Maturational changes in the survivability and fertility of fowl sperm during their passage through the male reproductive tract

The objective of this study was to examine whether domestic fowl (Gallus domesticus) sperm undergo maturation in their capacity for survival and fertilization in the male reproductive tract. Sperm collected from the testis, epididymis and the proximal, middle and distal vas deferens were simultaneously stored in vitro in minimum essential medium (MEM) at 39°C for 0, 3 and 6h, and at 4°C for 24 and 48h. Sperm membrane integrity was measured using the dual fluorescent stain SYBR-14/propidium iodide (PI). Aliquots of sperm from the various sites were subjected to artificial insemination (AI) into the uteri of hens to assess the duration of sperm survival in the oviduct and to determine the fertility status of the sperm. Testicular sperm exhibited a very low capacity to survive under in vitro liquid storage conditions, irrespective of the storage temperature used, and in the oviduct, and they had a low ability to fertilize the ovum. On the contrary, sperm from the distal vas deferens had a higher survival rate during in vitro storage periods, a longer life span in the oviduct, and high fertility. Survival and fertilizing capacity of the sperm recovered from the testes increased gradually (P<0.05) from the testes to the distal vas deferens. In conclusion, we suggest that fowl sperm may undergo functional maturation through a process of gradual changes in their survival and fertilization capacities during their passage through the successive parts of the male reproductive tract.     

Sequence heterogeneity of NS5A and core proteins of hepatitis C virus and virological responses to pegylated-interferon/ribavirin combination therapy

Both host and viral factors have been implicated in influencing the response to pegylated-interferon/ribavirin (PEG-IFN/RBV) therapy for hepatitis C virus (HCV) infection. Among the viral factors, sequence heterogeneity within NS5A and core regions has been proposed. This study aimed to clarify the relationship between virological responses to PEG-IFN/RBV therapy and sequence heterogeneity within NS5A, including the IFN/RBV resistance-determining region (IRRDR), the interferon sensitivity-determining region (ISDR) and the core region. Pretreatment sequences of NS5A and the core regions were analyzed in 57 HCV-1b-infected patients who were to be treated with PEG-IFN/RBV. Of 40 patients infected with HCV having an IRRDR with four or more mutations (IRRDR ≥ 4), 28 (70%) patients achieved a sustained virological response (SVR). On the other hand, only 4 (24%) of 17 patients infected with HCV having an IRRDR with three or fewer mutations (IRRDR ≤ 3) achieved a SVR (P = 0.001). Similarly, 22 (71%) of 31 patients infected with HCV and having an ISDR with one or more mutations (ISDR ≥ 1) achieved a SVR while 10 (38%) of 26 patients infected with HCV and having an ISDR without any mutations (ISDR = 0) achieved a SVR (P = 0.014). As for the core region, there was significant correlation between a single mutation at position 70 (Gln(70) ) and non-SVR (P = 0.02). Notably, Gln(70) was more prominently associated with the null response (P = 0.0007). In conclusion, sequence heterogeneity within the IRRDR and ISDR, and a single point mutation at position 70 of the core region of HCV-1b are likely to be correlated with virological responses to PEG-IFN/RBV therapy.     

Possible use of synthetic aggregation pheromones to control stinkbug Plautia stali in kaki persimmon orchards

• 1 The brown‐winged green stinkbug Plautia stali Scott (Hemiptera: Pentatomidae) is the most serious pest of all noxious stinkbugs in various orchards in Japan. An area‐wide integrated pest management programme using the species‐specific aggregation pheromone is desirable to control P. stali because nonspecific insecticides may kill arthropod natural enemies and induce the resurgence of other endemic pests.
• 2 The traditional mass‐trapping method is not expected to be effective as a result of huge migrations from cypress forests. Therefore, we chose an ‘attract‐and‐kill’ strategy of intensively installing poisonous eggplants with a synthetic aggregation pheromone lure as enclosures for the target orchards.
• 3 We found no overall control effect of the installation of poisonous eggplants, although regional differences were observed, which might originate from topological configurations or the distance from the source population in cypress forests.
• 4 The poisonous eggplants with an aggregation‐pheromone lure, however, changed the spatial distribution of fruit damage, appearing to induce the majority of the damage within a 100‐m range of the poisonous eggplants. Some damage, although at a low level, was found in regions 100–200 m away from the poisonous eggplants.
• 5 We postulate that such low‐damage regions were created because the majority of the bugs dispersed from the centre of the orchards to the areas with poisonous eggplants. The present study, in which the spatial scale of the damage spillover was estimated at approximately 150 m, has important implications for future strategies of attract‐and‐kill and possibly push–pull.

     

Overexpression,purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150?kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a β-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N’-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.     

Behavior of tricellulin during destruction and formation of tight junctions under various extracellular calcium conditions

Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca²⁺ depletion caused by EGTA treatment and Ca²⁺ repletion after Ca²⁺ starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca²⁺ depletion and repletion. In the early stage after Ca²⁺ repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca²⁺ repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca²⁺ repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.     

Investigation of factor affecting health‐related quality of life in head and neck cancer patients

doi: 10.1111/j.1741‐2358.2012.00662.x Investigation of factor affecting health‐related quality of life in head and neck cancer patients Objectives: Head and neck cancer (HNC) patients have profound illness of physical, social and psychological factors that affects quality of life (QOL). The purpose of this study is to investigate the factors affecting HRQL in patients with intra‐oral prostheses. Background: Some cross‐sectional studies have been performed to investigate HRQL in patients with HNC, but these studies did not report in detail how factors affect the HRQL of maxillectomy and mandibulectomy and/or glossectomy patients. Materials and Methods: The University of Washington Quality of Life version 4 questionnaires (Japanese version) was administered to 50 maxillectomy and 50 mandibulectomy and/or glossectomy patients with intra‐oral prostheses who were selected according to inclusion and exclusion criteria. Gathered data were statistically analyzed to investigate how a number of factors, namely, age, sex, pathologic diagnosis, neck dissection, resection size, radiotherapy and dental condition affect HRQL. Results: In the maxillectomy patients, there were no significant differences between malignant and benign tumor in pathological diagnosis or between dentate and edentulous in dental condition. Age, sex, neck dissection and radiotherapy affected HRQL. In the mandibulectomy and/or glossectomy patients, there was no significant difference between dentate and edentulous in dental condition. Age, sex, glossectomy, neck dissection and radiotherapy affected HRQL. Conclusions: The factors affecting HRQL in the maxillectomy patients were different from those in the mandibulectomy and/or glossectomy patients. Though they wore stable prostheses; we were still able to show that resection size, radiotherapy and neck dissection affected HRQL.     

Estrogen induces cell proliferation by promoting ABCG2-mediated efflux in endometrial cancer cells

Recently, it has reported that overeating of lipid-food has led to increase the amount of estrogen in vivo and the incidence of endometrial carcinomas. It is well-known that ATP-binding cassette transporter sub-family G2 (ABCG2) is highly expressed in cancer stem cells (CSCs). CSCs possess the ability for differentiation, tumorigenesis, stem cell self-renewal, and the efflux of anti-cancer drug and these abilities affect malignancy of cancer cells. However, little is known about the relationship between the expression of ABCG2 and malignancy of cancer cells. The present study aimed at understanding the regulatory mechanism underlying 17-β-estradiol (E₂)-induced cell proliferation under the control of ABCG2. E₂ increased cell viability with a peak at 1 μM and facilitated ABCG2 mRNA expression followed by the increase of ABCG2 expression level at plasma membrane. E₂-induced cell proliferation was inhibited by reserpine, an inhibitor of ABCG2, and the ABCG2 siRNA treatment. Thus, these results imply that ABCG2 plays an important role in the promotion of E₂-induced cell proliferation in Ishikawa cells.     

Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996     

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34⁺CD38⁺CD33⁺ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3⁺ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.