Targeting the Binding Interface on a Shared Receptor Subunit of a Cytokine Family Enables the Inhibition of Multiple Member Cytokines with Selectable Target Spectrum

The common γ molecule (γc) is a shared signaling receptor subunit used by six γc-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple γc-cytokines are pathogenically involved in a single disease, thus making the shared γc-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the γc and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the γc and cytokines, we successfully designed peptides that not only inhibit multiple γc-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three γc-cytokines without affecting the other three or non-γc-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of γc-cytokines the γc-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of γc-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases.     

Glutathione-dependent formaldehyde dehydrogenase homolog from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5 is a propanol-preferring alcohol dehydrogenase

Genome search of Bacillus subtilis revealed the presence of an open reading frame annotated as glutathione-dependent formaldehyde dehydrogenase/alcohol dehydrogenase. The open reading frame consists of 1137 nucleotides corresponding to a polypeptide of 378 amino acids. To examine whether the encoded protein is glutathione-dependent formaldehyde dehydrogenase or alcohol dehydrogenase, we cloned and characterized the gene product. Enzyme activity assays revealed that the enzyme exhibits a metal ion-dependent alcohol dehydrogenase activity but no glutathione-dependent formaldehyde dehydrogenase or aldehyde dismutase activity. Although the protein is of mesophilic origin, optimal temperature for the enzyme activity is 60°C. Thermostability analysis by circular dichroism spectroscopy revealed that the protein is stable up to 60°C. Presence or absence of metal ions in the reaction mixture did not affect the enzyme activity. However, metal ions were necessary at the time of protein production and folding. There was a marked difference in the enzyme activity and CD spectra of the proteins produced in the presence and absence of metal ions. The experimental results obtained in this study demonstrate that the enzyme is a bona-fide alcohol dehydrogenase and not a glutathionedependent formaldehyde dehydrogenase.     

A probabilistic neural network approach for modeling and classification of bacterial growth/no-growth data

In this paper, we propose to use probabilistic neural networks (PNNs) for classification of bacterial growth/no-growth data and modeling the probability of growth. The PNN approach combines both Bayes theorem of conditional probability and Parzen's method for estimating the probability density functions of the random variables. Unlike other neural network training paradigms, PNNs are characterized by high training speed and their ability to produce confidence levels for their classification decision. As a practical application of the proposed approach, PNNs were investigated for their ability in classification of growth/no-growth state of a pathogenic Escherichia coli R31 in response to temperature and water activity. A comparison with the most frequently used traditional statistical method based on logistic regression and multilayer feedforward artificial neural network (MFANN) trained by error backpropagation was also carried out. The PNN-based models were found to outperform linear and nonlinear logistic regression and MFANN in both the classification accuracy and ease by which PNN-based models are developed.     

Comprehensive analysis of cis- and trans-acting factors affecting ectopic Break-Induced Replication

Break-induced replication (BIR) is a highly mutagenic eukaryotic homologous DNA recombination pathway that repairs one-ended DNA double strand breaks such as broken DNA replication forks and eroded telomeres. While searching for cis-acting factors regulating ectopic BIR efficiency, we found that ectopic BIR efficiency is the highest close to chromosome ends. The variations of ectopic BIR efficiency as a function of the length of DNA to replicate can be described as a combination of two decreasing exponential functions, a property in line with repeated cycles of strand invasion, elongation and dissociation that characterize BIR. Interestingly, the apparent processivity of ectopic BIR depends on the length of DNA already synthesized. Ectopic BIR is more susceptible to disruption during the synthesis of the first ~35–40 kb of DNA than later, notably when the template chromatid is being transcribed or heterochromatic. Finally, we show that the Srs2 helicase promotes ectopic BIR from both telomere proximal and telomere distal regions in diploid cells but only from telomere proximal sites in haploid cells. Altogether, we bring new light on the factors impacting a last resort DNA repair pathway.     

Development and characterization of a new epithelial cell line PSF from caudal fin of Green chromide, <Emphasis Type="Italic">Etroplus suratensis</Emphasis> (Bloch, 1790)

A new cell line [pearlspot fin (PSF)] has been developed from caudal fin of Etroplus suratensis, a brackish/freshwater fish cultivated in India. The cell line was maintained in Leibovitz’s L-15 supplemented with 10% fetal bovine serum (FBS). The PSF cell line consisted predominantly of epithelial-like cells. The cells were able to grow at temperatures between 25°C and 32°C with optimum temperature of 28°C. The growth rate of PSF cells increased as the FBS proportion increased from 2% to 20% at 28°C with optimum growth at the concentration of 10% FBS. One marine fish virus (fish nodavirus) was tested on this cell line and found not susceptible. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached confluency on the third d after subculture. Polymerase chain reaction amplification of mitochondrial 16S rRNA and COI indicated identity of this cell line with those reported from this fish species, confirming that the cell line was of pearlspot origin. The cells were successfully cryopreserved and revived at the tenth, 25th, and 35th passages. The bacterial extracellular products from Vibrio cholerae MTCC 3904 were found to be toxic to PSF. Karyotyping analysis indicated that the modal chromosome number was 48.     

A maturation-related differential phosphorylation of the plasma membrane proteins of the epididymal spermatozoa of the hamster by endogenous protein kinases

When the plasma membranes of caput and cauda epididymal spermatozoa of hamster were evaluated for their ability to undergo phosphorylation, a differential phosphorylation of the membrane proteins was observed. In the plasma membranes of the caput epididymal spermatozoa (immature), twelve proteins were phosphorylated (100, 76, 67, 65, 55, 52, 47, 42, 38, 32, 30, and 20 kD), whereas in the plasma membranes of cauda epididymal spermatozoa (mature), a differential phosphorylation pattern was observed with respect to the 94, 67, 52, and 47 kD proteins. The 94 kD protein was found to be phosphorylated and the 67 kD protein was found to be not phosphorylated in cauda spermatozoal plasma membrane (Cd SPM) in contrast to this protein in caput spermatozoal plasma membrane (Cpt SPM). The 52 and 47 kD proteins were also more intensely phosphorylated in Cd SPM than Cpt SPM. The 100 kilodalton protein, although present in both Cpt and Cd sperm plasma membranes, was found to be phosphorylated at the tyrosine residues only in the Cd SPM, as indicated by the Western blot using antiphosphotyrosine antibody. Further, a differential phosphorylation of the substrate proteins present in the Cpt and Cd SPM was seen when Mg²⁺ in the assay buffer was replaced by other divalent cations. For instance, Zn²⁺ stimulated the phosphorylation of an 85 kD protein in cauda SPM and not in the caput SPM and Ca²⁺ stimulated the phosphorylation of a 76 kD protein only in the cauda SPM. The phosphoproteins in both the plasma membranes were found to be phosphorylated predominantly at the tyrosine residue. The differential phosphorylation of a 100 kD protein at tyrosine in the Cd SPM (Western blot), which is absent in the immature Cpt SPM, also indicated that certain proteins in the hamster spermatozoa are phosphorylated in a maturation-specific manner. Mol. Reprod. Dev. 47:341–350, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

Human aortic endothelial cell labeling with positive contrast gadolinium oxide nanoparticles for cellular magnetic resonance imaging at 7 Tesla

Positive T? contrast using gadolinium (Gd) contrast agents can potentially improve detection of labeled cells on magnetic resonance imaging (MRI). Recently, gadolinium oxide (Gd?O?) nanoparticles have shown promise as a sensitive T? agent for cell labeling at clinical field strengths compared to conventional Gd chelates. The objective of this study was to investigate Gado CELLTrack, a commercially available Gd?O? nanoparticle, for cell labeling and MRI at 7 T. Relaxivity measurements yielded r1 = 4.7 s?1 mM?1 and r?/r? = 6.2. Human aortic endothelial cells were labeled with Gd?O? at various concentrations and underwent MRI from 1 to 7 days postlabeling. The magnetic resonance relaxation times T? and T? of labeled cell pellets were measured. Cellular contrast agent uptake was quantified by inductively coupled plasma-atomic emission spectroscopy, which showed very high uptake compared to conventional Gd compounds. MRI demonstrated significant positive T? contrast and stable labeling on cells. Enhancement was optimal at low Gd concentrations, attained in the 0.02 to 0.1 mM incubation concentration range (corresponding cell uptake was 7.26 to 34.1 pg Gd/cell). Cell viability and proliferation were unaffected at the concentrations tested and up to at least 3 days postlabeling. Gd?O? is a promising sensitive and stable positive contrast agent for cellular MRI at 7 T.