Domains of neuronal heparan sulphate proteoglycans involved in neurite growth on laminin

Summary A single neuronal cell assay of neurite growth was utilized to determine types and domains of neuronal proteoglycans involved in neurite growth on laminin. Perturbations of biosynthesis and processing, enzymatic digestion with specific lyases, and competition with glycosaminoglycan side chains produced complementary data consistent with a molecular model implicating glycosaminoglycan (GAG) residues of heparan sulphate proteoglycans (HSPGs) in neurite growth. The observations suggest that HSPGs promote neurite growth on laminin by bridging between binding domains for HSPGs on laminin and on the neuronal cell surface, and that the bridge is tethered at both ends by noncovalent interactions between the binding domains and GAG side chains. Sulphation of the GAGs of HSPGs appears to be critical to the tethering and/or neurite growth-promoting activity of neuronal HSPGs.     

Fractionation and characterization of rat liver poly(A)-containing RNA.

Three fractions of poly(A)-containing RNA were separated from total rat liver RNA using poly(U)-Sepharose 4B affinity chromatography. The poly(A)-containing RNA fractions were released by thermal elution. Fraction 1, eluted under the mildest conditions, and had poly(A) tracts of approx. 200 AMP units in length which appeared to be associated with poly(U) sequences of 20-50 UMP in length. Fraction 1 appeared to be present mainly in the nucleus and, its size distribution was similar to that of fractions 2 and 3. Fractions 2 and 3 eluted at higher temperatures and were associated mainly with polysomal and microsomal fractions. Poly(U) sequences were absent in fractions 2 and 3 while their poly(A) sequences had a size distribution characteristic of those reported in the mRNA of other organisms.     

Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Macrophage cholesterol efflux and the active domains of serum amyloid A 2.1

Serum amyloid A 2.1 (SAA2.1) suppresses ACAT and stimulates cholesteryl ester hydrolase (CEH) activities in cholesterol-laden macrophages, and in the presence of a cholesterol transporter and an extracellular acceptor, there is a marked increase in the rate of cholesterol export in culture and in vivo. The stimulation of CEH activity by SAA2.1 is not affected by chloroquine, suggesting that it operates on neutral CEH rather than the lysosomal form. With liposomes containing individual peptides of SAA2.1, residues 1-20 inhibit ACAT activity, residues 74-103 stimulate CEH activity, and each of residues 1-20 and 74-103 promotes macrophage cholesterol efflux to HDL in culture media. In combination, these peptides exhibit a profound effect, so that 55-70% of cholesterol is exported to media HDL in 24 h. The effect is also demonstrable in vivo. [3H]cholesterol-laden macrophages injected intravenously into mice were allowed to establish themselves for 24 h. Thereafter, the mice received a single intravenous injection of liposomes containing intact SAA1.1, SAA2.1, peptides composed of SAA2.1 residues 1-20, 21-50, 51-80, 74-103, or SAA1.1 residues 1-20. Only liposomes containing intact SAA2.1 or its residues 1-20 or 74-103 promoted the efflux of cholesterol in vivo. A single injection of each of the active peptides is effective in promoting cholesterol efflux in vivo for at least 4 days.     

Review: amyloidogenesis-unquestioned answers and unanswered questions

Current assumptions and conclusions in several active areas of amyloid research are examined to see how consistent the data from chosen in vitro and in vivo model systems are with clinical and anatomic observations. These areas include the assembly of amyloid-like fibrils in vitro, the nucleation phenomenon, amyloid fibril structure in vivo and in vitro, common structural components of the amyloids, and the regression of tissue amyloid and proteolysis of amyloid proteins. Divergences and congruencies are highlighted, which in turn suggests caution in the interpretation of present data, greater collaboration and communication among investigators, and, additional areas and techniques for investigation.     

Synthesis of 4-deoxy analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-xylose and their effects on glycoconjugate biosynthesis

4-Deoxy analogues of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-xylose were synthesized and evaluated as inhibitors of glycoconjugate biosynthesis. Methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside (11) showed a reduction in [3H]GlcN and [14C]Leu incorporation into hepatocyte cellular glycoconjugates by 89 and 88%, of the control cells, respectively, at 20 mM, whereas the free sugars, 2-acetamido-2,4-dideoxy-alpha,beta-D-xylo-hexopyranoses (15), showed a reduction of [3H]GlcN and [14C]Leu incorporation by 75 and 64%, respectively, at 20 mM. The acetylated analogues of 11 and 15, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-beta-D-xylo-hexopyranoside and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha,beta-D-xylo-hexopyra noses, showed a greater inhibition of [3H]GlcN and [14C]Leu incorporation at 1 mM compared with their non-acetylated counterparts, but were toxic to hepatocytes at concentrations of 10 and 20 mM. Corresponding derivatives of 2-acetamido-2,4-dideoxy-L-threo-pentopyranose showed no biological effect up to 20 mM, suggesting that the C-6 substituent is important for the biological activity.     

Biocides formulation of essential oils having antimicrobial activity

Essential oils of fennel, peppermint, caraway, eucalyptus, geranium and lemon were tested for their antimicrobial activities against some plant pathogenic micro-organisms (Fusarium oxysporum, Alternaria alternate, Penicilium italicum Penicilium digitatum and Botyritus cinerea). Essential oils of fennel, peppermint, caraway were selected as an active ingredient for the formulation of biocides due to their efficiency in controlling the tested micro-organisms. Successful emulsifiable concentrates (biocides) were prepared from these oils using different emulsifiers (Emulgator B.L.M. Tween20 and Tween80) and different fixed oils (sesame, olive, cotton and soybean oils). Physico-chemical properties of the formulated biocide (spontaneous emulsification, emulsion stability test, cold stability and heat stability tests as well as viscosity, surface tension and pH) were measured. The prepared biocides were ready to be tested for application in a future work as a safe pesticide against different pathogens.     

A correlation between BCL-2 modifying factor,p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. Those genes that can detect colon - cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients. In this study, 50 pathologically diagnosed early cancer colon patients included and their tissue biopsy with 50 matched adjacent normal tissue, and 50 adenoma tissue specimens were analyzed for livin gene and BMF gene expressions using real time PCR. The relationship of those genes expressions with clinicopathological features, tumor markers, Time to Progression and overall survival for those patients were correlated in cancer colon group. In this study, there was a significant a reciprocal relationship between over expression of livin gene and down regulation of BMF and p53 genes in colon cancer cells. Livin mRNA was significantly higher, while BMF and p53 mRNA were significantly lower in colorectal cancer tissue compared to benign and normal colon tissue specimens (P < 0.001), however, this finding was absent between colon adenomas and normal mucosa. There was a significant association between up regulation of livin and down regulation of BMF and p53 expressions with more aggressive tumor (advanced TNM stage), rapid progression with metastasis and decreased overall survival in cancer colon patients, hence these genes can serve as significant prognostic markers of poor outcome in colon cancer patients. This work highlights the role of livin, BMF and p53 genes in colorectal tumorigenesis and the applicability of using those genes as a diagnostic and prognostic markers in patients with colon carcinoma and as a good target for cancer colon treatment in the future.