partial resistance to therapy. Low-level viremia below 50 copies/mL may represent less of a concern regarding impending drug failure of current HAART regimens. However, the archival drug-resistant virus may be relevant regarding future treatment strategies.

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Fibroblast growth factor receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least 22 exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from 16 CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed 21 other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately 16.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.

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We have cloned four acyl CoA synthetase (ACS) genes from Trypanosoma brucei strain 927. Each of these genes encodes a polypeptide about 78 kDa in size and all four contain the "ACS signature motif." Sequence alignments indicate that these proteins are 46%-95% identical in amino acid sequence. Interestingly, three of them share almost identical C-termini (about 215 amino acid residues). Southern blots suggest that these genes are present in a single copy, and Northern blots reveal that all four are expressed in both bloodstream and procyclic trypanosomes.

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The huntingtin-associated protein (HAP-1) interacts with the Huntington disease gene product, huntingtin. It is predominantly expressed in the brain and shows an increased affinity for mutant huntingtin. We have sequenced an 18,656bp genomic region encompassing the entire human HAP-1 gene and determined its genomic organisation, with 11 exons spanning 12.1kb. We have also found an intragenic polymorphism within intron 6 of HAP-1. We have recently shown that HAP-1 maps to a region of the genome which has been implicated in a variety of neurological conditions, including progressive supranuclear palsy (PSP), a late-onset atypical parkinsonian disorder. The detailed characterisation of the genomic organisation of HAP-1 and the presence of an intragenic polymorphism will be helpful in evaluating its role in different disorders, using candidate gene approaches.

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Manganese concentrates in the ventral mesencephalon of male Sprague-Dawley rats after intrathecal administration of MnCl2. We tested the hypothesis that Mn concentration in the central nervous system (CNS), particularly in the ventral mesencephalon, is decreased by inhibiting dopamine reuptake using cocaine or by decreasing dopamine concentrations using reserpine. The intrathecal administration of Mn (250 micrograms Mn/rat as MnCl2) caused the Mn concentration in the ventral mesencephalon to increase from 0.57 to 31.8 micrograms Mn/g. Cocaine administration (8.6 mg/kg i.p.) thirty minutes prior to MnCl2 decreased ventral mesencephalon Mn to 3.3 micrograms Mn/g. By giving reserpine (5 mg/kg i.p.) 24 hours prior to MnCl2 the ventral mesencephalon Mn concentration was decreased from 29.9 micrograms Mn/g to 3.7 micrograms Mn/g. Intrathecal MnCl2 decreased the dopamine concentration in the caudate putamen by 40% six hours after administration. Cocaine or reserpine decreased the Mn concentration in the ventral mesencephalon, occipital pole, frontal lobe and caudate putamen but did not change the Mn concentration in the cerebellum. The results indicate that the mechanism(s) by which Mn is concentrated in many brain regions can be inhibited by cocaine, a dopamine reuptake inhibitor, or by reserpine, a dopamine depleter, and suggest that the Mn concentration in the CNS is related to dopamine reuptake and/or concentration.

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A diet supplemented with (R)-lipoic acid, a mitochondrial coenzyme, was fed to old rats to determine its efficacy in reversing the decline in metabolism seen with age. Young (3 to 5 months) and old (24 to 26 months) rats were fed an AIN-93M diet with or without (R)-lipoic acid (0.5% w/w) for 2 wk, killed, and their liver parenchymal cells were isolated. Hepatocytes from untreated old rats vs. young controls had significantly lower oxygen consumption (P<0. 03) and mitochondrial membrane potential. (R)-Lipoic acid supplementation reversed the age-related decline in O2 consumption and increased (P<0.03) mitochondrial membrane potential. Ambulatory activity, a measure of general metabolic activity, was almost threefold lower in untreated old rats vs. controls, but this decline was reversed (P<0.005) in old rats fed (R)-lipoic acid. The increase of oxidants with age, as measured by the fluorescence produced on oxidizing 2',7'-dichlorofluorescin, was significantly lowered in (R)-lipoic acid supplemented old rats (P<0.01). Malondialdehyde (MDA) levels, an indicator of lipid peroxidation, were increased fivefold with age in cells from unsupplemented rats. Feeding rats the (R)-lipoic acid diet reduced MDA levels markedly (P<0.01). Both glutathione and ascorbic acid levels declined in hepatocytes with age, but their loss was completely reversed with (R)-lipoic acid supplementation. Thus, (R)-lipoic acid supplementation improves indices of metabolic activity as well as lowers oxidative stress and damage evident in aging.

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The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is likely to depend on knowledge of circulating variants of genes other than the commonly sequenced gag and env genes. In addition, full-genome data are particularly limited for HIV-1 subtype C, currently the most commonly transmitted subtype in India and worldwide. Likewise, little is known about sequence variation of HIV-1 in India, the country facing the largest burden of HIV worldwide. Therefore, the objective of this study was to clone and characterize the complete genome of HIV-1 from seroconverters infected with subtype C variants in India. Cocultured HIV-1 isolates were obtained from six seroincident individuals from Pune, India, and virtually full-length HIV-1 genomes were amplified, cloned, and sequenced from each. Sequence analysis revealed that five of the six genomes were of subtype C, while one was a mosaic of subtypes A and C, with multiple breakpoints in env, nef, and the 3' long terminal repeat as determined by both maximal chi2 analysis and phylogenetic bootstrapping. Sequences were compared for preservation of known cytotoxic T lymphocyte (CTL) epitopes. Compared with those of the HIV-1LAI sequence, 38% of well-defined CTL epitopes were identical. The proportion of nonconservative substitutions for Env, at 61%, was higher (P < 0.001) than those for Gag (24%), Pol (18%), and Nef (32%). Therefore, characterized CTL epitopes demonstrated substantial differences from subtype B laboratory strains, which were most pronounced in Env. Because these clones were obtained from Indian seroconverters, they are likely to facilitate vaccine-related efforts in India by providing potential antigens for vaccine candidates as well as for assays of vaccine responsiveness.

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Mitochondrial function and ambulatory activity were monitored after feeding old rats acetyl-L-carnitine (ALCAR). Young (3-5 mo) and old (22-28 mo) rats were given a 1.5% (wt/vol) solution of ALCAR in their drinking water for 1 mo, were sacrificed, and their liver parenchymal cells were isolated. ALCAR supplementation significantly reverses the age-associated decline of mitochondrial membrane potential, as assessed by rhodamine 123 staining. Cardiolipin, which declines significantly with age, is also restored. ALCAR increases cellular oxygen consumption, which declines with age, to the level of young rats. However, the oxidant production per oxygen consumed, as measured by 2',7'-dichlorofluorescin fluorescence levels, is approximately 30% higher than in untreated old rats. Cellular glutathione and ascorbate levels were nearly 30% and 50% lower, respectively, in cells from ALCAR-supplemented old rats than in untreated old rats, further indicating that ALCAR supplementation might increase oxidative stress. Ambulatory activity in young and old rats was quantified as a general measure of metabolic activity. Ambulatory activity, defined as mean total distance traveled, in old rats is almost 3-fold lower than in young animals. ALCAR supplementation increases ambulatory activity significantly in both young and old rats, with the increase being larger in old rats. Thus, ALCAR supplementation to old rats markedly reverses the age-associated decline in many indices of mitochondrial function and general metabolic activity, but may increase oxidative stress.

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The intrathecal administration of MnCl2 to young male rats caused dopamine depletion in the caudate-putamen and a decrease in spontaneous motor activity. Our experiments demonstrate that in the young rat: (a) the lateral choroid plexus protects the cerebrospinal fluid (CSF) from high concentrations of Mn in the blood by sequestering and thus preventing large amounts of this metal ion from entering the CSF. As blood Mn levels rise, the lateral choroid plexus may become overwhelmed and leak an increasing amount of Mn into the CSF. (b) The lateral choroid plexus does not remove Mn2+ from the CSF. (c) The injection of MnCl2 into the CSF of rats caused a rapid decrease in spontaneous motor activity which is dose-dependent and reversible under the present experimental conditions. Intrathecal Mn results in a substantial decrease in striatal dopamine but not homovanillic acid or 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations and is associated with an increase in the Mn concentration of the substantia nigra and caudate-putamen.

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Synaptic reaccumulation of the neurotransmitter dopamine is mediated by the dopamine transporter (DAT), a member of the family of twelve transmembrane domain, sodium- and chloride-dependent neurotransmitter transporters. Several DAT features, including its exclusive expression in dopaminergic neurons, implication in cocaine action, and prominent role in the mechanisms of Parkinsonism-inducing neurotoxins, make understanding of the DAT gene of interest. Isolation and characterization of the human and mouse DAT genes has allowed elucidation of similarities between each and other members of this transporter gene family. Sequences 5' to transcriptional start sites contain G-C rich, TATA-less, CAAT-less regions with striking conservation between human and mouse gene flanking regions. These studies suggest sequence elements that are candidates to contribute to the dopamine transporter's dopaminergic cell-specific expression.

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The efficacy of SCH 39304 (SCH) against Aspergillus fumigatus was assessed with an immunosuppressed, temporarily leukopenic rabbit model of invasive aspergillosis. Therapy with SCH at 10 or 15 mg/kg of body weight per day was begun 24 h after lethal challenge and compared with therapy with amphotericin B at 1.5 mg/kg/day. Compared with untreated controls, SCH reduced mortality and also reduced the tissue burden of A. fumigatus 100- to 1,000-fold in liver, kidney, and lung tissues. SCH at 15 mg/kg/day and amphotericin B eliminated A. fumigatus in liver, kidney, and lung tissues. In addition, both dosages of SCH significantly eliminated the organism from brain tissues, compared with controls. Both SCH and amphotericin B decreased or eliminated circulating aspergillus antigen. These results show that new azoles can be as effective as amphotericin B in eradicating the organism from tissues and offer promise in improving the treatment of invasive aspergillosis.

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Twenty-six Holstein calves were treated with hydroxyurea in order to induce neutropenia. Calves were given a daily dosage of hydroxyurea (70 mg/kg of body weight) for 4 consecutive days, and clinical signs, blood leukograms, hemograms, and platelet counts were monitored daily until the calves became neutropenic. Once a neutropenic state was induced, the calves were anesthetized and pulmonary function tests were performed. Subsequently, calves were euthanatized and complete necropsies were performed. Hydroxyurea treatment induced profound neutropenia in all calves by 8 days after initiating treatment, with mild decreases in circulating numbers of lymphocytes. Treatment did not cause clinical or pulmonary functional abnormalities. Severe pathologic changes were restricted to the bone marrow and consisted of partial to complete destruction of myeloid elements, with less severe effects on erythrocytic precursors and megakaryocytes. Hydroxyurea was useful for the induction of neutropenic states in calves and did not induce major toxic effects on other cells when given at 70 mg/kg.

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Acute lung injury was induced in 24 calves by intratracheal inoculation with Pasteurella haemolytica. Calves in groups 1 and 2 were neutrophil depleted, using hydroxyurea given IV. Group 1 calves (n = 7) were inoculated intratracheally with saline solution, and group 2 calves (n = 7) were inoculated with P haemolytica. Group 3 calves (n = 7) had normal numbers of neutrophils and were inoculated with P haemolytica. Group 4 calves (n = 3) were treated acutely with hydroxyurea IV, had normal numbers of neutrophils, and were inoculated with P haemolytica. After inoculation, calves with normal numbers of neutrophils (groups 3 and 4) became hypoxemic 2 hours after inoculation, and hypoxemia persisted until necropsy (6 hours after inoculation). These calves also developed tachypnea, bradycardia, neutropenia, and lymphopenia. Lung lesions consisted of necrosis of the alveolar walls, intra-alveolar hemorrhage, and a severe exudative and necrotizing bronchopneumonia, with accumulation of proteinaceous fluid in alveoli and lymphatics. In neutrophil-depleted calves (groups 1 and 2), blood gas values, heart and respiratory rates, and numbers of circulating leukocytes did not change after inoculation with saline solution or with P haemolytica. At necropsy, the lungs of neutrophil-depleted calves were grossly normal. Therefore, neutrophils were required for the acute lung injury induced by P haemolytica. The protective effect of neutrophil depletion was a specific effect of hydroxyurea because calves with high circulating concentrations of hydroxyurea and calves with normal numbers of neutrophils (group 4) developed lung injury.

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