Nutrition and Flavor Evaluation of Amino Acids in Guangyuan Grey Chicken of Different Ages, Genders and Meat Cuts.

The composition and content of amino acids in foodstuffs have a vital impact on the nutritional value and taste. With the aim of understanding the nutrition and flavor of Guangyuan grey chicken, the composition and content of amino acids in the pectoralis and thigh muscle of chickens at the age of 90 d, 120 d and 150 d were determine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an amino acid analyzer. A total of 17 amino acids were detected both in pectoralis and thigh muscle via the amino acid analyzer, of which the content of glutamate was the highest. Additionally, 21 deproteinized free amino acids were detected via LC-MS/MS. Among all samples, the content of glutamine in thigh muscle was the highest. The content of histidine in the pectoralis was the highest. In terms of the flavor amino acids (FAAs), the umami-taste and sweet-taste amino acids were higher in the thigh muscle of 120 d male chicken. From the perspective of protein nutrition, the essential amino acid was higher in pectoral muscle, and the composition was better. The results of the amino acid score showed that the content of leucine and valine were inadequate in Guangyuan grey chicken. Collectively, the content of amino acid in Guangyuan grey chicken was affected by age, gender and meat cut. This study confirms that meat of chicken in different ages, genders, and cuts presents different nutritional values and flavors owing to the variation of amino acids content.

Methioninase Gene Therapy.

Recombinant methioninase (rMETase) derived from Pseudomonas putida targets the elevated methionine (MET) requirement of cancer cells (methionine dependence) and has shown efficacy against a variety of cancer types in mouse models. To enhance the efficacy of rMETase, we constructed the pLGFP-METSN retrovirus encoding the P. putida methioninase (METase) gene fused with the green fluorescent protein (GFP) gene. pLGFP-METSN or control vector pLGFPSN was introduced into the human lung cancer cell line H460. The retrovirus-mediated METase gene transfer decreased the intracellular MET level of the cancer cells and consequently enhanced the efficacy of treatment with the rMETase protein. The rMETase gene was introduced into an adenovirus. rAd-METase transduction of human OVACAR-8 ovarian cancer cells and human fibrosarcoma HT1080 cells in vitro and in vivo resulted in high levels of METase expression up to 10% or more of the total protein of the cells, depending on the multiplicity of infection. The combination of rAd-METase and rMETase was synergistic to kill these cells. Normal fibroblasts, on the other hand, appeared relatively resistant to the METase gene in the presence of rMETase. Adenoviral METase-transduced cancer cells were used in combination with selenomethionine, releasing highly toxic methylselenol, which killed both the cancer cells containing the METase gene and bystanders. Methylselenol damaged the mitochondria via oxidative stress and caused cytochrome c release into the cytosol, thereby activating the caspase cascade and cancer-cell apoptosis. Adenoviral METase-gene/SeMET treatment also inhibited tumor growth in rodents and significantly prolonged their survival. AdMETase/SeMET therapy was effective against Bcl-2-overproducing A549 lung cancer cells, which were resistant to staurosporine-induced apoptosis, with a strong bystander effect. The combination of Ad-METase/SeMET and doxorubicin (DOX) delayed the growth of the H460 human lung cancer, growing subcutaneously in nude mice. These results demonstrate the potential of methionine restriction (MR) for cancer treatment.

Broad selective efficacy of recombinant methioninase and polyethylene glycol-modified recombinant methioninase on cancer cells In Vitro.

Elevated dependence on methionine of cancer cells is a cancer-specific metabolic defect. Recombinant L-methionine alpha,gamma-lyase (rMETase), an L-methionine depleting enzyme cloned from Pseudomonas putida, was shown to have efficacy on a broad series of cancer cell lines. Twenty-one different human tumor cell lines (4 lung, 4 colon, 4 kidney, 4 melanoma, 3 CNS, and 2 prostate) from the NCI Human Tumor Cell Line Screen and 7 human normal cell strains were treated with rMETase in vitro. We showed that rMETase had a mean IC(50) (units rMETase/ml) for the following cancer cell types: renal, 0.07; colon, 0.04; lung, 0.12; prostate, 0.01; melanoma, 0.19; and CNS, 0.195, which was approximately one order of magnitude lower than that for normal cell strains: skin fibroblasts, 4; aortic smooth muscle cells, 0.88; aortic endothelial cells, 0.8; keratinocytes, 0.75; and bronchial epithelial cells, 0.75. rMETase was also conjugated with polyethylene glycol (PEG). PEG-rMETase also had high cancer cell-killing activity. In vitro studies, animal studies and clinical trials have now shown that methionine restriction is an effective anticancer strategy. Cells from many different types of cancer are methionine dependent. The most effective strategy to deplete methionine is with the use of rMETase. PEG-rMETase offers additional advantages of increasing the circulating half-life and reducing the immunogenicity of rMETase which is a bacterial protein. The results of the current study demonstrate the broad clinical potential for rMETase and PEG-rMETase for cancer treatment.

Broad selective efficacy of rMETase and PEG-rMETase on cancer cells in vitro.

The elevated dependence on methionine of tumor cells is a cancer-specific metabolic defect. In current studies, the recombinant L-methionine alpha,gamma-lyase (rMETase), an L-methionine depleting enzyme cloned from Pseudomonas putida, was shown to have efficacy in a broad series of cancer cell lines. Twenty-one different human tumor cell lines (4 lung, 4 colon, 4 kidney, 4 melanoma, 3 CNS, and 2 prostate) from the NCI Human Tumor Cell Line Screen and 7 human normal cell strains were treated with rMETase in vitro. We showed that rMETase had mean IC(50) (units rMETase/ml) for the following cancer cell types: renal cancer, 0.07; colon cancer, 0.04; lung cancer, 0.12; prostate cancer, 0.01; melanoma, 0.19; and CNS cancer, 0.195, which was approximately one order of magnitude lower than that for normal cell strains: skin fibroblasts, 4; aortic smooth muscle cells, 0.88; aortic endothelial cells, 0.8; keratinocytes, 0.75, and bronchial epithelial cells, 0.75. rMETase was also conjugated with polyethylene lycol (PEG). PEG-rMETase also had high cell-kill activity. In vitro studies, animal studies and clinical trials have now shown that methionine restriction is an effective anticancer strategy. Cells from many different types of cancers are methionine dependent. The most effective strategy to deplete methionine is with the use of rMETase. PEG-rMETase offeres additional advantages of increasing the circulating half-life and reducing the immunogenicity of rMETase which is a bacterial protein. The results of the current study demonstrate the broad clinical potential for rMETase and rMETase for cancer treatment.

Serratia nematodiphila sp. nov., associated symbiotically with the entomopathogenic nematode Heterorhabditidoides chongmingensis (Rhabditida: Rhabditidae).

A novel red-pigmented, Gram-negative, motile, fluorescent, rod-shaped strain, DZ0503SBS1(T), with a single lateral flagellum, was isolated from the intestine of the nematode Heterorhabditidoides chongmingensis. Comparative 16S rRNA gene sequence analysis indicated that the strain is a member of the genus Serratia, sharing highest sequence similarities with Serratia marcescens subsp. sakuensis JCM 11315(T) (99.8 %), S. marcescens subsp. marcescens DSM 30121(T) (99.5 %) and Serratia ureilytica LMG 22860(T) (98.3 %). Similarities between the rpoB gene sequence of strain DZ0503SBS1(T) and those of S. marcescens subsp. sakuensis JCM 11315(T), S. marcescens subsp. marcescens DSM 30121(T) and S. ureilytica LMG 22860(T) were 98.0, 97.4 and 98.3 %, respectively. DNA-DNA hybridization values of strain DZ0503SBS1(T) with S. marcescens subsp. sakuensis JCM 11315(T), S. marcescens subsp. marcescens DSM 30121(T) and S. ureilytica LMG 22860(T) were 68.2, 65.1 and 53.0 %, respectively. The major isoprenoid quinone of strain DZ0503SBS1(T) was Q-8 and the predominant fatty acids were C(16 : 0) (34.76 %), cyclo-C(17 : 0) (20.03 %) and cyclo-C(19 : 0)omega8c (17.24 %). The cyclo-C(19 : 0)omega8c content (17.24 %) was significantly different from those found in S. marcescens subsp. sakuensis JCM 11315(T) and S. marcescens subsp. marcescens DSM 30121(T). Some characteristics of strain DZ0503SBS1(T), i.e. fluorescence and its symbiotic association with nematodes, have not been reported previously in any species of the genus Serratia. Phenotypic and biochemical characteristics and molecular data show that strain DZ0503SBS1(T) represents a novel species, for which the name Serratia nematodiphila sp. nov. is proposed; the type strain is DZ0503SBS1(T) (=KCTC 22130(T) =CGMCC 1.6853(T)).

In vivo fluorescence of medullary carcinoma of the thyroid: a technology with potential to improve visualization of malignant tissue at surgical resection.

Medullary carcinoma of the thyroid requires aggressive treatment because of its potential to metastasize and because of the current limitations of preoperative localization and systemic therapy. If these tumors could be made to fluoresce in vivo with tagged fluorophore antibodies against tumor antigens, surgeons would be able to obtain additional information in the operating room to facilitate a more complete resection. Based on the success of our previous work in breast and colon cancer models, we conducted an animal study of in vivo tumor fluorescence of a human medullary thyroid cell line in which bright tumor fluorescence is visible during dissection. To accomplish this, we used an inexpensive and commercially available handheld, blue (470 nm), light-emitting diode flashlight and filtered goggles (520 nm). This procedure, which we call the fluorescent antibody-assisted surgical technique (FAAST), is easy to perform, requires no complex or expensive technical equipment, and has the potential to be applied to a wide variety of tumors. To the best of our knowledge, this is the first experiment of its kind to be reported in the literature.

Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes.

During a recent soil sample survey in Eastern China, a new entomopathogenic nematode species, collected from the Chongming Islands in the southern-eastern area of Shanghai, was discovered. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, and described as Heterorhabditidoides chongmingensis gen. nov., sp. nov., for that it shares more morphological characteristics with heterorhabditids than with steinernematids. For males, the papillae formula of bursa is 1, 2, 3, 3, with constant papillae number in the terminal group, stoma tubular-shaped and about 1.5 head width; cheilorhabdions cuticularized, esophageal collar present and long, median bulb present. For infective juveniles, EP=90 (80-105)microm, ES=104 (92-120)microm, tail length=111 (89-159)microm, and a=19.1 (15-21). The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of heterorhabditids and other rhabditids. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer (ITS) sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae.

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide.

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xylophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xylophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those from the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal, Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xylophilus originated in North America and that the B. xylophilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.

Cloning and functional characterization of 5'-upstream region of metallothionein-I gene from crucian carp (Carassius cuvieri).

Metallothioneins are low molecular weight, cysteine-rich, metal-binding proteins, which can be induced by heavy metal ions, cytokines, stress, and hormones. To investigate the roles of the main cis-acting elements involved in the inducible expression of metallothionein gene in fish, the 5'-upstream region of crucian carp (Carassius cuvieri) metallothionein-I gene had been cloned and analyzed after our previous work on metallothionein-II. In its upstream region, several putative cis-acting elements, including nine metal regulatory elements (MREs), one antioxidant response element, one E-box, and three interleukin-6 responsive elements, etc. were found. The nine metal regulatory elements are confined in less than 1000 bp from ATG start codon and organized into two clusters with different roles to the induction of the metallothionein-I expression. Deletion mutant assays demonstrated that both the distal and proximal clusters of metal regulatory elements contributed to the basal expression of the metallothionein-I, but only the proximal cluster was the chief contributor to the metal fold induction. In transient luciferase reporter assays, Zn2+ and Cd2+ served as much stronger inducers than Cu2+ to the metallothionein-I expression. H2O2 also could activate the metallothionein-I promoter about two-fold, which was mediated by the antioxidant response element (TGACAACGC, -437/-445). In conclusion, our studies demonstrate the roles of metal regulatory element and antioxidant response element in the induction of crucian carp metallothionein-I gene, and provide the regulatory mechanism for the use of fish metallothionein as a biomarker for monitoring of metal contamination in waters.

Tumor cells genetically labeled with GFP in the nucleus and RFP in the cytoplasm for imaging cellular dynamics.

Dual-color fluorescent cells with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm were genetically engineered. The dual-color cancer cells enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of a series of human and rodent cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Using the Olympus OV100 Whole-Mouse Imaging System, total sub-cellular dynamics can be visualized in the living dual-color cells in real time in the live mouse after cell injection. Highly elongated cancer cells and nuclei in narrow capillaries were visualized where both the nuclei and cytoplasm deform. Both cytoplasm and nuclei were visualized to undergo extreme deformation during extravasation with cytoplasmic processing exiting vessels first and nuclei following along these processes. The dual-color cells described here thus enable the sub-cellular dynamics of cancer cell trafficking to be imaged in the living animal.

Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system.

With the use of dual-color fluorescent cells and a highly sensitive whole-mouse imaging system with both macro-optics and micro-optics, we report here the development of subcellular real-time imaging of cancer cell trafficking in live mice. To observe cytoplasmic and nuclear dynamics in the living mouse, tumor cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 whole-mouse imaging system with a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive whole-mouse imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time. This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development.

Cloning of crucian carp (Carassius cuvieri) metallothionein-II gene and characterization of its gene promoter region.

The genomic DNA of crucian carp (Carassius cuvieri) metallothionein-II (ccMT-II), with its upstream region, was obtained. The sequence analysis of its upstream region revealed several putative cis-acting elements including seven metal regulatory elements (MREs), three activator protein 1 (AP1), two glucocorticoid response elements (GREs), etc. The seven MREs locate into two clusters, a distal cluster with four MREs within -800/-600bp from the translation start site and a proximal cluster with three MREs close to TATA box. In transient luciferase gene expression assays, both of the distal and proximal cluster MREs have significantly shown synergistic effects in the transcription of ccMT-II gene; the proximal cluster of MREs serves as the major elements in metal inducing activity; Zn(2+) and Cd(2+) served as much stronger inducers than Cu(2+) shown in ccMT-II expression. The two GRE homologous sequences in ccMT-II promoter showed not to be inductive in either HepG2 or HEK293.

Nested genes in the human genome.

Here we studied one special type of gene, i.e., the nested gene, in the human genome. We collected 373 reliably annotated nested genes. Two-thirds of them were on the strand opposite that of their host gene. About 58% coding nested gene pairs were conserved in mouse and some were even maintained in chicken and fish, while nested pseudogenes were poorly conserved. Ka/Ks analysis revealed that nested genes were under strong selection, although they did not demonstrate greater conservation than other genes. With microarray data we observed that two partners of one nested pair seemed to be expressed reciprocally. A significant proportion of nested genes were tissue-specifically expressed. Gene ontology analysis demonstrated that quite a number of nested genes participated in cellular signal transduction. Based on these observations, we think that nested genes are a group of genes with important physiological functions.

Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration.

The mechanism of cancer cell deformation and migration in narrow vessels is incompletely understood. In order to visualize the cytoplasmic and nuclear dynamics of cells migrating in capillaries, red fluorescent protein was expressed in the cytoplasm, and green fluorescent protein, linked to histone H2B, was expressed in the nucleus of cancer cells. Immediately after the cells were injected in the heart of nude mice, a skin flap on the abdomen was made. With a color CCD camera, we could observe highly elongated cancer cells and nuclei in capillaries in the skin flap in living mice. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells and nuclei in the capillaries elongated to fit the width of these vessels. The average length of the major axis of the cancer cells in the capillaries increased to approximately four times their normal length. The nuclei increased their length 1.6 times in the capillaries. Cancer cells in capillaries over 8 microm in diameter could migrate up to 48.3 microm/hour. The data suggests that the minimum diameter of capillaries where cancer cells are able to migrate is approximately 8 microm. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

A functional promoter region of the CKLFSF2 gene is located in the last intron/exon region of the upstream CKLFSF1 gene.

The genes for CKLFSF1 (chemokine-like factor super family member 1) and CKLFSF2 (chemokine-like factor super family member 2) are very closely linked, within 312 bp of each other. Here, we present evidence that the last intron/exon region of the CKLFSF1 gene contains a novel eukaryotic promoter capable of directing the expression of the downstream gene, CKLFSF2. We identified two segments of the upstream region of the CKLFSF2 gene, 2146 bp (-2134/+12, relative to ATG +1) and 1483 bp (-2134/-652), that were capable of efficiently driving expression of a linked reporter gene upon transient transfection into several kinds of cell lines. The 1483 bp segment exhibited more than a two-fold increase in luciferase activity relative to the 2146 bp segment. By analyzing 5'-deletion mutants of the 1483 bp segment, we identified a 195 bp segment (-846/-625) located in the last intron/exon region of the CKLFSF1 gene that was critical for promoter activity. DNA decoy experiments revealed that a 122 bp (-846/-725) fragment markedly inhibited CKLFSF2 mRNA transcription. Furthermore, we found that the putative promoter region of the CKLFSF2 gene is separated from the transcription start site by about 500 bp. Accumulating reports suggest that introns have many functions, including the modulation of regulation and structure. This work provides evidence that a eukaryotic gene promoter sequence from one gene located in an intron/exon of another.

Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium.

Here we report a genetically modified bacteria strain, Salmonella typhimurium A1, selected for anticancer activity in vivo. The strain grows in tumor xenografts. In sharp contrast, normal tissue is cleared of these bacteria even in immunodeficient athymic mice. S. typhimurium A1 is auxotrophic (Leu/Arg-dependent) but apparently receives sufficient support from the neoplastic tissue to grow locally. Whether additional genetic lesions are present is not known. In in vitro infection, the GFP-expressing bacteria grew in the cytoplasm of PC-3 human prostate cancer cells and caused nuclear destruction. These effects were visualized in cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. In vivo, the bacteria caused tumor inhibition and regression of xenografts visualized by whole-body imaging. The bacteria, introduced i.v. or intratumorally, invaded and replicated intracellularly in PC-3 prostate cancer cells labeled with red fluorescent protein grafted into nude mice. By day 15, S. typhimurium A1 was undetectable in the liver, lung, spleen, and kidney, but it continued to proliferate in the PC-3 tumor, which stopped growing. When the bacteria were injected intratumorally, the tumor completely regressed by day 20. There were no obvious adverse effects on the host when the bacteria were injected by either route. The S. typhimurium A1 strain grew throughout the tumor, including viable malignant tissue. This result is in marked contrast to bacteria previously tried for cancer therapy that were confined to necrotic areas of the tumor, which may account, in part, for the strain's unique antitumor efficacy.

Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression.

We report here the genetic engineering of dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The parental HT-1080 and the derived dual-color clones had similar cell proliferation rates, suggesting that expression of GFP and/or RFP does not affect cell cycle progression. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse brain where extreme elongation of the cell body as well as the nucleus occurred. Dual-color cells in various positions of the cell cycle were visualized in excised mouse lungs after tail-vein injection of the dual-color cells. In the lung, the dual-color cells were observed frequently juxtaposing their nuclei, suggesting a potential novel form of cell-cell communication. The dual-color cells thus are a useful tool for visualizing living-cell dynamics in vivo as well as in vitro. Drugs that could specifically perturb these processes can now be readily screened in real time in vivo.

Molecular cloning and characterization of chemokine-like factor super family member 1 (CKLFSF1), a novel human gene with at least 23 alternative splicing isoforms in testis tissue.

Chemokine-like factor (CKLF) was isolated from PHA-stimulated U937 cells. It is composed of 152 amino acids and located on chromosome 16q22. Utilizing bioinformatics, based on CKLF cDNA and protein sequences, in combination with experimental validation, we identified a novel gene designated chemokine-like factor super family member 1 (CKLFSF1). CKLFSF1 maps on chromosome 16q22, and the full-length gene comprises of seven exons and six introns. Using RACE-PCR, we identified two potential alternative transcription start sites, 1A and 1B. Northern blot and RT-PCR analysis demonstrated that CKLFSF1 is predominantly expressed in human testis tissue, with only lower levels of expression in many other human tissues. RT-PCR and cDNA sequencing identified 23 alternatively spliced isoforms of CKLFSF1 in the testis tissue, which encode protein variants ranging from 36 to 169 amino acids in length. Immunohistochemistry analysis demonstrated that CKLFSF1 proteins are highly expressed in spermatocyte and in tissue fluid of human testes tissue. In light of these findings, we propose that CKLFSF1 may play an important role in spermatogenesis or testicular development.