Characterization of a novel yeast SNARE protein implicated in Golgi retrograde traffic

The protein trafficking machinery of eukaryotic cells is employed for protein secretion and for the localization of resident proteins of the exocytic and endocytic pathways. Protein transit between organelles is mediated by transport vesicles that bear integral membrane proteins (v-SNAREs) which selectively interact with similar proteins on the target membrane (t-SNAREs), resulting in a docked vesicle. A novel Saccharomyces cerevisiae SNARE protein, which has been termed Vti1p, was identified by its sequence similarity to known SNAREs. Vti1p is a predominantly Golgi-localized 25-kDa type II integral membrane protein that is essential for yeast viability. Vti1p can bind Sec17p (yeast SNAP) and enter into a Sec18p (NSF)-sensitive complex with the cis-Golgi t-SNARE Sed5p. This Sed5p/Vti1p complex is distinct from the previously described Sed5p/Sec22p anterograde vesicle docking complex. Depletion of Vti1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the Golgi. Temperature-sensitive mutants of Vti1p show a similar carboxypeptidase Y trafficking defect, but the secretion of invertase and gp400/hsp150 is not significantly affected. The temperature-sensitive vti1 growth defect can be rescued by the overexpression of the v-SNARE, Ykt6p, which physically interacts with Vti1p. We propose that Vti1p, along with Ykt6p and perhaps Sft1p, acts as a retrograde v-SNARE capable of interacting with the cis-Golgi t-SNARE Sed5p.     

Inescapable shock-induced potentiation of morphine analgesia.

Exposure to various stressors potentiates nociceptive and nonnociceptive responses to morphine. These phenomena have received little study despite their seeming generality and importance for understanding analgesia and opiate action. The present experiments characterize inescapable shock (IS)-induced potentiation of morphine analgesia. Rats were exposed to IS, equal escapable shocks (ESs), or restraint (control). Potentiation of analgesia (tail-flick [TF] test and hotplate test) was observed only in rats given IS 24 or 48 hr earlier, in agreement with previously reported learned-helplessness effects. Finally, no change in tail temperature or motor function was found that could be inaccurately interpreted as analgesia. The relevance of these findings to stressor-induced enhancement of morphine analgesia and potential substrates of IS effects are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

We evaluated a protocol involving two types of choice presentations for assessing leisure choice-making skills of seven older adults with severe disabilities. Initially when presented with pairs of objects representing choices, choice making was validated through demonstration of an object preference. A more complex choice-presentation format was then employed, involving pictures to represent choices. If the preference identified with objects was not demonstrated using pictures, a replication of the object format occurred to ensure changes in choice making using pictures was not due to a preference change. Five participants demonstrated choice-making skills using objects and two demonstrated choices using pictures. These results reflect the importance of assessing choice-making skills prior to presenting choice opportunities. Suggestions for future research focus on expanding the assessment protocol to include a wider array of choice-making skills and training staff to provide choices in a format commensurate with an individual's skill level.     

Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium

The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5, 5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.     

Activity of fotemustine in medulloblastoma and malignant glioma xenografts in relation to O6-alkylguanine-DNA alkyltransferase and alkylpurine-DNA N-glycosylase activity

Fotemustine is a chloroethylnitrosourea with antitumor activity in disseminated melanoma and adult primary brain tumors. Because new drugs are required for the treatment of medulloblastoma in children, we evaluated the preclinical antitumor activity of fotemustine in four s.c. medulloblastoma xenografts, in comparison with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Both drugs were administered as a single i.p. injection to nude mice bearing advanced-stage tumor. Fotemustine displayed significant antitumor activity in three of four medulloblastoma xenografts; two, IGRM34 and IGRM57, were highly sensitive, with 37 and 100% tumor-free survivors, respectively, more than 120 days after treatment at the highest nontoxic dose (50 mg/kg). Fotemustine was also highly active in a malignant glioma xenograft (IGRG88; five of six tumor-free survivors on day 177). Fotemustine proved to be significantly more active than BCNU in IGRM34 and the glioma xenograft IGRG88. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) was detected in all tumor xenografts, ranging in activity from 6 to 892 fmol/mg protein. The high in vivo sensitivity to fotemustine and BCNU observed in three xenografts was clearly associated with a low ATase activity (> 20 fmol/mg), whereas the two poorly sensitive or refractory medulloblastoma xenografts showed high ATase activity (> 500 fmol/mg). Alkylpurine-DNA N-glycosylase activity was detected in all tumor xenografts but at levels ranging only from 513 to 1105 fmol/mg/h; no consistent relationship was found between alkylpurine-DNA N-glycosylase activity and the in vivo sensitivity to the two chloroethylnitrosoureas. The improved activity and tolerance of fotemustine in comparison with BCNU in pediatric medulloblastoma xenografts strongly support the clinical development of this agent in children with brain tumors, in which ATase should be examined as a potential prognostic indicator.     

Increased chemosensitivity to doxorubicin of intrinsically multidrug-resistant human colon carcinoma cells by prolonged exposure to verapamil

Resistance modifying agents (RMA) such as verapamil (VER) have proved effective in reversing multidrug resistance (MDR) in many in vitro experimental models, but clinical results with RMA have been disappointing. To clarify this apparent discrepancy we have evaluated the cytotoxic effects of doxorubicin (DOX) plus VER in four human colon carcinoma (HCOC) cell lines (LoVo, DLD-1, SW948, SW1116). These lines were selected on the basis of their levels of mdr1 mRNA being similar to those expressed by HCC obtained from non-drug-treated patients. In all cell lines the sensitising effect of VER on DOX cytotoxicity was schedule-dependent and maximal potentiation of DOX cytotoxicity was obtained by exposure to VER for a time > or = the cells' population doubling time.     

Mucinous lesions of the breast. A pathological continuum

Mucocele-like tumor and invasive mucinous carcinoma of the breast may represent the two ends of the pathological spectrum of mucinous lesions of the breast, respectively. Little data exists on mucinous lesions that may be considered intermediate between mucocele-like tumor and invasive mucinous carcinoma. We studied 23 consecutive cases of invasive mucinous carcinoma of the breast and observed the following associated intermediate mucinous lesions: mucin-filled ducts (MFD) with unremarkable epithelium in 15 cases (65%), MFD with typical ductal hyperplasia in 9 cases (39%), MFD with atypical ductal hyperplasia in 5 cases (22%), and MFD with intraductal carcinoma in 13 cases (57%; micropapillary or cribriform types). Eighteen cases (78%) contained MFD with one of these four lesions and five cases (22%) contained all four lesions. Twenty-three consecutive cases of infiltrating ductal carcinoma-not otherwise specified (IDC-NOS), 21 cases of intraductal carcinoma, and 50 consecutive cases of surgically-excised breast tissue with fibrocystic change (FC), were similarly reviewed. Only one case (4%) of IDC-NOS, 1 case of intraductal carcinoma, and two cases (4%) of FC, contained small foci of MFD with intraductal carcinoma, intraductal carcinoma, and unremarkable epithelium, respectively. Our findings suggest the presence of a spectrum of mucinous lesions of the breast which represents a pathological continuum.