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K E Weaver

Publications and source records attributed to K E Weaver.

At least 19 recordsLinked to original sources
PubMed1998

Molecular characterization of Mycoplasma arthritidis variable surface protein MAA2.

Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2 genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed in Escherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidis clonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative -10 box. Full-sized recombinant MAA2 was expressed in E. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

Amino Acid Sequence↗
PubMed1996

Functional analysis of the Enterococcus faecalis plasmid pAD1-encoded stability determinant par.

The molecular organization and functional characteristics of the PAD1 replicon-encoded par stability determinant were examined. par encodes two convergently transcribed RNAS of approximately 210 and 65 nucleotides designated RNA I and RNA II, respectively. The sequence of RNA II is largely complementary to RNA I, suggesting that RNA II could regulate RNA I function as an anti-sense RNA. Results of functional studies are consistent with a role for par as a post-segregational killing system, the first to be identified in Gram-positive bacteria, with RNA I encoding the toxin and RNA II the antidote. These results include: (i) destabilization of par-containing replicons in the presence of a second complete par or the RNA II coding sequence in the same cell; (ii) par-dependent stabilization of a highly unstable vector at the expense of host-cell growth rate; and (iii) protection of cells from the toxic effects of overexpression of RNA I by RNA II supplied in trans.

Base Sequence↗
PubMed1995

Auditory brainstem responses of CBA/J mice with neonatal conductive hearing losses and treatment with GM1 ganglioside.

Exogenous administration of GM1 ganglioside to CBA/J mice with a neonatal conductive hearing loss ameliorates the atrophy of spiral ganglion neurons, ventral cochlear nucleus neurons, and ventral cochlear nucleus volume. The present investigation demonstrates the extent of a conductive loss caused by atresia and tests the hypothesis that GM1 ganglioside treatment will ameliorate the conductive hearing loss. Auditory brainstem responses were recorded from four groups of seven mice each: two groups received daily subcutaneous injections of saline (one group had normal hearing; the other had a conductive hearing loss); the other two groups received daily subcutaneous injections of GM1 ganglioside (one group had normal hearing; the other had a conductive hearing loss). In mice with a conductive loss, decreases in hearing sensitivity were greatest at high frequencies. The decreases were determined by comparing mean ABR thresholds of the conductive loss mice with those of normal hearing mice. The conductive hearing loss induced in the mice in this study was similar to that seen in humans with congenital aural atresias. GM1 ganglioside treatment had no significant effect on ABR wave I thresholds or latencies in either group.

Acoustic Stimulation↗
PubMed1995

Association of lysogenic bacteriophage MAV1 with virulence of Mycoplasma arthritidis.

Mycoplasma arthritidis causes a severe polyarthritis under natural conditions in rats and under experimental conditions in both rats and mice. Although the disease itself has been extensively studied, M. arthritidis virulence factors remain uncharacterized. Comparison of relative arthritogenicity of 20 strains of M. arthritidis revealed that the strains tended to fall into two groups, a highly arthritogenic group, inducing maximum arthritis scores of > or = 11 in rats, and a low-virulence group, inducing maximum scores of < 6. Chromosomal DNA from the more highly arthritogenic strains possessed sequences that hybridized by Southern analysis with a probe prepared from lysogenic M. arthritidis bacteriophage MAV1, while DNA from low-virulence strains did not. One of the low-virulence strains, 158, was experimentally lysogenized with MAV1. Lysogenized 158 showed a significant increase in arthritogenicity over nonlysogenized 158. These data suggest that MAV1 carries a factor that is important in pathogenesis of M. arthritidis-induced arthritis of rats.

Animals↗
PubMed1995

Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region.

pAD1 (60 kb) is a conjugative, hemolysin/bacteriocin plasmid in Enterococcus faecalis. It confers a mating response to the peptide sex pheromone cAD1 produced by recipient (plasmid-free) cells, leading to highly efficient plasmid transfer in broth matings. Control of the physiological response to cAD1 can been overridden by a reversible phase variation event at frequencies on the order of 10(-4) to 10(-3) per cell per generation (L. T. Pontius and D. B. Clewell, Plasmid 26:172-185, 1991). The variant forms are designated Dryc and Dry+, which reflects the colony morphologies of cells whose conjugation functions are switched on and off, respectively. Here we show that Dryc variants exhibit a structural change in a region between repA and repB that contains two clusters of 8-bp iterons. The change involved a 31- or 32-bp increase in size of this region. In three or four independent variants examined, one of the iteron clusters increased in size from 13 to 17 iterons. When iteron DNA was placed on a multicopy plasmid and introduced into a wild-type pAD1 derivative, the Dryc phenotype was generated. Since traA, a key negative regulator of conjugation, bears several centrally located iteron-like sequences with the same orientation, we speculate that the protein(s) that normally binds iterons (possibly RepA and/or RepB) blocks traA transcription in Dryc variants.

Bacterial Proteins↗
PubMed1995

Enterococcus faecalis plasmid pAD1 replication and maintenance.

A determinant has been identified which is required for the stable inheritance of a replicon isolated from the pheromone-responsive E. faecalis plasmid pAD1. This determinant, called par, is encoded on a DNA fragment no larger than 457 bp and directs the production of two small RNAs of approximately 250 (RNAI) and 145 (RNAII) nucleotides. Mutations affecting par RNA production also affected plasmid stability, indicating that these RNAs play a central role in par function. Putative transcription start sites were identified within par by DNA sequence analysis, but the sizes of the observed RNAs and the effects of transposon inserts on RNA production are not entirely consistent with predictions based on the hypothetical RNAs produced from these start sites. The absence of clearly functional open reading frames and the presence of various sequence repeat elements within par suggest that these RNAs may not be translated but may perform some structural role in the partition apparatus. The isolated par determinant was capable of producing both RNAs and stabilizing a heterologous vector, indicating that no other pAD1-encoded determinants are required for par function.

Base Sequence↗
PubMed1994

Identification and characterization of an Enterococcus faecalis plasmid pAD1-encoded stability determinant which produces two small RNA molecules necessary for its function.

A determinant, designated par, essential for stable maintenance for an autonomously replicating fragment of the Enterococcus faecalis plasmid pAD1, was identified by transposon mutagenesis, and its DNA sequence was determined. The position of flanking transposon inserts with no effect on stability indicates that par is encoded on no more than approximately 720 bp of DNA. This region contains no large open reading frames (> 62 amino acids) but does contain a number of direct and inverted repeats and GC- and AT-rich boxes. Disruption of these elements by transposon insertion, or deletion of the entire cluster of elements, resulted in a loss of plasmid stability but did not affect replication or copy number in E. faecalis. Characterization of selected mutants suggested that some manipulations of par may interfere with essential plasmid replication functions and/or be lethal to the host cell. Northern blot analysis revealed that two small RNA molecules of approximately 250 and 145 nucleotides homologous to par are produced by cells containing the complete pAD1 replicon. Mutations affecting par that resulted in a decrease in plasmid stability also resulted in changes in the pattern of production of the par RNAs, suggesting that these RNAs are important for par function.

Base Sequence↗
PubMed1993

Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.

Amino Acid Sequence↗
PubMed1991

Control of Enterococcus faecalis sex pheromone cAD1 elaboration: effects of culture aeration and pAD1 plasmid-encoded determinants.

Aeration of plasmid-free Enterococcus faecalis strains resulted in an 8- to 16-fold decrease in sex pheromone cAD1 activity in culture filtrates. Levels of two unrelated pheromones, cPD1 and cAM373, were unaffected by culture aeration. Aeration also resulted in a decrease in the expression of conjugative transfer functions observed in cells containing pAD1 traB mutations, verifying a link between traB function and pheromone "shutdown." Tests with a series of pAD1 mini-plasmids indicated that the product of the traB gene was involved in, but not sufficient for, pheromone shutdown; the cooperation of one or more other gene products encoded within the pheromone response control region was required.

Aerobiosis↗
PubMed1990

Total body irradiation and bone marrow transplantation for immunodeficiency disorders in young children.

Congenital immunodeficiency disorders such as severe combined immunodeficiency disease (SCID), Wiskott-Aldrich syndrome, and Chediak-Hegashi syndrome are almost uniformly fatal with most children dying before age one. Allogeneic bone marrow transplant (BMT) is the treatment of choice. Few of these children have matched donors. We use bone marrow processing techniques that allow us to utilize marrow from the parents. Children who lack HLA-identical donors are offered haploidentical, T-cell depleted parental BMTs. Some of these children do not have an immune deficiency severe enough to allow durable engraftment of processed mismatched bone marrow. Successful engraftment may necessitate the use of immunosuppression. Total body irradiation (TBI) is part of our intensive conditioning regimen for children with Wiskott-Aldrich and Chediak-Hegashi syndrome and most children with SCID who have undergone an unsuccessful prior mismatched, T-cell depleted BMT, or who have a high likelihood of donor marrow rejection based on pre-transplant immune function testing. TBI is considered extremely toxic therapy in infancy, with little information available on the acute and chronic effects. The 10 children presented in this report are among the youngest to have received TBI. Five patients were 2 to 6 months of age when they received TBI. The conditioning regimen for all patients was; antithymocyte globulin (25 mg/kg/day, x 3 days), cyclophosphamide (60 mg/kg/day, x 2 days), and TBI. 7.0 Gy TBI was given as a single dose AP-PA at approximately 15 cGy/min. Half value blocks shielded the brain, eyes and lungs. Six of 10 children were alive from 7 to 72 months post transplant.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow Transplantation↗
PubMed1990

Regulation of the pAD1 sex pheromone response in Enterococcus faecalis: effects of host strain and traA, traB, and C region mutants on expression of an E region pheromone-inducible lacZ fusion.

Pheromone-induced conjugal transfer of the hemolysin-bacteriocin plasmid pAD1 of Enterococcus faecalis is regulated by a cluster of determinants designated traA, traB, and regions C and E. The E region is believed to include a positive regulator that controls many structural genes related to conjugation. The pheromone-inducible Tn917-lac fusion NR5, located in the E region, is regulated by the products of traA, traB, and the C region. To more closely examine the effects of these genes on the induction of E region products, inserts in each of these genes were combined with the NR5 fusion in a novel approach involving triparental matings with a pAD1 miniplasmid and recombinational mutagenesis. Results indicate that (i) the traA gene product is a key repressor of the pheromone response; (ii) the traB gene product, in cooperation with a gene within or regulated by the E region, controls pheromone shutdown; (iii) a primary function of the C region gene product is in pheromone sensing, with secondary functions in pheromone shutdown and negative regulation; and (iv) the host in which the plasmid resides has a dramatic effect on the regulation of the NR5 fusion in traB and C region mutants. Numerous parallels were observed between the regulation of the NR5 fusion and the regulation of the aggregation and transfer response. These parallels aided in further defining the functions of particular regulatory determinants as well as further establishing the link between the regulation of the E region and the regulation of the aggregation and transfer response.

Cloning, Molecular↗
PubMed1989

Construction of Enterococcus faecalis pAD1 miniplasmids: identification of a minimal pheromone response regulatory region and evaluation of a novel pheromone-dependent growth inhibition.

Several pAD1 miniplasmids were constructed that consisted of all or a portion of the pAD1 EcoRI B fragment with pheromone-inducible "E region" lacZ transcriptional fusions. Miniplasmids containing the entire EcoRI B fragment (the "E miniplasmids") were found to regulate LacZ expression normally, indicating that sufficient information is present on this fragment to regulate a pheromone response. These plasmids also encoded normal replication functions. The E miniplasmids were further reduced by deleting a KpnI fragment. These "K miniplasmids" were able to perform most of the functions central to a pheromone response but failed to induce the fused transcripts to levels observed in the parental plasmids. This defect was found to be due to a pheromone-dependent growth inhibition of cells containing the K miniplasmids. Evidence indicated that this inhibition was due to transcriptional readthrough beyond the lacZ gene and into the putative replication region of the plasmid. Possible mechanisms of this inhibition as well as its potential usefulness in further examining the characteristics of the pheromone response are discussed.

Chromosome Mapping↗
PubMed1989

Sex pheromones and plasmid transfer in Enterococcus faecalis.

Plasmid-free Enterococcus faecalis excrete peptides (sex pheromones) which specifically induce a mating response in strains harboring certain conjugative plasmids. The response is characterized by the synthesis of a "fuzzy" surface material, visible by electron microscopy, which is believed to facilitate the aggregation of donors and recipients. Transconjugants which receive a specific plasmid shut down the production of endogenous pheromone; however, they continue to produce pheromones specific for donors harboring different classes of plasmids. In this review, we summarize what is known about the biochemistry and genetics of this phenomenon. Some emphasis is given to the hemolysin plasmid pAD1 and the regulation of its conjugal transfer.

Amino Acid Sequence↗
PubMed1989

Evaluation of abdominal pain.

This article discusses the approach to evaluation of abdominal pain in the Emergency Department setting. The physiologic differences among visceral, somatic, and referred pain are reviewed, as is the clinical significance of anorexia, nausea, and vomiting. The correlation of historical facts, interrelationships of symptoms, and pain patterns with specific intra-abdominal processes is reviewed. Recommendations for high-yield expedient laboratory studies to facilitate diagnosis are provided. Proficiency in the recognition and interpretation of these clinical symptoms, physical signs, laboratory data, ECGs, and radiographic findings is important when evaluating acutely ill, uncooperative, or unresponsive patients.

Abdomen↗
PubMed1988

Regulation of the pAD1 sex pheromone response in Enterococcus faecalis: construction and characterization of lacZ transcriptional fusions in a key control region of the plasmid.

Strains carrying the Enterococcus (formerly Streptococcus) faecalis plasmid pAD1 responded to exogenous sex pheromone by inducing a number of gene products which facilitated mating. A 7-kilobase region of pAD1 was identified which contained genes that are important for the regulation of this response. Using the transposon Tn917-lac delivery vector pTV32Ts, we generated a number of fusions that allowed us to examine transcription in this region. At least three transcriptional units were identified by grouping fusions by their phenotype, direction of transcription, and response to pheromone. Transcription from one set of fusions was sensitive to the presence of pheromone. Analysis of the patterns of protein production previously shown to be induced in the presence of pheromone provided more information on the function of the genes of interest. We postulate the existence of two negative regulatory proteins that act coordinately to repress the pheromone response, one of which may be involved in sensing or transmitting the pheromone signal, and at least one positive regulatory protein whose synthesis is dependent on the presence of pheromone. In addition, the isolation of a relatively small deletion mutant capable of producing cAD1. the pheromone specific for pAD1-containing cells, indicates that a factor(s) that is important for the shutdown of endogenous pheromone is also present in this region.

Bacterial Proteins↗
PubMed1988

Nutritional management of patients with ARC or AIDS.

Investigators are now predicting that 75 to 80 per cent of the 1.5 to 2.0 million HIV-positive patients in the United States will develop AIDS. The majority of AIDS patients will experience progressive weight loss and malnutrition prior to their death. Because nutritional therapy has clearly been demonstrated to have a beneficial effect on the clinical course and immunologic status of the critically ill general population, one must not disregard the potential positive benefits of nutritional therapy in the treatment of malnourished ARC/AIDS patients. As a result of the escalating cost of ARC/AIDS medical therapy and the predicted AIDS epidemic, ARC/AIDS nutritional therapy regimens must be simple to administer and cost effective. At SFGH, we have developed specific nutritional screening criteria in an attempt to identify those patients who would most benefit from nutritional therapy. Prior to initiating therapy, we interview each patient, perform a complete physical examination, conduct a thorough nutritional assessment, evaluate their gut function, and calculate their daily caloric and protein requirements. The selection of appropriate oral, enteral, and parenteral diets is crucial in the successful management of these patients. Because all ARC/AIDS patients differ in their nutritional requirements, diet tolerance, and degree of intestinal dysfunction, there is no single nutritional therapy regimen that can be utilized in the treatment of all these patients. Therefore, we recommend special individualized oral diets combined with food supplements and enteral and parenteral diets in the treatment of ARC/AIDS patients.

AIDS-Related Complex↗
PubMed1985

Complementation of a Rhodopseudomonas sphaeroides ribulose bisphosphate carboxylase-oxygenase regulatory mutant from a genomic library.

A genomic library containing HindIII partial digests of Rhodopseudomonas sphaeroides HR DNA was constructed in the broad-host-range cosmid cloning vector pVK102. With a portion of this library as donor in complementation studies with the ribulose 1,5-bisphosphate carboxylase-oxygenase regulatory mutant R. sphaeroides KW25/11, a fragment of DNA which is capable of partially complementing this mutant was isolated. In four independent matings, Aut+ transconjugants which contained a hybrid plasmid carrying the same 28-kilobase-pair insert were isolated. While complemented strains were capable of growing at rates equal to that of the wild type under photoautotrophic conditions, they were not able to match wild-type levels of ribulose 1,5-biphosphate carboxylase-oxygenase activity or of form I ribulose 1,5-bisphosphate carboxylase-oxygenase protein. In addition, there is some indication that recombination may be necessary for optimal complementation to occur. The size of the complementing fragment was further reduced to 2.7 kilobase pairs by using vectors constructed for subcloning.

Cloning, Molecular↗