A new species of the genus Achalinus (Squamata, Xenodermatidae) from southwest Hunan Province, China.

A new species of xenodermid snake, Achalinusnanshanensis H. Li, L.-Q. Zhu, Z.-Q. Zhang & X.-Y. Mo, sp. nov., is described based on three specimens collected from Nanshan National Park and Tongdao County of southwest Hunan Province. This new species is genetically distinct amongst its congeners with the mitochondrial COI uncorrected p-distance ranging from 4.4% (in A.yangdatongi) to 17.7% (in A.meiguensis). In addition, this new species can be distinguished from its congeners by a combination of the following morphological characters: (1) dorsal scales with 23 or 25 rows throughout and strongly keeled; (2) tail relatively longer so that TaL/ToL = 0.215-0.248; (3) length of suture between internasals significantly longer than that between prefrontals, LSBI/LSBP = 1.66-1.84; (4) single loreal scale present; (5) SPL 6 in number, with the fourth and fifth contacting eye; (6) IFL 6 in number, with the first three touching the first pair of chin shields; (7) TMP is 2-2-4/2-2(3)-4, with the anterior pair elongated and in contact with the eye; (8) ventrals 2 + 147-158; (9) subcaudals 64-77, unpaired; (10) dorsal body brownish black, with a bright yellow neck collar extending to the head and abdomen in the occipital region. The recognition of the new species increases the number of described Achalinus species to 28, of which 21 are found in China.

Microarray analyses of differentially expressed human genes and biological processes in ECV304 cells infected with rubella virus.

Changes in mRNA expression levels of ECV304 cells infected with the wild-type rubella strain were analyzed using a microarray system representing 18,716 human genes. Four hundred eighty-seven genes exhibited differential expression levels; 456 of these genes were up-regulated while 31 genes were down-regulated. We identified 53 biological processes that were significantly relevant to the RV-infection. Among these biological processes, 52 were one-gene processes and one was a process involving five genes: IFNA21 (interferon, alpha 21), interferon stimulated exonuclease gene 20 kDa (ISG20), zinc finger protein 175 (ZNF175), tripartite motif-containing 22 (TRIM22), and MX2 [myxovirus (influenza virus) resistance 2 (mouse)]. Except for ZNF175, gene annotation indicated four of these genes encoded interferon or interferon-induced genes. These results suggest that genes relevant to interferon-regulated pathways may be involved in the pathogenesis of rubella.

[Morphological changes of ECV304 cells infected by herpes simplex virus type 2].

OBJECTIVE: To observe the pathological changes and morphological alterations of ECV304 cells after the infection by herpes simplex virus type 2 (HSV-2) in vitro. METHODS: Passaged ECV304 cells were infected with HSV-2, TCID50 and morphological changes were observed by optical microscopy and tissue staining. RESULTS: One day after HSV-2 infection, swelling, rounding, and increase of thickened cytoplasmic granules occurred in the ECV304 cells, and on day 2, cell fusion was observed with weakened nuclear staining. CONCLUSION: ECV304 cells mostly undergo necrosis after HSV-2 infection without obvious evidence of cell apoptosis.

[Human cytomegalovirus induces apoptosis of ECV304 endothelial-like cells].

OBJECTIVE: To investigate the mechanisms for the cytopathic effect (CPE) of human cytomegalovirus (HCMV) in ECV304 endothelial-like cells. METHODS: PCR and indirect immunofluorescence were used to detect HCMV infection by examining immediate-early (IE) gene and protein expression of the virus in ECV304 cells. Phase-contrast and electron microscopies were performed to observe the morphological changes of the infected and uninfected cells, and DNA ladder analysis and flow cytometry were carried out to study HCMV-induced cell apoptosis. RESULTS: In HCMV-infected ECV304 cells, cytopathic effects were first observed at approximately 72 h post-infection. The cells with CPE changes exhibited detachment from the monolayer, cell rounding and shrinkage. The expression of the IE gene was detected. Chromatin condensation and nuclear fragmentation along with dramatic changes of the mitochondria were observed by electron microscopy at 96 h post-infection. Cellular DNA fragmentation was observed in the infected cells, which had cells apoptotic rates of 4.1% and 45.7% at 96 h and 144 h post-infection, respectively. CONCLUSION: HCMV can induce apoptosis of ECV304 endothelial-like cells.

The effects of different sample labelling methods on signal intensities of a 60-mer diagnostic microarray.

The effects of four different labelling methods on signal intensities of a 60-mer diagnostic microarray were studied. Eighty of virus-specific oligonucleotide probes for human influenza virus were prepared in an array of 15x16 spots. RNA samples from cultured human influenza virus strains were labelled with four different methods, including direct cDNA labelling (DL), universal primer labelling (UPL), direct cDNA labelling with restriction display (DL-RD), and Cy-dUTP incorporated cDNA labelling with restriction display (IL-RD) in a signal color format. The background-subtracted signal intensities from five replicate hybridization experiments of each labelling method were analyzed using one-way analysis of variance (one-way ANOVA) and linear regression techniques. The effect of sample labelling method on background-subtracted signal intensities was significant (p<0.001) and multiple comparisons showed the differences existed mainly between DL and the other three labelling methods. The sample labelling method explained about 4.3% of signal intensity. The results demonstrated that UPL and the RD-based methods are more efficient than the conventional DL method for sample labelling, an important variation factor affecting the signal intensities in diagnostic microarrays.

The VDR, COL1A1, PTH, and PTHR1 gene polymorphisms are not associated with bone size and height in Chinese nuclear families.

We tested the relationship of the ApaI, Eco31I, BstBI, and (AAAG)n polymorphisms in the vitamin D receptor (VDR), collagen type I alpha-1 (COL1A1), parathyroid hormone (PTH), and parathyroid hormone (PTH)/PTH-related peptide receptor (PTHR1) genes with variations in bone size (BS) and height. Population stratification, total-family association, and within-family association were used to test these relationships in 400 Chinese nuclear families with a total of 1256 individuals. The BS at hip and spine was measured using a Hologic QDR 2000 dual-energy X-ray absorptiometry (DXA) scanner. The minor allele frequencies were 29.2%, 36.0%, and 14.0% for the VDR-ApaI, COL1A1-Eco31I, and PTH-BstBI markers, respectively. (AAAG)5 and (AAAG)6 of the PTHR1 gene are two major alleles in the Chinese people. Significant population stratification was found between the spine BS and PTHR1-(AAAG)5 (P = 0.048) and PTHR1-(AAAG)6 (P = 0.023), as well as between PTHR1-(AAAG)5 and height (P = 0.048), but we did not detect any significant within-family association or total-family association between the VDR, COL1A1, PTH, and PTHR1 gene polymorphisms and the variations in BS and height in our sample. Our results do not support that the VDR, COL1A1, PTH, and PTHR1 genes have an important influence on the variation in BS and height in our Chinese population.

A novel sample labeling method with restriction display PCR for 60-mer oligonucleotide microarray.

OBJECTIVE: To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray. METHODS: Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases. RESULTS: Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes. CONCLUSION: Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.

The -1997 G/T polymorphism in the COLIA1 upstream regulatory region is associated with hip bone mineral density (BMD) in Chinese nuclear families.

Type I collagen is the most abundant protein of bone matrix, and the collagen type I alpha 1(COLIA1) gene has been considered one of the most important candidate genes for osteoporosis. In this study, we simultaneously tested linkage and/or association of the -1997 G/T polymorphism in the COLIA1 upstream regulatory region with the variation of bone mineral density (BMD) in 1263 subjects from 402 Chinese nuclear families, consisted of both parents and at least one healthy female offspring from 20 to 45 years of age. All the subjects were genotyped by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). BMD of the lumbar spine (L1-L4) and hip (respective and combined phenotype of the femoral neck, trochanter, and intertrochanter) was measured by dual-energy X-ray absorptiometry (DXA). By using the tests implemented in program QTDT (quantitative transmission disequilibrium test), we found significant within-family association (via TDT) between the -1997 G/T polymorphism with BMD variation at all the hip sites (respective and combined phenotypes, P < 0.05). The amount of BMD variation explained by the -1997G/T polymorphism was 1.6%, 2.0%, 1.2%, and 1.3% at the total hip, femoral neck, trochanter, and intertrochanter, respectively. Because of the limited number of sib pairs in this sample, we did not find evidence of linkage. In summary, the -1997 G/T polymorphism in the COLIA1 gene is likely to be in linkage disequilibrium with a nearby functional polymorphism affecting hip BMD, or the -1997 G/T polymorphism itself may have an important effect on the variation of hip BMD in our Chinese sample.

[A986S polymorphism of calcium-sensing receptor gene is not related to bone mineral density or bone size in premenopausal Chinese women].

OBJECTIVE: To investigate the association of a calcium-sensing receptor (CaSR) gene missense polymorphism, 986Ala/Ser (A986S), with bone mineral density (BMD) and bone size in healthy Chinese premenopausal women. METHODS: A total of 285 healthy Chinese premenopausal women (20.0 to 41.9 years of age) of Han nationality in the urban area of Shanghai were recruited for this study. The BMD and bone size of the spine and hip were measured by dual-energy X-ray absorptiometry. All the subjects were genotyped at the CaSR A986S site in exon 7 with polymerase chain reaction followed by restriction enzyme BsaHI digestion. The presence of the restriction fragment site was represented by alanine (A), while its absence by serine (S), rendering the genotypes AA, AS, and SS. RESULTS: The genotype AS was rare and SS absent in these Chinese women, and no significant differences in the BMD or bone size of either the spine or hip were found between the two genotypes. CONCLUSION: Given the important role of the CaSR in calcium metabolism, further studies with useful genetic markers may have better chances to define the association of the CaSR gene with bone phenotype variations.

Lack of association between the HindIII RFLP of the osteocalcin (BGP) gene and bone mineral density (BMD) in healthy pre- and postmenopausal Chinese women.

In Caucasian populations, the polymorphic restriction endonuclease HindIII marker of the osteocalcin (also known as BGP, for bone Gla protein) gene has recently been reported to be associated with bone mass, a major risk determinant of osteoporosis. In this study, we investigated the relationship between the BGP HindIII polymorphism and bone mineral density (BMD) in 388 premenopausal (31.18 +/- 5.92 years) and 169 postmenopausal (58.90 +/- 6.27 years) Chinese women. The BMD of spine and hip was measured by dual-energy X-ray absorptiometry (DEXA). All the study subjects were genotyped at the HindIII site of the BGP gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) detecting methods. The BGP alleles were designated according to the absence ( H) or presence ( h) of the HindIII restriction site. We did not find any significant difference in spine and hip BMD across BGP genotypes in either pre- or postmenopausal women or the combined group. Our result is not consistent with recent reports that the HindIII marker of the BGP gene is associated with osteoporosis. The different findings may reflect inter-population differences in the association (i.e., linkage disequilibrium) of molecular markers with BMD, and indicate the limit of using the HindIII marker of the BGP gene as a genetic marker to discern women susceptible to low BMD and thus osteoporosis in Chinese.

Estrogen receptor alpha gene polymorphisms and peak bone density in Chinese nuclear families.

PBD is an important determinant of osteoporotic fractures. Few studies were performed to search for genes underlying PBD variation in Chinese populations. We tested linkage and/or association of the estrogen receptor alpha gene polymorphism with PBD in 401 Chinese nuclear families. This study suggests the ER-alpha gene may have some minor effects on PBM variation in the Chinese population. Low peak bone density (PBD) in adulthood is an important determinant of osteoporotic fractures in the elderly. PBD variation is mainly regulated by genetic factors. Extensive molecular genetics studies have been performed to search for genes underlying PBD variation, largely in whites. Few studies were performed in Chinese populations. In this study, we simultaneously test linkage and/or association of the estrogen receptor alpha (ER-alpha) gene polymorphism with PBD in 401 Chinese nuclear families (both parents plus their female children) of 1260 subjects, with the 458 children generally between 20 and 40 years of age. All the subjects were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) at polymorphic PvuII and XbaI sites inside the ER-alpha gene. Bone mineral density was measured at the lumbar spine (L1-L4) and hip (femoral neck, trochanter, and intertrochanteric region). Raw bone mineral density values were adjusted by age, height, and weight as covariates. We detected marginally significant results for within-family association (transmission disequilibrium; p = 0.054) between the spine bone mineral density variation and the ER-alpha XbaI genotypes. For the hip bone mineral density variation, significant (p < 0.05) linkage results were generally found for the two intragenic markers. Analyses of the haplotypes defined by the two markers confer further evidence for linkage of the ER-alpha with the hip PBD variation. In conclusion, this study suggests that the ER-alpha gene may have minor effects on PBD variation in our Chinese population.

Polymorphisms of four bone mineral density candidate genes in Chinese populations and comparison with other populations of different ethnicity.

Studies on polymorphisms of candidate genes and their association with bone mineral density (BMD) have been reported in many populations, but few have been reported in Chinese populations. We investigated polymorphisms of the following five commonly used markers of four prominent BMD candidate genes with the purpose of identifying useful genetic markers for osteoporosis genetic research in Chinese: the Sp1 and RsaI polymorphisms of the collagen type 1 alpha l (Col1a1) gene, the -174G/C promoter polymorphism of the interleukin 6 (IL-6) gene, the Asn363Ser polymorphism of the glucocorticoid receptor (GR) gene, and the T --> C polymorphism in intron 5 of the transforming growth factor beta(1) (TGF-beta(1)) gene. We evaluated these polymorphisms using PCR-RFLP in samples of at least 124 random individuals. We compared the polymorphisms of these five markers with other populations using the chi(2) test and Fisher's exact two-tailed test. For the RsaI polymorphism, only three heterozygotes but no variant homozygote were identified. For the -174G/C polymorphic site, only one GC heterozygote and no CC homozygote were found. Alleles s, Ser, and A(1) at the Sp1, Asn363Ser, and T --> C marker sites that have been found to be polymorphic in other populations were not found in Chinese. Significant differences of allele and genotype frequency distributions were observed at these polymorphisms ( P < 0.001) after comparing with other populations. Our results suggest that variant alleles of the five markers are absent or too rare to be useful genetic makers in Chinese, despite the fact that they have been commonly used as polymorphic markers in osteoporosis genetic research in other populations.