Inhibitory effect of inducible nitric oxide synthase inhibitors on DMBA-induced hamster buccal–pouch squamous-cell carcinogenesis

We investigated the effects of two NOS inhibitors (AG and l-NAME) on DMBA-induced hamster buccal-pouch carcinogenesis. Six hundred Syrian golden hamsters were split into two divisions (I and II); divisions split into three groups (experimental groups A and B, control group C); and each group into subgroups of 20 (A1-A6, B1-B6 and C1-C3). The pouches of animals in groups A1-A3 were painted first with AG of differing concentrations (10, 20, and 30 micromol/ml) and then 30 min later with DMBA (0.5%), thrice weekly for 9 weeks. Subgroups A4-A6 only received AG treatment. Groups B1 to B6 were similarly treated with l-NAME. Animals in division II were treated in the same manner for 13 weeks. Post-mortem analysis revealed that both inhibitors can suppress the development of epithelial dysplasias and squamous-cell carcinomas. An associated increase in the numbers of epithelial hyperplasias was paralleled by a decrease in iNOS protein expression. This animal model can be employed to evaluate the potential use of iNOS inhibitors as novel therapeutic tools for oral squamous-cell carcinogenesis.     

Minimal surface as a model of beta-sheets

The purpose of this article is to present arguments based on experimental data that the beta-sheet structures in proteins are the result of the tendency to minimize surface areas. Thus, we propose the model that all beta-sheet structures are almost minimal surfaces, namely, their mean curvatures are nearly zero. To support this model, we chose 1740 disjoint beta-sheets with less than 10 strands from the all beta-protein class in a nonredundant 40% Structural Classification of Proteins (SCOP) database and applied the least-squares method to fit the minimal surface catenoid (and in some rare cases, the plane) to the beta-sheet structures. The fitting errors were extremely small: The error of 1729 beta-sheets with catenoid minimal surface is 0.90 +/- 0.55 A and the error of the remaining 11 flat sheets with the plane is 0.64 +/- 0.46 A. The fact that the commonly used models for some beta-sheet surfaces (i.e., the hyperboloid and strophoid) have very small mean curvatures (< 0.05) supports our model. Moreover, we showed that this model also includes the isotropically stressed configuration model proposed by Salemme, in which the intrastrand tendency of the individual chains to twist or coil is in equilibrium with the tendency of the interstrand hydrogen bonding to resist twisting of the sheet as a whole. As an application we used our model to quantify the two principal independent modes in the flexibility of beta-sheets, that is, the bending parameter of beta-sheets and the inclined angle of beta-strands in a sheet.     

Rapid cryopreservation of five mammalian and one mosquito cell line at −80°C while attached to flasks in a serum free cryopreservative

Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at −80°C while attached to the substratum of 25cm² tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.     

New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas aliena type strain KMM 3562T containing two residues of L-serine in the repeating unit

The structure of an acidic polysaccharide from Pseudoalteromonas aliena type strain KMM 3562(T) has been elucidated. The polysaccharide was studied by component analysis, (1)H and (13)C NMR spectroscopy, including 2D NMR experiments. A (1)H, (13)C band-selective constant-time heteronuclear multiple-bond connectivity experiment was used to determine amide linkages, between serine and uronic acid (UA) residues, via (3)J(H,C) correlations between Ser-alphaH and UA-C-6. It was found that the polysaccharide consists of pentasaccharide repeating units with the following structure: [carbohydrate structure]; see text.     

Synthesis and structural analysis of 6-deoxy-6-hydroxyphosphinyl-D-fructopyranose derivatives

3,4-Di-O-benzyl-6-deoxy-6-diethoxyphosphinyl-1,2-O-isopropylidene-beta-D-fructofuranose (13) was prepared from the known 1,2-O-isopropylidene-6-O-tosyl-beta-D-fructofuranose in five steps. Reduction of 13 with sodium dihydrobis(2-methoxyethoxy)aluminate, followed by the action of hydrochloric acid and then hydrogen peroxide, afforded the 6-deoxy-6-hydroxyphosphinyl-D-fructopyranose derivative. This was converted into the 1,2,3,4,5-penta-O-acetyl-6-deoxy-6-methoxyphosphinyl-D-fructopyranoses, whose structure and conformation were established by 1H NMR spectroscopy.     

Diastereoselective annulation of 4-hydroxypyran-2H-ones with enantiopure 2,3-dideoxy-alpha,beta-unsaturated sugar aldehydes derived from respective glycals

One-pot condensations of 4-hydroxypyran-2H-ones 1 and 2, respectively, with various enantiopure 2,3-dideoxy-alpha,beta-unsaturated carbohydrate enals in the presence of l-proline in EtOAc at room temperature generated pyrano-pyrones. It was observed that, while benzyl-protected carbohydrate enals on condensation with 1 or 2 under the above conditions produced an inseparable diastereomeric mixture in a ratio of 1:1, the acyl-protected carbohydrate enals on treatment with 1 or 2 under identical conditions yielded products with moderate to very high diastereoselectivity. A remarkable asymmetric induction was noticed from the C-4 stereogenic center of the acyl-protected carbohydrate enals. An almost complete diastereoselectivity was observed in those reactions that involved condensation of 1 with acetyl-protected enals 5 and 7. The reaction of 2 with 5 also proceeded diastereoselectively to furnish the corresponding annulated product. The reaction presumably took place by C-1,2-addition of the pyrone onto the iminium salt of the alpha,beta-unsaturated carbohydrate enal generated in situ, followed by beta-elimination and cyclization of the 1-oxatriene involving a 6pi-electron electrocyclic process to yield a 2H,5H-pyrano[3,2-c]pyran-5-one derivative.     

An efficient synthesis of methyl 1,3-O-isopropylidene-alpha-D-fructofuranoside and 2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal derivatives from sucrose

Acetalation of sucrose with 2,2-dimethoxypropane in 1,4-dioxane in the presence of p-toluenesulfonic acid, followed by acetylation, afforded methyl 4,6-di-O-acetyl-1,3-O-isopropylidene-alpha-D-fructofuranoside and 4-O-acetyl-2,3:5,6-di-O-isopropylidene-D-glucose dimethyl acetal as major products, while tosylation of the intermediate acetals provided methyl 6-O-tosyl-1,3-O-isopropylidene-alpha-D-fructofuranose.     

The structure of the capsular polysaccharide of Shewanella oneidensis strain MR-4

Capsular polysaccharides were extracted from Shewanella oneidensis strain MR-4, grown on two different culture media. The polysaccharides were analyzed using 1H and 13C NMR spectroscopy, and the following structure of the repeating unit was established: [structure: see text] where the residue of 4-amino-4,6-dideoxy-D-glucose (Qui4N) was substituted with different N-acyl groups depending on the growth media. All monosaccharides are present in the pyranose form. In the PS from cells grown on enriched medium (trypticase soy broth, TSB) aerobically it was N-acylated with 3-hydroxy-3-methylbutyrate (60%) or with 3-hydroxybutyrate (40%), whereas in the PS from cells grown on minimal medium (CDM) aerobically it was acylated mostly with 3-hydroxybutyrate (>90%).