A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Molecular determination of van genes among clinical isolates of enterococci at a hospital setting

Vancomycin-resistant enterococci (VRE) poses a formidable challenge to public health due to its inherent resistance to multiple antibiotics coupled with the ability to transfer genetic determinants to dangerous pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). The purpose of this study was to investigate the incidence of vancomycin resistance in enterococci among clinical isolates at a tertiary care military hospital in the eastern region of Saudi Arabia and to detect van genes using multiplex-PCR. Overall, 246 isolates of enterococci were collected from various clinical specimens. The isolates were identified, and antimicrobial susceptibility testing was done using the Vitek 2 system. Multiplex PCR was performed on the VRE isolates, thus identified to determine the van genes harbored. A total of 15 VRE were identified, of which 14 (93.3%) were Enterococcus faecium, and 1(6.7%) was Enterococcus casseliflavus with intrinsic vanC resistance. Of the 14 vancomycin-resistant Enterococcus faecium, 8 (57.1%) harbored vanB genes, while 6 (42.8%) harbored vanA genes. All the VRE were susceptible to linezolid and tigecycline. Our study detected a low prevalence (6.1%) of VRE among clinical isolates of enterococci and that the vanB gene predominates in such strains. Susceptibility profiles indicated that linezolid and tigecycline are still effective against these multidrug-resistant pathogens. Pus specimens yielded the highest percentage (53.3%) of isolates from which VRE was obtained, and this finding is novel among studies done in Saudi Arabia.     

Mechanism of non-enzymic transamination reaction between histidine and α-oxoglutaric acid

Non-enzymic transamination reactions at 85 degrees between various amino acids and alpha-oxoglutaric acid are catalysed by metal ions, e.g. Al(3+), Fe(2+), Cu(2+) and Fe(3+). The reaction is optimum at pH4.0. Of the 14 amino acids studied histidine is the most active. In the presence of Al(3+) histidine transaminates with alpha-oxoglutaric acid, forming glutamic acid and Al(3+)-imidazolylpyruvic acid complex as the end products. However, in the presence of Fe(2+) or Cu(2+) the products are glutamic acid and a 1:2 metal ion-imidazolylpyruvic acid chelate. The greater effectiveness of histidine in these reactions is attributed to the presence of the tertiary imidazole nitrogen atom, which is involved in the formation of stable sparingly soluble metal ion-imidazolylpyruvic acid complexes or chelates as end products of these reactions. Of the metal ions studied only Al(3+), Fe(2+), Fe(3+) and Cu(2+) are effective catalysts for the transamination reactions, and EDTA addition completely inhibits the catalytic effect of the Al(3+). Spectrophotometric evidence is presented to demonstrate the presence of metal ion complexes of Schiff bases of histidine as intermediates in the transamination reactions. These results may contribute to understanding the role of histidine in enzyme catalysis.     

Use of monoclonal antibody to immunochemically characterize variant-specific surface coat glycoprotein from Trypanosoma rhodesiense

Monoclonal antibodies were employed to study the molecular basis for charge heterogeneity in variant-specific surface coat glycoprotein prepared from clone CP3B4 of the Wellcome strain of Trypanosoma rhodesiense. Thirteen hybridomas secreting monoclonal antibodies specific for CP3B4 were obtained by fusing murine plasmacytoma cells to spleen cells from mice immunized with purified surface coat glycoprotein. The clone population of CP3B4 trypanosomes was shown to be homogeneous by means of immunofluorescent assays using culture supernatants from each of the 13 hybridomas. No cross-reactivity was found with other variant antigenic types of the same serodeme. Ascitic fluids were generated from 4 of the hybridomas and th molecular and epitopic specificities of the fluids or their IgG fractions were determined isoelectrofocusing of immunoprecipitates of radioiodinated glycoprotein antigen followed by autoradiography revealed that all 3 major components of the charge heterogeneous CP3B4 surface-coat glycoprotein were immunoprecipitated by each of the 4 monoclonal IgG fractions. Immunofluorescent staining of live trypanosomes was obtained with only one of the 4 ascitic fluids. The results show that charge heterogeneity does not derive from a heterogeneous population of parasites. Furthermore, the data indicate that there are at least 2 different epitopic specificities exhibited by the monoclonal antibodies tested and that each of the 3 charge heterogeneous components of the surface coat glycoprotein contains these epitopes. Charge heterogeneity of CP3B4 surface coat glycoprotein may be attributed to post-translational modification or to limited proteolysis.     

Countercurrent Distribution Studies on Hamycin

Hamycin, a polyene antifungal antibiotic, was isolated by use of countercurrent distribution. A pattern was obtained by plotting the absorption at 383 mmu of the contents of the various tubes against the tube numbers. The results indicated that the antibiotic contained three fractions, a major fraction (peak 2) comprising 48% of the total activity and two minor fractions (peak 1 and peak 3) comprising 3.62 and 11.32%, respectively, of the total activity. The solid material was isolated by pooling the contents of the tubes containing the major fraction, concentrating this in vacuo, and cooling. The antibiotic activities of the three peaks were evaluated by use of a cup-plate assay method with Paecilomyces varioti as test organism. All three components showed antibiotic activity; however, the preparation obtained from the major fraction showed approximately a 7-fold increase in antibiotic activity, a doubling of the E(1cm) (1%) value at 383 mmu, and approximately a 2.5-fold decrease in the amino acid content in comparison with the starting material. There was an apparent correlation obtained by plotting the curves of the absorption at 383 mmu of the different tubes comprising the major fraction and their biological activities.     

对“番泻甙”有代谢能力的几种肠内有益菌的分离和比较

本实验共分离到３属４种４２株肠内有益菌。上述菌株接种于１．０％“番泻甙”（Ｓｅｎｎｏｓｉｄｅｓ）Ｇａｍ ｂｒｏｔｈ中的培养结果表明,“番泻甙”对这些菌朱的增殖没有影响;并应用ＨＰＬＣ（高效液相色谱仪,直线梯度法）检测出４株具有“番泻甙”代谢能力的细菌,其中Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ ｂｒｅｖｅ２株、ＢＢｉｆｉｄｏｂａｃｔｅｒｉｕｍ ｌｏｇｎｕｍ１株、Ｅｎｔｅｒｏｃｏｃｃｕｓ ｆｅａｃａｌｉｓａ     

Determination of nicotine and cotinine in tobacco harvesters' urine by solid-phase extraction and liquid chromatography

A solid-phase extraction method using Drug Test-1 column containing chemically modified silica as a solid support for sample clean up and reversed phase ion-paired high-pressure liquid chromatography method have been developed for the simultaneous determination of nicotine and its metabolite cotinine from the urine samples. Mobile phase was consisted of acetate buffer (containing 0.03 M sodium acetate and 0.1 M acetic acid) pH 3.1 and acetonitrile (78:22% (v/v)) containing 0.02 M sodium octanosulfonate as an ion pair agent. pH of the mobile phase was adjusted to 3.6 with triethylamine for better resolution and to prevent peak tailing. The linearity was obtained in the range of 0.5-10 microg/ml concentrations of nicotine and cotinine standards. The correlation coefficients were 0.998 for cotinine and 0.999 for nicotine. The recoveries were obtained in the range of 79-97% with average value of 85% for nicotine and in the range of 82-98% with average value of 88% for cotinine. The limit of detection was 2 ng/ml for cotinine and 5 ng/ml for nicotine with 2 ml urine for extraction, calculated by taking signal to noise ratio 10:3. The intra-day co-efficient of variation (CV) were <4 and 7% and inter-day CV were <9 and 7% for nicotine and cotinine, respectively. The method was applied to the urine samples of tobacco harvesters, who suffer from green tobacco sickness (GTS) to check the absorption of nicotine through dermal route during the various processes of tobacco cultivation due to its good reproducibility and sensitivity.     

Bacillus subtilis as potential producer for polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.