Development and characterization of microsatellite markers for molecular genetic diversity in Atrina pectinata.

The pen shell, Atrina pectinata, is an economically valuable species that is widely distributed along the coastal waters of temperate and tropical areas, mainly growing in the Indian and Pacific Oceans. Eight novel microsatellite loci from the genome of A. pectinata were developed using fast isolation by amplified fragment length polymorphism of sequence containing repeats. The loci were screened in 30 wild individuals. The results showed that the number of alleles per locus and the polymorphism information content ranged from 2-6 and from 0.233-0.447, respectively. Observed and expected heterozygosities varied from 0.2069-0.7931 and 0.1887-0.5124, respectively. No significant deviations from Hardy-Weinberg equilibrium were detected. These microsatellite loci will be useful for further population studies of genetic diversity, population structure assessment, and conservation of A. pectinata.

Characterization and sequence analysis of the novel HLA-Cw*040105 allele in a Chinese Uygur individual.

A novel human leukocyte antigen (HLA) allele, HLA-Cw*040105, was identified in a Chinese Uygur individual. It differs from the closest allele Cw*04010101 by four nucleotides at position nt 60 (C>T) in exon 1, genomic nt 477 (A>T) in intron 2, nt 835(T>C), and nt 850 (C>T) in exon 3.

A novel HLA-C allele, Cw*0119, isolated from the Han Chinese.

A novel HLA-C allele, Cw*0119, which was identified from an individual of the Han Chinese, differs from Cw*010201 at codon 76 (AGC-->TGC) and codon 178 (CTG-->CCG).

Characterization of a novel DRB1*14 allele (DRB1*1449) in the Han-Chinese population.

We report here a novel human leukocyte antigen (HLA) allele, DRB1*1449, in the Han-Chinese population. The nature of the new allele was confirmed by the sequencing-based typing (SBT) method. Genomic DNA and six subclones containing DNA fragment of DRB1 exon 2 were sequenced in both forward and reverse directions. The exon 2 nucleotide sequence of DRB1*1449 is closely related to DRB1*1432 allele based on sequence homology. It has four nucleotide (nt) substitutions at positions 71, 196, 244 and 245 in exon 2, which lead to changes of amino acid sequences. The serological assignment of DRB1*1449 is DR14 based on the serological HLA typing result. This novel allele might be a result of recombination between DRB1*1402 and DRB1*140101 like alleles, which are very common among Chinese population.

A novel HLA allele, DRB1*1609, identified in the Chinese Han population*.

A novel human leucocyte antigen-DRB1*16 (HLA-DRB1*16) allele (DRB1*1609) has been identified by sequencing-based typing (SBT) in Chinese Han population. This new allele has identical nucleotide sequence to DRB1*160101 in exon 2, except for a single-nucleotide substitution from A to T at position 127. This change leads to an amino acid change from tyrosine to phenylalanin at residue 47 (Y47F). SBT was performed for cloned DRB1*16-specific polymerase chain reaction fragment. The serological phenotype of DRB1*1609 is equivalent to DR16 antigen.

A novel HLA-DRB1*15 allelic sequence isolated from the Han population of Guangdong, China.

A novel DRB1*15 allele, DRB1*1516, has been identified in a Guangdong Han individual. Its sequence was confirmed by sequencing of polymerase chain reaction products and clones. This allele differed by one nucleotide from DRB1*150101 at position 220 (G-->A), resulting in an amino acid substitution from Gly to Arg at codon 45.

Apoptosis induced by isoliquiritigenin in human gastric cancer MGC-803 cells.

Isoliquiritigenin, which is possibly a principal anti-tumor constituent of licorice, a traditional Chinese herb, was examined for apoptosis-inducing activity in human gastric cancer MGC-803 cells. Typical morphological and biochemical features of apoptosis including cell shrinkage, chromatin condensation, DNA ladder formation, and appearance of apoptotic peaks (subG(1)) were observed in MGC-803 cells with isoliquiritigenin treatment. Using Fluo-3 and Rh123 as fluorescent probes, respectively, it was found that the intracellular free calcium concentration increased and the mitochondrial transmembrane potential (Deltapsi(m)) decreased in a dose-dependent manner in apoptotic cells. These results suggest that isoliquiritigenin induced apoptosis of MGC-803 cells through calcium- and Deltapsi(m)-dependent pathways, indicating that it is potentially useful as a natural anti-cancer agent.

Induction of human tumor-loaded dendritic cells.

A preferred anti-cancer vaccine would be tumor-specific, simple to rapidly construct and safe to administer. It would permit immunization against a spectrum of the tumor's distinctive antigens, without requiring their prior identification. Toward these goals, we describe a modification of standard extracorporeal photopheresis (ECP) which initiates, within a single day, both monocyte-to-dendritic cell (DC) differentiation and malignant cell apoptosis. The transition of monocytes to immature DCs was identified by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were avidly phagocytic and engulfed apoptotic malignant T cells. Differentiating DCs were capable of stimulating significant proliferation of normal alloreactive lymphocyte responders, indicting increased expression of membrane MHC class II molecules. This approach provides a clinically practical means of developing tumor-loaded cells that have initiated the transition to DCs without the requirement of exogenous cytokines, excessive cellular manipulation or isolation. Construction of DC vaccines using this methodology can be generalized to other diseases and may offer a novel approach for improved cancer immunotherapy.

[Analysis of HLA-DQB1 polymorphism by PCR-SSO in Yichu of Yunnan Province].

HLA-DQB1 genes from 76 individuals of Yichu ethnic group in Yunnan Province were investigated, using PCR-SSO genotyping method. Of the 38 DQB1 alleles detected, DQB1 * 0301 (gene frequency: 36.18%-36.84%) was the most common gene. The frequencies of DQB1 * 0502(10.53%-11.18%), DQB1 * 0401 (9.21%), DQB1 * 0302(8.55%-9.21%), DQB1 * 0601(7.89%), DQB1 * 05031(6.58%), and DQB1 * 03032(5.92%-6.58%) are more than 5%. While DQB1 * 0504, DQB1 * 0604, DQB1 * 06052, DQB1 * 0606, DQB1 * 0607, DQB1 * 0608, DQB1 * 06112, DQB1 * 0613, DQB1 * 0615, DQB1 * 0203, DQB1 * 0305, DQB1 * 0306, DQB1 * 0307, and DQB1 * 0308 were not observed. Comparison of HLA-DQB1 allele frequencies of Yichu with those of 13 other Chinese ethnic groups showed some significant differences, suggesting Yichu is unique in the distribution of HLA alleles.

[Mitochondrial permeability transition pore regulates the apoptosis in MGC-803 induced by the extract of glycyrrhiza uralensis Fisch].

In our previous studies, we have discovered that the extract of glycyrrhiza uralensis Fisch (EGUF) can induce obvious apoptosis in gastric cancer cell Line MGC-803. Here, further investigation was carried on about the time-lapse changes of mitochondria transmembrane potential, intracellular free calcium ions, DNA electrophoresis, plasma membrane permeability and chromatin condensation during the apoptotic process of MGC-803 induced by EGUF and the influences of MPT-specific inhibitor Cyclosporin A(CsA) on these changes. Enhancement of plasma membrane permeability with PI staining, increase of intracellular free calcium ion and decrease of mitochondria transmembrane potential are early events in apoptotic cascades, prior to the appearances of apoptotic peak, chromatin condensation and DNA ladder. CsA significantly inhibited enhancement of plasma membrane permeability, change of intracellular free calcium ions and decrease of mitochondria transmembrane potential, also greatly delayed the progress of apoptosis. Thus, our results suggest that calcium and CsA-sensitive MPT is involved in the apoptosis of MGC-803 induced by EGUF.

[HLA-DRB1 gene polymorphism of Naxi ethnic group of Yunnan Province, China and its ethnological evolution analysis].

The HLA-DRB1 gene polymorphism of Naxi ethnic group of Yunnan Province, China was investigated for the first time using high resolution PCR-SBT method, which is based on sequences of HLA-DRB1 Intron 1 and Intron 2 with our improvement. From 60 individuals of Naxi 37 DRB1 alleles were detected. The distribution of allele frequencies is evenly spread for most of alleles detected in Naxi. However, the gene frequency for HLA-DRB1 * 12021 is 17.50%. The other common alleles (> 5%) were HLA-DRB1 * 1404(7.50%), 1504(5.83%), 04051(5.83%), 08032(5.83%), 09012(5%), 03011(5%), and they covered 35% of the total alleles detected from Naxi, and 52.49% after adding the frequency of HLA-DRB1 * 12021. HLA-DRB1 * 0305, 0438, 1123, 1132, 1310, 0812 were detected in Chinese for the first time, and were very rare in other ethnic groups worldwide. Using the HLA-DRB1 gene frequencies of various ethnic groups, we constructed the phylogenetic tree by Fitch-Margoliash and Least-Squares Distance Methods and Cavalli-Sforza's chord measure of genetic distance. In our dendrogram, the South China populations were clustered together, and Siberian, Japanese and Minority of North China were clustered together, in which two big clusters we thought represented the ancient Southern and Northern Mongolian. Our results showed Naxi was clustered with Lahu and Yao ethnic groups, which were in the cluster of South China population. Obviously Naxi belongs to the South China ethnic groups and can't trace its origin from northern ethnic groups with the HLA-DRB1 genetic data. But based on Naxi's historical folklore, it was down to Southern China from Northwest China. The preliminary analysis about its origin conundrum was discussed in this paper based on our genetic data in relationship with its history and ethnology studies.

Ultrastructural cytochemical study of enzymes expressed by signet ring cells in gastric cancer.

AIM: To study the ultrastructural localization of five marker enzymes (ALPase, ACPase, G6Pase, TPPase and CCOase) in gastric cancer signet ring cells to demonstrate their biologic behaviors. METHODS: Five marker enzymes were examined in signet ring cells of seven gastric cancer patients by ultrastructural enzyme cytochemical techniques. RESULTS: The number of corresponding organelles and the activities of marker enzymes, especially ACPase and TPPase, increased, leading to stronger mucus synthesis, secretion and digestion in gastric cancer signet ring cells. There was a lack of collagenous fibers in the stroma around the cancer nests. CONCLUSION: Signet ring cell carcinoma is very invasive with metastasis rates due to the secretion of proteolytic enzymes.

[Generation and immunochemical characterization of an anti-egg monoclonal antibody specific to Schistosoma japonicum].

Monoclonal antibodies (McAbs) were generated from mice immunized with soluble egg antigen (SEA) of Schistosoma japonicum. Five of which were specific to SEA of S. japonicum. The isotype of these McAbs was IgG1. Immunoblotting showed that the approximate molecule weight of the antigens recognized by the McAbs were 22 kDa, 116 kDa and greater than 200 kDa respectively. At least 3 isomorphs of the 22 kDa antigen recognized by 1E1 were found at pI 4.6-6 with 2-D Western blot. Moreover, immunoprecipitation using 125I-labeled S. japonicum SEA demonstrated that only one band corresponding to 30 kDa was recognized by 1E1.