Comparison of factor IX methylation on human active and inactive X chromosomes: implications for X inactivation and transcription of tissue-specific genes.

Maintenance of dosage compensation for housekeeping genes on the human X chromosome is mediated through differential methylation of clustered CpG nucleotides associated with these genes. To determine if methylation has a role in maintaining inactivity of X-linked genes which show tissue-specific expression, we examined the locus for blood clotting Factor IX. The analysis encompassed 91% of the HpaII and HhaI sites in the 41-kb region that includes the presumed promoter region, 5 kb of 5'- and 4 kb of 3'-flanking sequences. Although there are sex differences in methylation of the locus in leukocytes, the methylation pattern in liver, where the gene is expressed, is essentially the same for loci on the active and inactive X chromosome. The lack of differences in methylation of active and inactive genes makes it unlikely that methylation within the locus has a role in expression of the Factor IX gene. These findings, along with the absence of clustered CpG dinucleotides within the Factor IX locus, suggest that functional differences in DNA methylation related to X chromosome dosage compensation may be limited to CpG clusters. In any event, dosage compensation seems to be maintained regionally, rather than locus by locus.     

Molecular studies of marsupial X chromosomes reveal limited sequence homology of mammalian X-linked genes

To explore the extent to which the X chromosome has been conserved during mammalian evolution, we compared six loci that are X-linked in the human genome with the corresponding genes of the North American marsupial, the Virginia opossum (Didelphis virginiana). Our analysis shows that in the opossum genome there are sequences highly homologous to those of human cDNAs for housekeeping genes, glucose-6-phosphoribosyltransferase (HPRT), phosphoglycerate kinase A (PGK1), and alpha-galactosidase A (GLA). However, ornithine transcarbamylase and blood clotting Factor IX--tissue-specific genes that are X-linked in eutherians mammals--have no highly conserved homologs in the marsupial genome. By cloning opossum G6PD and HPRT, we found that these genes are X-linked in the opossum and that homologous sequences are limited to coding regions. As all genomic fragments hybridizing with the human GLA probe show dosage effects, it is likely that the opossum counterpart is X-linked. Finally, the pattern of hybridization suggests that the autosomal pseudogenes of HPRT and PGK1 in the opossum have remained highly homologous to the human X-linked genes.     

Pertussis toxin and target eukaryotic cells: binding, entry, and activation.

Pertussis toxin, a protein virulence factor produced by Bordetella pertussis, is composed of an A protomer and a B oligomer. The A protomer consists of a single polypeptide, termed the S1 subunit, which disrupts transmembrane signaling by ADP-ribosylating eukaryotic G-proteins. The B oligomer, containing five polypeptides, binds to cell receptors (most likely containing carbohydrate) and delivers the S1 subunit. Current knowledge suggests that expression of ADP-ribosyltransferase activity in target eukaryotic cells arises after 1) nucleotides and membrane lipids allosterically promote the release of the S1 subunit; and 2) the single disulfide bond in the S1 subunit is reduced by reductants such as glutathione. This model suggests conditions for the proper use of the toxin as an experimental reagent.     

Chitinase: a novel target for blocking parasite transmission?

In many species of blood-sucking arthropod, the internal tissues are covered by chitinous material that may hinder parasite invasion. To circumvent this potential barrier, therefore, parasites have developed mechanisms that involve the enzyme chitinase. In this review, Mohammed Shohobuddin and David C. Kaslow examine the relationship between chitinase and parasite transmission.     

Adaptations of glycogen metabolism in rat epididymal adipose tissue during fasting and refeeding.

It is well documented that adipose tissue glycogen content decreases during fasting and increases above control during refeeding. We now present evidence that these fluctuations result from adaptations intrinsic to adipose tissue glycogen metabolism that persist in vitro: in response to insulin (1 milliunit/ml), [3H]glucose incorporation into rat fat pad glycogen was reduced to 10% of control after a 3-day fast; incorporation increased 6-fold over fed control on the 4th day of refeeding following a 3-day fast. We have characterized this adaptation with regard to alterations in glycogen synthase and phosphorylase activity. In addition, we found that incubation of fat pads from fasted rats with insulin (1 milliunit/ml) increased glucose-6-P content, indicating that glucose transport was not the rate-limiting step for glucose incorporation into glycogen in the presence of insulin. In contrast, feeding a fat-free diet resulted in dramatic increases in glycogen content of fat pads without a concomitant increase in glucose incorporation into glycogen in response to insulin (1 milliunit/ml). Thus, fasting and refeeding appeared to alter insulin action on adipose tissue glycogen metabolism more than this dietary manipulation.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Comprehensive analysis of class I and class II HLA antigens and chronic hepatitis B virus infection

Following an acute hepatitis B virus (HBV) infection, clearance or persistence is determined in part by the vigor and breadth of the host immune response. Since the human leukocyte antigen system (HLA) is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are key determinants of viral clearance. HLA class I and II genes were molecularly typed in 194 Caucasian individuals with viral persistence and 342 matched controls who had cleared the virus. A single class I allele, A*0301 (odds ratio [OR], 0.47; 95% confidence interval [CI], 0.30 to 0.72; P = 0.0005) was associated with viral clearance. The class II allele DRB1*1302 was also associated with clearance (OR, 0.42; 95% CI, 0.19 to 0.93; P = 0.03), but its significance decreased in a multivariate model that included other alleles associated with disease outcome as covariates. B*08 was associated with viral persistence both independently (OR, 1.59; 95% CI, 1.04 to 2.43; P = 0.03) and as part of the conserved Caucasian haplotype A*01-B*08-DRB1*03. The B*44-Cw*1601 (OR, 2.23; 95% CI, 1.13 to 4.42; P = 0.02) and B*44-Cw*0501 (OR, 1.99; 95% CI, 1.22 to 3.24; P = 0.006) haplotypes were also associated with viral persistence. Interestingly, both the B*08 haplotype and DR7, which forms a haplotype with B*44-Cw*1601, have been associated with nonresponse to the HBV vaccine. The associations with class I alleles are consistent with a previously implicated role for CD8-mediated cytolytic-T-cell response in determining the outcome of an acute HBV infection.     

Favorable and unfavorable HLA class I alleles and haplotypes in Zambians predominantly infected with clade C human immunodeficiency virus type 1

The setpoint of viral RNA concentration (viral load [VL]) during chronic human immunodeficiency virus type 1 (HIV-1) infection reflects a virus-host equilibration closely related to CD8(+) cytotoxic T-lymphocyte (CTL) responses, which rely heavily on antigen presentation by the human major histocompatibility complex (MHC) (i.e., HLA) class I molecules. Differences in HIV-1 VL among 259 mostly clade C virus-infected individuals (137 females and 122 males) in the Zambia-UAB HIV Research Project (ZUHRP) were associated with several HLA class I alleles and haplotypes. In particular, general linear model analyses revealed lower log(10) VL among those with HLA allele B*57 (P = 0.002 [without correction]) previously implicated in favorable response and in those with HLA B*39 and A*30-Cw*03 (P = 0.002 to 0.016); the same analyses also demonstrated higher log(10) VL among individuals with A*02-Cw*16, A*23-B*14, and A*23-Cw*07 (P = 0.010 to 0.033). These HLA effects remained strong (P = 0.0002 to 0.075) after adjustment for age, gender, and duration of infection and persisted across three orders of VL categories (P = 0.001 to 0.084). In contrast, neither B*35 (n = 15) nor B*53 (n = 53) showed a clear disadvantage such as that reported elsewhere for these closely related alleles. Other HLA associations with unusually high (A*68, B*41, B*45, and Cw*16) or low (B*13, Cw*12, and Cw*18) VL were either unstable or reflected their tight linkage respecting disequilibria with other class I variants. The three consistently favorable HLA class I variants retained in multivariable models and in alternative analyses were present in 30.9% of subjects with the lowest (<10,000 copies per ml) and 3.1% of those with the highest (>100,000) VL. Clear differential distribution of HLA profiles according to level of viremia suggests important host genetic contribution to the pattern of immune control and escape during HIV-1 infection.     

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.