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Most basaltic explosive eruptions intensify abruptly, allowing little time to document processes at the start of eruption. One opportunity came with the initiation of activity from fissure 8 (F8) during the 2018 eruption on the lower East Rift Zone of Klauea, Hawaii. F8 erupted in four episodes. We recorded 28 minutes of high-definition video during a 51-minute period, capturing the onset of the second episode on 5 May. From the videos we were able to analyze the following in-flight parameters: frequency and duration of explosions; ejecta heights; pyroclast exit velocities; in-flight total mass and estimated mass eruption rates; and the in-flight total grain size distributions. Videos record a transition from initial pulsating outgassing, via spaced, but increasingly rapid, discrete explosions, to quasi-sustained, unsteady fountaining. This transition accompanied waxing intensity (mass flux) of the F8 eruption. We infer that all activity was driven by a combination of the ascent of a coupled mixture of small bubbles and melt, and the buoyant rise of decoupled gas slugs and/or pockets. The balance between these two types of concurrent flow determined the exact form of the eruptive activity at any point in time, and changes to their relative contributions drove the transition we observed at early F8. Qualitative observations of other Hawaiian fountains at Klauea suggest that this physical model may apply more generally. This study demonstrates the value of in-flight parameters derived from high resolution videos, which offer a rapid and highly time-sensitive alternative to measurements based on sampling of deposits post-eruption.

Relationships between live body condition score (BCS) and carcass fat depots have not been well established in equine. Our study was designed to quantify the relationship between BCS and fat depot measurements from equine carcasses. Live horses (n = 429) were evaluated immediately prior to immobilization at a commercial equine processor. Horses were independently assigned a BCS by a panel of three trained evaluators; BCS was evaluated by visual appraisal and manual palpation of the neck, withers, back, ribs, behind the shoulder, and tailhead. Median BCS frequencies were: 3.0 (n = 9), 4.0 (n = 43), 5.0 (n = 116), 6.0 (n = 86), 7.0 (n = 72), 8.0 (n = 76), and 9.0 (n = 27). Sex (stallion [n = 5], mare [n = 159], or gelding [n = 114]) and breed type (draft [n = 56], stock [n = 363], pony [n = 8], or mule [n =3]) were also denoted. Horses were processed for human consumption according to industry-accepted procedures under the supervision of the Canadian Food Inspection Agency. During the harvest process, all kidney–pelvic–heart (KPH) fat was trimmed from the carcass and weighed. After chilling, the marbling score was subjectively evaluated using beef grading standards. Carcass fat trim was weighed during the fabrication process. As BCS increased, hot carcass weight (HCW), absolute KPH weight, KPH expressed as a percentage of HCW, marbling score, neck fat depth, absolute weight of trimmed carcass fat, and trimmed carcass fat as a percentage of HCW increased (P < 0.01). A strong correlation (r = 0.74; P < 0.01) was detected between BCS and absolute KPH weight. Similarly, correlations between BCS and percentage of KPH (r = 0.65), neck fat depth (r = 0.60), absolute trimmed carcass fat (r = 0.58), trimmed carcass fat as a percentage of HCW (r = 0.54), marbling score (r = 0.54), and HCW (r = 0.52) were also detected (P < 0.01). These data indicate a strong relationship between subjective live BCS and objectively measured carcass fat depots in various equine breed types and sexes.

The unprecedented cost of the 2018 eruption in Hawai’i reflects an intersection of disparate physical and social phenomena: widely spaced, highly destructive eruptions, and atypically high population growth. These were linked and the former indirectly drove the latter with unavoidable consequences.

To assess the safety and efficacy of three new drugs in patients with antiretroviral failure and to correlate retrospectively baseline factors with virological response.Open-label, 48-week, single-arm, multi-center phase II trial conducted at nine US university or government clinics and private practices.Patients with HIV-1 RNAor =500 copies/ml despiteor =20 weeks of treatment with at least one protease inhibitor received abacavir 300 mg twice a day, amprenavir 1200 mg twice a day and efavirenz 600 mg once a day. Other antiretrovirals were prohibited until week 16 except for substitutions for possible abacavir hypersensitivity.HIV RNA at weeks 16 and 48.A total of 101 highly treatment-experienced patients enrolled; 60 were naive to non-nucleoside analog reverse transcriptase inhibitors (NNRTI). HIV RNA400 copies/ml was attained in 25 out of 101 (25%) patients at 16 weeks (35% of NNRTI-naive and 10% of -experienced patients) and 23 (23%) patients at 48 weeks (33% of naive and 7% of experienced patients). CD4 cells increased by a median of 15 x 10(6) and 43 x 10(6) cells/l at weeks 16 and 48, respectively. Drug-related rash occurred in 50 out of 99 (51%) of patients, and 17 out of 99 (17%) permanently discontinued one or more drugs as a result. Lower baseline viral load, fewer NNRTI-related mutations, absence of decreased abacavir (or =4-fold) and efavirenz (or =10-fold) susceptibility, and greater number of drugs to which virus was susceptible were associated with virological response at week 16.Abacavir, amprenavir and efavirenz durably reduced HIV RNA and increased CD4 cell counts in a subset of treatment-experienced adults. Baseline viral load and some genotypic and phenotypic markers of resistance correlated with HIV RNA response.

We describe the in vitro selection and characterisation of virus derived from B/Beijing/1/87 passaged in the presence of zanamivir. During zanamivir passage, the phenotype of virus isolates was either drug dependent or drug resistant in plaque reduction assays. The susceptibility of the neuraminidase of the drug-dependent isolates was unchanged from that of the wild-type enzyme. The drug-dependent isolates contained two mutations in the viral haemagglutinin: V90A, close to the proposed secondary sialic acid-binding site, and L240Q, close to the primary sialic acid-binding site. Virus isolates that were drug resistant contained the same mutations in the haemagglutinin but also contained the mutation E116G in the neuraminidase. For the drug-dependent viruses, zanamivir susceptibility could not be measured because plaque numbers increased with increasing drug concentration. The in vitro zanamivir susceptibility of drug-resistant viruses was lower than that of the wild-type virus by a factor of 275- to >2532-fold. Neuraminidase containing the E116G mutation has a 33-fold lower affinity for zanamivir than the wild-type enzyme. The finding that the same haemagglutinin mutations are found in both drug-dependent and drug-resistant viruses confirms that the same changes to the receptor binding function can contribute to both phenotypes. This observation demonstrates the interplay between the influenza virus haemagglutinin and neuraminidase in escape from zanamivir inhibition in vitro.

The nucleoside analog 2'-deoxy-2'-fluoroguanosine (2'-fluorodGuo) is phosphorylated by cellular enzymes and reversibly inhibits influenza virus replication in chick embryo cells within the first 4 h of infection. RNA hybridization studies revealed that primary and secondary transcription of influenza virus RNA were blocked at a compound concentration of 10 microM, but no inhibition of cell protein synthesis was seen even at high compound concentrations (200 microM). In vitro, the triphosphate of 2'-fluorodGuo is a competitive inhibitor of influenza virus transcriptase activity from disrupted virus, with a Ki of 1.0 microM. The cellular polymerases DNA polymerase alpha and RNA polymerase II were only weakly inhibited or were insusceptible to 2'-fluorodGTP. In kinetic studies with the influenza virus transcriptase, 2'-fluorodGTP, in the absence of GTP, blocked elongation of the virus RNA chain. Similarly, by using purified ribonucleoprotein complexes it was found that the addition of a single nucleotide of 2'-fluorodGTP to the virus RNA caused chain termination, which resulted in the blockage of further virus transcription. Furthermore, the specificity for influenza virus transcriptase was confirmed when the transcriptase from partially resistant virus was found to be 10-fold less susceptible to 2'-fluorodGTP (Ki = 13.1 microM).

beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.

The reverse transcriptase (RT) of HIV-1 has been mutagenized within the carboxyl-terminal domain which harbors the RNase H. Two amino acids highly conserved among all 14 known RT sequences but not in the bacterial RNase H have been mutagenized resulting in the mutant proteins N494D and Q475E. They were expressed as recombinant proteins, purified, and analyzed for their in vitro properties in comparison to the p66 homodimeric wild-type and a previously described H539N mutant. The N494D mutant closely resembles the wild-type RNase H, exhibits an endonuclease activity and a processive RNase H activity, gives rise to small RNA hydrolysis products, and acts in concert with the RT. The Q475E mutant is more defective and resembles the H539N mutant, exhibits a retarded endonuclease activity and an impaired 3'-->5' processive RNA cleavage activity, gives rise to predominantly larger RNA hydrolysis products, is less processive in the presence of competitor substrate, and is defective in its ability to hydrolyze the polypurine tract and homopolymeric hybrids. Short homopolymeric stretches cause a pausing of the RT of wild-type and mutants which results in a coordinated action of the RNase H. Pausing of the RT correlates with RNase H cleavages about 20 nucleotides behind the point of synthesis. The defects of the mutant enzymes can be interpreted on the basis of the known crystallography data.

We investigated the prophylactic and therapeutic efficacy of an intravenous (IV) formulation of zanamivir in a macaque infection model for highly pathogenic influenza A (H5N1) virus. Antiviral efficacy was dose-dependent, with no reduction in viral load observed at 2 mg/kg, but a significant reduction observed at 10 mg/kg (p = 0.039) and at 20 mg/kg in the combined prophylactic and therapeutic groups (p = 0.049) with both prophylaxis (commencing 12 h before infection) and therapy (commencing 4 h after infection) showing similar reductions in viral load. Combined gross pathology and microscopic pneumonia scores in the treated animals relative to untreated controls were significantly reduced at 10 mg/kg (p = 0.02) and at 20 mg/kg in the prophylaxis group (p, = 0.02), but were not significant in the treatment group (p = 0.145). in this new animal model for evaluation of influenza antivirals, despite variability observed between individual animals, IV zanamivir showed evidence of efficacy against highly pathogenic H5N1 virus. (C) 2008 Elsevier B.V. All rights reserved.

To evaluate the antiretroviral activity and safety of multiple escalating doses of amprenavir administered alone, and in combination with abacavir in HIV-1-infected adults.Sixty-two HIV-1-infected subjects were enrolled in a multicentre, open-label, non-randomized, dose-escalating trial.Subjects were assigned to one of six dose groups and received amprenavir 300 mg twice daily, 300 mg three times daily, 900, 1050, or 1,200 mg twice daily for 4 weeks. One dose group received amprenavir 900 mg twice daily in combination with abacavir 300 mg twice daily for 4 weeks. Antiretroviral activity was assessed by measuring changes from baseline in plasma HIV-1 RNA levels and CD4 cell counts. Safety was evaluated by monitoring clinical adverse events and changes in laboratory values. Genotypic and phenotypic analyses were performed using ABI sequencing and the recombinant virus assay, respectively.At week 4, amprenavir monotherapy (900, 1,050, or 1,200 mg twice daily) resulted in marked decreases in plasma HIV-1 RNA levels (1.3-1.6 log10 copies/ml), and substantial increases in CD4 cell counts in the two dose groups who received 1,050 mg twice daily (118 x 10(6) cells/mm3) or 1,200 mg twice daily (114 x 10(6) cells/mm3). Amprenavir/abacavir resulted in median plasma HIV-1 RNA reductions of 1.8 log10 copies/ml, and median CD4 cell count increases of 138 x 10(6) cells/mm3. Amprenavir was reasonably well tolerated with few treatment-limiting adverse events. No known active site mutations associated with amprenavir resistance were selected in any of the dose groups, and no significant phenotypic resistance to amprenavir developed during 4 weeks of therapy.The antiviral effect of amprenavir monotherapy increased with escalating doses, and all amprenavir doses were reasonably well tolerated over 4 weeks of therapy. Amprenavir/abacavir combination therapy elicited a potent antiviral effect. The three highest doses of amprenavir (900, 1,050 and 1,200 mg twice daily) were selected to design subsequent Phase II and III studies that confirmed the safety profile and efficacy of amprenavir in combination regimens and led to the approval of amprenavir in the USA in 1999.

Objective To evaluate the antiretroviral activity and safety of multiple escalating doses of amprenavir administered alone, and in combination with abacavir in HIV-1-infected adults. Design Sixty-two HIV-1-infected subjects were enrolled in a multicentre, open-label, non-randomized, dose-escalating trial. Methods Subjects were assigned to one of six dose groups and received amprenavir 300 mg twice daily, 300mg three times daily, 900, 1050, or 1200 mg twice daily for 4 weeks. One dose group received amprenavir 900 mg twice daily in combination with abacavir 300 mg twice daily for 4 weeks. Antiretroviral activity was assessed by measuring changes from baseline in plasma HIV-1 RNA levels and CD4 cell counts. Safety was evaluated by monitoring clinical adverse events and changes in laboratory values. Genotypic and phenotypic analyses were performed using ABI sequencing and the recombinant virus assay, respectively. Results At week 4, amprenavir monotherapy (900, 1050, or 1200 mg twice daily) resulted in marked decreases in plasma HIV-1 RNA levels (1.3–1.6 log10 copies/ml), and substantial increases in CD4 cell counts in the two dose groups who received 1050 mg twice daily (118x106 cells/mm3) or 1200 mg twice daily (114x106 cells/mm3). Amprenavir/abacavir resulted in median plasma HIV-1 RNA reductions of 1.8 log10 copies/ml, and median CD4 cell count increases of 138x106 cells/mm3. Amprenavir was reasonably well tolerated with few treatment-limiting adverse events. No known active site mutations associated with amprenavir resistance were selected in any of the dose groups, and no significant phenotypic resistance to amprenavir developed during 4 weeks of therapy. Conclusions The antiviral effect of amprenavir monotherapy increased with escalating doses, and all amprenavir doses were reasonably well tolerated over 4 weeks of therapy. Amprenavir/abacavir combination therapy elicited a potent antiviral effect. The three highest doses of amprenavir (900, 1050 and 1200 mg twice daily) were selected to design subsequent Phase II and III studies that confirmed the safety profile and efficacy of amprenavir in combination regimens and led to the approval of amprenavir in the USA in 1999.

Recent drug regimens have had much success in the treatment of human immunodeficiency virus (HIV)-infected individuals; however, the incidence of resistance to such drugs has become a problem that is likely to increase in importance with long-term therapy of this chronic illness. An analysis and understanding of the molecular interactions between the drug(s) and the mutated viral target(s) is crucial for further progress in the field of AIDS therapy. The protease inhibitor amprenavir (APV) generates a signature set of HIV type 1 (HIV-1) protease mutations associated with in vitro resistance (M46I/L, I47V, and I50V [triple mutant]). Passage of the triple-mutant APV-resistant HIV-1 strain in MT4 cells, in the presence of increasing concentrations of saquinavir (SQV), gave rise to a new variant containing M46I, G48V, I50V, and I84L mutations in the protease and a resulting phenotype that was resistant to SQV and, unexpectedly, resensitized to APV. This phenotype was consistent with a subsequent kinetic analysis of the mutant protease, together with X-ray crystallographic analysis and computational modeling which elucidated the structural basis of these observations. The switch in protease inhibitor sensitivities resulted from (i) the I50V mutation, which reduced the area of contact with APV and SQV; (ii) the compensating I84L mutation, which improved hydrophobic packing with APV; and (iii) the G-to-V mutation at residue 48, which introduced steric repulsion with the P3 group of SQV. This analysis establishes the fine detail necessary for understanding the loss of protease binding for SQV in the quadruple mutant and gain in binding for APV, demonstrating the powerful combination of virology, molecular biology, enzymology, and protein structural and modeling studies in the elucidation and understanding of viral drug resistance.

We investigated HIV-1 reverse transcriptase (RT) polymorphisms of plasma isolates from 98 HIV-1-infected study subjects with2 years of antiretroviral therapy who were failing their current protease inhibitor (PI)-containing regimen. In 1 patient, we detected a virus with a heavily mutated beta3-beta4 connecting loop of the HIV-1 RT fingers subdomain, consisting of a single aspartate codon insertion between positions 69 and 70 and five additional variations: 64N, K65, K66, 67G, 68Y, T69, Ins D, 70R, W71, R72, K73, 74I. Mutants with the recently described 2-aa insertions between codons 68 and 70 of RT were detected in another 3 patients. Among the four isolates with the 1- or 2-aa insertions, the novel genotype was the most refractory to therapy and displayed the highest level of phenotypic resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Follow-up samples demonstrated that the novel mutant represents a stable genetic rearrangement and that the amino acid insertions can coexist with nonnucleoside analogue reverse transcriptase inhibitors (NNRTI) mutations resulting in phenotypic resistance to both NRTIs and NNRTIs. An increasing number of HIV-1 isolates containing various insertions in the beta3-beta4 hairpin of the HIV-1 RT fingers subdomain appear to emerge after prolonged therapy with different NRTIs, and these polymorphisms can confer multiple drug resistance against NRTIs.

Abacavir (1592U89) is a nucleoside inhibitor of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT). Resistance to abacavir was studied with abacavir alone and with abacavir in combination with other nucleoside analogues in cell culture, in virus isolates from zidovudine/lamivudine clinical trials, and in the first dose-escalating 12-week clinical trial (CNA2001) to evaluate abacavir clinical potency. Abacavir alone in vitro selected for mutations at HIV RT codons K65R, L74V, Y115F, and M184V. However, abacavir combined with zidovudine selected against virus with the M184V mutation. Abacavir therapy in vivo resulted in large decreases in HIV load (1 log), even in 1 subject who had the M184V mutation at baseline. A total of 51% of subjects showed new mutations at any of codons K65R, L74V, and M184V after abacavir monotherapy, compared with 11% who received zidovudine/abacavir. Small changes (2- to 4-fold) in abacavir susceptibility were detected. On stopping therapy, reselection of the pretherapy sequence occurred within 4 weeks.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol(IsoddA)isthemostantivirallyactivemember of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1*position to the 2*position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3*-azido-3*-deoxythymidine), 2*,3*-dideoxyinosine, or 5-fluoro-2*deoxy-3*-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with aKiof 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 mM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subcloneP5A)celllinesbutwere12and11 mMforhumangranulocyte-macrophage(CFU-GM)anderythroid (BFU-E) progenitor cells, respectively. When given orally to rats and mice, the compound was very well absorbed and rapidly eliminated. However, there was no detectable brain penetration by IsoddA in rats. Catabolic metabolites were not detected, and this is consistent with the observed resistance of the compound to metabolic degradation by adenosine deaminase.

Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.

Single-dose treatments (5 to 40 mg/kg of body weight given intraperitoneally) of ferrets with 2'-deoxy-2'-fluoroguanosine or its prodrug, 2,6-diamino-purine-2'-fluororiboside, 1 h after infection with influenza A virus significantly inhibited replication of virus in the upper respiratory tract, resulting in amelioration of fever and nasal inflammation. Replication of virus in the lower respiratory tract was also reduced > 100-fold, but three doses were required to prevent replication in the lungs. In ferrets infected with influenza B virus, single-dose treatment (40 mg/kg given intraperitoneally) produced a similar but reduced response in comparison with that in ferrets infected with influenza A virus, indicating that dosing was not optimal for this virus.

Monoclonal antibodies to human immunodeficiency virus (HIV)-2 reverse transcriptase have been raised with the ultimate goal of generating Fab fragments for future co-crystallization studies. A number of mouse monoclonal antibodies to recombinant HIV-2 reverse transcriptase have been obtained and characterized in terms of the possible epitopes they recognise together with cross-reactivity with a related reverse transcriptase. The antibodies were shown to fall into three groups that recognize different regions of the reverse transcriptase enzyme. One antibody, which recognizes part of the RNase H domain, demonstrated cross-reactivity between the HIV-1 and HIV-2 reverse transcriptase.

The reverse transcriptase (RT) of HIV-1 has been mutagenized within the carboxyl-terminal domain which harbors the RNase H. Two amino acids highly conserved among all 14 known RT sequences but not in the bacterial RNase H have been mutagenized resulting in the mutant proteins N494D and Q475E. They were expressed as recombinant proteins, purified, and analyzed for their in vitro properties in comparison to the p66 homodimeric wild-type and a previously described H539N mutant. The N494D mutant closely resembles the wild-type RNase H, exhibits an endonuclease activity and a processive RNase H activity, gives rise to small RNA hydrolysis products, and acts in concert with the RT. The Q475E mutant is more defective and resembles the H539N mutant, exhibits a retarded endonuclease activity and an impaired 3'--5' processive RNA cleavage activity, gives rise to predominantly larger RNA hydrolysis products, is less processive in the presence of competitor substrate, and is defective in its ability to hydrolyze the polypurine tract and homopolymeric hybrids. Short homopolymeric stretches cause a pausing of the RT of wild-type and mutants which results in a coordinated action of the RNase H. Pausing of the RT correlates with RNase H cleavages about 20 nucleotides behind the point of synthesis. The defects of the mutant enzymes can be interpreted on the basis of the known crystallography data.

The C-termini of p66 and p51 forms of HIV-1 reverse transcriptase have been engineered to contain a Glu-Glu-Phe sequence recognized by a monoclonal antibody to α-tubulin YL12. Mututed RTs were purified in a single step using peptide elution from columns of immobilized YL12. The known sequence requirements of the YL12 epitope arc consistent with protein eluting from the column with an intact C-terminus. Kinetic parameters of these mutated RTs are essentially unchanged from wild-type enzyme. The p15 RNaseH domain has been purified using this method and shown to have low enzyme activity compared to the parental p66 subunit.

The process of cytoplasmic cleavage has been studied in thin sections of rat erythroblasts and the cells of mouse leukemia and Walker 256 carcinoma of the rat. The development of the cleavage furrow begins in relation to the mid-body, which, earlier, appears on the equatorial plane in association with the continuous fibers of the spindle. The earliest evidence of a cleavage furrow is the presence of a vesicle or vesicles close to the mid-body. Subsequently, many smaller vesicles are seen in the equatorial plane. The cleavage furrow probably develops by the fusion of these vesicles so that a new plasma membrane is formed between the daughter cells, and extends from the telophase intercellular bridge to the cell margin. During the stage of formation of the vesicles, cisternae, believed to be part of the endoplasmic reticulum, assume an intimate relationship with the cleavage plane, and they may perhaps be involved in the formation of the vesicles.

The development of the mid-body has been studied in mitotic erythroblasts of the rat bone marrow by means of thin sections examined with the electron microscope. A differentiated region on the continuous spindle fibers, consisting of a localized increase in density, is observed at the equatorial plane. The mid-body seems to develop by the aggregation of such denser lengths of spindle fiber. Its appearance precedes that of the cleavage furrow. A plate-like arrangement of fibrillary material lies transversely across the telophase intercellular bridge. Later, this material becomes amorphous and assumes the form of a dense ring closely applied to a ridge in the plasma membrane encircling the middle of the bridge. Although the mid-body forms in association with the spindle fibers, it is a structurally distinct part, and the changes which it undergoes are not shared by the rest of the bundle of continuous fibers.

The objectives of this program are to assess the magnitude of the resources and locate areas of highest potential for peat deposits in South Carolina. The energy potential of these peat resources is also being evaluated. This report presents the results of progress made during the last quarter in: assessing data and prioritizing peat areas to be surveyed; procurement of equipment and supplies; and preliminary peat resource assessment. A summary of the results of all new field surveys conducted during the quarter is included. Approximate locations of potential major peat deposits have been identified. Preliminary sampling studies indicate that Pigeon Bay may have the thickest and best quality peat in Berkeley County. Probes indicate peats up to 12 feet thick are located near the Black River in Georgetown County. Samples from areas designated as organic soils by the USDA were analyzed for moisture, organic, and ash content. (DMC)

We describe the production of eight monoclonal antibodies reactive with human immunodeficiency virus type 1 reverse transcriptase (RT) by immunization of mice with purified recombinant RT. These antibodies were found to react with one or the other of two regions of the enzyme and were found to be useful in immunodeficiency purification of large amounts of the enzyme. One epitope located at the C terminus of the enzyme was of particular interest, since it was present in only the larger, 66-kilodalton (kDa) RT species and not its smaller, 51-kDa counterpart. To define this epitope, a series of mutants was made which synthesized C-terminally truncated RT. These mutants indicated that the same region of the enzyme, when deleted, both removed the C-terminal epitope and drastically reduced RT activity, indicating the importance of this region in the function of the enzyme; however, even the 51-kDa enzyme component had demonstrable activity.

South Carolina has few indigenous energy resources. Most widely known and utilized are hydropower, wood, and solar. Peat is a material composed of partially decomposed organic matter that, after burial for long periods of time, may eventually become coal. Peat is utilized as an energy resource for the production of electricity and for home heating in Europe and the Soviet Union. There are peat deposits in South Carolina, but peat has never been used as an energy resource within the state. This report presents the results of the two years of a planned four-year study of the quantity and energy potential of peat in South Carolina. In this year's survey two activities were undertaken. The first was to visit highly probable peat deposits to confirm the presence of fuel-grade peat. The second was to survey and characterize in more detail the areas judged to be of highest potential as major resources. The factors carrying the greatest weight in our determination of priority areas were: (1) a description of peat deposits in the scientific literature or from discussions with state and federal soil scientists; (2) mention of organic soils on soil maps or in the literature; and (3) information from farmersmore » and other local citizens.« less

The clinical success of zidovudine has established the human immunodeficiency virus (HIV) reverse transcriptase (RT) as a valid target for the design of drugs to treat acquired immunodeficiency syndrome. In order to facilitate structural studies of this enzyme, expression systems in Escherichia coli, which allow the production of large amounts of RT, have been established. Using this recombinant material the RT has been purified and crystallized. Crystallographic studies currently underway are aimed at elucidating the three-dimensional structure of HIV RT. The availability of a bacterial expression system has enabled structural/functional studies of the RT by site-directed mutagenesis. These studies have identified amino acid residues that are essential for activity of the enzyme and might be involved in substrate binding. It is hoped that structural information of this nature will allow the rational design of HIV RT inhibitors.