Macrophage migration inhibitory factor (MIF) is an important cytokine related to host defenses and autoimmune diseases. Here, we reported two full-length cDNA clones isolated from Chinese amphioxus (Branchiostoma belcheri tsingtaunese). Amino acid sequences analysis and structure prediction of these two molecules, called Bbt-MIF-I and Bbt-MIF-II, respectively, indicated that several conservative domains existed in the two amphioxus MIFs and their sequences were highly homologous to their counterparts of other species. Intriguingly, the Bbt-MIFs gene is present in multi-copy per haploid genome, which is very unusual compared with vertebrate's MIF gene given the known genome duplication theory. The genomic copy number, expression pattern of MIF gene and phylogenetic analysis of MIF proteins all suggested that a leap forward happened for MIF gene during the evolution from invertebrate to vertebrate. Considering the crucial role of MIF in innate immunity, MIF might serve as one of key molecular markers of evolution of immune system.

A novel coronavirus called the severe acute respiratory syndrome (SARS)-associated coronavirus (CoV) has been identified as the causal agent of SARS. 1-3 To understand the humoral immunity to this virus, we studied the profile of IgM and IgG antibody responses to SARSCoV. IgM and IgG antibodies were analyzed by an indirect enzyme-linked immunosorbent assay in 20 patients with SARS from week 1 of their illness to week 12 and in 103 healthy contacts. All 20 patients tested negative for IgM and IgG at week 1 after the onset of symptoms. Of these patients, 16 tested positive for IgM and 17 tested positive for IgG at week 2 (Fig. 1). All 20 patients were IgG-positive after week 3 and continued to have high levels of IgG up to three months after the onset of symptoms. The IgG titers were low at the beginning of week 2 (mean, 1:40, with the cutoff for a positive result being 1:10), increased to an average of 1:256 at week 3, and peaked at 1:640 at week 12. The IgM titers peaked during the acute or early convalescent phase and they declined with IgM disappearing by the end of week 12. All 103 healthy contacts tested negative for IgM and IgG. Our results suggest that 100 percent of patients had antibody responses to SARS-CoV during the convalescent phase. The SARS-specific IgG antibody persisted for a long time, but the SARS-specific IgM remained measurable for a much shorter period, suggesting that IgG antibody to SARSCoV may represent the primary humoral immune response protecting patients against SARS. The profile of antibodies against SARS-CoV was consistent with common findings with regard to acute viral infectious diseases such as hepatitis A. 4 The profile of anti-SARS antibodies may be helpful in the diagnosis and in epidemiologic surveys. The presence of high titers of IgG antibody to SARS-CoV in the patients at the convalescent stage also suggests that a live attenuated or inactivated vaccine could be developed for active immunization and a concentrated human SARS-specific IgG antibody for passive immunization could be developed for the treatment of SARS.

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser97-Trp98, which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately Mr=12,800 displayed an obvious inhibition of papain (Ki value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltagedependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.

A cDNA library of male Chinese seahorse (Hippocampus kuda Bleeker) was constructed to investigate the molecular pro?le of seahorse as one of the most famous traditional Chinese medicine materials,and to reveal immunological and physiological mechanisms of seahorse as one of the most primitive vertebrates at molecular level. A total of 3372 expressed sequence tags (ESTs) consisting of 1911 unique genes (345 clusters and 1566 singletons) were examined in the present study. Identification of the genes related to immune system, paternal brooding and physiological regulation provides not only valuable insights into the molecular mechanism of immune system in teleost fish but also plausible explanations for pharmacological activities of Chinese seahorse. Furthermore, the occurrence of high prevalent C-type lectins suggested that a lectin-complement pathway might exert a more dominant function in the innate immune system of teleost than mammal. Carbohydrate recognition domain (CRD) without a collagen-like region in the lectins of seahorse was likely an ancient characteristic of lectins similar to invertebrates.

Exon 2 and intron 2 of the HLA DQB1 gene from 20 individuals were cloned and sequenced and eight alleles were obtained. Based on our analysis, the nucleotide diversity of the 5' end of intron 2 was higher than the synonymous nucleotide diversity of exon 2, which may be due to the lower GC content and the'hitch-hiking effect'. In contrast, the opposite phenomenon was observed for the 3' end of intron 2, which may be the result of the recombination between the 3' end and 5' end of intron 2 and the subsequent genetic drift. The results indicated that different regions of intron 2 in the DQB1 gene had different evolutionary patterns.

In the present study, DNA typing for HLA-DRB1, DQB1 and DPB1 was performed using polymerase chain reaction-sequencing based typing (PCR-SBT) method in 144 random selected Jing ethnic individuals inhabiting in South China. Allele frequencies and two-locus haplotypes (DRB1-DQB1) were statistically analyzed and 20 DPB1 alleles, 27 DRB1 and 20 DQB1 were detected. The most frequent DPB1 allele was DPB1*0501 with the percentage of 36.9% followed by DPB1*1301 (15.7%), DPB1*0401 (11.0%) and DPB1*020102 (9.8%). Among the 27 detected DRB1 alleles, DRB1*120201 (13.8%) was most commonly observed followed by DRB1*150201, *030101 and *090102 alleles with the frequencies of 9.4%, 9.1% and 8.3%, respectively. Among the 20 detected DQB1 alleles the most predominant one was DQB1*030101/0309 (19.9%). DQB1*050201 (19.1%), DQB1*0201/0202 (16.1%) and DQB1*050101 (12.3%) were also frequently observed in Jing population. Statistical analysis of two-locus haplotypes showed that DRB1*120201-DQB1*030101/DRB1*120201-DQB1*0309 (HF=9.4%, D=6.65

Amphioxus, a cephalochordate, is the closest living relative to the vertebrates. In order to investigate the molecular mechanisms of the early embryogenesis of amphioxus, we constructed a neurula embryo cDNA library of Chinese amphioxus (Branchiostoma belcheri tsingtauense) and generated 5235 expressed sequenced tags in the present study. The initial ESTs consisted of 638 clusters and 1855 singletons, which revealed approximately 2493 unique genes in the data set. Of these sequences, 35.52% ESTs matched to known genes, 12.76% matched to other ESTs, and 51.71% had no match to any known sequences in GenBank. Interestingly we found homologous genes related to neural development and human disease. Bioinformatic analysis showed the direct evidence that the gene homologue found only in vertebrates in previous studies also exists in the amphioxus genome. This study provides a preliminary view of the gene information involved in the development of neurula embryos of Chinese amphioxus and helps our understanding of vertebrate evolution at gene level.

Acutiaporberine is a novel ether-linked bisalkaloid isolated from the traditional Chinese medicinal herb Thalictrum acutifolium (Hand.-Mazz.) Boivin (TAB). The present study demonstrates for the first time, by means of nuclear staining, DNA agarose gel electrophoresis, and flow cytometry, that acutiaporberine induces apoptosis in PLA-801 cells, a cultured human non-small cell lung cancer (NSCLC) cell line. An immunohistochemical assay and western immunoblot analysis showed down-regulation of the bcl-2 gene and upregulation of the bax and c-myc genes in the acutiaporberine-treated cells. The observations also indicate that acutiaporberine induces apoptosis of PLA-801 cells in a concentration-and time-dependent manner. These results suggest that acutiaporberine may be a potential, natural apoptosis-inducing agent for NSCLC.