Histochemical detection of l-gulonolactone: phenazine methosulfate oxidoreductase activity in several mammals with special reference to synthesis of vitamin C in primates

Summary An improved detection of activity of l-gulonolactone oxidase, which is responsible for the final oxidative step in the synthetic process of l-ascorbate from glucose in animals, was achieved using phenazine methosulfate and cyanide. Cold acetone fixation eliminated non-specific deposition of formazan on lipid droplets. The specificity of the method was tested and proven by a biological control, histochemical controls, inhibitors and activators. By application of the method, strong reactivity was found in the cytoplasm of centrilobular parenchymal cells of livers of the opossum, rat, ground squirrel and flying squirrel. Staining of dog liver was moderate and centrilobular. Prosimians were strongly positive: The centrilobular localization was found in the tree shrew and galago; slow lorises and some pottos showed strong reactivity in centrilobular cells and some peripheral cells as well. These prosimians seem to be able to synthesize l-ascorbate as many lower mammals are. On the contrary, true simians (i.e. the squirrel monkey, spider monkey, rhesus monkey and chimpanzee) were negative as guinea pigs were, suggesting their probable inability for l-ascorbate synthesis.Visiting scientist from the Department of Anatomy, Tokyo Medical and Dental University, Tokyo, Japan. T. R. Shanthaveerappa in previous publications, also fellow, Department of Anesthesiology, Emory University.     

Histochemical studies on the distribution of some enzymes of the glycolytic pathways in the olfactory bulb of the squirrel monkey (Saimiri sciureus)

Summary Detailed histochemical studies on the distribution of glycolytic enzymes have been made in the olfactory bulb of the Squirrel Monkey. The olfactory glomeruli, mitral cells, tufted cells, glial cells and nerve fibers are well equipped with the enzymes of the glycolytic pathways. Granule cells do not have the ability to synthesize or breakdown glycogen, but they have the Embden-Meyerhof-Parnas pathway and the Warburg-Dickens pathway. The synapses of the olfactory glomeruli may have the ability to break-down glycogen for an energy source. Small glial cells found in the olfactory glomeruli may be a special type of oligodendrocyte. Glial cells found abundantly in and around the olfactory glomeruli may be energy donators to the synapses of the olfactory glomeruli. It is suggested that oligodendrocytes and astrocytes of the olfactory bulb may have different branching enzymes.Visiting scientist from Anatomy Department, Tokyo Medical and Dental University, Tokyo, Japan. T. R. Shanthaveerappa in previous publications.     

Suppression of Oxygen Evolving System in Spinach Chloroplast Membranes due to Release of Manganese on Exposure to Osmotic Stress in vitro in Presence of Magnesium

When spinach chloroplast membranes were exposed to osmotic stress in vitro, by incubation in 1.0 M sorbitol + 10 mM MgCl₂ their oxygen evolving system was suppressed. The possible reasons for such inactivation of PS II mediated oxygen evolution were examined. There were conformational changes in the chloroplast membranes, as indicated by their absorption spectra. The pattern of sensitivity to DCMU was not altered. The sensitivity of PS II to water stress remained, even after a pre-wash treatment with NaCI (which removed 18 and 24 kD proteins) but not when the thylakoids were pretreated with NH₂0H or CaCl₂ (removed manganese and 33 kD). The manganese content of thylakoid membranes was markedly reduced under osmotic stress in presence of magnesium. We suggest that exposure of chloroplasts to 1.0 M sorbitol in presence of Mg₂₊ released manganese from thylakoid membranes, thereby leading to a suppression in oxygen evolution.     

Hydrophobic surfaces in oligosaccharides: linear dextrins are amphiphilic chains.

Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D-glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation.     

A possible cause of non-disjunction of additional chromosome 21 in Down syndrome

Summary A possible cause of non-disjunction of chromosome 21 in Down Syndromes has been cytogenetically evaluated by examining the parents by Ag-staining technique. In all the cases studied so far, the contributing parents have active ribosomal cistrons on both chromosomes 21 i.e. both chromosomes are stained positively by silver staining. These results show that the active NORs might play an essential role in meiotic non-disjunction. Furthermore, the preliminary results demonstrate that the acrocentric associations of homologous and non-homologous nature involving chromosome 21 are the most frequent in the contributing parent which may further indicate the role of multiple cellular factors affecting the associations in promoting the non-disjunction in addition to active NORs. The possible mechanisms regarding the non-disjunction of chromosome 21 have been described.Presented at the 34th Annual Meeting of the American Society of Human Genetics, Norfolk, VA, USA     

Liposomal Delivery Systems for Encapsulation of Ferrous Sulfate: Preparation and Characterization

Liposomal delivery systems for water-soluble bioactives were prepared using the pro-liposome and the microfluidization technologies. Iron, an essential micronutrient as ferrous sulfate and ascorbic acid, as an antioxidant for iron were encapsulated in the liposomes. Liposomes prepared by the microfluidization technology using 6% (w/w) concentration of the lipid encapsulated with ferrous sulfate and ascorbic acid had particle size distributions around 150 to 200 nm, whereas liposomes from the pro-liposome technology resulted in particle sizes of about 5 μm. The encapsulation efficiency of ferrous sulfate was 58% for the liposomes prepared by the microfluidization using 6% (w/w) lipid and 7.5% of ferrous sulfate concentrations, and it was 11% for the liposomes from pro-liposome technology using 1.5% (w/v) lipid and 15% of ferrous-sulfate concentration. Both the liposomes exhibited similar levels of oxidative stability, demonstrating the feasibility of microfluidization-based liposomal delivery systems for large-scale food/nutraceutical applications.     

Search for α3β2/3γ2 subtype selective ligands that are stable on human liver microsomes

Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA_A) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β_(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100 mg/kg and was devoid of sedative properties.     

Directionlet transform based sharpening and enhancement of mammographic X-ray images

Due to difficulty in detecting the low contrast and noisy nature of X-ray mammography images, they have to be enhanced to obtain a clear and good view. Though Sharpening Technique (ST) is used to enhance the contrast, it introduces noise in the enhancement process, and they do not include anisotropic features. This paper proposes a ST, which uses multiscale linear and anisotropic geometrical features obtained from directionlet transform (DT). The newly formulated method that combines multidirectional geometrical information has various tunable parameters and improved noise control by means of multiscale features. The DT that uses skewed and elongated directional basis functions not only captures the point singularities, but also links them into linear structure. The performance of the proposed DT ST is compared with non-linear unsharp masking (NLUSM). While the DT and LoG based sharpened images are given to the input of standard AHE, their performance is improved. Enhancement Measure and structural similarity measure are used to analyze the performance of the proposed method. Though the images are enhanced, the quality of the image is not degraded. As a specific application, the enhanced images are used to detect the microcalcification and spiculated masses in mammograms.     

Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain

Glycosylation in the CH2 domain of Fc is required for immunoglobulins G (IgGs) to exhibit immune effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent (Ab-dependent) cellular cytotoxicity (ADCC). We recently established that glycosylated Abs are more resistant to papain digestion than non-glycosylated IgGs (Biochem. Biophys. Res. Commun. 2006, 341, 797-803). To test whether specific Fc glycan structures affect Ab resistance to papain, we used in vitro glycoengineering methods to prepare homogeneous Ab glycoforms terminated with either sialic acid (G2S2), beta-galactose (G2), or N-acetylglucosamine (G0) and subjected them to papain digestions. Analyses of aliquots taken at different times during the digestions by matrix-assisted laser desorption-time-of-flight-mass spectroscopy (MALDI-TOF-MS) and high-performance liquid chromatography (HPLC) methods showed that the G0 glycoform was at least two times more resistant to papain digestion than the G2 and G2S2 glycoforms. The increased resistance of the G0 glycoform over the G2 and G2S2 glycoforms was independent of the specific Ab analyzed. A mouse/human chimeric version of Ab1, a fully human version of Ab2, and a humanized version of Ab3 exhibited a similar pattern of glycoform-dependent resistance. These data suggest that terminal sugars of Fc glycans may play important roles in Ab stability and affect resistance to proteases in addition to impacting Ab effector functions.