Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines,a novel class of herpesvirus antiviral agents

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.     

Three-dimensional numerical approach to investigate the substrate transport and conversion in an immobilized enzyme reactor

This numerical study evaluates the momentum and mass transfer in an immobilized enzyme reactor. The simulation is based on the solution of the three-dimensional Navier-Stokes equation and a scalar transport equation with a sink term for the transport and the conversion of substrate to product. The reactor consists of a container filled with 20 spherical enzyme carriers. Each of these carriers is covered with an active enzyme layer where the conversion takes place. To account for the biochemical activity, the sink term in the scalar transport equation is represented by a standard Michaelis-Menten approach. The simulation gives detailed information of the local substrate and product concentrations with respect to external and internal transport limitations. A major focus is set on the influence of the substrate transport velocity on the catalytic process. For reactor performance analysis the overall and the local transport processes are described by a complete set of dimensionless variables. The interaction between substrate concentration, velocity, and efficiency of the process can be studied with the help of these variables. The effect of different substrate inflow concentrations on the process can be seen in relation to velocity variations. The flow field characterization of the system makes it possible to understand fluid mechanical properties and its importance to transport processes. The distribution of fluid motion through the void volume has different properties in different parts of the reactor. This phenomenon has strong effects on the arrangement of significantly different mass transport areas as well as on process effectiveness. With the given data it is also possible to detect zones of high, low, and latent enzymatic activity and to determine whether the conversion is limited due to mass transfer or reaction resistances.     

Cranial integration in Homo: singular warps analysis of the midsagittal plane in ontogeny and evolution

This study addresses some enduring issues of ontogenetic and evolutionary integration in the form of the hominid cranium. Our sample consists of 38 crania: 20 modern adult Homo sapiens, 14 sub-adult H. sapiens, and four archaic Homo. All specimens were CT-scanned except for two infant H. sapiens, who were imaged by MR instead. For each specimen 84 landmarks and semi-landmarks were located on the midsagittal plane and converted to Procrustes shape coordinates. Integration was quantified by the method of singular warps, a new geometric-statistical approach to visualizing correlations among regions. The two classic patterns of integration, evolutionary and ontogenetic, were jointly explored by comparing analyses of overlapping subsamples that span ranges of different hypothetical factors. Evolutionary integration is expressed in the subsample of 24 adult Homo, and ontogenetic integration in the subsample of 34 H. sapiens. In this data set, vault, cranial base, and face show striking and localized patterns of covariation over ontogeny, similar but not identical to the patterns seen over evolution. The principal differences between ontogeny and phylogeny pertain to the cranial base. There is also a component of cranial length to height ratio not reducible to either process. Our methodology allows a separation of these independent processes (and their impact on cranial shape) that conventional methods have not found.     

Multifaceted roles of Lys166 of ribulose-bisphosphate carboxylase/oxygenase as discerned by product analysis and chemical rescue of site-directed mutants.

Ab initio calculations [King, W. A., et al. (1998) Biochemistry 37, 15414-15422] of an active-site mimic of D-ribulose-1,5-bisphosphate carboxylase/oxygenase suggest that active-site Lys166 plays a role in carboxylation in addition to its functions in the initial deprotonation and final protonation steps. To test this postulate, the turnover of 1-(3)H-labeled D-ribulose 1,5-bisphosphate (RuBP) by impaired position-166 mutants was characterized. Although these mutants catalyze slow enolization of RuBP, most of the RuBP-enediol undergoes beta-elimination of phosphate to form 2,3-pentodiulose 5-phosphate, signifying deficiencies in normal carboxylation and oxygenation. Much of the remaining RuBP-enediol is carboxylated but forms pyruvate, rather than 3-phospho-D-glycerate, due to incapacity in protonation of the terminal aci-acid intermediate. As a further test of the postulate, the effects of subtle perturbation of the Lys166 side chain on the carboxylation/oxygenation partitioning ratio (tau) were determined. To eliminate a chemically reactive site, Cys58 was replaced by a seryl residue without any loss of activity. The virtually inactive K166C-C58S double mutant was chemically rescued by aminoethylation or aminopropylation to reinsert a lysyl-like side chain at position 166. Relative to the wild-type value, tau for the aminoethylated enzyme was increased by approximately 30%, and tau for the aminopropylated enzyme was decreased by approximately 80%. Thus, two lines of experimentation support the theoretically based conclusion for the importance of Lys166 in the reaction of RuBP-enediol with gaseous substrates.     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.     

The prevalence of connexin 26 ( GJB2) mutations in the Chinese population

Mutations in GJB2, encoding gap junction beta 2 protein (connexin 26), are responsible for the commonest form of non-syndromic recessive deafness in many populations. It has been reported recently that the most common 35delG mutation in GJB2 is exceptionally low in Japanese and Korean populations, but another deletion, 235delC, is relatively frequent. Since the Chinese constitute approximately one fifth of the global population, the frequency of GJB2 mutations in the population has important implications for understanding worldwide causes of genetic deafness. To determine whether GJB2 mutations are an important cause of deafness in Chinese, we conducted mutation screening for GJB2 in 118 deaf Chinese probands, including 60 from simplex and 58 from multiplex families with non-syndromic deafness, and 150 normal hearing Chinese controls. Four mutations, including 235delC, 299-300delAT, V37I, and 35delG, were found in the patients. Thirty-nine percent of the probands had a GJB2mutation. Of the 118 probands, 19 carried two definitely pathogenic mutations: three among the 58 multiplex cases (5.2%) and 16 among the 60 simplex cases (26.7%). Twenty-seven probands (22.9%) were found to carry only single GJB2 mutations. None of them had mutations in exon 1 of GJB2 and or the 342-kb deletion of GJB6. The 235delC mutation was the most prevalent mutation (20.3% of alleles), accounting for 81% of the pathologic alleles in multiplex cases and 67% in simplex cases. Analysis of the affected haplotypes in the patients with the homozygous 235delC mutation yielded evidence for a single origin of the mutation. The carrier frequency of the 235delC mutation in control subjects with normal hearing was 1.3%. The 35delG mutation was only noted as a heterozygous change in two simplex cases (1.2% of alleles). These results indicated that mutations in GJB2 are a major cause of inherited and sporadic congenital deafness in the Chinese population. The 235delC mutation, rather than 35delG, is the most common mutation found in the Chinese deaf population. Our data support the view that specific combinations of GJB2 mutation exist in different populations.     

Chk1 suppresses bypass of mitosis and tetraploidization in p53-deficient cancer cells

Many cancer cells are unable to maintain a numerically stable chromosome complement. It is well established that aberrant cell division can generate progeny with increased ploidy, but the genetic factors required for maintenance of diploidy are not well understood. Using an isogenic model system derived by gene targeting, we examined the role of Chk1 in p53-proficient and -deficient cancer cells. Targeted inactivation of a single CHK1 allele in stably diploid cells caused an elevated frequency of mitotic bypass if p53 was naturally mutated or experimentally disrupted by homologous recombination. CHK1-haploinsufficient, p53-deficient cells frequently underwent sequential rounds of DNA synthesis without an intervening mitosis. These aberrant cell cycles resulted in whole-genome endoreduplication and tetraploidization. The unscheduled bypass of mitosis could be suppressed by targeted reversion of a p53 mutation or by exogenous expression of Cdk1. In contrast, the number of tetraploid cells was not increased in isogenic cell populations that harbor hypomorphic ATR mutations, suggesting that suppression of unscheduled mitotic bypass is a distinct function of Chk1. These results are consistent with a recently described role for Chk1 in promoting the expression of genes that promote cell cycle transitions and demonstrate how Chk1 might prevent tetraploidization during the cancer cell cycle.     

Escherichia coli K12 does not colonize the gastrointestinal tract of Fischer-344 rats

Summary The colonizing potential ofEscherichia coli K12 containing a vector coding for somidobove (bovine somatotropin) was determined. Treated male and female Fischer-344 rats were given a single oral gavage inoculum of sucrose with/without tetracycline (15 g/ml). Untreated control animals received similar drinking water regimes. All animals survived until termination. There were no clinical signs of toxicity observed and no treatment-related effect upon body weight, food consumption, or efficiency of food utilization. Fresh fecal samples were collected from each rat every 24 h following inoculation and the population of the marked strain was quantitated until no bacterial colonies were observed for two consecutive days. While all inoculated rats were positive at 24 h, by 72 and 96 h all had become negative for the test (marked) strain, as were the corresponding control group throughout the test. The frozen stock of the marked strain used as the positive control demonstrated that the agar plates were selective for the test strain. Fourteen days following inoculation, all groups of rats were killed and the gastrointestinal tracts removed and treated to recover the marked strain. There was no evidence of the marked strain in the gastrointestinal tract of any rat from any group. Thus, theE. coli K12 host/vector system used in this experiment does not colonize the gastrointestinal tract of Fischer-344 rats.     

Effect of potassium on drought resistance of Hibiscus rosa-sinensis cv. Leprechaun: Plant growth,leaf macro- and micronutrient content and root longevity

As competition for the limited water supply available for irrigation of horticultural crops increases, research into crop management practices that enhance drought resistance, plant water-use efficiency and plant growth when water supply is limited has become increasingly essential. This experiment was conducted to determine the effect of potassium (K) nutritional status on the drought resistance of Hibiscus rosa-sinensis L. cv. Leprechaun (Hibiscus). All the treatments were fertilized with Hoagland's nutrient solution, modified to supply K as K₂SO₄, at 0 mM K (K₀), 2.5 mM K (K_2.5), and 10 mM K (K₁₀), under two irrigation regimes (drought stressed [DS] and non-drought stressed [non-DS]). Regular irrigation and fertigation were adopted for 54 days, and drought stress treatment (initiated on day 55) lasted for 21 days; while non-DS control plants continued to receive regular irrigation and fertigation. Following the 21-day drought stress period, plants were labeled with ⁸⁶Rb⁺ to determine the percentage of post-drought stress live roots. Both K deficiency (K₀) and drought stress reduced shoot growth, but drought stress increased root growth and thus the root:shoot ratio. At K₀, plants were K-deficient and had the lowest leaf K, Fe, Mn, Zn, Cu, B, Mo and Al, and highest Ca concentrations. Although the percentage of live roots was decreased by drought stress, K_2.5 and K₁₀ plants (with similar percent live roots) had greater root survival ratio after drought treatment than the K-deficient plants. These observations indicate that adequate K nutrition can improve drought resistance and root longevity in Hibiscus rosa-sinensis.     

Aging of cylinders excised from pulp tissues of the ;golden delicious' apple

Aging cylinders excised from `Golden Delicious' apple (Pyrus malus L.) pulp, like the intact fruit, exhibit some characteristic phenomena such as rise in respiration (climacteric), ethylene synthesis, enzymic changes, and increase in ribosomes and mRNA. Aging of cylinders of pulp tissues may offer a useful physiological tool for the study of maturation and senescence.