Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Molecular characterization of porcine MMP19 and MMP23B genes and its association with immune traits

MMP19 and MMP23B belong to the Matrix metalloproteases (MMPs) family, which are zinc-binding endopeptidases that are capable of degrading various components of the extracellular matrix. They are thought to play important roles in embryonic development, reproduction and tissue remodeling, as well as in cell proliferation, differentiation, migration, angiogenesis, apoptosis and host defense. However, they are poorly understood in pigs. Here, we obtained the full length coding region sequence and genomic sequence of the porcine MMP19 and MMP23B genes and analyzed their genomic structures. The deduced amino acid sequence shares similar precursor protein domains with human and mouse MMP19 and MMP23B protein, respectively. Using IMpRH panel, MMP19 was mapped to SSC5p12-q11 (closely linked to microsatellite DK) and MMP23B was mapped to SSC8q11-q12 (linked to microsatellite Sw2521). Quantitative real-time PCR showed that MMP19 was abundantly expressed in the liver, while MMP23B was strongly expressed in the ovarian and heart. Furthermore, both genes were all expressed increasingly in prenatal skeletal muscle during development. Three SNPs were detected by sequencing and PCR-RFLP methods, and association analysis indicated that C203T at exon 5 of MMP19 has a significant association with the blood parameters WBC (G/L) and IgG2 (mg/mL) (P<0.05), SNP C131T at exon 3 of MMP23B is significantly associated with the blood parameters HGB (g/L) and MCH (P<0.05), and A150G in exon 4 has no significant association with the economic traits in pigs.     

Rab23 在皮肤鳞状细胞癌中的表达

目的:研究Rab23在皮肤鳞状细胞癌(SCC)中的表达及意义。方法:用免疫组化S-P法分别检测30例皮肤SCC、15例正常皮肤组织标本中Rab23的表达。结果:Rab23在皮肤SCC和正常皮肤中阳性率分别为90%和13.3%,二者差异有统计学意义(P<0.05)。结论:Rab23在皮肤SCC中高表达可能在皮肤SCC的发生发展过程中发挥作用。     

Current status of the E23K Kir6.2 polymorphism: implications for type-2 diabetes

The ATP-sensitive potassium (K_ATP) channel couples membrane excitability to cellular metabolism and is a critical mediator in the process of glucose-stimulated insulin secretion. Increasing numbers of K_ATP channel polymorphisms are being described and linked to altered insulin secretion indicating that genes encoding this ion channel could be susceptibility markers for type-2 diabetes. Genetic variation of K_ATP channels may result in altered -cell electrical activity, glucose homeostasis, and increased susceptibility to type-2 diabetes. Of particular interest is the Kir6.2 E23K polymorphism, which is linked to increased susceptibility to type-2 diabetes in Caucasian populations and may also be associated with weight gain and obesity, both of which are major diabetes risk factors. This association highlights the potential contribution of both genetic and environmental factors to the development and progression of type-2 diabetes. In addition, the common occurrence of the E23K polymorphism in Caucasian populations may have conferred an evolutionary advantage to our ancestors. This review will summarize the current status of the association of K_ATP channel polymorphisms with type-2 diabetes, focusing on the possible mechanisms by which these polymorphisms alter glucose homeostasis and offering insights into possible evolutionary pressures that may have contributed to the high prevalence of K_ATP channel polymorphisms in the Caucasian population.This work was supported by funding from the Canadian Diabetes Association (CDA) in honor of Gordon M. Stevenson, the Alberta Heritage Foundation for Medical Research (AHFMR), and the Canadian Institutes of Health Research (CIHR). M.J.R. is supported by AHFMR and CDA Scholarships. P.E.L. received salary support as an AHFMR Scholar and CIHR New Investigator     

The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.     

Prevalence of Burkholderia sp. nodule symbionts on four mimosoid legumes from Barro Colorado Island, Panama

Sequences of 16S rRNA and partial 23S rRNA genes and PCR assays with genotype-specific primers indicated that bacteria in the genus Burkholderia were the predominant root nodule symbionts for four mimosoid legumes (Mimosa pigra, M. casta, M. pudica, and Abarema macradenia) on Barro Colorado Island, Panama. Among 51 isolates from these and a fifth mimosoid host (Pithecellobium hymenaeafolium), 44 were Burkholderia strains while the rest were placed in Rhizobium, Mesorhizobium, or Bradyrhizobium. The Burkholderia strains displayed four distinct rRNA sequence types, ranging from 89% to 97% similarity for 23S rRNA and 96.5-98.4% for 16S rRNA. The most common genotype comprised 53% of all isolates sampled and was associated with three legume host species. All Burkholderia genotypes formed nodules on Macroptilium atropurpureum or Mimosa pigra, and sequencing of rRNA genes in strains re-isolated from nodules verified identity with inoculant strains. Sequence analysis of the nitrogenase alpha-subunit gene (nifD) in two of the Burkholderia genotypes indicated that they were most similar to a partial sequence from the nodule-forming strain Burkholderia tuberum STM 678 from South Africa. In addition, a PCR screen with primers specific to Burkholderia nodB genes yielded the expected amplification product in most strains. Comparison of 16S rRNA and partial 23S rRNA phylogenies indicated that tree topologies were significantly incongruent. This implies that relationships across the rRNA region may have been altered by lateral gene transfer events in this Burkholderia population.     

PIKE/nuclear PI 3-kinase signaling in preventing programmed cell death

PI 3-kinase enhancer (PIKE) is a nuclear GTPase that enhances PI 3-kinase (PI3K) activity. Nerve growth factor (NGF) treatment leads to PIKE activation by triggering the nuclear translocation of PLC-gamma1, which acts as a physiological guanine nucleotide exchange factor (GEF) for PIKE. PI3K occurs in the nuclei of a broad range of cell types, and various stimuli elicit PI3K nuclear translocation. While cytoplasmic PI3K has been well characterized, little is known about the biological function of nuclear PI3K. Surprisingly, nuclei from 30 min NGF-treated PC12 cells are resistant to DNA fragmentation initiated by the activated cell-free apoptosome, and both PIKE and nuclear PI3K are sufficient and necessary for this effect. Moreover, pretreatment of the control nucleus with PI(3,4,5)P3 alone mimics the anti-apoptotic activity of NGF by selectively preventing apoptosis, for which nuclear Akt is required but not sufficient. Recently, a nuclear PI(3,4,5)P3 receptor, nucleophosmin/B23, has been identified from NGF-treated PC12 nuclear extract. PI(3,4,5)P3/B23 complex mediates the anti-apoptotic effects of NGF by inhibiting DNA fragmentation activity of caspase-activated DNase (CAD). Thus, PI(3,4,5)P3/B23 complex and nuclear Akt effectors might coordinately mediate PIKE/nuclear PI3K signaling in promoting cell survival by NGF.     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].