cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

Comparison of statistical methods for classification of ovarian cancer using mass spectrometry data

MOTIVATION: Novel methods, both molecular and statistical, are urgently needed to take advantage of recent advances in biotechnology and the human genome project for disease diagnosis and prognosis. Mass spectrometry (MS) holds great promise for biomarker identification and genome-wide protein profiling. It has been demonstrated in the literature that biomarkers can be identified to distinguish normal individuals from cancer patients using MS data. Such progress is especially exciting for the detection of early-stage ovarian cancer patients. Although various statistical methods have been utilized to identify biomarkers from MS data, there has been no systematic comparison among these approaches in their relative ability to analyze MS data. RESULTS: We compare the performance of several classes of statistical methods for the classification of cancer based on MS spectra. These methods include: linear discriminant analysis, quadratic discriminant analysis, k-nearest neighbor classifier, bagging and boosting classification trees, support vector machine, and random forest (RF). The methods are applied to ovarian cancer and control serum samples from the National Ovarian Cancer Early Detection Program clinic at Northwestern University Hospital. We found that RF outperforms other methods in the analysis of MS data.     

Idle time in the washing and iron concentration in leachate removed: two basic parameters in the desulphurization of coal in a packed column

Column biodesulphurization of coal is at the experimental stage and is influenced by many variables including temperature, pH, particle size, concentration of iron in solution, among others. Idle time in the washing process and the concentration of dissolved iron in the purged leachate are two variables with a definite effect on the yield of the desulphurization system. In the laboratory, several trials were run with columns packed with coal for different idle times: 1, 2, 3, 5, 6 and 7 days, and for different concentrations of iron in the purged leachate: 500 to 4,000 mg/l. The optimal values for the two variables; that is, those allowing for the highest desulphurization yields, were idle times of 3 and 5 days, which give an elimination of 56% and 49% of pyritic sulphur, respectively, and 3,000 mg/l of iron concentration in the purged leachate, giving a decrease in pyritic sulphur in coal of 57%.     

Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity

To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K(4)K(20)-S4 was 2-3-fold more potent against protozoa and RBC, yet K(4)K(20)-S4 was more potent by 2 orders of magnitude against bacteria. K(4)-S4 had similar behavior as K(4)K(20)-S4, but K(20)-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K(20)-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K(4)-S4 and K(4)K(20)-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity.     

Epidermal growth factor receptor dimerization monitored in live cells

We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing beta-galactosidase (beta-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as beta-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. beta-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and beta-gal complementation. These results demonstrate that beta-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.     

Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess

Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA.