Bacterial disinfectant resistance—a challenge for the food industry

The focus on hygiene in the food industry has resulted in an increasing use of chemical disinfection and it has been speculated that this will impose a selective pressure and contribute to the emergence of disinfectant-resistant microorganisms. The frequency of strains with a low-level resistance to quaternary ammonium compounds (QAC) is relatively high for Listeria monocytogenes (10%), Staphylococcus spp. (13%) and Pseudomonas spp. (30%) and lower for lactic acid bacteria (1.5%) and coliforms (1%) isolated from food and food processing industry. In general, bacteria isolated after disinfection are more resistant and represent a potential food safety or food spoilage problem. Adaptation to disinfectants may be accompanied by cross-resistance to related disinfectants. We have recently found a genetic linkage between resistance to QAC and antibiotics in food associated staphylococci, and there is a growing concern about cross-resistance between antibiotics and disinfectants.Disinfectant resistance can in most cases be prevented by effective cleaning and disinfection procedures. More effort should be made to avoid build-up of resistance in food production environments.     

Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways

Prostate cancer (PCA), the most frequently diagnosed malignancy in men, represents an excellent candidate disease for chemoprevention studies because of its particularly long latency period, high rate of mortality and morbidity. Infusion of black tea and its polyphenolic constituents have been shown to possess antineoplastic effects in androgen dependent PCA in both in vivo and in vitro models including transgenic animals. In the present study, we report that black tea polyphenol, Theaflavins (TF)-induced apoptosis in human prostate carcinoma, LNCaP cells is mediated via modulation of two related pathways: up-regulation of p53 and down-regulation of NF-kappa B activity, causing a change in the ratio of pro-and antiapoptotic proteins leading to apoptosis. The altered expression of Bcl-2 family member proteins triggered the release of cytochrome-C and activation of initiator capsase 9 followed by activation of effector caspase 3. Furthermore, TF also affected the protein expression of mitogen activated protein kinases (MAPK) pathways. Our results demonstrated that TF treatment resulted in down-regulation of phospho-extracellular signal-regulated protein kinase (Erk1/2) and phospho-p38 MAPK expressions. We conclude that TF induces apoptosis in LNCaP cells by shifting the balance between pro-and antiapoptotic proteins and down-regulation of cell survival pathways leading to apoptosis. Further extending this work, we also showed that TF induces apoptosis in androgen independent PCA cell line, PC-3 through caspases and MAPKs mediated pathways. Thus, effect of TF on PCA cell lines seems to be irrespective of their androgen status.     

Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS

Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca(2+), KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and G?-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K(+) depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and G?-6850 (each 10(-5) M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs' SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS.     

Design of cyclic and d‐amino acids containing peptidomimetics for inhibition of protein‐protein interactions of HER2‐HER3

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2‐positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2‐mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18 ) exhibited antiproliferative activity in HER2‐overexpressing lung cancer cell lines with IC₅₀ values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d ‐amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18 , compound 32 , a cyclic, d ‐amino acid‐containing peptidomimetic, was found to have an IC₅₀ value in the nanomolar range in HER2‐overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d ‐amino acid analogs of 18 , compound 32 , binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.     

Identification and structural characterization of FYVE domain-containing proteins of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

FYVE domains have emerged as membrane-targeting domains highly specific for phosphatidylinositol 3-phosphate (PtdIns(3)P). They are predominantly found in proteins involved in various trafficking pathways. Although FYVE domains may function as individual modules, dimers or in partnership with other proteins, structurally, all FYVE domains share a fold comprising two small characteristic double-stranded β-sheets, and a C-terminal α-helix, which houses eight conserved Zn²⁺ ion-binding cysteines. To date, the structural, biochemical, and biophysical mechanisms for subcellular targeting of FYVE domains for proteins from various model organisms have been worked out but plant FYVE domains remain noticeably under-investigated.     

Solid-phase synthesis and bioevaluation of lupeol-based libraries as antimalarial agents

The use of the triterpenoid lupeol as a scaffold for the synthesis of lupeol-based libraries is described. Lupeol was anchored to a solid support (Rink amide/Sieber Amide) through aliphatic dicarboxylic acid moieties, which also served as a site for introducing diversity. The resulting polymer linked 3beta-O (resin-alkanoyl)-lup-20(29)-ene 3 was used to generate key intermediates 3beta-O (resin-alkanoyl)-30-bromo-lup-20(29)-ene 4 and 3beta-O (resin-alkanoyl)-30-amino-lup-20(29)-ene 6 for the generation of libraries based on disubstituted lupeol derivatives. A 96-member library was screened for its in-vitro antimalarial activity against Plasmodium falciparum.     

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5?h in human serum compared to the half-life of 2?h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.     

Effects of farmyard manure and fertilizers on yield, fibre quality and nutrient balance of rainfed cotton (Gossypium hirsutum)

Two-year field experiments were conducted to evaluate the effect of fertilizer with or without farmyard manure (FYM) application on cotton productivity and fibre quality. A partial nutrient balance was calculated by the difference method (nutrient applied--crop removal). Seed cotton yield was improved with addition of FYM (5 Mg ha(-1)). Application of both N and P resulted in significant improvements in seed cotton yield than the control and without N plots (PK). Uniformity ratio and ginning outturn (GOT) was greater in the FYM amended plots than the plots without FYM. Nitrogen and P balance was positive in the fertilizer-N and P applied plots whereas K balance was negative in spite of the addition of fertilizer-K. Potassium balance was positive only when FYM was applied. These studies suggest that it is advantageous to apply FYM as it improves fibre yield by way of improved GOT and maintains a positive nutrient balance.     

Studies on lactic dehydrogenase and sorbitol dehydrogenase release in relation to deep freezing of buffalo semen in certain extenders

Forty seminal ejaculates from five mature buffalo bulls (n=8 from each bull), exhibiting more than 70% initial sperm motility, were frozen in the following three extenders: egg-yolk sodium citrate glycerol (EYCG); tris-egg yolk glycerol (TYG); and citric acid whey glycerol (CAWG). The extenders were evaluated for the release of intracellular enzymes, lactic dehydrogenase (LDH) and sorbitol dehydrogenase (SODH) from the spermatozoa during freezing (in fresh semen, after dilution and after equilibration) and post freezing (24 h and 7 d after freezing. It was found that the release of LDH and SODH enzymes was significantly lower in TYG than in EYCG and CAWG extenders. The most critical stage, at which the enzyme release was maximal, was between equilibration and 24 h post freezing in all three extenders.