Molecular cloning and single nucleotide polymorphism analysis of IGF2a genes in the common carp (Cyprinus carpio).

We studied whether two IGF2 transcripts in common carp are similar to those found in zebrafish. The full-length IGF2a cDNA contains a 5'-terminal untranslated region (UTR) of 105 bp, a 3'-terminal UTR of 1358 bp and an open reading frame of 612 bp, which encodes a 206-amino acid protein. A 6614-bp full-length IGF2a DNA molecule, including the 5'-flanking region, was isolated. Genomic DNA structure analysis revealed that the IGF2a gene contains four exons and three introns. Bioinformatics analysis indicated that the proteins encoded by IGF2a genes in common carp have one signal peptide and one apparent transmembrane region. Bootstrapping was performed 1000 times to obtain support values for each branch. The common carp IGF2a were clustered in one group, while the outgroup (common carp IGF1) clustered in another group. We identified two new single nucleotide polymorphisms in intron 2 of the gene. One polymorphism, A/N, can be found only in the Huanghe carp. The other polymorphism, C/N, can be found in both male Huanghe carp × female Heilongjiang carp and male Huanghe carp × female Jian carp. The second polymorphism, C/N, is primarily transferred from the male and may be related to heterosis.

Hydrothermal synthesis of Zn2SnO4 as anode materials for Li-ion battery.

Spinel Zn2SnO4 particles with the cubic shape are prepared via a hydrothermal reaction under mild conditions. The hydrothermal conditions, such as alkaline concentration, reaction temperature, and duration time, have an important influence on the product structure and the performance of the electrode prepared with the product. The optimized product is cube-shaped Zn2SnO4 crystalline, which is prepared with 0.4 M of NaOH solution at 200 degrees C for 24 h. These cube-shaped Zn2SnO4 particles with the spinel structure exhibit a large electrochemical capacity of 988 mA h/g and a relatively good capacity retention as anode materials for Li-ion battery. The structures of the as-prepared product and specimens taken from the electrodes after charging-discharging cycles are analyzed by X-ray diffraction, scanning electron microscopy, and transition electron microscopy techniques. In particular, it is found for the first time that the spinel Zn2SnO4 structure exists to a great extent after the first cycle and contributes to the extremely high reversible capacity during the following cycles.

Separation of Chinese Leishmania isolates into five genotypes by kinetoplast and chromosomal DNA heterogeneity.

Leishmaniasis remains endemic in China, especially in the west and northwest frontier regions in central Asia. Epidemic outbreaks of both visceral and cutaneous forms of the disease have become a serious concern in view of such events occurring in neighboring countries. In the present study, we have begun to characterize available parasites as an initial step in understanding the epidemiology of leishmaniasis in central Asia. Nineteen Leishmania isolates collected since the 1950s from epidemiologically different foci in China were separated into five genotypes (Groups I-V) based on their polymorphisms in both kinetoplast (kDNA) and nuclear (nDNA) DNAs. Both kDNA and nDNA are conserved in Group I, which consists of six isolates, i.e., five cases of human kala-azar and one case of canine leishmaniasis isolated from three distant foci more than 30 years apart. In contrast, both kDNA and nDNA are heterogeneous in Group II, consisting of 10 isolates scattered in the plain area from the eastern coast to the western desert. This group includes five kala-azar cases, one post-kala-azar dermal leishmaniasis case, two sand fly isolates, and two canine isolates. The remaining three groups (III-V), two from great gerbils (Rhombomys opimus) and one from a kala-azar case, differ among themselves and from the aforementioned groups. Groups I, II/III, IV, and V contain isolates that have been recognized epidemiologically or typed isoenzymatically as L. donovani s.l., L. infantum s.l., L. turinica, and L. gerbilli, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Serodiagnosis of Asian leishmaniasis with a recombinant antigen from the repetitive domain of a Leishmania kinesin.

rK39 is a recombinant product of the 39 amino acid repeats found in a kinesin-like gene of visceral Leishmania spp. This and other antigens were compared for immunodiagnostic potential by enzyme-linked immunosorbent assay with sera from confirmed cases of Asian cutaneous and visceral leishmaniasis. In preliminary trials, rK39 proved superior to 2 purified Leishmania antigens, a cytosolic protein (p36) and a membrane protein (gp63), for immunodiagnosis of visceral leishmaniasis. Of the 53 visceral cases from China and Pakistan assayed, 52 were seropositive (98%) at a 10(-1) dilution with 36 ng of rK39. End point titrations of 27 highly positive samples yielded anti-rK39 antibody titres ranging from c. 10(-3) to beyond 10(-4). Antigen titrations with one positive serum further revealed that rK39 was 25-fold more sensitive than Leishmania whole cell soluble lysates. 31 cutaneous leishmaniasis cases from Turkey assayed for anti-rK39 antibody gave reactions ranging from negative or marginally positive to positive. In Brazil, all cutaneous and mucocutaneous leishmaniasis cases gave negative results in this assay.

[The extent and pattern of infiltration along the bronchial wall in central type lung cancer].

Gross and microscopic study was made in fifty specimens emphasizing on the bronchial wall of lung cancer. The extent and pattern of infiltration along the proximal bronchial wall in the central type lung cancer were observed. The cancerous tissue extended proximally from less than 1.2 cm in the majority of cases, to a maximum of 1.6 cm from the cancer base. The lesions can be divided into three gross types: infiltrating, polypoid and nodular. In the infiltrating type, the cancerous tissue spread along the bronchial wall far and may involve multiple layers in the wall. In the polypoid type, the infiltration did not reach far as compared with the other types. None in the polypoid type was over 0.4 cm. The nodular type comprising 54% (27/50), was more common than the others. In order to improve the operative result, infiltrating pattern and distance along the bronchial wall are important references for resection of central type lung cancer.

[Application of monoclonal antibodies against Leishmania donovani, II. Detection of circulating antigen in sera of visceral leishmaniasis before and after treatment].

Monoclonal antibodies L12F7 against the target antigen of L. donovani promastigotes were used for the detection of circulating antigen (CAg) from sera of visceral leishmaniasis patients. The results showed that of 118 serum samples from visceral leishmaniasis tested, 105 were positive (88.9%). 55 normal serum samples were negative. No cross reaction with sera from patients with vivax malaria, schistosomiasis, leprosy and brucellosis, was found. The authors suggested that this assay may be used as a sensitive and new serodiagnostic test for detecting existing infection of visceral leishmaniasis for epidemiological survey and for assessment of cure after effective treatment.

[Scanning electron microscopic study on the influence of monoclonal antibody on the invasion of macrophage by Leishmania parasites].

The interaction of promastigotes of Leishmania with mouse peritoneal macrophage in vitro was studied by scanning electron microscopy. The results showed that when promastigotes had been incubated with 1/1,000 McAb for 1 h, morphological changes could be seen in most promastigotes, some promastigotes were lysed and some promastigotes although adhered to the macrophage, but no penetration could be seen. When 1/1,000 McAb had been inactivated at 56 degrees C for 1 h and then incubated with promastigotes, scanning electron microscopic examination showed the promastigotes closely adhered to the surface of macrophage with their anterior end "buried" within macrophage. The results of the present study suggested that the mechanism of promastigote-macrophage adhesion was ligand-receptor binding interaction (Figs. 1-11).

Leishmania mexicana: the circular DNA 1 (CD1) element contains genes encoding nucleotide-binding protein.

OBJECTIVE: To determine the nucleotide sequence of cloned CD1 fragments from Leishmania mexicana and find ORFs predicted to have protein coding function. METHODS: CD1 element was separated by CHEF and recovered by agarase, and the digested CD1 fragments were cloned into pZero vector. Nucleotide sequences were determined by the dideoxy chain termination method with the automatic sequencing system ALF using the M13 universal primers. Sequences were analyzed using GCG-PCGENE computer programs. RESULTS: The sequence with 4,385 nucleotides was determined and two ORFs were considered to have protein coding function (encoding nucleotide-binding protein). CONCLUSION: Genes encoding nucleotide-binding protein were identified from the amplified CD1 element of Leishmania mexicana.

[Identification of L. donovani promastigotes from Phlebotomus by monoclonal antibody].

It has been reported by the authors that monoclonal antibody L12G9 produced from target antigens of L. donovani promastigotes, was very useful for detecting promastigotes from artificially infected sandflies. In the present study, detection of promastigotes from artificially infected sandflies by McAb showed that the positive rate correlated with the infection duration of sandflies. 4 days after feeding on infected Chinese hamsters, the sandflies were lightly infected with L. donovani promastigotes with a positive rate of 15.9%, but 10 days later, the sandflies were heavily infected, the positive rate being 100%. Observation has been made on the relationship between the number of promastigotes and mouse blood dilution. The results showed that satisfactory results could be obtained by using monoclonal antibodies in the detection of L. donovani, the number of promastigotes should be over 1 x 10(7)/ml, and the blood meals in sandflies completely digested. If very few promastigotes were present in naturally infected sandflies before identification by monoclonal antibody, the parasites must be grown in NNN medium. Positive result could then be obtained.

[ELIB of specific antigens in in vitro cultivated germinal cells of Echinococcus granulosus].

Specific antigens are detected in cyst fluid and cyst wall of Echinococcus granulosus, as well as germinal cells cultivated in vitro by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme linked immunotransfer blot technique (ELIB), using the sera from mice infected with protoscoleces of E. granulosus for at least 10 months. A specific reaction band of 52 kDa or 38 kDa was detected in soluble protein of germinal cells and cyst fluid, respectively, but these two reaction bands were present concurrently in the cyst wall. The sera from 7 normal controls, 7 hydatidosis patients and 3 cysticercosis patients were used to study the specific antigen from the germinal cells. The results noted that the reaction band of 52 kDa was seen in all the sera from hydatidosis patients, while the sera from normal human controls and cysticercosis patients showed no reaction band.

Rapid screening with a recombinant antigen (rK39) for diagnosis of visceral leishmaniasis using dipstick.

OBJECTIVE: To evaluate the diagnostic value of the recombinant antigen of 39 amino acid repeats encoded by a kinesin-like gene of Leishmania changasi (rK39) in serodiagnosis of visceral leishmaniasis (VL). METHODS: In Kashi, Xinjiang, 13 VL patients with splenomegaly and bone marrow aspirate culture positive were subjected to dipstick assay. A drop of whole blood or serum from patient was placed at the absorbing pad at the bottom of the dipstick. Flooding of the bottom protein with buffer allows serum proteins to migrate upwards, producing the positive band and Western blot analysis of rK39 subsequently performed with the sera collected. RESULTS: The end-point titers of anti-rK39 antibodies of these sera were determined by ELISA and found to fall within the range of 10(-2) to 10(-4), which were consistent with the intensity of their reaction with rK39 in dipstick assay. The positive sera could also recognize the specific rK39 band as analyzed by Western blot analysis. CONCLUSION: The rK39 dipstick assay is more rapid, specific, sensitive and less invasive than the conventional methods of diagnosis for VL in the areas of low endemicity.

Discovery and study of Leishmania turanica for the first time in China.

Reported are the results of multidisciplinary studies on Leishmania turanica, which was isolated from the auricular tissues of naturally infected great gerbils in Xinjiang, China. Discussed are the biology of the parasite, its molecular biology, its pathogenicity in rodents and humans and its vectors. This was the first time that L. turanica had been reported in China. L. turanica is highly pathogenic in BALB/c mice, with the resulting systemic infection being lethal, and it causes dermal lesions in Meriones unguiculatus. L. turanica parasitizes the macrophages in the interstitium of the testes of Cricetulus barabensis, and entirely destroys the Leydig's cells of severely infected animals. Inoculation of L. turanica can induce simian and human cutaneous leishmaniasis. The cell membrane and flagella of the promastigotes of L. turanica have rather active ACPase. The major vectors of L. turanica were Phlebotomus mongolensis and P. andrejevi.

Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene.

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.