Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum.

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.     

Linkage of the acetylcholine transporter-vesamicol receptor to proteoglycan in synaptic vesicles.

The relationship of the acetylcholine transporter-vesamicol receptor (AcChT-VR) to proteoglycan in Torpedo electric organ synaptic vesicles was investigated. The cholate-solubilized VR was immunoprecipitated by a monoclonal antibody directed against the SV1 epitope located in the glycosaminoglycan portion of the proteoglycan. AcChT that was photoaffinity-labeled with a tritiated high-affinity analogue of AcCh [cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate] and then denatured in sodium dodecyl sulfate also immunoprecipitated. The labeled AcChT exhibited a M(r) range of 100,000-200,000. Proteoglycan did not engage in detectable nonspecific reversible aggregation that might mask the presence of another subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vesicles permeabilized with cholate, the enzymes keratanase and testicular hyaluronidase inactivated binding of vesamicol and destroyed the SV1 epitope without detectable proteolysis. Other glycosaminoglycan-degrading enzymes were without effect. The results demonstrate that the AcChT-VR and proteoglycan are very strongly linked and that glycosaminoglycan-like polysaccharide controls the conformation of the VR. The unexpected linkage to proteoglycan suggests that AcChT-VR in intact terminals might communicate with extracellular matrix and participate in stabilization and operation of the synapse.     

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.     

Lysosomal interconnections and horseradish peroxidase compartmentalization in three-dimensionally reconstructed bovine alveolar macrophages

Following a 1-h incubation of bovine alveolar macrophages in 1 to 2 mg/ml exogenous horseradish peroxidase (HRP), ultrathin sections revealed vacuolar interconnections among both labeled and unlabeled vacuoles constituting the lysosomal compartment. Four entire cells and their vacuolar components were subsequently computer resconstructed from serial transmission electron micrographs and measured using a morphometric technique. HRP-labeled and unlabeled vacuoles ranged in size from 0.5 micron to greater than or equal to 4.0 microns in diameter and occupied up to 25% of the cytoplasmic volume. HRP-containing vacuoles were distributed throughout each cell in a clumped distribution (P less than 0.05) and occupied up to 75% of the total vacuole compartment. Up to 60% of all vacuoles were interconnected through a series of openings formed by membrane fusions (average pore diameter 0.42 micron), which resulted in a labyrinth of vacuoles comprising up to 55% of the total volume of the lysosomal compartment. The area of open interconnections resulting from vacuolar fusions represented less than 1% of the total surface area of the lysosomal membrane. Rotation of a three-dimensionally reconstructed macrophage about the Y-axis revealed an interconnected vacuolar network of 75 fused vacuoles in a chain up to 21 microns in length. We have demonstrated that HRP-labeled vacuoles interconnect with each other as well as with preexisting unlabeled vacuoles. As a result of such interconnections, individual vacuoles become contributing members of a large, continuous, lysosomal compartment in bovine alveolar macrophages.     

Crown ether mediated cadmium halide dimers and polymers

The reactions of cadmium halides with the 15-membered macrocyclic crown ethers, 15-crown-5 and benzo-15-crown-5, have been carried out and six new complexes have been isolated and structurally characterized. Metal to ligand stoichiometries of 1:1, 2:1, 3:1 and 3:2 have been observed with a variety of different formulations. Examples of charge separated ion pairs ([(NH₄)(benzo-15-crown-5)₂]₂[Cd₂I₆]), halogen bridged monomers, dimers or polymers ([Cd(15-crown-5)(OHMe)(μ-Br)CdBr₃], [Cd(15-crown-5)(μ-Br)₂CdBr(μ-Br)]₂(isolated from the same reaction mixture) and [(CdCl₂)₂CdCl₂(15-crown-5)]_n), and hydrogen bonded finite chains or polymers ([(Cd(OH₂)₂(15-crown-5)][CdI₃(OH₂)]₂·2(15-crown-5)·2CH₃CN and [CdI₂(OH₂)₂(THF)]·benzo-15-crown-5) have been isolated. Three different types of 15-crown-5 coordination modes have been observed in these complexes. In-cavity coordination resulting in pentagonal bipyramidal geometries about Cd²⁺ was observed in [(CdCl₂)₂CdCl₂(15-crown-5)]_n, [Cd(15-crown-5)(OHMe)(μ-Br)CdBr₃], and [Cd(OH₂)₂(15-crown-5)][CdI₃(OH₂)]₂·2(15-crown-5)·2CH₃CN, [Cd(15-crown-5)(μ-Br)₂CdBr(μ-Br)]₂ displays out-of-cavity coordination with one etheric donor distorted into an axial position of a distorted pentagonal bipyramid. The third coordination mode is secondary sphere coordination via hydrogen bonding which is observed for [Cd(OH₂)₂(15-crown-5)][CdI₃(OH₂)]₂·2(15-crown-5)·2CH₃CN. The good fit of Cd²⁺ within the cavity of 15-crown-5 results in shorter bonding contacts and a more narrow distribution in Cd---O values (2.273(7)-2.344(6) Å) than observed for cadmium halide complexes of 18-crown-6 (Cd---O = 2.69(1)–2.81(1) Å).     

Ectoparasitic Acugutturid Nematodes of Adult Lepidoptera

Noctuidonema guyaneme is an interesting ectoparasite of adult Lepidoptera that feeds on hosts from at least five families with its long stylet. Noctuidonema guyanense spends its entire life on the adult moth and is sustained as it is passed from moth to moth during host mating. Overlapping host generations are essential for parasite survival. This nematode occurs throughout tropical and subtropical America and is transported by at least one of its hosts, Spodoptera frugiperda, during migration to northern sites in the United States each spring. Noctuidonema guyanense debilitates its hosts. Research conducted to help determine the biological control importance of this nematode is reviewed. Two additional species, N. daptria and N. dibolia, are now known for Noctuidonema.     

A novel human type I hair keratin gene: evidence for two keratin hHa3 isoforms

We present the nucleotide and amino acid sequence for a novel human type I hair keratin, which could be identified through its high sequence homology and strict carboxyterminal length identity as a human ortholog of the murine hair keratin mHa3. Our hHa3 sequence differs, however, from that of a previously described hHa3 hair keratin (published only as an amino acid sequence; [13]) in 24 amino acid positions, 8 of which occur in the middle of the carboxyterminal domain. PCR of genomic DNA from 25 normal human subjects using a primer pair derived from sequence segments located in the 3-region of our hHa3 clone that encode conserved amino acid sequences in both keratins, resulted in the amplification of two distinct products of 0.38 kbp and 1.0 kbp. DNA sequence analysis of the cloned PCR products allowed identification of the 0.38 kb sequence as that originating from Yuet al. [13] and the 1.0 kb sequence as that being derived from our data. The difference in fragment length was due to unique intron 6 sequences, indicating that these two keratin species are encoded by genes of their own. Moreover, extensive Southern blot analyses with DNA from 25 unrelated individuals of different races using a 3-noncoding sequence from our keratin and the intron 6 sequence of the keratin of Yuet al. [13], as hybridization probes showed that both keratin genes are present as single copy sequences occurring ubiquitously and without gross alterations in the human genome. Collectively, these data demonstrate that the human type I hair keratin described in this paper represents an isoform of the previously described hHa3 keratin. We propose that these hHa3 isoforms be named in chronological order of discovery hHa3-I and hHa3-II.     

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.     

CENTRAL MOMENTS AND PROBABILITY DISTRIBUTION OF COLLESS'S COEFFICIENT OF TREE IMBALANCE

The great increase in the number of phylogenetic studies of a wide variety of organisms in recent decades has focused considerable attention on the balance of phylogenetic trees—the degree to which sister clades within a tree tend to be of equal size—for at least two reasons: (1) the degree of balance of a tree may affect the accuracy of estimates of it; (2) the degree of balance, or imbalance, of a tree may reveal something about the macroevolutionary processes that produced it. In particular, variation among lineages in rates of speciation or extinction is expected to produce trees that are less balanced than those that result from phylogenetic evolution in which each extant species of a group has the same probability of speciation or extinction. Several coefficients for measuring the balance or imbalance of phylogenetic trees have been proposed. I focused on Colless's coefficient of imbalance (7) for its mathematical tractability and ease of interpretation. Earlier work on this statistic produced exact methods only for calculating the expected value. In those studies, the variance and confidence limits, which are necessary for testing the departure of observed values of I from the expected, were estimated by Monte Carlo simulation. I developed recursion equations that allow exact calculation of the mean, variance, skewness, and complete probability distribution of I for two different probability-generating models for bifurcating tree shapes. The Equal-Rates Markov (ERM) model assumes that trees grow by the random speciation and extinction of extant species, with all species that are extant at a given time having the same probability of speciation or extinction. The Equal Probability (EP) model assumes that all possible labeled trees for a given number of terminal taxa have the same probability of occurring. Examples illustrate how these theoretically derived probabilities and parameters may be used to test whether the evolution of a monophyletic group or set of monophyletic groups has proceeded according to a Markov model with equal rates of speciation and extinction among species, that is, whether there has been significant variation among lineages in expected rates of speciation or extinction.     

Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation.

Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA.