Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

Summary We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the on-off model (E.B. George et al.,J. Membrane Biol.,103:245–253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a sigmoidally shaped increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.     

Non-involvement of lignin peroxidases and manganese peroxidases in 2,4,5-trichlorophenoxyacetic acid degradation byPhanerochaete chrysosporium

Summary Phanerochaete chrysosporium (ME-446) mineralized 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in high N medium and in malt extract medium in which lignin peroxidases (LIPs) and manganese peroxidases (MNPs) are not produced; furthermore,per mutant of ME-446, which lacks LIPs and MNPs, mineralized 2,4,5-T as well as the wild type. These results indicate that LIPs and MNPs are not required for 2,4,5-T degradation byP. chrysosporium.     

A lumped parameter mathematical model of the splanchnic circulation.

A lumped parameter mathematical model to describe the propulsion of blood in the splanchnic circulation was developed by integrating the principles of mechanics and physiology. A set of governing equations by derived by specifically considering the contractility of the portal vein, hepatic vein, liver sinusoids, and of the draining lymphatics. These equations were then simulated on a computer. The present simulation results substantiate previous experimental observations that hepatic venous pressure leads to portal hypertension and increased liver interstitial fluid volume.     

High-performance anion exchange-chromatography of neutral milk oligosaccharides and oligosaccharide alditols derived from mucin glycoproteins.

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH approximately 13) has been used to separate neutral oligosaccharides from human milk as well as oligosaccharide alditols isolated by alkaline borohydride degradation of O-linked glycoproteins having blood group A and H activities. Due to the diminished retention times of the alditols compared to their reducing counterparts, a very low base concentration (approximately 15 mM) was used in the fractionation of oligosaccharide alditols. The method appears to be ineffective in fractionation of monosaccharide alditols. Although the retention times generally increased with increasing oligosaccharide chain length, linkage of Fuc alpha-(1----2) to galactose and by Fuc alpha-(1----3) or Fuc alpha-(1----4) to glcNAc may decrease the retention times of both the alditols and the reducing oligosaccharides. Branching generally increased the retention times for oligosaccharide alditols. The retention times of isomers differing in the position of fucose substitution (LNF-1 vs LNF-2) differed greatly while those of the linkage isomers LNF-2 and LNF-3 were similar but distinct. Pulsed amperometric detection is sensitive at the picomole level both for these underivatized oligosaccharides and alditols. On-line desalting with an ion-exchange membrane has been found to be effective in preparative chromatography of these oligosaccharides for NMR spectroscopy and mass spectrometry.     

Polymorphism of the 3'' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III.

The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf.     

Uncoupling Warburg effect and stemness in CD133<Superscript>+ve</Superscript> cancer stem cells from Saos-2 (osteosarcoma) cell line under hypoxia

Cancer stem cells (CSCs) which are known to be residing deep inside the core of the tumor in its hypoxia niche is responsible for relapse of cancers. Owing to this hypoxic niche, the residing CSCs simultaneously fuel their stemness, cancerous and drug resistance properties. Attributes of CSCs are still not properly understood in its hypoxia niche. Addressing this, we sorted CSCs from Saos-2 (osteosarcoma) cell line using CD133 antibody. The CD133^+ve CSCs exhibited quiescent cell proliferation in DNA doubling, Ca²⁺ signaling and cell cycle analysis. CD133^+ve CSCs exhibited increased production of ATP and lactate dehydrogenase (LDH) activity under hypoxia. CD133^+ve cells exhibited decreased glucose uptake compared to ATP levels under hypoxia. Moreover, there was only negligible LDH activity in CD133^+ve cells under normoxia which do not rely on Warburg effect. Stemness markers (such as c-Myc, SOX2, Oct4 and TERT), metastasis marker (CD44) and drug resistance marker (ABCG2) were highly expressed in CD133^+ve cells. In summary, both CD133^+ve/?ve cells of Saos-2 (osteosarcoma) cell line did not exhibit Warburg effect under normoxic condition. Moreover, this significantly indicates an uncoupling between stemness and Warburg effect in CD133^+ve. This work provides a novel insight into the metabolic and functional features of CSCs in a hypoxic environment which could open new avenues for therapeutic strategies aimed to target CSCs.     

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV “O” 100 GPID₅₀ challenge virus.