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Density (rho), apparent molar volume (V(phi)), and viscosity (eta) of 0.0010 to 0.0018% (w/v) of bovine serum albumin (BSA), egg albumin, and lysozyme in 0.0002, 0.0004, and 0.0008 M aqueous RbI and CsI, and (dodecyl)(trimethyl)ammonium bromide (DTAB) solutions were obtained. The experimental data were regressed against composition, and constants are used to elucidate the conformational changes in protein molecules. With salt concentration, the density of proteins is found to decrease, and the order of the effect of additives on density is observed as CsI > RbI > DTAB. The trend of apparent molar volume of proteins is found as BSA > egg-albumin > lysozyme for three additives. In general, eta values of BSA remain higher for all compositions of RbI than that of egg-albumin for CsI and DTAB. These orders of the data indicate the strength of intermolecular forces between proteins and salts, and are helpful for understanding the denaturation of proteins.

Open Access online publishing is the trend of the future for unrestricted rapid and international dissemination of knowledge. Several journals are published on acquired immune deficiency syndrome (AIDS) research, but none of them appear to be Open Access. To eliminate or to abate the scourge of AIDS, it is important that the knowledge acquired through research be disseminated as soon as possible. The Open Access journal, AIDS Research and Therapy, is intended to fill this knowledge gap by online publication of basic, preclinical, and clinical research articles.

A new efficient reusable universal polymer support for oligonucleotide synthesis, based on a non-ammoniacal cleavable linker, is described. Twenty six cycles of oligonucleotide synthesis have been carried out without compromising the quality of the fully deprotected oligonucleotides.

In this paper, we introduce nonlinear large-signal scattering ( [Formula: see text]) parameters, a new type of frequency-domain mapping that relates incident and reflected signals. We present a general form of nonlinear large-signal [Formula: see text]-parameters and show that they reduce to classic [Formula: see text]-parameters in the absence of nonlinearities. Nonlinear large-signal impedance ( [Formula: see text]) and admittance ( [Formula: see text]) parameters are also introduced, and equations relating the different representations are derived. We illustrate how nonlinear large-signal [Formula: see text]-parameters can be used as a tool in the design process of a nonlinear circuit, specifically a single-diode 1 GHz frequency-doubler. For the case where a nonlinear model is not readily available, we developed a method of extracting nonlinear large-signal [Formula: see text]-parameters obtained with artificial neural network models trained with multiple measurements made by a nonlinear vector network analyzer equipped with two sources. Finally, nonlinear large-signal [Formula: see text]-parameters are compared to another form of nonlinear mapping, known as nonlinear scattering functions. The nonlinear large-signal [Formula: see text]-parameters are shown to be more general.

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5'- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5'-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.

A new heterobifunctional reagent, N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) has been developed, useful for making bioconjugates and immobilization of biomolecules, viz., oligonucleotides, peptides, proteins, etc., on a variety of carbon-containing solid surfaces. Its trifluoroethanesulfonate ester group reacts with aminoalkyl or mercaptoalkyl functions present in biomolecules, and the anthraquinone structure reacts with a variety of carbon-containing polymers under ultraviolet irradiation (365 nm). The reagent has been used in two ways. First, the reagent, NTPAC, was first brought in contact with the above said supports and exposed to long wavelength ultraviolet light (365 nm), thereby generating active trifluoroethanesulfonate ester functions on the support, which subsequently react with appropriate mercaptoalkyl- or aminoalkyl-containing biomolecules to fix them on the supports. In another route, the proposed reagent was allowed to react first with proteins or 5'-aminoalkyl- or mercaptoalkyl-modified oligonucleotides to form the appropriate biomolecule-anthraquinone conjugate, which was then brought in contact with a variety of carbon-containing polymers, viz., modified controlled pore glass (CPG), modified glass microslides, cross-linked polystyrene, nylon, cross-linked polysaccharides, polypropylene (PP), polyethylene (PE), etc., and exposed to long wavelength ultraviolet light (365 nm), resulting in immobilization of the conjugates on the support. Both of the routes work satisfactorily and we could successfully immobilize a number of enzymes and modified oligonucleotides on a variety of supports.

Short synthetic oligonucleotides are finding wide variety of applications in area of genomics and medicinal chemistry. Since the isolation of nucleic acids to the mapping of human genome, chemical synthesis of nucleic acids has undergone tremendous advancements. Further improvements in this area such as, introduction of high throughput synthesizers, better coupling reagents, improved polymer supports, newer sets of protecting groups for exocyclic amino groups of nucleic bases and introduction of universal polymer supports have completely revolutionized the entire field of nucleic acids chemistry. Most of these developments have been targeted to assemble these molecules more efficiently in a cost-effective manner and rapidly. Preparation of oligonucleotide conjugates has further helped in identifying the newer areas of their applications. A number of conjugates with biological and abiological ligands have been discussed in this article along with their possible wide spectrum of applications. Recently developed microarray technology, which refers to attachment of short oligonucleotides on a solid/polymeric surface, has proved to be useful for screening of genetic mutations, study of polymorphism, as diagnostics, etc. The major developments in these areas are presented in the review.

A simple method has been devised to construct oligonucleotide array on a variety of surfaces using commonly available reagents and chemistry with good efficiency and accuracy. The method involves the generation of hydroxyl functionalities on glass, polypropylene, polyethylene, and commonly used surfaces for construction of oligonucleotide arrays followed by their activation with trifluoroethanesulfonyl chloride (tresyl chloride). The activated surface in the subsequent reaction is used to covalently immobilize oligonucleotides in regioselective fashion to create an oligonucleotide array. The surface bound tresyl sulfonate esters allow the immobilization of oligonucleotides specifically via their 3'- or 5'-end having mercaptohexyl- or aminohexyl functionalities. The constructed oligonucleotide arrays were successfully used to analyze oligonucleotides by hybridization technique.

Temperature-responsive cellulose has been obtained by graft copolymerization of N-isopropylacrylamide (NIPAAm) monomer using ceric ammonium nitrate (CAN) as initiator at 25.0 +/- 0.1 degrees C in acidic medium. Kinetic and grafting parameters were evaluated at different concentrations of NIPAAm ranging from 1.25 x 10(-3) to 12.5 x 10(-3) mol dm(-3) and varying concentrations of CAN from 1.5 x 10(-3) to 9.0 x 10(-3) mol dm(-3) at constant concentration of nitric acid (2.5 x 10(-2) mol dm(-3)). The graft copolymerization of NIPAAm onto cellulose has shown a significant increasing trend below lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAAm) and shown low energy of activation (18.0 kJ mol(-1)) for graft copolymerization within the temperature range of 10-35 degrees C as determined with Arrhenius plot. The PNIPAAm-grafted cellulose has shown improved thermal stability and shown temperature-dependent degree of swelling. Variation in degree of swelling of PNIPAAm-grafted cellulose as a function of temperature has been used to determine LCST of PNIPAAm-grafted cellulose. The contact angle (theta) has shown variation on increasing the graft yield and temperature. On the basis of experimental observations, the reaction steps for graft copolymerization have been proposed and a rate expression has been derived.

Polyamine-assisted deprotection conditions have been developed for the rapid and clean cleavage of oligonucleotide chains from a cis-diol group bearing universal polymer support, making it compatible with modern oligonucleotide synthesis via all types of phosphoramidite synthons, including base labile protecting group bearing synthons as well. The synthesized oligonucleotides were found to be comparable with the corresponding standard oligomers with respect to their retention time on HPLC, mass on MALDI-TOF and biological activity in PCR amplification.

Ceric ammonium nitrate (CAN) in the presence of nitric acid has been used as efficient initiator for graft copolymerization of the ethyl acrylate onto cellulose at 35.0 +/- 0.1 degrees C. Graft copolymerization of ethyl acrylate onto cellulose has taken place through the radical initiation process. The graft yield and other grafting parameters have been evaluated by varying concentration of ethyl acrylate from 2.5 x 10(-1) to 15.0 x 10(-1) mol dm(-3) and ceric ammonium nitrate from 5.0 x 10(-3) to 25.0 x 10(-3) mol dm(-3) at constant concentration of the nitric acid (8.0 x 10(-2) mol dm(-3)). The rate of graft copolymerization has shown 1.5 order with respect to the concentration of the ceric ammonium nitrate. The graft copolymerization data obtained at different temperatures were used to calculate the energy of activation, which has been found to be 28.9 kJ mol(-1) within the temperature range from 20 to 50 degrees C. The effect of addition of cationic and anionic surfactants on graft copolymerization has also been studied. On the basis of the experimental observations, reaction steps have been proposed and a suitable rate expression for graft copolymerization has been derived.

A rapid and high yielding method for the synthesis of precursors of synthons for DNA synthesis, N-protected 2'-deoxyribonucleosides is described, which occur under mild conditions using microwave irradiation. The desired material, N-protected nucleosides, was obtained in 93-96% yield in few minutes. The final products were then characterized by 1H-NMR and MALDI-TOF and compared with the standard samples. The method is amenable to small to moderate scale of synthesis.

Semi-interpenetrating polymer network (IPN) microspheres of chitosan and poly(ethylene glycol) PEG were prepared for controlled release of drugs. A new method for the chemical crosslinking of chitosan microspheres containing isoniazid (INH) as a model drug is proposed and evaluated. The method consists of the exposure of microspheres to the vapor of crosslinking agent that act in gaseous phase under mild conditions. The structural analysis of the microspheres was carried out by FTIR-analysis. The swelling behavior, hydrolytic degradation, structural changes of the microspheres and loading capacity (LC) of the microspheres for INH were investigated. The prepared microspheres have shown 93% drug loading capacity, which suggested that these semi-IPN microspheres are suitable for controlled release of drugs in an oral sustained delivery system., (Copyright 2001 Kluwer Academic Publishers)

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV(DH12)) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups (P = 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV(DH12) strongly correlated with the level of NAbs directed against the virus on the day of challenge (P = 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV(DH12) challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) (P = 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.

The graft copolymerization of acrylonitrile and ethyl methacrylate from their binary mixtures onto cellulose has been carried out in the presence of Ce(IV) ions in acidic media at 30 +/- 0.1 degrees C. The graft copolymerization has shown an increasing trend on increasing the feed molarity of the comonomers and on increasing the reaction time. The analysis of grafted cellulose prepared with different feed compositions (f(AN)) has clearly indicated the existence of the synergistic effect of ethyl methacrylate on acrylonitrile which normally shows poor affinity for grafting on cellulose while used individually. The elemental analysis and IR data were used to determine the composition of grafted copolymers and reactivity ratios (r(1), r(2)) of the monomers using the Mayo and Lewis method. The reactivity ratios for acrylontrile and ethyl methacrylate have been found to be 0.68 and 1.15, respectively, which supported the alternate arrangement of the monomer blocks of individual monomers in the grafted chains. The rate of grafting has shown a square dependence on the concentration of the comonomers within the studied range of feed molarity from 0.5 to 2.5 mol dm(-3). The grafting data were used to calculate the number of grafted chains (N(g)), frequency of grafting (G(F)) per molecule of the cellulose and efficiency of grafting (G(E)). The variation in these parameters as a function of feed molarity, feed composition, and ceric ion concentration has been determined. The rate of ceric ion disappearance as a function of feed molarity and reaction time has provided strong support to consider the participation of ceric ions in the formation of free radicals at the backbone of the cellulose. On the basis of the experimental observations, reaction steps for grafting of monomers on cellulose have been proposed.

Objective: To evaluate the effect of varying ingestion of drinking water containing high fluorides and its effect on serum parathyroid hormone., Design: Cross sectional clinical study., Setting: S.M.S. Medical College, Jaipur., Subject: 200 children were selected from four areas (50 from each area) consuming water containing 2.4, 4.6, 5.6 and 13.5 mg/l of fluoride. All children were in an age group of 6 to 12 years., Methods: All children were graded for clinical, radiological and dental fluorosis and biochemical estimations were made for serum calcium, serum and urinary fluoride and serum parathyroid hormone., Results: Serum calcium levels were well within normal range in the patients of all areas but an increase in serum parathyroid levels (S. PTH) was noted. The increased S. PTH was well correlated with increase in fluoride ingestion. The severity of clinical and skeletal fluorosis was observed to increase with increase in S. PTH concentration., Conclusions: High Fluoride ingestion has a definite relationship with increased parathyroid hormone secretion, which may be responsible for maintaining serum calcium levels and may have a role in toxic manifestations of fluorosis.

Potential response of cadmium(II) ion selective electrode based on cyanocopolymer matrices and 8-hydroxyquinoline as ionophore has been evaluated by varying the amount of ionophore, plasticizer and the molecular weight of the cyanocopolymer. The sensitivity, working range, response time, and metal ions interference have shown a significant dependence on the concentration of ionophore, plasticizer and molecular weight of cyanocopolymers. The electrodes prepared with 2.38x10(-2) mol kg(-1) of ionophore, 1.23x10(-2) mol dm(-3) of plasticizer and 2.0 g of cyanocopolymer (molecular wt., 59 365) have shown a Nernstian slope of 29.00+/-0.001 mV per decade activities of Cd(2+) ions with a response time of 12+/-0.007 s. Electrodes have shown an appreciable selectivity for Cd(2+) ions in the presence of alkali and alkaline earth metal ions and could be used in a pH range of 2.5-6.5. The cyano groups of the copolymers contributed significantly to enhance the selectivity of the electrode. The electrode has shown an appreciable average life of 6 months without any significant drift in the electrode potential and found to be free from leaching of membrane ingredients. Electrode response is explained considering phase boundary model based on thermodynamic considerations.

Beads and microgranules carriers have important potential applications for the administration of therapeutic molecules. A novel approach for the preparation of chitosan beads and microgranules is presented. The present work is an investigation of the in vitro release kinetics of diclofenac sodium (DFS) from chitosan beads and microgranules. The in vitro release profiles of DFS from chitosan beads and microgranules are monitored using Shimadzu 1601 UV-VIS spectrophotometer. Drug release behavior of beads and microgranules has been compared. The release rate of DFS from the beads has been found to be slower in comparison to the microgranules. It may also be noted that the percent and amount of the drug release were much higher in acidic solution than in basic solution, probably due to the swelling properties of the matrix at acidic pH.

Phosphorylation of the negative-sense RNA virus phosphoproteins is highly conserved, implying functional significance. Sendai virus (SV) phosphoprotein (P) is constitutively phosphorylated at S249. Abrogation of the SV P primary phosphorylation causes phosphorylation of P at alternate sites, creating a problem in determining the function of phosphorylation. We have now identified the alternate phosphorylation sites using two-dimensional phosphopeptide analysis of several deletion and point mutants of the P protein. The alternate phosphorylation sites were mutagenized to create P with (S249combo) or without (combo) primary phosphorylation. The combo protein has less than 10% phosphorylation compared with the wild-type P or S249combo. Functional analysis of the mutant proteins using a Sendai virus minigenome replication system showed that the combo P protein was as proficient in supporting minigenome replication as the wild-type P in cell cultures. These studies suggest that like the primary, the alternate phosphorylation of the P protein is also dispensable for virus replication in cell cultures. Interestingly, the ability of the multiple site mutant of P (combo mutant has eight serine residues changed to alanine residues) to support efficient virus RNA synthesis suggests that the P protein has a high flexibility at least in its sequence and perhaps also in structure., (Copyright 2000 Academic Press.)

Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis., (Copyright 1999 Academic Press.)

Two sets of deprotection conditions have been evolved for the deprotection of oligodeoxyribonucleotides and their cleavage from commercially available cis -diol group-bearing universal polymer supports. In the first case, oligodeoxyribonucleotides anchored on the universal support were subjected to one of the standard deprotection conditions followed by treatment with aqueous 0.5 M sodium chloride + 0.2 M sodium hydroxide solution for 30 min at room temperature. In the second case, oligonucleotides bound to the universal support were treated with methanolic sodium hydroxide solution under microwave radiation to obtain fully deprotected oligomers within 4 min. Under both conditions, the cleavage of oligonucleotides from the support and their deprotection occurred quantitatively without any side product formation. The cleaved oligonucleotides were found to be identical in all respects (retention time on HPLC and biological activity in PCR) to the corresponding standard oligo-nucleotides.

RNA-dependent RNA polymerases of single-stranded, negative-sense RNA viruses comprise a phosphoprotein (P) and a large protein. The constitutive phosphorylation of the P protein in these viruses is highly conserved, yet the functional significance of phosphorylation is enigmatic. To approach this problem, phosphorylation sites were determined in two closely related paramyxovirus P proteins. Sendai virus (SV) is a prototypic paramyxovirus. Previously, using a phosphopeptide mapping technique, the primary constitutive phosphorylation site of SV P protein was mapped to Ser-249. Phosphorylation at Ser-249 is dependent on the presence of Pro-250. Human parainfluenza virus type 1 (HPIV-1) P protein has 66% similarity to SV P protein and its predicted secondary structure is highly similar to that of SV P protein. However, there is no obvious conserved phosphorylation site in HPIV-1 P protein. Using the phosphopeptide mapping strategy, the constitutive phosphorylation sites of HPIV-1 P protein were mapped. The HPIV-1 P protein is primarily phosphorylated at Ser-120. Phosphorylation at Ser-120 is dependent on the presence of Pro-121. It also has a minor phosphorylation site at Ser-184. The sequence at Ser-184 does not match any consensus phosphorylation target site for the known kinases. Significantly, the P proteins from both viruses are constitutively and primarily phosphorylated at one serine and the phosphorylation of that serine is dependent on the presence of a proline on its carboxyl side.

A novel method for the deprotection of oligodeoxyribonucleotides under microwave irradiation has been developed. The oligodeoxynucleotides having base labile, phenoxyacetyl (pac), protection for exocyclic amino functions were fully deprotected in 0. 2 M sodium hydroxide (methanol:water : : 1:1, v/v) = A and 1 M sodium hydroxide (methanol:water : : 1:1, v/v) = B using microwaves in 4 and 2 min, respectively. The deprotection of oligodeoxyribonucleotides carrying conventional protecting groups, dAbz, dCbzand dGpac, for exocyclic amino functions was achieved in 4 min in B without any side product formation. The deprotected oligonucleotides were compared with the oligomers deprotected using standard deprotection conditions (29% aq. ammonia, 16 h, 55 degrees C) with respect to their retention time on HPLC and biological activity.

The polycistronic Sendai virus P/C mRNA is translated into five proteins (P, C', C, Y1 and Y2) from distinct start sites in virus-infected cells. The translation mechanism(s) of these proteins from two overlapping open reading frames in the P/C mRNA are poorly understood [Gupta, Ono and Xu (1996) Biochemistry 35, 1223-1231]. While investigating the initiation mechanism of C' from an ACG start site, we found that C' synthesis was resistant to inhibitors of peptide chain elongation such as cycloheximide (CHX) and anisomycin, but not to pactamycin (an inhibitor of chain initiation) or puromycin (a peptide chain terminator). Moreover, low levels (less than 30 microg/ml) of CHX significantly stimulated C' synthesis. Whereas C' synthesis was stimulated, synthesis of the P and C proteins, which are translated from the same mRNA, decreased by more than 95%. Stimulation of C' synthesis by CHX is not related to its initiation at an ACG codon. Mutation of ACG to alternative start sites had no effect on the CHX-stimulated C' synthesis. Similarly, C' synthesis was preferentially stimulated when Sendai virus-infected cells were exposed to hypotonic growth medium. These results suggest that the P/C mRNA may exist in at least two reversible conformations: whereas one conformation allows synthesis of the P and C proteins, the alternative conformation allows synthesis of the C' protein. It might be that low concentrations of CHX somehow increase the alternative conformation, which increases C' synthesis. The C' protein synthesis is reminiscent of the synthesis of stress-related proteins. Perhaps Sendai virus has evolved a novel mechanism to express both non-stress-related and stress-related proteins from the same mRNA.

The Sendai virus polycistronic P/C mRNA encodes the P and C proteins from alternate overlapping reading frames. To determine the functions of these proteins in virus replication, hammerhead ribozymes were targeted to cleave the 5'-untranslated region of the P/C mRNA. Both cell-free and intracellular assays were employed to determine ribozyme efficacy. To appropriately compare activities between cell-free and intracellular assays, identical ribozymes were synthesized in vitro as well as expressed in cells. Ribozyme parameters, namely hybridization arm length (HAL) and nonhybridizing extraneous sequences (NES), were found to have rate-determining properties. In cell-free reactions, ribozymes with 13-mer HAL were up to 10-fold more efficient than those with 9-mer HAL. Ribozymes with 9-mer HAL were relatively ineffective in transfected cells. Minimizing the number of NES increased ribozyme efficiency in vitro. However, ribozymes with minimal NES were essentially inert intracellularly. The NES at the termini of the most effective intracellular ribozyme, Rz13st ( approximately 95% inhibition of the p gene expression), were predicted to fold into stem-loop structures. These structures most likely increase ribozyme stability as evidenced by the 8-fold higher resistance to ribonuclease T2 digestion of Rz13st compared with Rz13B. Our results suggest that when designing effective intracellular ribozymes, parameters that enhance formation of productive ribozyme:substrate duplexes and that increase RNA stability should be optimized.

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

Stroke, though considered a thromboembolic disorder, is known to be associated with hyperlipidaemia. In Western country, some workers have performed studies exploring the role of lipids in stroke in their country. Such a study is lacking in Indian population. This study was therefore conducted to observe the role of lipids in stroke by evaluating 13 parameters of lipids in 48 patients of non haemorrhagic cerebral stroke hospitalised in acute condition and compared with those of 70 age matched normal subjects. Results revealed that phospholipids and arachidonic acid were significantly altered in patients of acute stroke.

Previously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in virions was primarily and constitutively phosphorylated on serine(s) in a single tryptic phosphopeptide TP1. By two-dimensional thin-layer electrophoresis and chromatography analysis of tryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is serine249. Interestingly, when serine249 was deleted or mutagenized alternate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (delta 208-236). Mutagenesis of proline250 to alanine abrogated phosphorylation at serine249 suggesting that proline250 is essential for the primary phosphorylation of the P protein. Conceivably, serine249 phosphorylation is mediated by a proline-directed protein kinase. This finding is unusual because a majority of the P proteins from other negative-strand RNA viruses have been shown to be phosphorylated primarily by casein kinase II. Our results demonstrate that the P protein has a strong potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all potential sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural integrity of the Sendai virus P protein.

The polycistronic P/C mRNA of Sendai virus encodes five proteins (C', P, C, Y1, and Y2) each of which initiates from a distinct start site. Two major proteins, P and C, are expressed in approximately equimolar amounts from two consecutive AUGs in overlapping reading frames. To better understand the mechanism of expression of the C protein from a downstream AUG, site-directed mutants of the P/C mRNA were created and expressed in COS1 cells. The secondary structure of the mRNA was examined to determine whether the mRNA structure played any role in the synthesis of the C protein. Our results ruled out any significant involvement of the 5' UTR, sequence contexts, secondary structure, distance between the start sites, and sequences downstream to the C-AUG. However, they are consistent with the concept that the synthesis of the C protein is primarily dependent on the orientation of its reading frame, i.e., +1 in relation to the upstream P reading frame. The downstream reading frame was translated poorly when it occurred in +2 orientation in relation to the upstream reading frame. Interestingly, all the known functional bicistronic mRNAs with overlapping reading frames from cytoplasmic RNA viruses have their downstream reading frame in +1 orientation relative to the upstream frame. We propose that the evolutionary conservation of the downstream reading frame in +1 orientation in these bicistronic mRNAs is important for its efficient translation.

Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotide-directed site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with > 90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient.

Expression of the platelet derived growth factor (PDGF) B-chain, the product of the c-sis proto-oncogene, is regulated both at the transcriptional and translational level. Previous studies have shown that the long 5' untranslated region (UTR) of the c-sis mRNA strongly inhibits synthesis of the PDGF-B chain. However, the assignments of down-regulatory regions within the 5' UTR were ambiguous. Expression of several site-directed point and deletion mutants of the 5' UTR of the c-sis mRNA in COS1 cells revealed that the UTR inhibited PDGF-B chain synthesis in a more complex manner than indicated by the previous studies. Abrogation of the three upstream short open reading frames by mutating each of the AUGs did not have any effect on the synthesis of the PDGF-B chain. Expression of deletion mutants revealed two partially overlapping regions, nucleotides 1-651 and 475-1022, each of which independently inhibited c-sis mRNA translation as effectively as the entire 5' UTR. Each of these regions contains a potentially strong stem-loop structure and a GC-rich element. These elements of the alternate down-regulatory regions could interact within the same region and/or with the elements of the other regulatory region to block c-sis mRNA translation. We show, in contrast to the previous reports, that the inhibition of c-sis mRNA translation cannot be attributed exclusively to any particular predicted secondary structure or a GC-rich element within the 5' UTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation status of the Sendai virus P protein was examined during virus infection and compared with cell-free phosphorylation. P protein from Sendai virus-infected (VI) and P/C gene-transfected (PT) mammalian cells and from purified virions (PV) was phosphorylated at only serine residues. In contrast, cell-free phosphorylation of the P protein with virion-associated protein kinase (VAPK) occurred at both threonine and serine. Tryptic phosphopeptide maps of the P protein from VI, PT, and PV showed that the phosphorylation was primarily localized on one peptide (TP1), while VAPK phosphorylated the P protein on several peptides. There was no change in the steady-state phosphopeptide map of the P protein during virus replication, indicating that the TP1 is constitutively phosphorylated. Inhibition of cellular phosphatases (PP1 and PP2A) by okadaic acid (OA) in virus-infected cells caused a sixfold increase in the P protein phosphorylation, solely at serine residues. The phosphopeptide map of the OA-P protein revealed that phosphorylation occurred on several peptides, but the OA-P map was significantly different from the VAPK-P map. However, additional phosphorylation of the P protein did not block its association with nucleocapsids. These results suggest that the Sendai virus P protein is constitutively phosphorylated primarily at one locus but has the potential for phosphorylation at additional sites. Further, our results do not show any correlations between the intracellular and cell-free phosphorylation of the P protein and, therefore, question the validity of cell-free phosphorylations.

Sendai virus P/C mRNA, human erythrocyte membrane protein 4.1 mRNA and PDGF-B chain mRNA were used to test whether translation initiation from non-AUG codons in COS1 cells was mRNA species dependent. Site-directed mutants of the authentic translation start sites of these mRNAs to alternate start codons showed that while P/C mRNA is capable of initiating translation from non-AUG start sites the other two mRNAs are not. Our study shows that translation initiation from non-AUG codons is mRNA species dependent and suggests that higher order structure of an mRNA determines the non-AUG translation start site.

The effect of single oral dose of 1 gm gugulipid was studied on bioavailability of single oral dose of propranolol (40 mg) and diltiazem (60 mg) in 10 and 7 normal healthy male volunteers respectively. It was a randomised within group crossover study. Blood samples were collected at hourly intervals upto 8 hrs. Gugulipid significantly reduced (P < .01) peak plasma concentration (Cmax) and area under curve (AUC 0-8 hrs) of both the drugs in normal volunteers. Such interaction in patients receiving propanolol or diltiazem with gugulipid may lead to diminished efficacy or nonresponsiveness due to significant reduction in bioavailability.

We show that the Sendai virus C' and C proteins exist as both phosphorylated and nonphosphorylated forms, while the Y1 and Y2 proteins are not phosphorylated in virus-infected cells. Phosphorylation occurs primarily on serine residues, most likely at the N-terminus of the C' and C proteins. Phosphatases PP1 and PP2A significantly modulate the phosphorylation status of the C proteins as evidenced by okadaic acid inhibition of these phosphatases. The other Sendai virus proteins including the cotranslationally expressed P protein are not necessary for the appropriate phosphorylation of the C' and C proteins. Differential phosphorylation and potential for the modulation of phosphorylation suggests regulatory functions for the C proteins.

Screens aid in identifying some severe irritants or corrosives and eliminating them from consideration for in vivo eye irritation testing. Products may be evaluated for ocular irritation potential in a stepwise progression as follows: (1) products at pH extremes of 2 or below or of 11.5 or above may be considered to be ocular irritants; (2) based on chemical structure-activity considerations, some products may be judged to have ocular irritation potential; (3) validated and accepted in vitro systems may possibly be used as a screen in the future; (4) when a test material demonstrates severe acute dermal toxicity (lethality at < or = 200 mg/kg body weight), further testing for either dermal or ocular irritation may not need to be undertaken; (5) if a substance shows a primary dermal irritation index of 5 or above, it may be considered to be an ocular irritant; (6) materials that are not removed from consideration based on these proposed screens may then be considered for testing for ocular irritation in rabbits under accepted procedures. In a survey given to participants in the workshop, a high percentage believed that screens should be used. However, opinions on the use of the individual screens varied between the different interested groups attending, with the possible future use of in vitro screens for specific product lines having the highest percentage of agreement (57-100%).

An in vivo test protocol and an evaluation and classification system for the determination of eye irritation potential of chemicals and mixtures (substances) is proposed. The protocol uses two or three rabbits and reduces distress in test animals. The test substances are classified as non-irritant, irritant or severe irritant to meet regulatory needs. They may be classified on the basis of past experience with similar compounds or mixtures. Screens such as structure-activity relationships, pH extremes, validated and accepted in vitro tests, severe dermal irritation (primary dermal irritation index > or = 5) or severe dermal toxicity (lethality at < 200 mg/kg body weight) should be used to classify irritant or severe irritant materials when one or more of the screens can provide convincing evidence. For suspected severe irritant materials, the proposed in vivo test permits the use of one rabbit and instillation of 0.01 ml (0.01 g) of the test material into the cornea. Materials that are not classified irritant or severe irritant by screens or severe irritant by one rabbit test are tested in two or three rabbits; 0.1 ml (0.1 g) is instilled into the conjunctival sac. The responses (corneal opacity, iritis and conjunctival redness) are scored according to the modified Draize scoring system at 24, 48 and 72 hr and 7 days post-instillation. A rabbit is considered positive when corneal opacity of 1 or above, iritis of 1 or above or conjunctival redness of 2 or above is present at 24, 48 or 72 hr post-instillation. The material is classified as a severe irritant when the rabbit in the one-animal test or two or more rabbits in the standard test have responses of corneal opacity of 3 or above and iritis of 2 at 24, 48 or 72 hr, or positive responses on day 7 after instillation. The material is classified as an eye irritant when two or more rabbits are positive but the responses are not severe and they clear 7 days after instillation. The material is classified as a non-irritant when no more than one rabbit is positive. The opinions expressed in this article are those of the authors and do not necessarily reflect the views of US Federal agencies.

US regulatory agencies have used six animals in eye irritation tests. Analyses of eye irritation tests on pesticides (n = 48), consumer products and cosmetics (n = 53), Marzulli and Ruggles database (n = 139), and cleaning products and ingredients (n = 30) have greatly extended previous investigations of the merit of reducing animal sample size in the eye test. Given the existing scoring system for positive animal responses (corneal opacity > or = 1, iritis > or = 1, conjunctival redness > or = 2 and conjunctival chemosis > or = 2), the accuracy of the classification systems currently used by these agencies was determined. The US Consumer Product Safety Commission, US Food and Drug Administration, and US Occupational Safety and Health Administration use a classification system by which a substance is designated as an irritant when at least four of six animals give a positive response. This decision rule leads to a very high accuracy of at least 99% with essentially no false positive and false negative judgments. In contrast, the system used by the US Environmental Protection Agency pesticide program, in which only one or more of six treated animals result in an irritant decision, has an accuracy of only 50-80% with very high false positive rates. Analyses indicated that test sample size could be reduced to three and still preserve very good accuracy, whereas two-animal and one-animal tests did not give satisfactory responses. A two-stage test, in which two animals are tested and evaluated in the first stage before the need for testing one more animal in the second stage is determined, also demonstrated good operating characteristics. Both the one-stage/three-animal test and the two-stage test deserve consideration.

Pretreatment of the eyes of rabbits with a topical anaesthetic can be viewed as a refinement of the test for eye irritation. It reduces pain at the time of test-material administration, decreases animal distress and permits easier application of the test agent to the eye. In some cases, however, use of an anaesthetic either alone or in combination with the test substance may alter ocular responses or provide little benefit. Although anaesthetic pretreatment may result in decreased pain at the time of test-compound administration, it does not affect possible pain after the effects of the anaesthetic have dissipated. Some anaesthetics are themselves irritating to eyes. In addition, anaesthetics reduce blinking and tearing, thereby maintaining the test-material concentration at the surface of the eye longer. Corneal permeability may also be increased with pretreatment use of an anaesthetic, and may bring the test agent into contact with more structures of the eye. Some anaesthetics delay healing after ocular injury. All of these varied effects may result in increased irritation to the eye. Overall, pretreatment with anaesthetics has usually resulted in a tendency for slightly higher irritation scores; eye irritancy classification is usually unaffected.

Scoring of the rabbit eye test and the resulting evaluation and classification should provide useful information about the likelihood that a test material may cause injury on contact with the human eye. When an animal test is necessary, a rabbit eye test based on the following characteristics is proposed for deriving the maximum information from the fewest animals. The ocular effects of interest should include corneal opacity, iritis and conjunctival redness. Animals should be scored for each ocular effect at 24, 48 and 72 hr after the test substance is administered. If an animal is negative at all three scoring times, it can be removed from the test at 72 hr. If it shows a positive effect at a scoring time but the lesion clears at 72 hr, it can be removed at 72 hr. If it shows a positive effect that does not clear at 72 hr, it should be scored again on day 7 when the test ends. However, if an animal shows severe effects at one or more scoring times, it can be removed from the test at 72 hr. An animal is positive if any one of the following criteria is observed at 24, 48 or 72 hr: corneal opacity of 1 or above, iritis of 1 or above, or conjunctival redness of 2 or above. Severe ocular effects (noted at 24, 48 or 72 hr) that may endanger sight deserve special recognition for the classification of chemicals and include corneal opacity of 3 or above, or iritis of 2. This proposal is consistent with the opinions of the majority of respondents who attended the Workshop on Updating Eye Irritation Test Methods, Proposals for Regulatory Consensus. The most notable exception was the suggestion by respondents to add conjunctival chemosis as one of the scoring parameters.

A proposal encompassing considerations and criteria for the development of in vitro alternatives to the eye irritation test has been developed and is presented here. Two factors need to be considered initially in developing an alternative test. The first is to determine whether the alternative assay is to be used as a screen or as a replacement for the eye irritation test. Less stringent acceptance criteria are required for an assay used as a screen than for that used as a replacement test. A screen is a preliminary test for the assessment of eye irritation. It is used for making preliminary decisions or establishing the direction for further testing. Screens answer fewer and less complex questions than a replacement test would, since the results from screens are usually confirmed by more definitive testing. A replacement test, however, must provide the same answers as in vivo methods for the assessment of eye irritation and must provide data for making a definitive toxicological assessment of eye irritation. The second factor to be considered is knowledge of the in vivo assay intended to be replaced. This knowledge should include the procedural aspects of the test and the regulatory information it provides. The following may be considered as criteria for in vitro tests used as screens or as replacements for the eye irritation test in rabbits: rationale (there should be a clear statement regarding the rationale for the use of a particular test in relation to the availability of other tests); relevance (the in vitro endpoint should have biological or physiological relevance to the effect to be detected in vivo); and validational (intralaboratory as well as interlaboratory validation must be conducted).

The Draize rabbit eye test was developed to provide a method for assessing the irritation potential of materials that might come in contact with human eyes. The method involves the instillation of 0.1 ml of a test liquid (100 mg solid) into the conjunctival sac of an animal's eye. A refinement of the Draize test is the low-volume eye test in which 0.01 ml of a substance is placed directly on the cornea of the eye. Studies indicate that the low-volume method provides a better correlation to human eye irritation experience for some substances. The Interagency Regulatory Alternatives Group (IRAG) proposes that the low-volume eye test can be used to substantiate the irritancy of suspect severe ocular irritants that have not been eliminated by various pre-eye test 'screens'. A substance testing positive by the low-volume method can be classified as an irritant; one that tests negative will require further testing by the use of the 0.1-ml volume procedure. For all other definitive testing, the Draize test (0.1 ml) should be used. Results from a questionnaire distributed at the IRAG workshop showed that many workshop participants thought that the low-volume test should be used as an eye irritation screening procedure.

Oligonucleotide conjugated with water-soluble meso-tetra(4-carboxyphenyl) porphine (TPPC4) has been prepared by a supporting synthesis and novel solid-phase conjugation strategy. The conjugates could be used in dual fashion: i) on formation of iron complex, target DNA could be site-specifically cleaved on incubation with dithiothreitol; ii) on incubation of RR 1022 rat epithelial cell culture with non-metalized oligonucleotide TPPC4 conjugate, cytotoxic effect was detected after irradiation with laser light at 635 nm.

A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization.

Human tonsillar lymphocytes immunized in vitro with diphtheria toxoid were fused with the heteromyeloma cell line SP2/HPT. Hybrids were selected on hypoxanthine-thymidine aminopterin (HAT) medium and assayed for antibody production both by ELISA and Western blotting. Four stable hybridoma cell lines producing 3-7 micrograms/mL of human IgG1 antibodies were isolated. In Western blotting studies these antibodies show binding to the B fragment of diphtheria toxin. Cells from the subclone P2-13/G35 were injected i.p. in pristane-primed nude mice, and ascites fluid was collected after 4-6 weeks. The monoclonal antibody neutralized the cytotoxicity of diphtheria toxin in the Vero cell assay system. This human monoclonal antibody may have a potential in the immunotherapy of diphtheric disease.