A novel testis-specific GTPase serves as a link to proteasome biogenesis: functional characterization of RhoS/RSA-14-44 in spermatogenesis

Most Rho family GTPases serve as key molecular switches in a wide spectrum of biological processes. An increasing number of studies have expanded their roles to the spermatogenesis. Several members of Rho family have been confirmed to be essential for mammalian spermatogenesis, but the precise roles of this family in male reproduction have not been well studied yet. Here we report a surprising function of an atypical and testis-specific Rho GTPase, RSA-14-44 in spermatogenesis. Featured by unique structural and expressional patterns, RSA-14-44 is distinguished from three canonical members of Rho cluster. Thus, we define RSA-14-44 as a new member of Rho GTPases family and rename it RhoS (Rho in spermatogenic cells). RhoS associates with PSMB5, a catalytic subunit of the proteasome, in a series of stage-specific spermatogenic cells. More importantly, RhoS does not directly modulate the cellular proteasome activity, but participates in regulating the stability of "unincorporated" PSMB5 precursors. Meanwhile, our data demonstrate that the activation of RhoS is prerequisite for negatively regulating the stability of PSMB5 precursors. Therefore, our finding uncovers a direct and functional connection between the Rho GTPase family and the pathway of proteasome biogenesis and provide new clues for deciphering the secrets of spermatogenesis.     

Plasma-mediated release of morphine from synthesized prodrugs

Two morphine prodrugs (‘PDA’ and ‘PDB’) were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5–10 min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse > rat > guinea pig > human). Morphine’s release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.     

How myosin VI coordinates its heads during processive movement

A processive molecular motor must coordinate the enzymatic state of its two catalytic domains in order to prevent premature detachment from its track. For myosin V, internal strain produced when both heads of are attached to an actin track prevents completion of the lever arm swing of the lead head and blocks ADP release. However, this mechanism cannot work for myosin VI, since its lever arm positions are reversed. Here, we demonstrate that myosin VI gating is achieved instead by blocking ATP binding to the lead head once it has released its ADP. The structural basis for this unique gating mechanism involves an insert near the nucleotide binding pocket that is found only in class VI myosin. Reverse strain greatly favors binding of ADP to the lead head, which makes it possible for myosin VI to function as a processive transporter as well as an actin-based anchor. While this mechanism is unlike that of any other myosin superfamily member, it bears remarkable similarities to that of another processive motor from a different superfamily--kinesin I.     

How does cholecystokinin stimulate exocrine pancreatic secretion? From birds, rodents, to humans

The field of cholecystokinin (CCK) stimulation of exocrine pancreatic secretion has experienced major changes in the recent past. This review attempts to summarize the present status of the field. CCK production in the intestinal I cells, the molecular forms of CCK produced and subsequently circulated in the blood, the presence or absence of CCK receptors on the isolated pancreatic acinar cells and the associated signaling for acinar cell secretion, and the actual circuits and sites of action for CCK regulation of exocrine pancreatic secretion in vivo are reviewed in different animal species with an emphasis on birds, rodents, and humans. Clear differences in the relative importance of neural and direct modes of CCK action on pancreatic acinar cells were identified. Rodents seem to be endowed with both modes of action, whereas in humans the neural mode may predominate. In birds, such as duck, the direct mode needs further assistance from pituitary adenylate cyclase-activating peptide/VIP receptors. However, much further work needs to be directed to the neural mode to map out all sites of CCK action and details of the full circuits, and we foresee a major revival for this field of research in the near future.     

Layer-by-layer hydroxymethyl ferrocene modified sensor for one-step flow/stop-flow injection amperometric immunoassay of alpha-fetoprotein

A rapid one-step flow/stop-flow injection amperometric immunoassay for alpha-fetoprotein (AFP) using a novel home-produced electrochemical sensor was proposed. The sensor was prepared using layer-by-layer adsorption of positively charged poly(allylamine) (PAA) and negatively charged hydroxymethyl ferrocene on a screen-printed electrode (SPE). The electrochemistry of the immobilized ferrocene moieties showed a surface-controlled electrode process. Based on an electrochemical enzyme-linked immunoassay with the immobilized ferrocene moieties as an electron transfer mediator between the electrode and the horseradish peroxidase (HRP)-labeled anti-AFP antibody, a calibration curve with two linear ranges from 5 to 20 and 20 to 150 ng ml-1 and a detection limit of 2 ng ml-1 for AFP determination was obtained under the optimized conditions of 0.891 ml min-1 flow rate, 20 microl injection volume and +25 mV applied potential. The sensor showed good repeatability and reproducibility and retained more than 95% of its original signal after 15 days of storage. The proposed method eliminated the need for washing and addition of any substrate or mediator. The complete assay could be handled in less than 25 min with a one-step injection of a 40 microl sample solution. The proposed method would be valuable for the diagnosis and monitoring of carcinoma and its metastasis.     

Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining

DNA double strand breaks (DSBs) formed during S phase are preferentially repaired by homologous recombination (HR), whereas G₁ DSBs, such as those occurring during immunoglobulin class switch recombination (CSR), are repaired by non-homologous end joining (NHEJ). The DNA damage response proteins 53BP1 and BRCA1 regulate the balance between NHEJ and HR. 53BP1 promotes CSR in part by mediating synapsis of distal DNA ends, and in addition, inhibits 5’ end resection. BRCA1 antagonizes 53BP1 dependent DNA end-blocking activity during S phase, which would otherwise promote mutagenic NHEJ and genome instability. Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair. Here, we ask whether increased expression of RNF168 or 53BP1 impacts physiological versus mutagenic NHEJ. We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR. As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors.