An acyclic phosphonate prodrug of HPMPC is effective against VZV in skin organ culture and mice.

Varicella zoster virus (VZV) causes chicken pox and shingles and is prevalent worldwide. Acyclovir and penciclovir (and its prodrugs) are first-line treatments for VZV infections, but they are not highly potent against VZV and resistance may arise in immunocompromised people on long-term therapy. HPMPC (cidofovir) is active against VZV, but cidofovir is not approved for treating VZV diseases, is nephrotoxic, and is not orally bioavailable. Here, we present the synthesis and evaluation of USC-373, a phosphonate prodrug of HPMPC with activity against VZV and other DNA viruses. In cultured fibroblasts, it was potent against VZV Ellen laboratory strain and was not overtly toxic, with EC50 of 4 nM and CC50 of 0.20 µM, producing a selectivity index of 50. In ARPE-19 cells, USC-373 was effective against VZV-ORF57-Luc wild type strain and the acyclovir-resistant isogenic strain. In human skin organ culture, USC-373 formulated in cocoa butter and applied topically prevented VZV-ORF57-Luc spread without toxicity. In NuSkin mice with human skin xenografts, one daily dose of 3 mg/kg was effective by the subcutaneous route, and one daily dose of 10 mg/kg was effective by the oral route. Remarkably, a 10 mg/kg oral dose given every other day was also effective. USC-373 was well tolerated and mice did not lose weight or show signs of distress. The prodrug modifications of USC-373 increase the potency and oral bioavailability compared to its parent nucleoside analog, HPMPC.

Pathology of upper respiratory tract disease of gopher tortoises in Florida.

Between August 1993 and September 1995, 24 gopher tortoises (Gopherus polyphemus) were received for pathological evaluations from various locations in Florida (USA). All tortoises were examined for clinical signs of upper respiratory tract disease (URTD) including nasal and ocular discharge, palpebral edema, and conjunctivitis. Of the 24 tortoises, 10 had current or previously observed clinical signs of URTD and 14 did not. A blood sample was drawn for detection of anti-mycoplasma antibodies by ELISA, and nasal lavage samples were collected for culture and detection of Mycoplasma agassizii gene sequences by polymerase chain reaction (PCR). Of the 14 clinically healthy tortoises, eight were sero-, culture- and PCR-negative, and six were seropositive for antibodies against M. agassizii. Of those six, five were culture- and/or PCR-positive for M. agassizii, and one was culture- and PCR-negative. Of the 10 ill tortoises, nine were seropositive by the ELISA and one was in the suspect range. Nine of the ill tortoises, including the suspect tortoise, were culture- and/or PCR-positive for M. agassizii, and one was culture- and PCR-negative. For histologic evaluation and discussion, the eight sero-, culture-, and PCR-negative tortoises were designated URTD-negative, and the other 16 were classified as URTD-positive. Histologic evaluation of the upper respiratory tract (URT) indicated the presence of mild to severe inflammatory, hyperplastic, or dysplastic changes in 14 URTD-positive tortoises. Seven of eight URTD-negative tortoises had normal appearing nasal cavities; one had mild inflammatory changes. Transmission electron microscopy revealed an organism consistent with Mycoplasma spp. on the nasal mucosal surface of tortoises with clinical signs and lesions of URTD. Additionally, gram-negative bacteria were isolated more frequently from the nasal cavities of URTD-positive tortoises than URTD-negative tortoises. Because clinical signs of URTD were never observed in six of the URTD-positive tortoises, we also conclude that subclinical URTD can occur in gopher tortoises.

Genetic localization of an autosomal dominant leukodystrophy mimicking chronic progressive multiple sclerosis to chromosome 5q31.

The hereditary leukodystrophies represent a group of neurological disorders, in which complete or partial dysmyelination occurs in either the central nervous system (CNS) and/or the peripheral nervous system. Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive, neurological disorder characterized by symmetrical widespread myelin loss in the CNS, and the phenotype is similar to that of chronic progressive multiple sclerosis. We report clinical, neuroradiological and neuropathological data from the originally reported ADLD family. Furthermore, we have localized the gene that causes ADLD to a 4 cM region on chromosome 5q31. Linkage analysis of this family yielded a LOD score of 5.72 at theta = 0.0 with the microsatellite marker D5S804. Genetic localization will lead to cloning and characterization of the ADLD gene and may yield new insights into myelin biology and demyelinating diseases.

Enumerative and binomial sampling plans for armored scale (Homoptera: Diaspididae) on kiwifruit leaves.

The spatial dispersion of armored scale insects; greedy scale, Hemiberlesia rapax (Comstock); and latania scale, Hemiberlesia lataniae (Signoret), was investigated on kiwifruit, Actinidia deliciosa (A. Chevalier) C. F. Liang et A. R. Ferguson, leaves in New Zealand. A universal description for dispersion was determined using Taylor's power law, which encompassed a wide range of different orchards, blocks, block sizes, sampling times, scale control practices, regions and seasons. Scale density significantly altered dispersion, especially at the high densities found on unsprayed kiwifruit. Most commercially managed kiwifruit blocks had low densities of <0.5 scale per leaf and had a slightly aggregated scale dispersion. Wilson and Room's binomial model, which incorporates a clumping pattern as a function of density, gave a significant relationship between the proportion of infested leaves and scale density. The optimal leaf sample sizes were estimated for predetermined levels of sampling reliability. Where population estimates require a high degree of precision and enumerative sampling methods are used, 2,500 leaves should be sampled when scale densities are near the current spray threshold of 4% infested leaves and 500 leaves at 20% infested leaves. For management-decision sampling, where a lower level of precision was acceptable, enumerative sampling would require that 400 leaves be sampled at 4%; or 85 leaves at 20% infested leaves. With binomial sampling to achieve an equivalent level of precision an increased sample size of 6-11% is required.

Reduction of cyclic and acyclic diazene derivates by Azotobacter vinelandii nitrogenase: diazirine and trans-dimethyldiazene.

Nitrogenase reduces N2 to NH3, but the mechanistic details are unclear. Diazene (N2H2), a proposed 2e-/2H+ intermediate on the reduction pathway, is labile under typical enzyme assay conditions, and no firm evidence is available on whether or not it can be reduced by or inhibit nitrogenase. In this paper, we compare the interactions of Azotobacter vinelandii (Av) nitrogenase with two diazene analogues: diazirine, a photolabile diazene containing the azo (-N=N-) group in a strained, three-membered ring, and trans-dimethyldiazene, a diazene containing an unstrained trans-disubstituted N=N bond. Diazirine is reduced by nitrogenase under specific conditions to methane, methylamine, and ammonia in a ratio of ca. 1:2:4-5 with a Km value for all three products similar (0.05-0.09 mM) to that of dinitrogen (0.06-0.12 mM). The Km value of diazirine does not depend on the ratio of nitrogenase Fe protein (Av2) to nitrogenase MoFe protein (Av1) at Av2:Av1 ratios of 0.71 and 14.9. Diazirine potently and competitively inhibits acetylene reduction by Av nitrogenase with Ki = 0.03 mM and is predicted to inhibit H2 evolution completely at pressures >> Km. The experimental Henry's Law constant (1.50 M/atm) determined for trans-dimethyldiazene in H2O shows that it has about 20-fold higher solubility than diazirine in water at 30 degrees C. trans-Dimethyldiazene is reduced by nitrogenase under specific conditions to ammonia, methane and methylamine in a ratio of ca. 1:1:1 with Km values for the three products of 0.51-0.58 M. The product ratio does not change significantly when the component ratio (Av2:Av1 ) is varied over 2.06-13.62. trans-Dimethyldiazene reduction is inhibited noncompetitively by CO and C2H2 with Ki values of ca. 0.0008 and 0.006 atm, respectively. The results are discussed with respect to the stereoelectronic differences between the two azo substrates. A "random-edge" reduction is compared with alternative schemes for the diazirine reduction. For trans-dimethyldiazene, initial C-N cleavage is proposed to yield CH4 and a bound CH3N2H species, which is then reduced to CH3NH2 and NH3.

Taxonomic analysis of the tortoise mycoplasmas Mycoplasma agassizii and Mycoplasma testudinis by 16S rRNA gene sequence comparison.

The nucleotide sequences of the 16S rRNA genes of two mycoplasmas, Mycoplasma agassizii (proposed sp. nov.) and Mycoplasma testudinis, isolated from tortoises were determined and used for taxonomic comparisons. Signature nucleotide sequence motifs and overall sequence similarities to other mollicutes positioned these mycoplasmas in the M. hyorhinis and M. pneumoniae phylogenetic groups, respectively. A third, previously unrecognized tortoise mycoplasma was detected by 16S rRNA gene amplification and sequence analysis and was positioned in the M. fermentans phylogenetic group. The 16S rRNA gene of Acholeplasma laidlawii was similarly detected in a tortoise isolate, showing that diverse mollicutes can share the same family of reptilian host.

Absolute configuration of (+)-[fluoro(hydroxyphenylphosphinyl)methyl]-phosphonic acid, a specific inhibitor of Na(+)-gradient-dependent Na(+)-phosphate cotransport across renal brush border membrane, by X-ray crystallographic analysis of its (-)-quinine salt.

Racemic [fluoro(hydroxyphenylphosphinyl)methyl]phosphonic acid (1) and its individual enantiomers [(+), 98% ee; (-), 67% ee] were previously shown to inhibit Na(+)-gradient-dependent Na(+)-phosphate cotransport across renal brush border membrane, without measurable stereospecificity. Resolution of 1 was effected by fractional recrystallization of its (-)-quinine salts. The more levorotatory, diquinine product 2, corresponding to (+)-1, has now been analyzed by X-ray crystallography and found to be composed of the S enantiomer of 1. This result confirms the absence of stereochemical preference in inhibition of the cotransporter by the enantiomers of 1 and provides the first absolute configuration assignment of an asymmetrical alpha-halomethylene pyrophosphate analogue.

Some phosphonic acid analogs as inhibitors of pyrophosphate-dependent phosphofructokinase, a novel target in Toxoplasma gondii.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was identified previously in Toxoplasma gondii as the only kinase that phosphorylates fructose-6-P to fructose-1,6-bisP. Since such an enzyme is not present in mammals, it was considered to be a good target for prospective selective inhibitors of the parasite. We have examined the effects of several phosphonic acid derivatives, analogs of pyrophosphate, on PPi-PFK activity, as well as on the replication of T. gondii in human foreskin fibroblast (HFF) cells. The most active compound in inhibiting PPi-PFK was tetrasodium carbonyldiphosphonate. Several bisphosphonates and related arylhydrazones showed inhibition of the enzyme, but with higher IC50 values. Although several phosphonoacetic acid derivatives also inhibited PPi-PFK, as a group they were less potent than the bisphosphonate derivatives. Comparison among the structures of various inhibitors and their effects against PPi-PFK indicates that a carbonyl (C=O) or amino (C=N) group between two phosphoryl moieties is associated with more potent enzyme inhibiton. Tetrasodium carbonyldiphosphonate did not show a significant effect against replication of T. gondii cells, probably because, as a charged molecule, it could not cross the cell membrane to reach the intracellular parasite. Tetraisopropyl carbonyldiphosphonate 2,4-dinitrophenylhydrazone showed some selective inhibitory effect against replication of the parasite in the HFF cells and protected the mammalian cells from damage by T. gondii. The results indicate that carbonyldiphosphonic acid is a good prototype compound that is amenable to chemical manipulation, which, in turn, may optimize selective inhibition of T. gondii PPi-PFK and increase accessibility to the intracellular parasite.

Alpha-halo [(phenylphosphinyl)methyl]phosphonates as specific inhibitors of Na(+)-gradient-dependent Na(+)-phosphate cotransport across renal brush border membrane.

Certain phosphonocarboxylate analogues of phosphate are known to inhibit Na(+)-phosphate (Pi) cotransport in renal brush border membrane (BBM), but previously tested potential inhibitors incorporating structurally versatile aryl functionality were inactive. In this work, a series of novel alpha-halogenated [(phenylphosphinyl)methyl]phosphonates [PhpXYMP: X, Y = H, F (2); F, F (3); H, Cl (6); Cl, Cl (4); H, Br (7); Br, Br (5); and Cl, Br (8)] were prepared via synthesis of the corresponding triethyl esters, acid hydrolysis, and isolation as pyridine salts. The compounds were evaluated as inhibitors of Na(+)-gradient-dependent 32Pi uptake by rat renal cortex BBM vesicles (BBMV) in vitro. The PhpFMP racemate 2 had higher activity (-49% delta inhibition) than other members of the series (-22 to -39% delta inhibition). pKa values of 1.5-2.0, 2.7, and 7.1 were estimated for 2 using a 31P delta vs pH plot, indicating that in the activity assays it exists as both dianion and trianion, with the latter form predominant. PhpFMP had no significant inhibitory effect on Na(+)-gradient-dependent uptake of D-glucose or L-proline in the same BBMV, and did not inhibit BBM alkaline phosphatase. Kinetic analysis showed that PhpFMP acts as a strictly competitive inhibitor of Na(+)-Pi cotransport with Ki = 0.358 +/- 0.021 mM (n = 3). The racemate 2 was resolved as its (-)-quinine salt into enantiopure (+)-2 [Na+ salt, [alpha]25D = +6 degrees (aqueous MeOH)] and a Na+ salt of 2 enriched in (-)-2. The two compounds did not differ significantly as inhibitors of Na(+)-gradient dependent 32Pi uptake by rat renal cortex BBM vesicles (BBMV) in vitro. The results are discussed in terms of structural requirements for inhibition.

Unique and independent parameters (UIP) formulation for thermodynamic models of complex protein-ligand systems.

A method for reformulating the thermodynamic (delta G) description of complex equilibria is presented. The purpose of this reformulation is to take a system of N complexes which is completely defined by N delta Gs, and recast it in terms of a new set of N delta Gs. This reformulation is an extension of the concept of interaction energy (J. Wyman, Adv. Protein Chem. 19 (1964) 223-286). The new delta Gs obtained by this reformulation reflect the intrinsic properties of the binding sites and the hierarchical nature of potential interactions between them. A simple set of rules are developed which allow for the description of complex protein-ligand binding schemes and these rules are used to derive schemes for hemoglobin O2 binding. This reformulation represents the foundation for the theoretical description of the coupling of energy in protein-ligand systems as illustrated by the theoretical analysis of allosterism in a dimeric protein presented in the following paper. This reformulation also provides the foundation for the analysis of data pertaining to complex equilibria.

A method for preparing analytically pure sodium dithionite. Dithionite quality and observed nitrogenase-specific activities.

Sodium dithionite (Na2S2O4) is widely used as a reductant in biochemical studies, but has not been available in its pure form. A convenient, detailed procedure is given for the recrystallization of commercial dithionite from 0.1 M NaOH-methanol under anaerobic conditions. Twice-recrystallized dithionite had a purity of 99 +/- 1% by UV spectroscopy (A315) and elemental analysis. The influence of dithionite quality on the apparent reduction activities of the nitrogenase components (Av1 and Av2) from Azotobacter vinelandii was investigated.

Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta.

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.

Reduction of cyclopropene by NifV- and wild-type nitrogenases from Klebsiella pneumoniae.

The nitrogenase from wild-type Klebsiella pneumoniae reduces cyclopropene to cyclopropane and propene in the ratio 1:2 at pH 7.5. We show in this paper that the nitrogenase from a nifV mutant of K. pneumoniae also reduces cyclopropene to cyclopropane and propene, but the ratio of products is now 1:1.4. However, both nitrogenases exhibit the same Km for cyclopropene (2.1 x 10(4) +/- 0.2 x 10(4) Pa), considerably more than the Km for the analogous reaction with Azotobacter vinelandii nitrogenase under the same conditions (5.1 x 10(3) Pa). Analysis of the data shows that the different product ratio arises from the slower production of propene compared with cyclopropane by the mutant nitrogenase. During turnover, both nitrogenases use a large proportion of the electron flux for H2 production. CO inhibits the reduction of cyclopropene by both K. pneumoniae proteins, but the mutant nitrogenase exhibits 50% inhibition at approx. 10 Pa, whereas the corresponding value for the wild-type nitrogenase is approx. 110 Pa. However, H2 evolution by the mutant enzyme is much less affected than is cyclopropene reduction. CO inhibition of cyclopropene reduction by the nitrogenases coincides with a relative increase in H2 evolution, so that in the wild-type (but not the mutant) the electron flux is approximately maintained. The cyclopropane/propene production ratios are little affected by the presence of CO within the pressure ranges studied at least up to 50% inhibition.

A rapid, sensitive high-performance liquid chromatography analysis of ammonia and methylamine for nitrogenase assays.

A dansyl chloride precolumn derivatization method has been developed for high-performance liquid chromatography analysis of NH3 and/or CH3NH2 produced by nitrogenase-catalyzed reduction of substrates such as N2 (NH3) or diazirine (NH3, CH3NH2). The dansyl chloride reagent can be used immediately after preparation and is stable at 4 degrees C for 1 month. The derivatization products from NH3 and CH3NH2 are prepared by direct treatment of the assay mixture (30 min of incubation) and are then stable in air and ambient light at room temperature for at least 1 day. They are separated isocratically within several minutes on a mu Bondapak C-18 reversed-phase column (Altex) at 2000-2500 psi using an eluent of 7:7:3 H2O:methanol:acetonitrile and are detected fluorometrically (368 nm excitation; 500 nm emission). The NH3 sensitivity is limited by the background NH3 in the reagents and under practical conditions is ca. 0.02 nmol (20-microliters injection volume). Methylamine sensitivity is an order of magnitude greater. The detector response for either product is linear to at least 2.4 nmol. The method is compared to alternative analytical procedures with respect to sensitivity, convenience, and absence of interferences.

alpha-Cl-alpha-Br-phosphonoacetic acid is a potent and selective inhibitor of Na+/Pi cotransport across renal cortical brush border membrane.

We found that alpha-Cl-alpha-Br-phosphonoacetate (ClBrPAA) is a competitive, solute-specific inhibitor of Na+/Pi cotransport across renal cortical brush border membrane. Inhibition by ClBrPAA (Ki = 62 microM) is more than three times more effective than inhibition by phosphonoformate (PFA), the most potent Na+/Pi cotransport inhibitor known to date, and 26 times more effective than the parent compound, phosphonoacetate (PAA). These observations indicate that substitution of bromine and chlorine atoms at the alpha-carbon of PAA greatly enhances its efficacy as a competitive inhibitor of Na+/Pi cotransport. As ClBrPAA is much less inhibitory than PAA and PFA towards viral DNA polymerases and did not inhibit human alpha-DNA polymerase (ref. 10), the results also demonstrate that Na+/Pi cotransport inhibition can be dissociated from inhibition of DNA polymerases by phosphonocarboxylate compounds.

Circular dichroism and magnetic circular dichroism of reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

Studies of the circular dichroism (CD) and magnetic circular dichroism (MCD) of the dithionite-reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase (Av1) are reported. CD and MCD are measurable at room temperature across a wide spectral range, from the near-UV to the near-IR. The visible-near-UV CD is insignificantly affected by moderate variations in pH, temperature, ionic strength, and buffer, providing evidence against conformational change in the range studied. Mg2+ and ATP also cause no observable change in the visible-near-UV CD. Both CD and MCD in the visible-near-UV are unaffected by 30% inactivation by O2. However, the CD and MCD spectra of uncrystallized Av1 differ very significantly from those of crystallized Av1; in particular, the MCD spectrum is very sensitive to the presence of heme impurities. The identicality in both CD and MCD spectra of the reduced molybdenum-iron proteins from Azotobacter vinelandii and Klebsiella pneumoniae shows that these proteins contain metal clusters, identical in number, structure, and protein environment. While the absorption, CD, and MCD spectra of reduced Av1 are typical in many respects of simpler iron-sulfur proteins and are most similar to the [Fe4S4(SR)4]3- clusters found in reduced bacterial ferredoxins, significant differences exist. It is concluded, therefore, that the clusters present are not identical with those previously characterized, a conclusion earlier arrived at from electron paramagnetic resonance, Mössbauer, and EXAFS spectroscopies.

Low stereoselectivity in methylacetylene and cyclopropene reductions by nitrogenase.

The stereochemistry of reductions catalyzed by nitrogenase in 2H2O has been investigated by using allene, methylacetylene, and cyclopropene as substrates. Deuterium labeling patterns in the reduction products were determined by mass spectroscopy, infrared spectroscopy, 2H-decoupled 220-MHz 1H NMR, and 1H-decoupled 30.7-MHz 2H NMR. Reduction of allene gave pure [2,3-2H2]propene. Reduction of methyl acetylene gave a 1.8:1.0 mixture of [cis- and [trans-1,2-2H2]propene. (Similar reduction of acetylene reportedly gave virtually all [cis-1,2-2H2]ethylene.) Reduction of cyclopropene gave [cis-1,2-2H2]cyclopropane and a mixture of [2H2]propenes. The major propene 2H2 isomers formed were [trans-1,3-2H2]-propene (approximately 2), [cis-1,3-2H2]propene (approximately 1) and [2,3-2H2]propene (approximately 1). Cyclopropene appears to be unique as a nitrogenase substrate in that it simultaneously undergoes parallel reductions, one of which proceeds with high stereoselectivity while the other proceeds with low stereoselectivity. The weakly selective stereochemistry observed in these reductions is not consistent with a completely concerted dual proton-dual electron transfer mechanism. The results provide a basis to probe stereochemical effects in nitrogenase and in biomimetic model systems.

Circular dichroism and magnetic circular dichroism of nitrogenase proteins.

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from Azotobacter vinelandii (Av) and Klebsiella pneumoniae (Kp) have been obtained in the near infrared-visible-near ultraviolet spectral region. Previously, visible CD was reported to be absent or barely detectable in nitrogenase proteins; MCD spectra have not been reported. The chiroptical spectra can be measured in solution at room temperature, an advantage relative to spectroscopic methods requiring cryogenic sample temperatures. Absorption spectra were also obtained. The CD and MCD are markedly more structured, and thus interpretively more useful, than the corresponding absorption spectra. The dithionite-reduced MoFe proteins (Av1, Kp1) have nearly identical CD and MCD, demonstrating identical numbers and types of metal centers in similar protein environments. The CD and MCD cannot be explained solely in terms of contributions from known 4-Fe or 2-Fe clusters; the near-infrared MCD is inconsistent with the presence of known 4-Fe clusters. CD and MCD spectra of Lauth's violet-oxidized Kp1 are also reported. The reduced Fe proteins (Av2, Kp2) have similar CD and MCD, again indicating significant conservation of chromophore environment. The spectra clearly demonstrate the presence of a reduced bacterial ferredoxin-like (C(3-)) 4-Fe cluster. No obvious evidence of additional chromophores is observed. CD, MCD, and absorption spectra of Av1-oxidized Av2 are reported. The absorption spectrum shows the expected shoulder near 390 nm. The CD and MCD are characteristic of a C(2-) 4-Fe cluster; in particular, the diagnostic near-infrared MCD peak is observed at approximately 8300 cm(-1). The CD of Av2 oxidized in the presence and absence of MgATP are radically different, providing the first direct evidence for MgATP interaction with Fe protein in this oxidation state.