Cloning and characterization of ApRab4, a recycling Rab protein of Aiptasia pulchella, and its implication in the symbiosome biogenesis.

The biogenesis of Symbiodinium symbiosome in the host cells of the sea anemone, Aiptasia pulchella, involves retention of ApRab5 on and exclusion of ApRab11 from the organelle. One predicted consequence of this differential Rab association is the constant membrane fusion of symbiosomes with endocytic vesicles in the absence of parallel membrane retrieval and the subsequent formation of spacious symbiosomes, which nevertheless, contradicts the common perception. To solve this discrepancy, we determined whether membrane fusion occurs between symbiosomes and endocytic vesicles, and whether ApRab11-independent recycling is involved in symbiosome biogenesis. By using the biotin-avidin detection system, we found evidence for symbiosome-endocytic vesicle fusion. Cloning and characterization of ApRab4, an A. pulchella homolog of Rab4, showed that ApRab4 is associated with both the early endocytic and the perinuclear recycling compartments, and its normal function is required for the organization of the recycling compartments. Immunostaining localized ApRab4 to the symbiosome membrane, partially overlapping with ApRab5-decorated microdomains. Significantly, a treatment that impaired Symbiodinium photosynthesis also abolished symbiosome association of ApRab4. Furthermore, ApRab4 was quickly recruited to newly formed phagosomes, but prolonged association only occurred in those harboring live zooxanthelllae. We propose that ApRab4 retention on the symbiosome is an essential part of the mechanism for the biogenesis of Symbiodinium symbiosome.

ApRab3, a biosynthetic Rab protein, accumulates on the maturing phagosomes and symbiosomes in the tropical sea anemone, Aiptasia pulchella.

Symbiosome biogenesis and function are central to the endosymbiotic interaction between symbiotic dinoflagellates and their host cnidarians. To understand these important organelles, we have been conducting studies to identify and characterize symbiosome-associated proteins of the Rab family, key regulatory components of vesicular trafficking and membrane fusion in eukaryotic cells. Our prior studies have implicated three endocytic Rab proteins in the regulation of symbiosome biogenesis. Here, we show that ApRab3 is a new member of the Rab3 subfamily, associating with symbiosomes and accumulating on the maturing phagosomes in the A. pulchella digestive cells. ApRab3 is 78% identical to human Rab3C, and contains all Rab 3-specific signature motifs. EGFP-ApRab3-labeled vesicular structures tended to either align along the cell peripheral, or aggregate at one side of the nucleus. ApRab3 specifically co-distributed with the TGN marker, WGA, but not other organelle-specific markers tested. Immunofluorescence staining with a specific peptide antibody showed similar results. Significantly, an expression of a constitutively active mutant caused the enlargement and random dispersion of EGFP-ApRab3-decorated compartments in PC12 cells. Together, these data suggest that ApRab3 is a new member of the Rab3 subfamily, participating in the biosynthetic trafficking pathway, and symbiosome biogenesis involves an interaction with ApRab3-positive vesicles.

ApRab11, a cnidarian homologue of the recycling regulatory protein Rab11, is involved in the establishment and maintenance of the Aiptasia-Symbiodinium endosymbiosis.

Endosymbiotic association of the Symbiodinium dinoflagellates (zooxanthellae) with their cnidarian host cells involves an alteration in the development of the alga-enclosing phagosomes. To uncover its molecular basis, we previously investigated and established that the intracellular persistence of the zooxanthella-containing phagosomes involves specific alga-mediated interference with the expression of ApRab5 and ApRab7, two key endocytic regulatory Rab proteins, which results in the selective retention of the former on and exclusion of the later from the organelles. Here we examined the role of ApRab11, a cnidarian homologue of the key endocytic recycling regulator, Rab11, in the Aiptasia-Symbiodinium endosymbiosis. ApRab11 protein shared 88% overall sequence identity with human Rab11A and contained all Rab-specific signature motifs. Co-localization and mutagenesis studies showed that EGFP-tagged ApRab11 was predominantly associated with recycling endosomes and functioned in the recycling of internalized transferrin. In phagocytosis of latex beads, ApRab11 was quickly recruited to and later gradually removed from the developing phagosomes. Significantly, although ApRab11 immunoreactivity was rapidly detected on the phagosomes containing either newly internalized, heat-killed zooxanthellae, or resident zooxanthellae briefly treated with the photosynthesis inhibitor DCMU, it was rarely observed in the majority of phagosomes containing either newly internalized live, or healthy resident, zooxanthellae. It was concluded that through active exclusion of ApRab11 from the phagosomes in which they reside, zooxanthellae interfere with the normal recycling process required for efficient phagosome maturation, and thereby, secure their intracellular persistence, and consequently their endosymbiotic relationship with their cnidarian hosts.

Important genetic etiology of infertile Chinese males: chromosome abnormality and deletion of DAZ gene copy in the AZFc region of Y chromosome / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the correlation of male infertility with abnormality of chromosomal quantity and construction and with the deletion of DAZ gene copy in the AZFc region of Y chromosome.</p><p><b>METHODS</b>Included in the study were 247 azoospermic and 206 severe oligozoospermic patients, as well as 210 fertile men as controls. Multi-PCR and PCR-RFLP were used to analyze the deletion of DAZ gene copies in the AZFc region of Y chromosome. Chromosomal quantity and construction were detected by G-band in the 453 patients.</p><p><b>RESULTS</b>In the azoospermic and severe oligozoospermic patients, the incidences of chromosomal abnormality were 12.6% and 8.3%; the rates of complete DAZ deletion were 7.7% and 11.2%, and the rates of DAZ1/DAZ2 deletion were 7.3% and 4.9% respectively, but no deletion was detected in the controls.</p><p><b>CONCLUSION</b>There is a high frequency of chromosomal abnormality and DAZ gene copy deletion in patients with azoospermia and oligospermia, which suggests that chromosomal abnormality and partial and complete deletion of DAZ gene copy might be important genetic causes of Chinese male infertility.</p>

Clinical study of sildenafil in the treatment of premature ejaculation complicated by erectile dysfunction / 中华男科学杂志

<p><b>OBJECTIVES</b>To evaluate the efficacy and safety of sildenafil citrate in the treatment of premature ejaculation (PE) complicated by erectile dysfunction (ED).</p><p><b>METHODS</b>Forty-five patients of PE complicated by ED received flexible doses of sildenafil from 50 to 100 mg for 1 to 3 months. Intravaginal ejaculatory latency time (IELT) and sexual satisfaction ratio (SSR) of partner were recorded to evaluate the effect of PE treatment, as well as the general efficacy and satisfaction of ED treatment. And the difference of IIEF-5 before and after the treatment were compared.</p><p><b>RESULTS</b>Twenty-seven patients had their PE improved and the effective rate was 60%. Forty patients reported the improvement in erection and the percentage of erectile improvement was 88.88%. All the 27 patients with improvement of PE achieved effective erection through the administration of 50 mg sildenafil and the satisfaction rate reached 81.48%. On the other hand, only 1 case (5.56%) reported satisfaction over the treatment in the 18 patients who did not obtain improvement of PE. Between the PE improvement group and non-improvement group, there were significant differences (P < 0.001) shown in IIEF-5 scores before and after the treatment. Mild or moderate side effects were reported in 9 patients(20%), who recovered without any treatment.</p><p><b>CONCLUSIONS</b>To premature ejaculation patients with ED, sildenafil can safely and effectively improve their erectile function, the satisfaction over the ED treatment outcome means that their PE symptoms could be alleviated.</p>