Bioinformatics Analysis the Complete Sequences of Cytochrome b of Takydromus sylvaticus and Modeling the Tertiary Structure of Encoded Protein

Cytochrome b (cyt b) gene complete sequences (1143bp) of Takydromus sylvaticus were sequenced. In order to clarify the phylogenetic position of the Takydromus sylvaticus, we investigated the phylogeny of 15 Takydromus spp. distributed in East-Asia by Maximum Parsimony (MP), Bayesian Inference (BI), and Maximum Likelihood (ML) methods using DNA fragments of cyt b genes. The results supported that the Platyplacopus merged into Takydromus and negated the validity of Platyplacopus. Furthermore, the prediction of tertiary structures of cyt b exhibited the CD loop region contain two short helices forming a hairpin arrangement, namely cd1 and cd2. Thermostability analysis shows that the CD-loop region is unstable thermodynamically and may provide mobility to amino acids located at the heme, and might provide high flexibility to the top of ISP (iron-sulfur protein) and the cavity region of Qo binding site. It suggested that the two short helices of CD loop region of cyt b was a dominating portion for ISP binding site.     

Spatial relationship among individuals of the Japanese lacertidTakydromus tachydromoides (Sauria,Lacertidae)

The home range ofTakydromus tachydromoides was studied in a grassland area from April 1977 to November 1978. The mean size of home range did not differ markedly between sexes; 136.5 m² for males and 130.8 m² for females. Home ranges of adults overlapped greatly in each sex, and the lizard was considered to be non-territorial. Individuals showed return movement to a definite area (sleeping site) within the home range, and the home range did not shift within a year or between years. Characteristics of the home range of this grassland-inhabiting lizard were discussed in relation to resource abundance and predation pressure.     

Peculiarities of osmoregulation of circulating red blood cells in steno-and euryhaline marine fish species under hypoosmotic conditions

The peculiarities of osmoregulation of circulating red blood cells of the stenohaline giant gobyGobius cobitis and the euryhaline toad gobyGobius batrachocephalus have been studied under experimental conditions. In the giant goby, volume of the red blood cells increased steadily by 10.6–18.1% (p < 0.05) after reduction of the medium salinity from 15–17 to 6.0–6.8‰ and this volume increase remained during the entire experimental period (40–45 days). Lysis of red blood cells was noticed in some cases, which was indicated by a decrease of the number of red blood cells and an increase of concentration of free hemoglobin in the blood plasma. No similar reactions were observed in the euryhaline toad goby; the mean cell volume did not change statistically significantly. The volume regulation resulted in K⁺ efflux from red blood cells. The blood red cells of the toad goby had a high resistance to osmotic stress. The Na⁺,K⁺-ATPase activity in the red blood cell membranes of the toad goby was higher by 18.8% (p < 0.001) than in the giant goby.     

Peculiarities of osmoregulation of circulating red blood cells in steno- and euryhaline marine fish species under hypoosmotic conditions

The peculiarities of osmoregulation of circulating red blood cells of the stenohaline giant gobyGobius cobitis and the euryhaline toad gobyGobius batrachocephalus have been studied under experimental conditions. In the giant goby, volume of the red blood cells increased steadily by 10.6–18.1% (p (WENA) 0.05) after reduction of the medium salinity from 15–17 to 6.0–6.8‰ and this volume increase remained during the entire experimental period (40–45 days). Lysis of red blood cells was noticed in some cases, which was indicated by a decrease of the number of red blood cells and an increase of concentration of free hemoglobin in the blood plasma. No similar reactions were observed in the euryhaline toad goby; the mean cell volume did not change statistically significantly. The volume regulation resulted in K⁺ efflux from red blood cells. The blood red cells of the toad goby had a high resistance to osmotic stress. The Na⁺,K⁺-ATPase activity in the red blood cell membranes of the toad goby was higher by 18.8% (p (WENA) 0.001) than in the giant goby.     

A mathematical model for drug administration by using the phagocytosis of red blood cells

 A mathematical model for the delivery of drug directly to the macrophages by using the phagocytosis of senescent red blood cells is proposed. The model is based on the following assumption: At time t=0 a preassigned red blood cell population n(0, a)=φ(a), a>0, loaded by the drug, is injected in the blood circulation. Among the cells of that population only those with an age a≧ā (ā=120 days) will be phagocytosed by macrophages. Of course, the lifetime of the drug must be higher than ā. Within the red blood cells it cannot be metabolized, neither can it diffuse through their membranes. The emphasis of the paper is on the mathematical properties and on the formulation of the control problem. Received 15 December 1994; received in revised form 20 July 1995     

Antifungal effects of Ficus sycomorus and Pergularia tomentosa aqueous extracts on some organs in Bufo regularis treated with Aspergillus niger

The antifungal efficacy of Ficus sycomorus and Pergularia tomentosa plant extracts on Bufo regularis experimentally infected with Aspergillus niger was studied. After an oral administration of the pathogen for 15 days, the blood, kidney and liver were examined. Treatment with A. niger produced a reduction in red blood count cells and hemoglobin content. Also, both livers and kidneys revealed marked destruction and degenerative changes. These changes included congestion of blood vessels, leukocytic infiltration, and cytoplasmic vacuolization of the hepatocytes. As well as complete destruction of the cellular boundaries of the tubular epithelia, inflammatory leukocytes between the intertubular spaces, destruction and necrosis in renal tubule cells and the swollen glomeruli with wide glomerular spaces were seen. Pretreatment with F. sycomorus and P. tomentosa plant extracts 1 h prior the administration of A. niger for two weeks improved blood parameters and protected against hepatic and renal damage as observed from histological examination and reduced spore numbers in culture media on these organs.     

Isolation and characterization of 12 tetra‐repeated microsatellite loci from the Formosan grass lizard (Takydromus formosanus)

Twelve tetra‐repeated microsatellite loci were developed for the island‐endemic grass lizard Takydromus formosanus (Squamata: Lacertidae). We characterized these loci by genotyping 51 individuals sampled from six localities. The number of alleles per locus ranged between 12 and 23, whereas the observed heterozygosity ranged between 0.686 and 0.941. None of these loci showed gametic disequilibrium or sex linkage. With such high polymorphism, we believe that these loci should be suitable for fine‐scaled analysis of population structure, hybridizations or individual identification of grass lizards.     

Tail vibration of the Japanese grass lizardTakydromus tachydromoides as a tactic against a snake predator

Behavioral responses of the Japanese lacertid lizard,Takydromus tachydromoides, to its natural predator, the Japanese striped snake,Elaphe quadrivirgata, were examined under laboratory conditions.T. tachydromoides often thrashed its tail rapidly from side to side for short intervals (less than 0.5 s), usually just before fleeing away from an approaching snake. This tail vibration behavior appears to distract the snake away from the vulnerabl parts of the body to the tail, as tail vibration occurred more frequently when the head of the snake was closer to the lizard's head than its tail. Distraction of snake attack by this action is suggested to be an antipredator tactic, because it was demonstrated that the vibration increased the chance of escape.     

Haematopoietic stem cell content of murine bone marrow, spleen, and blood. Limiting dilution analysis of diffusion chamber cultures

Suspensions of mouse bone marrow cells, spleen cells, and blood leucocytes were cultured in diffusion chambers in dilution series in order to establish the minimum concentrations of haematopoietic stem cells (HSC). The observed frequencies of empty chambers after seven days of culture conformed to the expected frequencies of a null response in a Poisson distribution. The proportions of empty chambers could therefore be used to estimate the concentrations of HSC in the cell suspensions. The following numbers of HSC per 10⁵ cells were found (with 95% confidence limits): Bone marrow: 50 (44–56). Spleen: 3.5 (2.8–4.3). Blood leucocytes: 1.4 (1.2–1.8). The mean (± standard error) HSC-content per femur, spleen, and milliliter blood when pooling cells from three to six donor mice was 8240 ± 600, 7660 ± 490, and 56 ± 6.5 respectively. For comparison, the HSC concentrations were also determined with the spleen colony technique; the ratio between the HSC-concentrations of bone marrow, spleen, and blood determined with the diffusion chamber technique was similar to that determined with the spleen colony technique.     

Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured CD34⁺ cells were compared. The average expansion of total cells and CD34⁺ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of CD3⁺ cells declined and the percentage of CD33⁺ cells increased during culture. The engraftment levels of fourteen-day cultured CD34⁺ cells were higher than those of fresh and seven-day cultured CD34⁺ cells. Fourteen-day cultured CD34⁺ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured CD34⁺ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.     

Immunomodulation property of hexane fraction of leaves of Cinnamomum tamala Linn. in rats

The leaves of Cinnamomum tamala Linn. (CT) (Lauraceae) clinically used in Ayurveda as antidiabetic and diuretic, but no reports are available towards immunomodulating property. Its hexane fraction (CTH) was orally given to rats for 10 days and delayed type of hypersensitivity (DTH), antibody producton against sheep red blood cells (SRBCs), mitotic index in bone marrow cells and concanavalin A (Con A) mediated proliferation of lymphocytes were assessed. Further on 30 days treatment, change in body weight (BW), spleen weight, thymus weight, bone marrow cellularity and hematological changes were observed. It inhibited significantly the DTH response (IC₅₀ 1475 ± 57.19 mg kg^?1 BW), antibody production, suppressed mitotic index in bone marrow cells along with the suppression of lymphocyte proliferation against Con A (IC₅₀ 63.33 ± 1.95 µg mL^?1). In all experiments, cyclophasphamide and dexamethasone had been used as reference drug for in vivo and in vitro studies, respectively. On 30 days treatment, the CTH (800 mg kg^?1 BW and above) significantly suppressed growth rate, increase of spleen and thymus weight and low bone marrow cellularity. In hematological examination, it inhibited total white blood cell and lymphocytes count and increased per cent of polymorphs. Thus, it could be suggested that the fraction possesses immunosuppressive property at doses, higher than 800 mg kg^?1 BW in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Stable‐hydrogen isotope turnover in red blood cells of two migratory thrushes: application to studies of connectivity and carry‐over effects

ABSTRACT Understanding turnover rates of stable isotopes in metabolically active tissues is critical for making spatial connections for migratory birds because samples provide information about pre‐migratory location only until the tissue turns over to reflect local values. We calculated stable‐hydrogen isotope (δ²H) turnover rate in the red blood cells of two long‐distance migratory songbirds, Bicknell's Thrushes (Catharus bicknelli) and Swainson's Thrushes (Catharus ustulatus), using samples collected at a breeding site in New Brunswick, Canada. Blood from both species captured early in the breeding site was more positive in δ²H than blood sampled later in the summer, but did not match blood values for wintering Bicknell's Thrushes. An asymptotic exponential model was used to estimate turnover of red blood cell δ²H and yielded a half‐life estimate of 21 days and 14 days for Bicknell's and Swainson's thrushes, respectively. Red blood cells of both species approached the local breeding site value one month after the first individuals were detected at the site. For Bicknell's Thrushes, estimated δ²H in blood at arrival (?72‰) was closer to blood collected at wintering sites (mean ?61‰) than to expected breeding site δ²H (?120‰). Discrimination values calculated for red blood cells collected at the breeding site for both species were greater than expected based on studies using keratin. Turnover during migration currently limits the use of blood sampled early in the breeding season for connectivity/carry‐over effect studies. However, direct tracking technology such as geolocators can provide information about migration duration, timing, and stopovers that can be used to improve isotopic turnover equations for metabolically active tissues.     

Heat tolerance and altitudinal distribution of a mountainous lizard, Takydromus hsuehshanensis, in Taiwan

1.

We investigated whether heat tolerance has a crucial impact on the altitudinal distribution of a high-altitude lizard, Takydromus hsuehshanensis (>1800 m altitude).     

Ontogenetical development of the chick and duck subcommissural organ

Summary The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used.In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct; day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. The duck SCO appears to follow a similar pattern of development.Supported by Grant I/60 935 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged     

Invasion and destruction of a murine fibrosarcoma by <Emphasis Type="Italic">Salmonella</Emphasis>-induced effector CD8 T cells as a therapeutic intervention against cancer

We have developed a new vaccination strategy by using the Salmonella type III secretion system (T3SS) to translocate heterologous antigens into the cytosol of host cells. This leads to an efficient antigen-specific CD8 T cell induction. Recently, we have demonstrated the use of Salmonella’s T3SS for the immunoprophylaxis of a solid tumor. The murine fibrosarcoma WEHI 164 was transfected with the DNA sequence encoding the MHC class I-peptide p60_217–225 from Listeria monocytogenes. In the present study, we used this tumor model to investigate the potential of vaccination with recombinant Salmonella in a therapeutic setting. BALB/c mice were subcutaneously challenged with WEHI-p60 cells. Simultaneously or 4 days later, these mice received either an orogastric or intravenous immunization with Salmonella translocating p60. Interestingly, 71–80% of the intravenously and 50–52% of the orogastrically immunized mice showed a complete tumor regression after 14 days. In addition, the distribution of tetramer-positive p60_217–225-specific CD8 T cell subpopulations in blood and tumor tissue was analyzed. Co-staining with CD62L and CD127 revealed that the frequencies of p60_217–225-specific effector and effector memory CD8 T cells in blood and in fibrosarcoma tissue were related to the kinetics of tumor regression. In summary, our study demonstrates that therapeutic vaccination with Salmonella leads to efficient induction of tumor-invading effector CD8 T cells that may result in significant tumor regression.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Interrelationships and evolution of the east Asian grass lizards, Takydromus (Squamata: Lacertidae)

The east Asian lacertid lizard genus, Takydromus , is well-defined but its relationships to other primitive Palaearctic lacertids are still uncertain and, although it has similarities to the lower Miocene Miolacerta, Takydromus does not appear to be identical with this. The 16 or 17 recognizable species are reviewed and a key provided for their identification; the remaining area of uncertainty about species boundaries is the Takydromus sexlineatus group in Assam. A phylogeny for Takydromus is estimated using a data set of 35 morphological characters drawn from external features, skeleton and soft-parts. There are two main constituent clades which may be informally recognized as Takydromus s.s. and Platyplacopus. Platyplacopus is subtropical and perhaps relict, whereas Takydromus ranges from temperate to tropical areas. The two clades have different ranges of eggs per clutch. In both of them, elongate forms that climb extensively in flimsy vegetation have evolved and have independently acquired similar features that confer performance advantages in this situation. Interestingly, the order of assembly of tliese features is markedly different.     

Detection of heterozygotes for galactokinase deficiency in a human population

A simple method for assaying galactokinase in red blood cells has been described. The assay is proportional with respect to time and enzyme concentration within the limits studied. The pH optimum of the enzyme is between pH 7.8 and 8.2. In whole blood kept at 4 C, the enzyme is stable for at least 8 days, but activity is rapidly lost when the cells are washed and frozen. Employing this method, blood samples from 642 individuals were assayed for galactokinase activity. Six individuals had activity about one-half the normal value and were considered to be heterozygotes for galactokinase deficiency. Family studies on four of the individuals further substantiated this conclusion. The calculated gene frequency indicates that approximately 1 in 46,000 individuals would be expected to have galactokinase deficiency. This work was supported by a grant from the Children's Bureau.     

Fetal erythropoiesis in steel mutant mice : I. A morphological study of erythroid cell development in fetal liver

A method of definitive identification of mutant (S1/S1^d) and wild-type (+/+) mouse embryos in segregating litters is described, based on the total number of circulating erythrocytes in a unit volume of embryonic blood and the relative proportion of nonnucleated vs. nucleated red blood cells. Evidence is presented that from days 13–17 of gestation, S1/S1^d embryos have many fewer fetal liver derived nonnucleated erythrocytes whereas the number of yolk sac-derived nucleated red blood cells is similar between S1/S1^d and +/+. Erythroid precursor cells at various stages of maturation in mutant fetal livers are studied by light and electron microscopy, and their fine structure is found to be identical to those present in normal embryos. The number of hemoglobin-containing mature erythroblasts in mutant fetal livers is far fewer than that of the normal, whereas the number of immature erythroid precursors present in a unit area of fetal liver is not significantly different between S1/S1^d and +/+. It is suggested that the mutant S1 gene product(s) interferes with or fails to support the differentiation of immature erythroid precursors into hemoglobin synthesizing cells.     

Prophylactic Effect of Enterococcus faecalis FK-23 Preparation on Experimental Candidiasis in Mice

The prophylactic effects of heat-killed cells of Enterococcus faecalis FK-23 (FK-23 preparation) on experimental candidiasis were investigated in normal and leukopenic mice. In cyclophosphamide-induced leukopenic mice, oral or intraperitoneal administration of the FK-23 preparation at a daily dose of 1.25 or 5 mg/mouse for 3 consecutive days prior to Candida albicans infection significantly prolonged survival periods of the infected mice, and decreased viable counts of C. albicans recovered from their kidneys. In normal mice, the FK-23 preparation administered at dosages ranging from 0.63 to 10 mg/mouse/day for 3 consecutive days was ineffective, while in leukopenic mice, the FK-23 administered orally caused a facilitated recovery in the number of white blood cells including neutrophils. Furthermore, intraperitoneal administration of the FK-23 preparation into mice augmented the anti-Candida activity of immunocompromised peritoneal exudate cells obtained from the animals. These results suggested the potential usefulness of the FK-23 preparation as a prophylactic agent for the management of patients with opportunistic fungal infections.