Modulation of immune cell proliferation and chemotaxis towards CC chemokine ligand (CCL)-21 and CXC chemokine ligand (CXCL)-12 in undenatured whey protein-treated mice

Whey protein concentrates (WPCs) enhance innate mucosal immunity during early life and have a protective role in some immune disorders. To further elucidate the potential benefits of this protein, the present study investigated the effect of dietary supplementation with WPCs on blood parameters, plasma cytokine profiles, and immune cell proliferation and chemotaxis. A total of 45 male mice were equally distributed into three experimental groups and treated daily for 21 days as follows: group I was a control group that was orally supplemented with distilled water, group II was orally supplemented with undenatured WP (100 mg/kg body weight), and group III was orally supplemented with bovine serum albumin (100 mg/kg body weight). We found that the plasma cytokine levels of interleukin (IL)-1α, IL-1β, IL-10 and tumor necrosis factor-α and the levels of reactive oxygen species, cholesterol, triglycerides and the lipid profile were significantly decreased in the WP-treated group compared to the control group. In contrast, the levels of IL-2, IL-4, IL-7, IL-8 and glutathione were significantly elevated, and consequently, the ability of peripheral blood mononuclear cells to proliferate in response to stimulation with different antigens was significantly increased in the WP-treated group. Moreover, the in vitro chemotaxis of B, T and bone-marrow-derived dendritic cells toward CC chemokine ligand-21 and CXC chemokine ligand-12 was significantly increased, by twofold, in WP-treated mice compared to the control group. Taken together, our data reveal the benefits of WP supplementation in enhancing immune cell proliferation and migration to the secondary lymphoid organs.     

Cellular and Molecular Mechanisms Underlie the Anti-Tumor Activities Exerted by Walterinnesia aegyptia Venom Combined with Silica Nanoparticles against Multiple Myeloma Cancer Cell Types

Multiple myeloma (MM) is a clonal disease of plasma cells that remains incurable despite the advent of several novel therapeutics. In this study, we aimed to delineate the impact of snake venom extracted from Walterinnesia aegyptia (WEV) alone or in combination with silica nanoparticles (WEV+NP) on primary MM cells isolated from patients diagnosed with MM as well as on two MM cell lines, U266 and RPMI 8226. The IC₅₀ values of WEV and WEV+NP that significantly decreased MM cell viability without affecting the viability of normal peripheral mononuclear cells (PBMCs) were determined to be 25 ng/ml and 10 ng/ml, respectively. Although both WEV (25 ng/ml) and WEV+NP (10 ng/ml) decreased the CD54 surface expression without affecting the expression of CXCR4 (CXCL12 receptor) on MM cells, they significantly reduced the ability of CXC chemokine ligand 12 (CXCL12) to induce actin cytoskeleton rearrangement and the subsequent reduction in chemotaxis. It has been established that the binding of CXCL12 to its receptor CXCR4 activates multiple intracellular signal transduction pathways that regulate MM cell chemotaxis, adhesion, and proliferation. We found that WEV and WEV+NP clearly decreased the CXCL12/CXCR4-mediated activation of AKT, ERK, NFκB and Rho-A using western blot analysis; abrogated the CXCL12-mediated proliferation of MM cells using the CFSE assay; and induced apoptosis in MM cell as determined by PI/annexin V double staining followed by flow cytometry analysis. Monitoring the expression of B-cell CCL/Lymphoma 2 (Bcl-2) family members and their role in apoptosis induction after treatment with WEV or WEV+NP revealed that the combination of WEV with NP robustly decreased the expression of the anti-apoptotic effectors Bcl-2, Bcl_XL and Mcl-1; conversely increased the expression of the pro-apoptotic effectors Bak, Bax and Bim; and altered the mitochondrial membrane potential in MM cells. Taken together, our data reveal the biological effects of WEV and WEV+NP and the underlying mechanisms against myeloma cancer cells.     

Synthesis, anti-inflammatory activity and ulcerogenic liability of novel nitric oxide donating/chalcone hybrids

A group of novel nitric oxide (NO) donating chalcone derivatives was prepared by binding various amino chalcones with different NO donating moieties including; nitrate ester, oximes and furoxans. Most of the prepared compounds showed significant anti-inflammatory activity using carrageenan-induced rat paw edema method compared with indomethacin. The prepared compounds exhibited more protection than indomethacin in regard to gastric toxicity. Histopathological investigation confirmed the beneficial effects of the NO releasing compounds in reducing ulcer formation. The incorporation of the NO-donating group into the parent chalcone derivatives caused a moderate increase in the anti-inflammatory activity with a marked decrease in gastric ulcerations compared to their parent chalcone derivatives.     

Synthesis of the Human Insulin Gene: Protein Expression,Scaling Up and Bioactivity

Abstract

Optimized Synthetic human insulin gene was preferred to easy of cloning, plasmid stability, and protein expression away from the native sequence and its rare codons. Two steps to obtain the insulin, so we assembled the gene of 293 bp using a battery of overlapped synthetic oligos, then cloned into pET101directional TOPO expression vector downstream to the T7 promoter. The proinsulin products were produced as inclusion bodies in E. coli at a level of 10%. The batch cultivation of the strain yielded 6 g/L, while the high cell density of fed‐batch cultivation yielded 46 g/L. The proinsulin purification yielded 110 mg/gram cell weight, and 1.3 mg/gram of a bioactive insulin. The native insulin was generated by enzymatic conversion of chemically processed proinsulin. The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS‐PAGE, RP‐HPLC, and bioactivity. The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.     

A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V_1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.     

Recognition of 2-keto-3-deoxyoctonate in bacterial cells and lipopolysaccharides by the sialic acid binding lectin from the horseshoe crab Carcinoscorpiusrotundacauda

The sialic acid binding loctin carcinoscorpin agglutinates $\text{Escharichia}\text{coli}\text{K12}\text{and}\text{Salmonella}\text{minnesots}$ R595 cells. This interaction can be inhibited by the saccharides namely 2-keto-3-deoxyoctonate and the disaccharide D-(N-acetylneuraminyl) (2→6)2-acetamide-2-deoxy-D-galactitol. N-acetylneuraminic acid is shown to be a poor inhibitor. The same behaviour is seen when purified lipopolysaccharides from these two Gram negative bacteria are used. $\text{Vibrio}\text{cholerae}$, a Grum negative bectarium devoid of 2-keto-3-deoxyoctonate and $\text{Staphylococcus}\text{sureus}$ a typical Gram positive bacterium failed to agglutinate in the presence of the lectin. The results suggest that the 2-keto-3-deoxyoctonate residues might represent the physiological substrate for the sialic acid binding lectin from the horseshoa crab.     

Spatial and seasonal variation of the zooplankton in the coastal zone and main khors of Lake Nasser (Egypt)

A prominent feature of Lake Nasser is the presence of khors (dendritic side extensions). We studied the zooplankton of the larger khors and coastal zone of the main lake in 1996 and 1997, and found an assemblage of rotifers, cladocerans and copepods that was partly tropical, partly temperate, at relatively high biomasses. Spatial differences were weak, but the upstream khors (Toushka and Korosko) were consistently richer than the downstream khors (El-Ramla and Kalabsha), with a rather sudden transition around km 150 at El Madiq. Summer standing crops were higher than those in winter by a factor 2–3. The zooplankton of the littoral of the main channel showed the same spatial pattern as that in the khors, being more abundant in spring (average 82300 ind m^–3) than in autumn (average 72700 ind m^–3). Zooplankton dry weight increased from 4 g m^–2 at khor El-Ramla to 7 g m^–2 at khor Toushka. These rather high values had low variation. The number of species, diversity and evenness all showed a high degree of similarity among the khors and in the littoral of the main lake. The lake fish fauna is poor, lacking a pelagic planktivore. The predominance of medium-sized Copepoda (one calanoid, two cyclopoids) in the zooplankton suggests that fish predation is moderate. This is confirmed by the persistence of two Daphnia species at low abundance, although rather strong variations in time suggest that Cladocera suffer from summer predation by invertebrates (copepods) as well as vertebrates (mostly larval fish). Because the zooplankton is underutilised by higher trophic levels, we suggest to assess the benefits of introducing a pelagic zooplanktivorous fish.     

Total, free and conjugated sterolic forms in three microalgae used in mariculture

Total, free and conjugated forms (steryl esters, steryl glycosides and acyl steryl glycosides) of sterols from three microalgae that are extensively used in mariculture (Tetraselmis chuii, Nannochloropsis salina and Skeletonema costatum) were examined. The results revealed that cholesterol is the only common fraction detected in all investigated species and distributed in free and all conjugated forms. However, the total sterol content of T. chuii was about 325 microg/g dry wt, most of it was concentrated amongst 24-methylcholesta-5,24-diene-3beta-ol and 24-methylcholest-5-en-3beta-ol. On the other hand, the majority of the fractions were distributed in the free form. The total sterol content of N. salina was about 180 microg/g dry wt, cholesterol was the major fraction that was detected. Nevertheless, the dominant distribution forms were esterified. While in S. costatum, the total sterol content was 76 microg/g dry wt, approximately most fractions are quantitatively alike and dominated in the free form. Furthermore, our study shows clearly that most sterols are not distributed regularly within each form, a result that encouraged us to suggest a distribution of specific sterol fraction as a free or conjugated can be used as a serving tool in chemotaxonomic studies.     

A rapid method for computing the inverse of the gametic covariance matrix between relatives for a marked Quantitative Trait Locus

The inverse of the gametic covariance matrix between relatives, G^-1, for a marked quantitative trait locus (QTL) is required in best linear unbiased prediction (BLUP) of breeding values if marker data are available on a QTL. A rapid method for computing the inverse of a gametic relationship matrix for a marked QTL without building G itself is presented. The algorithm is particularly useful due to the approach taken in computing inbreeding coefficients by having to compute only few elements of G. Numerical techniques for determining, storing, and computing the required elements of G and the nonzero elements of the inverse are discussed. We show that the subset of G required for computing the inbreeding coefficients and hence the inverse is a tiny proportion of the whole matrix and can be easily stored in computer memory using sparse matrix storage techniques. We also introduce an algorithm to determine the maximum set of nonzero elements that can be found in G^-1 and a strategy to efficiently store and access them. Finally, we demonstrate that the inverse can be efficiently built using the present techniques for very large and inbred populations.