2011 Vol. 32, No. 3
2011, 32(3): 241-247. doi: 10.3724/SP.J.1141.2011.03241
The phylogenetic relationships among subspecies of Pomatorhinus ruficollis are not clear. In this paper we sequenced two mitochondrial genes (Cyt b and ND2, in total 2 184 bp) and examined twenty individuals representing ten of the eleven subspecies of P. ruficollis in China.Molecular phylogenetic trees were reconstructed using maximum parsimony, maximum likelihood and Bayesian inference methods, with P. ruficollis and P. schisticeps as in-groups, P. montanus, P. ochraceiceps, P. ferruginosus and Xiphirhynchus superciliaris as out-groups. Results indicated that P. ruficollis was paraphyletic, in which P. schisticeps was embedded, and was divided into three phylogeographic groups: southwestern China group, southeastern China group and central China group. Our results also suggested discordances between phylogenetic relationships and morphological characteristics, and clarified some intraspecific taxonomic debates.
Phylogenetic evaluation of the taxonomic status of Papilio maackii and P. syfanius (Lepidoptera: Papilionidae)
2011, 32(3): 248-254. doi: 10.3724/SP.J.1141.2011.03248
The taxonomic status of Papilio maackii and P. syfanius has long been disputed. We conducted a molecular phylogenetic study to evaluate the taxonomic status of P. maackii and P. syfanius. A total of twenty-four P. maackii individuals from six localities and sixteenP. syfanius individuals from two localities were analyzed. We sequenced the partial region of the CO-I gene (about 579bp) and partial CO-II gene sequence (about 655bp) of the two species. The Kimura-2-Parameter distances among P. maackii and P. syfanius ranged from 0 to 0.6%. Fifteen haplotypes were obtained based on the combined data set. The results strongly supported that all P. maackii individuals and all P. syfanius individuals formed a large clade, and could not be divided into separated clades. This research indicated that the two species have only very recently undergone speciation.
2011, 32(3): 255-261. doi: 10.3724/SP.J.1141.2011.03255
To assess the genetic diversity between randomly and selectivelybred populations, we sequenced 438 bp of the mitochondrial DNA control region from 102 pigs. These samples represented four native pig breeds, one nucleus and one conservation herd from Yunnan, China. Twenty haplotypes with sixteen polymorphic sites were identified. The number of haplotypes in the nucleus herd of Saba pig and the conservation herd of Banna miniature pig were restricted to three and one, respectively, while the randomly bred pig populations exhibited over six haplotypes. Notably, haplotype diversity in randomly bred populations was significantly greater than the selectively bred populations (h=0.732 vs. 0.425 and 0, exact test, P≤0.0036). These findings demonstrate that selective breeding generated low genetic diversity compared to randomly bred pig breeds. A timely intervention and well programmed breeding approach would stop further genetic diversity reduction in the nucleus and conservation herds of native pig breeds. Otherwise, selective breeding would dramatically reduce genetic diversity in only several years, indicating that sharp contradictions exist between breeding, conservation and genetic diversity. Genetic relationships are discussed based on net genetic distances among pig populations.
An extension strategy of Discovery Studio 2.0 for non-bonded interaction energy automatic calculation at the residue level
2011, 32(3): 262-266. doi: 10.3724/SP.J.1141.2011.03262
Non-bonded interaction forces play crucial roles in molecular recognition and binding in biological systems.However, it is difficult for traditional methods to automatically calculate and batch the non-bonded energy at the residue level. In recent years, many studies have focused on non-bonded interactions and developed tools to calculate and analyze such interactions. In this study, we present a highly automated approach for the calculation of non-bonded energy. Our strategy invoked protocols relevant to non-bonded interactions within Discovery Studio 2.0 (DS2.0, Accelrys Inc.) bottom module using Perl script, and determined the direct command line operation of calculating non-bonded interaction energy batches without accessing the graphical interface of DS. This approach extended the DS2.0 module and was applied to a recent study of complex structure analysis.
2011, 32(3): 267-276. doi: 10.3724/SP.J.1141.2011.03267
Thirteen hsp70 genes with complete conserved domains were identified in Tetrahymena thermophila, and expression of five similar and non-intron hsp70 genes were analyzed. Under heat shock conditions of 37, 39 and 41ºC, hsp70-2 mRNA had the highest relative expression level, suggesting it is closely related to heat shock. The basal level of constitutive T. thermophilahsp70-4 gene was high during 20 physiological/developmental stages of growth, starvation and conjugation, and it changed little upon exposure to heat shock: evidence that hsp70-4 is an hsc70 gene. The hsp70-4 cDNA is 2 208 bp long, and contains an open reading frame of 1 959 bp encoding 635 amino acids. Microarray data indicated that T. thermophilahsp70-3 gene probably participated in early starvation (0–12 h) stress and late conjugation (6–10 h) events, such as new macronuclear and micronuclear anlagen formation and old macronuclear elimination. However, hsp70-5 gene possibly participates in late starvation (12–15 h) stress and early conjugation (0–6 h) events such as micronuclear meiosis, micronuclear exchange and pronuclear fusion. Blast2GO indicated that they participated in dissimilar biological processes, suggesting hsp70-3 and hsp70-5 perform different functions.
2011, 32(3): 277-284. doi: 10.3724/SP.J.1141.2011.03277
Betaine homocysteine methyltransferase (BHMT, EC 126.96.36.199) catalyzes the transfer of a methyl group from betaine to homocysteines (Hcy) to form dimethylglycine and Met, respectively. A full-length cDNA of the BHMT in Lateolabrax japonicus was amplified using RT-PCR and SMART RACE methods. The cDNAof the BHMT in L. japonicus is 1 461 bp in size, with 72 bp 5'-UTR, 183 bp 3'-UTR and 1206 bp ORF, encoding a protein of 401 amino acids with a molecular weight of 44.32 kD and pI 7.21. The sequence analysis indicated that the deduced amino acid sequence of BHMT shared high identity (77% – 93%) with nine other species; the highest was 93% with Perca flavescens. Semi-quantitativeRT-PCR was used to characterize the expression of BHMT in ten tissuesincluding muscle, heart, eye, brain, gill, liver, intestine, kidney, adipose tissue and spleen. The results showed that BHMT is only expressed in the liver, intestines and kidney. BHMT mRNA in these three tissues declined after the fish were transferred from sea water to a higher salinity environment and induced when transferred to a lower salinity environment.BHMT gene in liver, intestine and kidney can also be induced after intraperitoneal injection of betaine. Our results show that betaine can induce the transcription of BHMT in fish, and BHMT play pivotal roles in adaptation to osmotic change.
We cloned the full length of tilapia prepro-orexin cDNA using RT-PCR and rapid-amplification of cDNA ends (RACE). The full-length of prepro-orexin cDNA was 648 bp containing an open reading frame of 423 bp. The 140 amino acid prepro-orexin protein included a 37 AA signal peptide, a 43 AA Orexin-A and, and 28 AA Orexin-B and the end of the 32 AA peptide of unknown function. The expression of prepro-orexin on tissue distribution, peri-prandial changes, starvation and re-feeding were quantified by real-time PCR. We found that prepro-orexin mRNA was present in all tissues tested and that the highest level was observed in hypothalamus. Expression levels were significantly higher at mealtime (0 h) than before (−2 h, −1 h) and after (+1 h, +2 h) mealtime. Fasting for 2, 4, 6 and 8 d caused significant increases in prepro-orexin mRNA expression in the hypothalamus, and after re-feeding, expression levels of prepro-o rexin mRNA returned to the same level compared to that in the fed group.
Correlation between cardiovascular risk factors and the expression of cardiovascular disease related genes in cynomolgus monkeys
2011, 32(3): 293-299. doi: 10.3724/SP.J.1141.2011.03293
The correlation between cardiovascular risk factors and cardiovascular disease related genes plays an important role in early-warning risk and early diagnosis. Thirty middle-aged male crab-eating macaques were fed a moderately atherogenic diet (0.053 mg cholesterol/kJ and 40% of calories as saturated fat) for twelve months. According to cardiovascular risk factors, we selected low-risk and high-risk crab-eating macaques, then analyzed the expression of 113 cardiovascular related genes by real-time PCR. A total of 65 genes were detected in peripheral blood leukocytes by real-time PCR. Sixteen up-regulated genes and nineteen down-regulated genes were detected in low-risk and high-risk crab-eating macaques compared to normal crab-eating macaques (P<0.05), in addition to fifteen genes that showed unique expression patterns (P<0.05). We also detected 42 genes in human peripheral blood leukocytes. The expression patterns of 22 genes were consistent between human and crab-eating macaques. These results narrowed the scope of genes for further research.
2011, 32(3): 300-306. doi: 10.3724/SP.J.1141.2011.03300
A total of 138 middle and aged cynomolgus monkeys (Macaca fascicularis) (above 10 years) were classified into three groups based on fasting plasma glucose (FPG) values, specifically low FPG, normal FPG, and high FPG group. Total cholesterol (TCHO), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) were detected in blood by automatic biochemical analyzer. The mRNA expressions of 37 diabetes-associated genes were analyzed with Real-time PCRin monocytes isolated from monkey peripheral blood.No significant correlation between the four serum lipid indictors HDL-C, LDL-C, TCHO, TG and FPG (P＞0.05) were found. However, the expressions of ACE, ACLY, PRKCB1, SLC2A4, SNAP23, VAPA, IGF2BP2, and IFNG were significantly enhanced when FPG increased (P<0.05).
2011, 32(3): 307-310. doi: 10.3724/SP.J.1141.2011.03307
To screen spontaneous diabetic mellitus and explore methods for its rapid identification, the basal and inferred levels of blood glucose of 440 overweight, middle- and old-aged cynomolgus monkeys were analyzed. Diagnostic diabetes was further validated by the oral glucose tolerance test (OGTT) and urine glucose. The average level of blood glucose of these cynomolgus monkeys was (3.88±0.98) mmol/L, which was lower than the level for suspected diabetes (5.0 mmol/L). Of them, 56 (12.72%) monkeys were identified with levels of blood glucose greater than 5.0 mmol/L and diagnosed as the diabetic subjects. This population showed impaired glucose tolerance using the OGTT and 39 of the 56 (69.23%) had glucose positive urine. The methods for screening diabetic mellitus used in this study were simple, quick, and limited the harm to animals. However, the incidence of diabetes was higher in these tested monkeys than in the regular human population in China (9.7%), suggesting that these methods are useful for screening diabetic disease in a large population but not suitable for all cynomolgus monkeys.
Inhibitory effect of ferulic acid on in?ammatory response in microglia induced by lipopolysaccharides
2011, 32(3): 311-316. doi: 10.3724/SP.J.1141.2011.03311
Ferulic acid (FA) is a natural compound that expresses antioxidant and anti-inflammatory activities. Microglial cells are innate immune cells that reside within the central nervous system (CNS). Activated microglia mediated neuronal immunity contributes to the neurodegeneration associated with Alzheimer's disease. In this study, we investigated the inhibitory effect of FA on neuroinflammation in BV-2 microglial cells induced by lipopolysaccharides (LPS). Our study showed that FA significantly suppressed the production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), and decreased induced type II nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein in LPS-stimulated BV-2 microglia cells in a dose dependent manner. We hypothesized that this was achieved by suppressing the protein level of Toll-like receptor 4 (TLR4).
Estrogen receptor expression and vitellogenin synthesis induced in hepatocytes of male frogs Rana chensinensis exposed to bisphenol A
2011, 32(3): 317-322. doi: 10.3724/SP.J.1141.2011.03317
The effects of bisphenol A (BPA) on estrogen receptor (ER) and vitellogenin (VTG) in hepatocytes of male amphibians have attracted significant attention in recent years. Adult male frogs Rana chensinensis were exposed to different concentrations of 10-7, 10-6 and 10-5 mol/L BPA consecutively for 10, 20, and 30 d, respectively. We used 10-9, 10-8 mol/L 17β-esradiol (E2) as positive controls. The expressions of ER mRNA in the hepatocytes were detected by in situ hybridization, and ER and VTG protein were detected by immunohistochemistry. The results showed that positive effects of ER mRNA were detected in all BPA and E2 treatment groups. Compared with the control group, the expression level of ER and VTG protein increased significantly in the hepatocytes of R. chensinensis. Both ER and VTG expression exhibited BPA dose-dependence for the same exposure time. For the same concentration of BPA treatment, VTG synthesis markedly increased with prolonged treatment, whereas the expression of ER did not significantly fluctuate. It is suggested that BPAcaused the synthesis of VTG by inducing ER up-regulation in the hepatocytes of male R. chensinensis, but its estrogenic activity was much lower than E2.
2011, 32(3): 323-328. doi: 10.3724/SP.J.1141.2011.03323
Interleukin-1β (IL-1β) is an important proinflammatory cytokine that plays a key role in injuries and diseases of the central nervous system (CNS). The voltage-gated Na+channel is the most important ion channel of neurons, and is essential for regenerative action potential (AP). The Na+ channel also contributes to many diseases of the brain. However, relations between IL-1β and central Na+ channels remain unreported. In this study, whole cell patch-clamp recording was used to investigate the acute effects of IL-1β (10 ng/mL) on voltage-dependent Na+ currents and AP of cultured cortical neurons from rats. Results showed that the half-activation voltage of Na+ channels and the threshold of AP, but not the amplitude, slope factor of activation, and inactivation properties, were affected by IL-1β. These data suggest that increased IL-1β in injury and disease may upregulate the excitability of neurons, and thereby exacerbate neurotoxicity.
Rat orbital frontal (orbital frontal cortex, OFC) GABA B receptor mechanisms in stress and depression
2011, 32(3): 329-336. doi: 10.3724/SP.J.1141.2011.03329
Stress-induced depression is a kind of functional and structural disability of the brain and involves many neurotransmitters and regions of the brain. A number of studies suggest involvement of γ-Aminobutyric acid (GABA) in the orbital frontal cortex (OFC) in the mechanism of stress-associated depression-like behavior in rodents. However, little work has been done on the relationship between GABA and neural plasticity of the OFC under stress. Here we examine the effect of the GABA in the OFC during acute forced swim stress (FSS). We found remarkable depression-like behavior in FSS and an open field test (OFT), and we observed a marked decrease in Kalirin-7 expression and the basal dendritic spine density of layer V pyramidal neurons in OFC after FSS. GABA administration reversed these changes, which were inhibited by CGP35348, an antagonist of GABA-B receptors. These results suggest an anti-depression effect of GABA in the OFC, which may be mediated by GABA-B receptor. The anti-depression effect of GABA is related to the plasticity of the dendritic spine density. This discovery may be helpful in the development of new therapies to treat depression.
Effects of urethane on the response properties of visual cortical neurons in young adult and old cats
2011, 32(3): 337-342. doi: 10.3724/SP.J.1141.2011.03337
Previous studies have shown that visual cortical neurons in old mammals exhibit higher spontaneous activity, higher responsiveness to visual stimuli, and lower selectivity for stimulus orientations and motion directions than did neurons in young adult counterparts. However, whether the responsive difference in cortical neurons between young and old animals resulted from different effects induced by anesthetics has remained unclear. To clarify this issue, we recorded the response properties of individual neurons in the primary visual cortex of old and young adult cats while systematically varying the anesthesia level of urethane, a widely used anesthetic in physiology experiments. Our results showed that cumulatively administrating 50 mg and 100 mg of urethane upon the minimal level of urethane required to anesthetize an old or young adult cat did not significantly alter the degree of neuronal response selectivity for stimulus orientations and motion directions nor significantly change the visually-driven response and spontaneous activity of neurons in old and young adult cats. Cumulatively administrating 150 mg of urethane decreased neuronal responsiveness similarly in both age groups. Therefore, urethane appears to exert similar effects on neuronal response properties of old and young adult animals.
Morphologic differentiation analysis for rounded caudal fin group of Oreonectes (Balitoridae) from China
2011, 32(3): 343-347. doi: 10.3724/SP.J.1141.2011.03343
Morphological characteristics were compared between three recorded species of rounded caudal fin Oreonectesand specimens collected from Guan’an of Guangxi. The results showed the Oreonectessp. from Guan’an was distinguished from O. anphthalmus. Although some morphologic differentiations existed among Oreonectes sp., O. polystigmus, and O. platycephalus, their differentiations were not enough to distinguish between them. Consequently, we used multivariate morphometrics to determine their morphologic differentiation. Twenty-six frame characteristics and 20 general characteristics of 63 specimens were measured. The resultsof the Principal Component Analysis showed that Oreonectessp. and O. polystigmus,and Oreonectessp. and O. platycephalus occupied obviously different areas in the scatter plot. It is suggested that the specimens collected from Guan’an may be a cryptic new species of Oreonectes. However, its taxonomic status should be decided by evidence from anatomical and molecular biological studies.
2011, 32(3): 349-352. doi: 10.3724/SP.J.1141.2011.03349
A new record species of Chaetodontidae, Heniochus diphreutes, was found by comparative analysis of the morphological characteristics in a taxonomic revision of the family Chaetodontidae in China. It was distinguished from its relative species H. acuminatus by a combination of the following: Dorsal fin usually with 12 spines (vs. 11); 2−3 rows of teeth on both jaws (vs. 5−7); ventral profile of head convex (vs. almost straight); anal fin more angular, and black area on posterior part of anal fin usually extending anteriorly to longest soft ray (vs. more round, and usually not extending anteriorly to longest soft ray); and snout shorter than eye (vs. longer).