Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Early embryonic development of the vulnerable Shalyni barb,Pethia shalynius (Yazdani & Talukdar, 1975)

The present study provides the first detailed early embryonic development of the Shalyni barb, Pethia shalynius (Yazdani & Talukdar, 1975), a vulnerable cyprinid fish occurring in streams and lentic waters of Meghalaya, northeast India. Induced spawning by synthetic hormone injection in May 2019 was conducted to a pair of mature female and male P. shalynius under controlled conditions in a well-aerated aquarium. Fertilized eggs were spherical, 0.75–0.80 mm (approx.) in diameter, transparent, unpigmented and non-adhesive. A total of 22 developmental stages could be categorized under seven broad periods, viz. the zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatchling. The first cleavage occurred at 15 min post fertilization (mpf), followed by blastulation at 01:23 hr post-fertilization (hpf), gastrulation at 04:20 hpf, initial somite formation at 07:00 hpf, and pharyngula period at 19:20 hpf, respectively. Embryos hatched between 26–27 hpf and the newly-hatched larvae ranged 2.2–2.5 mm in total length. For naturally-declining populations of this vulnerable fish species, inferences drawn from the present study will help provide a baseline data for its conservation and management, and aid the research fields of developmental biology, biotechnology, molecular biology as well as taxonomy of this species.     

A detailed model and Monte Carlo simulation for predicting DIP genome length distribution in baculovirus infection of insect cells

Baculoviruses have enormous potential for use as biopesticides to control insect pest populations without the adverse environmental effects posed by the widespread use of chemical pesticides. However, continuous baculovirus production is susceptible to DNA mutation and the subsequent production of defective interfering particles (DIPs). The amount of DIPs produced and their genome length distribution are of great interest not only for baculoviruses but for many other DNA and RNA viruses. In this study, we elucidate this aspect of virus replication using baculovirus as an example system and both experimental and modeling studies. The existing mathematical models for the virus replication process consider DIPs as a lumped quantity and do not consider the genome length distribution of the DIPs. In this study, a detailed population balance model for the cell‐virus culture is presented, which predicts the genome length distribution of the DIP population along with their relative proportion. The model is simulated using the kinetic Monte Carlo algorithm, and the results agree well with the experimental results. Using this model, a practical strategy to maintain the DIP fraction to near to its maximum and minimum limits has been demonstrated.     

Phosphonoformate Esters of Anti-HIV Nucleosides: 3′-Azido-3′-deoxythymidine and 2′,3′-Dideoxycytidine Derivatives Containing a Small 5′-(0-Alkoxycarbon-ylphosphinyl) or 5′-(O-Cholesterylcarbonylphosphinyl) Substituent

Abstract

A convenient general method of synthesis of 5′-O-(alkoxycarbonyl)phosphonate esters of 2′,3′-dideoxyribonucleosides is presented, using the 5′-O-(methoxycarbonyl)phosphinyl, 5′-0-(ethoxycarbonyl)phosphinyl, and 5′-O-(cholesterylcarbonyl)phosphinyl derivatives of 3′-azido-3′-deoxythymidine (AZT) and the 5′-0-(ethoxycarbonyl)phosphinyl derivative of 2′,3′-dideoxycytidine (ddC) as examples. Reaction of trimethyl phosphonoformate, methyl phosphonoformate, or dimethyl cholesterylcarbonylphosphonate with phosphorus pentachloride in carbon tetrachloride, followed by direct condensation of the resulting phosphonyl chloride with the nucleoside, gave the fully esterified phosphonoformate derivatives, which on treatment with sodium iodide in tetrahydrofuran underwent selective cleavage of the P-OMe or P-OEt groups, leaving the carboxylate esters intact. The resulting products were converted from sodium salts to ammonium salts by ion-exchange chromatography.     

Preparation, application and testing of permanent antibacterial and antiviral coatings

Protocols for the synthesis of the microbicidal polycation N,N-dodecyl,methyl-polyethylenimine and coating (painting) of glass slides with this polycation's butanol solution are described. Subsequently detailed are the procedures for validating that the resultant coated slides are essentially 100% lethal to the human bacterial pathogens, Staphylococcus aureus and Escherichia coli, as well as to two common strains of influenza virus. The time required to prepare and apply the cationic polymer and to test its microbicidal efficiency is conservatively estimated to be <4 weeks.     

Cell culture and animal models of viral hepatitis. Part I: hepatitis B

Despite the existence of a preventative vaccine, HBV represents a substantial threat to public health, suggesting the need for research to develop new treatments to combat the disease. The authors review the available in vitro and in vivo models, including recently developed transgenic and chimeric mouse models.     

Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase in rabbit reticulocyte lysates

Heat shock protein 90 (Hsp90), an abundant and ubiquitous cytoplasmic protein has recently been indicated to participate in the regulation of protein synthesis by interacting with the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase, also known as the heme-regulated inhibitor (HRI). However, there exists an ambiguity on the exact nature of its action. In this investigation, the interaction of Hsp90 and HRI has been examined bothin vitro using purified proteins, andin situ in rabbit reticulocyte lysates subjected to heat shock and treatment with N-ethylmaleimide (NEM), a sulfhydryl reagent known to induce stress response. During heat shock or NEM-treatment of reticulocyte lysates, Hsp90 co-immunoprecipitated with activated HRI by anti-HRI monoclonal antibodies. Furthermore, the amount of Hsp90 being associated with HRI was a function of duration of heat shock and was correlated with the extent of HRI activation. Interestingly, simultaneous heat shock and NRM-treatment of reticulocyte lysates led to maximal association of HRI and Hsp90, leaving nearly no free HRI in the lysates.In vitro, with the purified proteins, the autokinase and the eIF-2α kinase activities of HRI were enhanced when HRI was pre-incubated with Hsp90, both in the presence and absence of hemin. These data, therefore, clearly demonstrate that Hsp90 interacts with HRI during stress, and that this association leads to activation of HRI and thereby inhibition of protein synthesis at the level of initiation. Considering the ubiquitous nature of Hsp90 and the presence of HRI or HRI-like eIF-2α kinase activity in a number of organisms, it is highly possible that Hsp90 may universally mediate down regulation of global protein synthesis during stress response.