Identification of in vitro phosphorylation sites in the growth cone protein SCG10. Effect Of phosphorylation site mutants on microtubule-destabilizing activity

SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitro phosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone.     

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).     

Cathepsin E expression by normal and premalignant cervical epithelium

We have investigated the expression of the aspartic proteinase cathepsin E and HLA-DR and the presence of HPV16 in normal squamous epithelium (n = 8) and low-grade (n = 21) and high-grade (n = 14) intraepithelial squamous lesions of the uterine cervix. Immunohistochemistry of cervical biopsies revealed that up-regulation of cathepsin E expression was related to increasing severity of the cervical intraepithelial neoplasia (CIN). Up-regulation of protein was associated with increased message as assessed by in situ hybridization. Langerhans cells and the majority of koilocytes did not express detectable cathepsin E levels. Although there was also an up-regulation of HLA-DR expression by cervical keratinocytes in cervical intraepithelial neoplasia lesions, as determined by immunohistochemistry, no significant correlation was found between HLA-DR and cathepsin E expression in these lesions; neither was expression of cathepsin E correlated to the presence of HPV16, detected by polymerase chain reaction. The expression of cathepsin E, an aspartic proteinase that is reported to play a role in antigen processing for presentation by class II major histocompatibility complex molecules, is associated with cellular dedifferentiation in cervical intraepithelial neoplasia.     

Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.     

Stochastic model for a wavelike exothermal reaction in condensed heterogeneous systems

The labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Overlap of resonances in the imino-imino region of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multi-pathway approach for obtaining sequence-specific assignments wherein all possible NOE connectivities to the labile protons are utilized, including those from the 2H of adenine, 5CH3 of thymine, and 5H of cytosine. Resonance assignments are obtained for all slowly exchanging imino and cytosine amino protons. Base-pairing up to and including the junction point is found in all four arms of both Holliday junctions. Several cross-arm NOE connectivities are identified and can be used to infer the geometry of the helical stacking domains. The two Holliday junctions studied, which differ only by the exchange of two base pairs at the branch point, appear to have opposite arm stacking geometries. These assignments form an important part of the critical background for detailed NMR analysis of Holliday junction structure and dynamics.     

Patient preference for self-collected cultures for group B streptococcus in pregnancy

The purpose of this study was to examine the factors which affect the level of fatigue among patients participating in clinical trials in which this symptom had been assessed with the EORTC QLQ-C30. Data were assembled from 2390 patients in ten clinical trials in which the QLQ-C30 had been used to assess baseline and on-study quality of life. The relationship between the level of fatigue reported by the patients on the fatigue scale of this questionnaire and patient and disease characteristics was assessed in univariate and multivariate cross-sectional analyses. In addition, changes in fatigue scores were compared in a longitudinal analysis among patients on two arms of an anti-emetic trial whose emesis control was markedly different. Baseline fatigue levels differed substantially among patients taking part in the different trials. Factors associated with greater fatigue severity on univariate analysis included: female gender, presence of metastatic disease, and poorer performance status. In addition, on multivariate analyses the oldest patients were found to have less fatigue, as were patients with breast cancer, while patients with ovarian and lung cancer experienced greater fatigue. Patients on the arm of the anti-emetic trial in which emesis was better controlled showed significantly less increase in fatigue after receiving chemotherapy. The fatigue scale of the QLQ-C30 appears to provide a useful approach to assessing this important symptom. The relationships found between fatigue and patient and disease characteristics need further exploration as does the degree to which the QLQ-C30 fully captures this dimension of quality of life.     

Analysis of some herbal plants from India used in the control of diabetes mellitus by NAA and AAS techniques

Elemental analysis of some herbal plants used in the control of diabetes has been done by the techniques of Neutron Activation Analysis (NAA) and Atomic Absorption Spectroscopy (AAS). The elements Mn, Na, K, Cl, Al, Cu, Co, Pb, Ni, Cr, Cd, Fe, Ca, Zn and Hg are found to be present in different plants in various proportions.     

Study of the beta -delayed neutron decay of 18N

The shortage of suitable liver donors for children has motivated the use of ABO-incompatible (ABO-I) grafts for transplantation in urgent situations. However, survival after ABO-I liver grafts has been reported at about 30% as compared with 80% in cases of ABO-identical or -compatible liver grafts. This difference has been attributed to antibody-mediated, hyperacute or chronic liver rejection, due to preformed ABO antibodies (alloantibodies). In this study, we report our results with ABO-I livers in children without alloantibodies at the time of transplantation. From January 1988 to June 1993, 143 OLT were performed in 122 children. Eight children received 8 ABO-I liver grafts. Of these, 7 patients were included in the study. All 7 were alloantibody free before OLT. Five children were spontaneously alloantibody free, while in 2 children, the plasma alloantibodies were eliminated before and after transplantation using intravenous infusion of specific blood group antigens of the donor blood group (soluble antigens). Immunosuppression consisted of a triple-drug treatment combining CsA, AZA, and steroids. The follow-up period was between 10 and 48 months. One child died from a surgical complication. Six children survived, but 1 died 10 months later from intestinal obstruction. There were no graft losses and no episodes of hyperacute or chronic rejection. The graft and patient survival rate was 71%. There was a 28% incidence of rejection, but all were mild (requiring steroid boluses only). Our results suggest that the absence of ABO alloantibodies at the time of and after transplantation can protect ABO-I liver grafts against antibody-mediated rejection, whether hyperacute or chronic, and that soluble antigens are effective in eliminating alloantibodies in children.     

Working memory and work with memory: visual-spatial and further components of processing

Empirical and theoretical evidence for the concept of working memory is considered. We argue that the major weakness of this concept is its loose connection with the knowledge about background perceptive and cognitive processes. Results of two relevant experiments are provided. The first study demonstrated the classical chunking effect in a speeded visual search and comparison task, the proper domain of a large-capacity very short term sensory store. Our second study was a kind of extended levels-of-processing experiment. We attempted to manipulate visual, phonological, and (different) executive components of long-term memory in the hope of finding some systematic relationships between these forms of processing. Indeed, the results demonstrated a high degree of systematicity without any apparent need for a concept such as working memory for the explanation. Accordingly, the place for working memory is at all the interfaces where our metacognitive strategies interfere with mostly domain-specific cognitive mechanisms. Working memory is simply our work with memory.