Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.     

Molecular Genetics of Cystinuria: Identification of Four New Mutations and Seven Polymorphisms, and Evidence for Genetic Heterogeneity

A cystinuria disease gene (rBAT) has been recently identified, and some mutations causing the disease have been described. The frequency of these mutations has been investigated in a large sample of 51 Italian and Spanish cystinuric patients. In addition, to identify new mutated alleles, genomic DNA has been analyzed by an accurate and sensitive method able to detect nucleotide changes. Because of the lack of information available on the genomic structure of rBAT gene, the study was carried out using the sequence data so far obtained by us. More than 70% of the entire coding sequence and 8 intron-exon boundaries have been analyzed. Four new mutations and seven intragenic polymorphisms have been detected. All mutations so far identified in rBAT belong only to cystinuria type I alleles, accounting for ~44% of all type I cystinuric chromosomes. Mutation M467T is the most common mutated allele in the Italian and Spanish populations. After analysis of 70% of the rBAT coding region, we have detected normal sequences in cystinuria type II and type III chromosomes. The presence of rBAT mutated alleles only in type I chromosomes of homozygous (type I/I) and heterozygous (type I/III) patients provides evidence for genetic heterogeneity where rBAT would be responsible only for type I cystinuria and suggests a complementation mechanism to explain the intermediate type I/type III phenotype.     

Trophic relationships and seasonal utilization of salt-marsh creeks by zooplanktivorous fishes

Synopsis In a high salinity estuary at North Inlet, South Carolina, co-occurrence and possible competition among adults of four dominant zooplanktivorous fishes were minimized by seasonal adjustments in lateral and vertical distributions as well as in dietary preferences. In winter, Atlantic silversides, Menidia menidia, occupied the entire water column while other planktivores were rare or absent from the estuary, and they consumed large prey such as mysid shrimps and fish larvae. An immigration of bay anchovies, Anchoa mitchilli, in the spring resulted in a redistribution of species with Atlantic silversides shifting to the surface waters and bay anchovies dominating the lower half of the water column. Both fishes consumed mostly copepods in the spring, but each favored a different species. There was little similarity in the large prey items consumed by the two fishes. Striped anchovies, Anchoa hepsetus, arrived in mid-summer and were most abundant at the surface while bay anchovies continued to dominate the bottom waters. Atlantic silversides were rare in all summer collections. The diets of the two anchovies were similar, but vertical separation during the period of maximum zooplankton abundance probably minimized competition. Rough silversides, Membras martinica, which were obligate surface dwellers, shared the upper water column with striped anchovies, but the two species had very different diets during their period of co-occurrence. Although seasonal changes in fish diets reflected shifts in zooplankton composition and all fishes consumed a variety of prey types, preferences for some prey taxa and total avoidance of others were indicated. Electivity indices indicated an especially strong selection for fiddler crab megalopae by all fishes in the summer and fall. All fishes, except rough silversides, which fed almost exclusively on copepods and crab zoeae, consumed large prey items when they were available. Fine scale partitioning of the food resources was apparent in the selection of different copepod and insect species by the fishes. Spatial and temporal separation in the distribution and/or dietary preferences of the zooplanktivores fishes probably reduces the potential for resource competition. Given the high abundances and selectivity of the planktivores, significant impacts on some zooplankton populations probably result.     

Active site kinetics of horse serum cholinesterase

Horse serum Cholinesterase hydrolyzes choline and p-nitrophenol esters at different rates. Deacylation and acylation seem to be the rate-limiting step for charged and uncharged substrates respectively. Activation energy is similar for the acetic, propionic, and butyric esters of thiocholine, but it is higher for p-nitrophenylpropionate. Inhibition by the tetramethyl-ammonium ion is competitive. Tetraethyl-, tetrapropyl-, and tetrabutyl-ammonium ions are mixed-type inhibitors. The pH studies demonstrated the existence of a residue, pK = 6.33, involved in catalysis.     

Ultrastructural changes in Blastomyces dermatitidis after in vitro exposure to lidocaine

Electron microscopic examination of yeasts of Blastomyces dermatitidis, exposed in vitro to concentrations of lidocaine that occur when the drug is used for topical anesthesia, showed that lidocaine rapidly damaged intracellular structures. The extent of damage was dependent on the concentration of drug and length of exposure. The observed ultrastructural changes were very similar to those reported for other drugs that directly damage membranes. This relationship suggests that the antifungal effect of lidocaine is the result of direct membrane damage.     

Nitrogen fixation efficiency,natural15N abundance,and morphology of mesquite (Prosopis glandulosa) root nodules

Summary The relative nitrogen fixation efficiencies (RE 1-[H₂ evolved÷C₂H₂ reduced]·100) of four mesquite (Prosopis glandulosa var.torreyana) rhizobia (Strains WR 1001, WR 1002, L5, L9) and a cowpea rhizobia (Strain 176A32) on mesquite were evaluated in a glasshouse experiment. Plant yield, shoot N accumulation, and the natural¹⁵N abundance (¹⁵N) of nodule tissue were determined. Strain WR 1002 failed to nodulate mesquite and strain L5 produced ineffective nodules. Among the three effective strains (WR 1001, L9, 176A32) the cowpea strain (176A32) and strain L9 had significantly higher RE than strain WR 1001. Differences in RE, however, were not accompanied by significantly higher plant yield and shoot N accumulation. The difference in¹⁵N abundance between foliar tissue and nodules (nodules minus leaves) was 0.47 ¹⁵N for the ineffective L5 nodules, while for the effective WR 1001, L9, and 176A32 nodules, respectively, this difference was 8.35, 7.81, and 8.35 ¹⁵N. This indicates a similar relationship between N₂-fixing effectiveness and natural¹⁵N enrichment of nodules that was previously observed in soybeans (Glycine max, L. Merr.). Strains WR 1001 and L9 produced elongate, indeterminate nodules typical for mesquite. The ineffective L5 nodules had few infected cells and an abundance of cortical amyloplasts. Mesquite nodules produced by the cowpea strain were spherical and were somewhat more similar in internal morphology to determinate nodules typical of cowpea than indeterminate nodules normally associated with mesquite.     

Liquid preservation of highly purified human granulocytes

Highly purified human granulocytes isolated from continuous flow centrifugation leukapheresis concentrates by counterflow centrifugation-elutriation were stored at 4 °C in concentrations of 6 × 10⁶ to 1 × 10⁷ granulocytes per milliliter for up to 14 days. The in vitro physiological function assays of phagocytosis, oxygen consumption associated with phagocytosis, bacterial growth inhibition, chemotaxis, and five enzyme analyses indicated good storage survival for up to 4 days. Stored granulocytes separated from other blood cells have greater storage stability than granulocytes stored as leukapheresis concentrates. After 14 days of storage a small percentage of granulocytes still maintained all physiological functions, with the exception of chemotaxis. Of the five enzymes assayed, only the enzyme activity of leucine aminopeptidase decreased significantly by the 14th day of storage. The storage stability of each physiological function assayed decreased as follows: bacterial growth inhibition (most stable), phagocytosis, oxygen consumption, and chemotaxis (least stable).     

The genome of Zea mays,its organization and homology to related grasses

The pattern of genome organization of Zea mays has been analyzed, and the relationship of maize to possible progenitor species assessed by DNADNA hybridization. Reassociation of 470 and 1,350 bp fragments of maize DNA to various C₀t values demonstrates that the genome is composed of 3 major kinetic classes: highly repetitive, mid-repetitive, and unique. Mini-C₀t curves of the repetitive sequences at short fragment length indicate that the highly repetitive sequence class is 20% of the genome and is present at an average reiteration frequency of 800,000 copies; the mid-repetitive sequence class is 40% of the genome and is present at an average reiteration frequency of 1,000 copies. Thermal denaturation studies show that the highly repetitive sequences are 12% divergent and mid-repetitive sequences are 6% divergent. Most of the genome is organized in two interspersion patterns. One, approximately one-third of the genome, is composed of unique sequences of average length 2,100 bp interspersed with mid-repetitive sequences; the other, also one-third of the genome, is mid-repetitive sequences interspersed with highly repetitive sequences. The repetitive sequences are 500 to 1,000 bp by electron microscopic measurement. The remaining third of the genome is unique sequences farther than 5,000 bp from a palindromic or repetitive sequence. Hybridization of maize DNA from Midwestern Dent to popcorn and related grasses indicates that both the unique and repetitive sequence elements have diverged. Teosinte and popcorn are approximately equally divergent from Midwestern Dent whereas Tripsacum is much more divergent. The divergence times calculated from the depression of T_m in heterologous duplexes indicate that the divergence within Zea mays and between maize and near relatives is at least an order of magnitude greater than expected. This high degree of divergence may reflect the pressures of domestication of maize.     

Non-random X-chromosome expression early in mouse development

We have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1), in order to investigate the randomness of X-chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk-1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk-1^a/Pgk-1^b heterozygous conceptuses, and in the one informative 5 1/2-day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (X^m), so soon after the time of X-chromosome inactivation, favors the possibility that the preferential expression of X^m is a consequence of primary non-random X-chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK-1 band, corresponding to the allozyme produced by the Pgk-1 allele on X^m, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.