附睾局部组织肾素血管紧张素系统(英文)

附睾内液体微环境对精子的成熟和贮藏是相当重要的。附睾液体的形成取决于附睾上皮的吸收与分泌功能,而先前的实验已经证明：这些吸收与分泌的活动是受到除神经激素以外旁分泌与自分泌的调控。虽然肾素血管紧张素系统（ＲＡＳ）在很多组织上旁分泌与自分泌的作用已多有报导,但其在附睾的存在及作用仍鲜为人知。本综述总结了本实验室在这方面的研究结果,通过使用不同的实验方法,例如,免疫组织化学,放射免疫分析,分子生物学及短路电流电生理学方法,我们得到的结果显示了ＲＡＳ主要成员在附睾的分布（Ｆｉｇ．１）和表达,并阐明了血管紧张素ＩＩ对附睾阴离子分泌的调控作用（Ｆｉｇｓ．２＆３）。本综述还就血管紧张素ＩＩ对附睾旁分泌与自分泌的作用及其机制（Ｆｉｇ．４）,以及对精子功能的作用进行了讨论。     

A Conserved Negative Regulatory Region in αPAK: Inhibition of PAK Kinases Reveals Their Morphological Roles Downstream of Cdc42 and Rac1

αPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129–1143, 1997) resembling those of Cdc42^G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in αPAK). αPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42^G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding αPAK^83-149 potently inhibited GTPγS-bound Cdc42-mediated kinase activation of both αPAK and βPAK. Coexpression of this PAK inhibitor with Cdc42^G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1^G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of αPAK^83-149 completely blocked the phenotypic effects of hyperactive αPAK^L107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.     

Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase Acts as a Cdc42 Effector in Promoting Cytoskeletal Reorganization

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ~190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCKα and Cdc42^V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCKα resulted in actin and myosin reorganization. Expression of kinase-dead MRCKα blocked Cdc42^V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCKα mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCKα plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCKα mutants. These results suggest that MRCKα may act as a downstream effector of Cdc42 in cytoskeletal reorganization.     

RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.     

Ascorbic acid in pollen: Conversion of L-galactono-1,4-lactone to L-ascorbic acid by Lilium longiflorum

The L-ascorbic acid (AA) content of pollen from three cultivars of Lilium longiflorum Thunb. was 260–280 μg/g fresh wt. of pollen. During germination ascorbic acid content gradually decreased reaching 70% of the original value at 6 h. Pollen germinated in media containing 0.29 M D-glucose (an osmoregulator and carbon source) failed to synthesize ascorbic acid but pollen germinated in 0.29 M pentaerythritol (a non-metabolizable osmoregulator) supplemented with L-galactono-1,4-lactone (L-GalAL) did form ascorbic acid, dependent upon the concentration of the lactone. Lycorine inhibited germination but had negligible effect on the conversion of L-GalAL to AA.     

Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

BackgroundCirculating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.MethodsWe measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.ResultsThe average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005).ConclusionsCirculating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.     

TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways

Connective tissue growth factor (CTGF), which is also called CCN2, is a secreted matricellular protein. CTGF regulates various important cellular functions by interacting with multiple molecules in the microenvironment. In the ovary, CTGF is mainly expressed in granulosa cells and involved in the regulation of follicular development, ovulation and luteinization. TGF-β1 has been shown to up-regulate CTGF expression in rat and hen granulosa cells. However, the underlying molecular mechanisms of this up-regulation remain undefined. More importantly, whether the stimulatory effect of TGF-β1 on CTGF expression can be observed in human granulosa cells remains unknown. In the present study, our results demonstrated that TGF-β1 treatment up-regulates CTGF expression in both immortalized human granulosa cells and primary human granulosa cells. Using a siRNA-mediated knockdown approach and a pharmacological inhibitor, we demonstrated that the inhibition of Smad2, Smad3 or ERK1/2 attenuates the TGF-β1-induced up-regulation of CTGF. This study provides important insights into the molecular mechanisms that mediate TGF-β1-up-regulated CTGF expression in human granulosa cells.