Identification of second-site mutations that enhance release and spread of vaccinia virus

The spread of most strains of vaccinia virus in cell monolayers occurs predominantly via extracellular enveloped virions that adhere to the tips of actin-containing microvilli and to a lesser extent via diffusion of released virions. The mechanism by which virions adhere to the cell surface is unknown, although several viral proteins may be involved. The present investigation was initiated with the following premise: spontaneous mutations that increase virus release will be naturally selected by propagating a virus unable to spread by means of actin tails. Starting with an A36R deletion mutant that forms small, round plaques, five independent virus clones with enhanced spread due to the formation of comet or satellite plaques were isolated. The viral membrane glycoprotein genes of the isolates were sequenced; four had mutations causing C-terminal truncations of the A33R protein, and one had a serine replacing proline 189 of the B5R protein. The comet-forming phenotype was specifically reproduced or reversed by homologous recombination using DNA containing the mutated or natural sequence, respectively. Considerably more extracellular enveloped virus was released into the medium by the second-site mutants than by the parental A36R deletion mutant, explaining their selection in tissue culture as well as their comet-forming phenotype. The data suggest that the B5R protein and the C-terminal region of the A33R protein are involved in adherence of cell-associated enveloped virions to cells. In spite of their selective advantage in cultured cells, the second-site mutants were not detectably more virulent than the A36R deletion mutant when administered to mice by the intranasal route.     

New tool use by wild Sumatran orangutans (Pongo pygmaeus abelii)

Two forms of tool use by wild Sumatran orangutans are reported from the Agusan Monitoring Station, a new research site in Indonesia. One form, a branch "hook" used in locomotion, has not been reported previously in wild orangutans. The second form, a leaf "pad" used to protect the hands and feet from thorns while feeding, shares similarities in form and function with a tool type used by orangutans at Ketambe, a nearby research site. Both instances of tool use occurred in areas of disturbance, and appear to be spontaneous inventions under novel conditions, although habitual use of the tool in other ecological contexts is plausible. A summary of the distribution of tool use types in wild orangutans is presented.     

Phylogenetic relationships in the Gesnerioideae (Gesneriaceae) based on nrDNA ITS and cpDNA trnL-F and trnE-T spacer region sequences

The Gesnerioideae includes most of the New World members of the Gesneriaceae family and is currently considered to include five tribes: Beslerieae, Episcieae, Gesnerieae, Gloxinieae, and Napeantheae. This study presents maximum parsimony and maximum likelihood phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer regions (ITS), and the chloroplast DNA trnL intron, trnL-trnF intergenic spacer region, and trnE-trnT intergenic spacer region sequences. The ITS and cpDNA data sets strongly support the monophyly of a Beslerieae/Napeantheae clade; an Episcieae clade; a Gesnerieae clade; a Gloxinieae clade minus Sinningia, Sinningia relatives, and Gloxinia sarmentiana; and a Sinningia/Paliavana/Vanhouttea clade. This is the first study to provide strong statistical support for these tribes/clades. These analyses suggest that Sinningia and relatives should be considered as a separate tribe. Additionally, generic relationships are explored, including the apparent polyphyly of Gloxinia. Chromosome number changes are minimized on the proposed phylogeny, with the exception of the n = 11 taxa of the Gloxinieae. Scaly rhizomes appear to have been derived once in the Gloxinieae sensu stricto. The number of derivations of the inferior ovary is unclear: either there was one derivation with a reversal to a superior ovary in the Episcieae, or there were multiple independent derivations of the inferior ovary.     

An analysis of telephone interview data collected in 1992 from 820 women who reported problems with their breast implants to the food and drug administration

How health care providers deal with concerns and feelings of women who have problems with their breast implants affects the women's satisfaction with their breast implants, yet in 1992 little was known about the concerns and feelings of these women. A qualitative analysis of in-depth telephone interviews conducted in 1992 with 820 women from all regions of the United States who reported problems with their breast implants to the Food and Drug Administration and responded to an invitation to be interviewed provided data. Respondents were primarily 40 to 69 years of age at the time of interview, Caucasian, married, and educated beyond high school. The sample was almost equally divided in reason for breast implants, with 65 percent being dissatisfied with their breast implants. Nearly all of the women had heard of problems with silicone gel-filled implants. Their main sources of information were television, newspapers, and magazines rather than their physicians or the breast implant manufacturers. Some women tried to avoid hearing the reports, and many tried to put the reported problems out of their minds. However, a majority (88.7 percent) wanted more information. The women expressed feelings of anger, regret, and worry, and repeatedly said they needed more information. Women who contacted the Food and Drug Administration about breast implant problems needed accurate and honest information from health care professionals. They wanted their physicians to explore their symptoms, fears, and concerns.     

Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica

Root-knot nematodes (Meloidogynejavanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.     

Post-cranial prey transport mechanisms in the black pinesnake, Pituophis melanoleucus lodingi: an x-ray videographic study

Most previous studies of snake feeding mechanisms have focused on the functional morphology of the highly specialized ophidian jaw apparatus. Although some of these studies have included observations of post-cranial movements during feeding, the functional roles of these movements have remained poorly understood. In this study, we used x-ray videography to examine post-cranial prey transport mechanisms in a colubrid snake, Pituophis melanoleucus lodingi. We found that prey transport in this species progresses through four distinct phases, three of which are characterized by either undulatory or concertina-like movements of the anterior portion of the trunk. In the first phase of transport (the oral phase), unilateral movements of the jaws are used to pull the head forward around the prey. In the second phase (the orocervical phase), unilateral jaw movements continue, but are augmented by concertina-like movements of the anterior portion of the trunk. In the third phase (the cervical phase), prey transport occurs exclusively through concertina-like movements of the neck. Finally, in the fourth phase (the thoracic phase), prey is transported to the stomach via undulatory movements of the trunk. Our observations of feeding behavior in a phylogenetically diverse sample of fourteen other snake species demonstrate that similar post-cranial transport mechanisms are used by a wide variety of alethinophidian snakes that feed on large, bulky prey.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

A critical appraisal of clinical practice guidelines for the treatment of lower-limb osteoarthritis

Clinical practice guidelines are important tools to assist clinical decision-making. Recently, several guidelines addressing the management of osteoarthritis (OA) have been published. Clinicians treating patients with OA must ensure that these guidelines are developed with consistency and methodological rigour. We undertook a qualitative summary and critical appraisal of six medical treatment guidelines for the management of lower-limb OA published in the medical literature within the past 5 years. A review of these six guidelines revealed that each possesses strengths and weakness. While most described the scope and intended patient populations, the guidelines varied considerably in the rigour of their development, coverage of implementation issues, and disclosure of conflicts of interest.