Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.     

Fallacies of preoperative lymphoscintigraphy in detecting sentinel node in breast cancer

BACKGROUND: Preoperative lymphoscintigraphy is one of the three methods of evaluating sentinel nodes in patients with breast cancer; however, it has been reported to have a high false negative rate. CASE PRESENTATIONS: We report here two cases where the preoperative lymphoscintigraphy was found to be fallacious. A 44-year-old female with T2N0 breast cancer underwent preoperative lymphoscintigraphy with Tc99 sulfur colloid which failed to show any uptake in axilla or internal mammary chain. Intraoperative scintigraphy with blue dye and hand held gamma probe identified sentinel lymph node in axilla. Another patient with T2N0 lesion underwent preoperative lymphoscintigraphy which showed a sentinel lymph node in axilla and another in supraclevicular fossa. Intraoperative scintigraphy failed to show supraclevicular node however axillary node was correctly identified. CONCLUSION: These two cases further strengthen the need to carry out triple test in identification of sentinel lymph node in patients with breast cancer. It also demonstrates the fallacies of preoperative lymphoscintigraphy.     

Theoretical model of the aqua-copper [Cu(H2O)5]+cation interactions with guanine

Pentaaqua complexes of Cu(I) with guanine were optimized at the DFT B3PW91/6-31G(d) level. For the most stable structures, vibration frequencies and NBO charges were computed followed by energy analyses. The order of individual conformers was very sensitive to the method and basis sets used for the calculation. Several conformers are practically degenerated in energy. The inclusion of an entropy term changes the order of the conformers stability. Water molecules associated at the N9 position of guanine are favored by the inclusion of the entropy correction. Bonding energies of Cu–O(aqua) interactions were estimated to be about 60 kcal mol^–1 and for Cu–N7 bonding in the range of 75–83 kcal mol^–1. The broad range in Cu–N interaction energies demonstrates the role of induction effects caused by water molecules associated at the various sites of guanine. The charge distribution of the guanine molecule is changed remarkably by the coordination of a Cu(I) cation, which can also change the base-pairing pattern of the guanine.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

Prediction of methylated CpGs in DNA sequences using a support vector machine

DNA methylation plays a key role in the regulation of gene expression. The most common type of DNA modification consists of the methylation of cytosine in the CpG dinucleotide. At the present time, there is no method available for the prediction of DNA methylation sites. Therefore, in this study we have developed a support vector machine (SVM)-based method for the prediction of cytosine methylation in CpG dinucleotides. Initially a SVM module was developed from human data for the prediction of human-specific methylation sites. This module achieved a MCC and AUC of 0.501 and 0.814, respectively, when evaluated using a 5-fold cross-validation. The performance of this SVM-based module was better than the classifiers built using alternative machine learning and statistical algorithms including artificial neural networks, Bayesian statistics, and decision trees. Additional SVM modules were also developed based on mammalian- and vertebrate-specific methylation patterns. The SVM module based on human methylation patterns was used for genome-wide analysis of methylation sites. This analysis demonstrated that the percentage of methylated CpGs is higher in UTRs as compared to exonic and intronic regions of human genes. This method is available on line for public use under the name of Methylator at http://bio.dfci.harvard.edu/Methylator/.     

Hemoglobin is expressed in alveolar epithelial type II cells

Hemoglobin is the main oxygen carrying heme protein in erythrocytes. In an effort to study the differential gene expression of alveolar epithelial type I and type II cells using DNA microarray technique, we found that the mRNAs of hemoglobin alpha- and beta-chains were expressed in type II cells, but not in type I cells. The microarray data were confirmed by RT-PCR. The mRNA expression of both chains decreased when type II cells trans-differentiated into type I-like cells. Immunocyto/histochemistry revealed that hemoglobin protein was specifically localized in type II cells of a lung cell mixture and rat lung tissue. The endogenous synthesis of hemoglobin in alveolar epithelial cells suggests that hemoglobin may have unidentified functions other than oxygen transport in the lung.     

Ligand-induced coupling versus receptor pre-association: cellular automaton simulations of FGF-2 binding

The binding of basic fibroblast growth factor (FGF-2) to its cell surface receptor (CSR) and subsequent signal transduction is known to be enhanced by heparan sulfate proteoglycans (HSPGs). HSPGs bind FGF-2 with low affinity and likely impact CSR-mediated signaling via stabilization of FGF-2-CSR complexes via association with both the ligand and the receptor. What is unknown is whether HSPG associates with CSR in the absence of FGF-2. In this paper, we determine conditions by which pre-association would impact CSR-FGF-2-HSPG triad formation assuming diffusion-limited surface reactions. Using mean-field rate equations, we show that (i) when [HSPG] is much higher than [CSR], the presence of pre-formed complexes does not affect the steady state of FGF-2 binding, and (ii) when the concentrations are comparable, the presence of pre-formed complexes substantially increases the steady-state concentration of FGF-2 bound to CSR. These findings are supported by explicit cellular automaton simulations, which justify the mean-field treatment. We discuss the advantages of such a two-receptor system compared to a single-receptor model, when the parameters are comparable. Further, we speculate that the observed high concentration of HSPG in intact cells ([HSPG]-100[CSR]) provides a way to ensure that the binding levels of FGF-2 to its signaling receptor remains high, irrespective of the presence of pre-formed CSR-HSPG complexes on the cell surface, while allowing the cell to finely tune the response to FGF-2 via down-regulation of the signaling receptor.     

Virus persistence in an animal model of multiple sclerosis requires virion attachment to sialic acid coreceptors

Persistent Theiler's virus infection in the central nervous system (CNS) of mice provides a highly relevant animal model for multiple sclerosis. The low-neurovirulence DA strain uses sialic acid as a coreceptor for cell binding before establishing infection. During adaptation of DA virus to growth in sialic acid-deficient cells, three amino acid substitutions (G1100D, T1081I, and T3182A) in the capsid arose, and the virus no longer used sialic acid as a coreceptor. The adapted virus retained acute CNS virulence, but its persistence in the CNS, white matter inflammation, and demyelination were largely abrogated. Infection of murine macrophage but not oligodendrocyte cultures with the adapted virus was also significantly reduced. Substitution of G1100D in an infectious DA virus cDNA clone demonstrated a major role for this mutation in loss of sialic acid binding and CNS persistence. These data indicate a direct role for sialic acid binding in Theiler's murine encephalomyelitis virus persistence and chronic demyelinating disease.