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Biomedical subjects

K A White

Publications and source records attributed to K A White.

At least 19 recordsLinked to original sources

Spreading disease through social groupings in competition.

Many animal populations live in social groups which avoid contact with other conspecific groups for at least part of the year. This may give rise to competition between groups for items such as shelter, land and mates. We couple intra-specific group competition with disease dynamics to investigate how infectious diseases may spread through population subgroups, particularly with reference to the contact rates between groups. Our model uses a nonlinear systems of ODEs for which steady-state analysis is carried out in the simplest two-group system. This indicates that coexistence of social groups is possible with the disease or that competitive exclusion occurs with one group dying out whilst the other retains disease. Moreover, we show that in certain circumstances the model can exhibit multistability and we discuss the ecological implications of this result in relation to contact between social groups.

Animals↗

Regulatory activity of distal and core RNA elements in Tombusvirus subgenomic mRNA2 transcription.

Positive-strand RNA viruses that encode multiple cistrons often mediate expression of 3'-encoded open reading frames via RNA-templated transcription of subgenomic (sg) mRNAs. Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus that transcribes two such sg mRNAs during infections. We have previously identified a distal element (DE), located approximately 1100 nucleotides upstream from the initiation site of sg mRNA2 transcription, part of which must base pair with a portion of a core element (CE), located just 5' to the initiation site, for efficient transcription to occur (Zhang, G., Slowinski, V., and White, K. A. (1999) RNA 5, 550-561). Here we have analyzed further this long distance RNA-RNA interaction and have investigated the regulatory roles of other subelements within the DE and CE. Our results indicate that (i) the functional base-pairing interaction between these elements occurs in the positive strand and that the interaction likely acts to properly position other subelements, (ii) two previously undefined subelements within the DE and CE are important and essential, respectively, for efficient sg mRNA2 accumulation, and (iii) the production of (-)-strand sg mRNA2 can be uncoupled from the synthesis of its (+)-strand complement. These data provide important insight into the mechanism of sg mRNA2 transcription.

RNA, Messenger↗

An RNA domain within the 5' untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication.

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5'-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication.

5' Untranslated Regions↗

In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

Amino Acid Sequence↗

A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic acid (Kdo) kinase. Possible involvement of kdo phosphorylation in bacterial virulence.

The lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue derivatized with either a phosphate or an ethanolamine pyrophosphate moiety at the 4-OH position. In previous studies, we identified a kinase unique to H. influenzae extracts that phosphorylates Kdo-lipid IV(A), a key precursor of lipopolysaccharide in this organism. We have now identified the gene encoding the Kdo kinase by using an expression cloning approach. A cosmid library containing random DNA fragments from H. influenzae strain Rd was constructed in Escherichia coli. Extracts of 472 colonies containing individual hybrid cosmids were assayed for Kdo kinase activity. A single hybrid cosmid directing expression of the kinase was found. The kinase gene was identified by activity assays, sub-cloning, and DNA sequencing. When the putative kinase gene was expressed in E. coli behind a T7 promoter, massive overproduction of kinase activity was achieved ( approximately 8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IV(A) as the substrate, were the same as observed with H. influenzae membranes. Unexpectedly, the kinase gene was identical to a previously characterized open reading frame (orfZ), which had been shown to be important for establishing bacteremia in an infant rat model (Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbiol. 22, 951-965). However, based solely on the genome sequence of H. influenzae Rd, no biochemical function had been assigned to the product of orfZ, which we now designate kdkA ("Kdo kinase A"). Although Kdo phosphorylation may be critical for bacterial virulence of H. influenzae, it does not appear to be required for growth.

Amino Acid Sequence↗

A deacylase in Rhizobium leguminosarum membranes that cleaves the 3-O-linked beta-hydroxymyristoyl moiety of lipid A precursors.

Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4'-phosphate groups but is derivatized with a galacturonic acid substituent at position 4'. R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked beta-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleave E. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVA and (3-deoxy-D-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked beta-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.

Carbohydrate Sequence↗

Enhancer-like properties of an RNA element that modulates Tombusvirus RNA accumulation.

Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus tomato bushy stunt virus contain four noncontiguous segments (regions I-IV) derived from the viral genome. Region I corresponds to 5'-noncoding sequence, regions II and III are derived from internal positions, and region IV represents a 3'-terminal segment. We analyzed the internally located region III in a prototypical DI RNA to understand better its role in DI RNA accumulation. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduced levels ( approximately 10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of region III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region III-mediated RNA stabilization, (5) DI RNAs lacking region III permit the efficient accumulation of head-to-tail dimers and are less effective at suppressing helper RNA accumulation, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication. A scanning-type mutagenesis strategy was used to define portions of region III important for its stimulatory effect on DI RNA accumulation. Interestingly, the results revealed several differences in the requirements for activity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this element in the reproductive cycle of the virus.

Base Sequence↗

Modelling density-dependent resistance in insect-pathogen interactions.

We consider a mathematical model for a host-pathogen interaction where the host population is split into two categories: those susceptible to disease and those resistant to disease. Since the model was motivated by studies on insect populations, we consider a discrete-time model to reflect the discrete generations which are common among insect species. Whether an individual is born susceptible or resistant to disease depends on the local population levels at the start of each generation. In particular, we are interested in the case where the fraction of resistant individuals in the population increases as the total population increases. This may be seen as a positive feedback mechanism since disease is the only population control imposed upon the system. Moreover, it reflects recent experimental observations from noctuid moth-baculovirus interactions that pathogen resistance may increase with larval density. We find that the inclusion of a resistant class can stabilise unstable host-pathogen interactions but there is greatest regulation when the fraction born resistant is density independent. Nonetheless, inclusion of density dependence can still allow intrinsically unstable host-pathogen dynamics to be stabilised provided that this effect is sufficiently small. Moreover, inclusion of density-dependent resistance to disease allows the system to give rise to bistable dynamics in which the final outcome is dictated by the initial conditions for the model system. This has implications for the management of agricultural pests using biocontrol agents-in particular, it is suggested that the propensity for density-dependent resistance be determined prior to such a biocontrol attempt in order to be sure that this will result in the prevention of pest outbreaks, rather than their facilitation. Finally we consider how the cost of resistance to disease affects model outcomes and discover that when there is no cost to resistance, the model predicts stable periodic outbreaks of the insect population. The results are interpreted ecologically and future avenues for research to address the shortfalls in the present model system are discussed.

Animals↗

Subgenomic mRNA regulation by a distal RNA element in a (+)-strand RNA virus.

Subgenomic (sg) mRNAs are synthesized by (+)-strand RNA viruses to allow for efficient translation of products encoded 3' in their genomes. This strategy also provides a means for regulating the expression of such products via modulation of sg mRNA accumulation. We have studied the mechanism by which sg mRNAs levels are controlled in tomato bushy stunt virus, a small (+)-strand RNA virus which synthesizes two sg mRNAs during infections. Neither the viral capsid nor movement proteins were found to play any significant role in modulating the accumulation levels of either sg mRNA. Deletion analysis did, however, identify a 12-nt-long RNA sequence located approximately 1,000 nt upstream from the site of initiation of sg mRNA2 synthesis that was required specifically for accumulation of sg mRNA2. Further analysis revealed a potential base-pairing interaction between this sequence and a sequence located just 5' to the site of initiation for sg mRNA2 synthesis. Mutant genomes in which this interaction was either disrupted or maintained were analyzed and the results indicated a positive correlation between the predicted stability of the base-pairing interaction and the efficiency of sg mRNA2 accumulation. The functional significance of the long-distance interaction was further supported by phylogenetic sequence analysis which revealed conservation of base-pairing interactions of similar stability and relative position in the genomes of different tombusviruses. It is proposed that the upstream sequence represents a cis-acting RNA element which facilitates sg mRNA accumulation by promoting efficient synthesis of sg mRNA2 via a long-distance RNA-RNA interaction.

Amino Acid Sequence↗

Bordetella pertussis waaA encodes a monofunctional 2-keto-3-deoxy-D-manno-octulosonic acid transferase that can complement an Escherichia coli waaA mutation.

Bordetella pertussis lipopolysaccharide (LPS) contains a single 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue, whereas LPS from Escherichia coli contains at least two. Here we report that B. pertussis waaA encodes an enzyme capable of transferring only a single Kdo during the biosynthesis of LPS and that this activity is sufficient to complement an E. coli waaA mutation.

Bordetella pertussis↗

A primary determinant of cap-independent translation is located in the 3'-proximal region of the tomato bushy stunt virus genome.

Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the prototype member of the genus Tombusvirus. The genomes of members of this genus are not polyadenylated, and prevailing evidence supports the absence of a 5' cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3' terminus of the TBSV genome was implicated as a determinant of translational efficiency (S.K. Oster, B. Wu and K. A. White, J. Virol. 72:5845-5851, 1998). In the present report, we provide evidence that a 3'-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation. Our results indicate that (i) a 5' cap structure can substitute functionally for the absence of region 3.5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3.5 from viral and chimeric mRNAs has no appreciable effect on message stability; (iii) region 3.5 represents part of a larger 3' proximal element, designated as the 3' cap-independent translational enhancer (3'CITE), that is required for proficient cap-independent translation; (iv) the 3'CITE also facilitates cap-dependent translation; (v) none of the major viral proteins are required for 3'CITE activity; and (vi) no significant 3'CITE-dependent stimulation of translation was observed when mRNAs were tested in vitro in wheat germ extract under various assay conditions. This latter property distinguishes the 3'CITE from other characterized plant viral 3'-proximal cap-independent translational enhancers. Additionally, because the 3'CITE overlaps with cis-acting replication signals, it could potentially participate in regulating the initiation of genome replication.

3' Untranslated Regions↗

Crisis of conscience: reconciling religious health care providers' beliefs and patients' rights.

In this note, Katherine A. White explores the conflict between religious health care providers who provide care in accordance with their religious beliefs and the patients who want access to medical care that these religious providers find objectionable. Specifically, she examines Roman Catholic health care institutions and HMOs that follow the Ethical and Religious Directives for Catholic Health Care Services and considers other religious providers with similar beliefs. In accordance with the Directives, these institutions maintain policies that restrict access to "sensitive" services like abortion, family planning, HIV counseling, infertility treatment, and termination of life-support. White explains how most state laws protecting providers' right to refuse treatments in conflict with religious principles do not cover this wide range of services. Furthermore, many state and federal laws and some court decisions guarantee patients the right to receive this care. The constitutional complication inherent in this provider-patient conflict emerges in White's analysis of the interaction of the Free Exercise and Establishment Clauses of the First Amendment and patients' right to privacy. White concludes her note by exploring the success of both provider-initiated and legislatively mandated compromise strategies. She first describes the strategies adopted by four different religious HMOs which vary in how they increase or restrict access to sensitive services. She then turns her focus to state and federal "bypass" legislation, ultimately concluding that increased state supervision might help these laws become more viable solutions to provider-patient conflicts.

Catholicism↗

Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis.

The Escherichia coli msbA gene, first identified as a multicopy suppressor of htrB mutations, has been proposed to transport nascent core-lipid A molecules across the inner membrane (Polissi, A., and Georgopoulos, C. (1996) Mol. Microbiol. 20, 1221-1233). msbA is an essential E. coli gene with high sequence similarity to mammalian Mdr proteins and certain types of bacterial ABC transporters. htrB is required for growth above 32 degreesC and encodes the lauroyltransferase that acts after Kdo addition during lipid A biosynthesis (Clementz, T., Bednarski, J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102). By using a quantitative new 32Pi labeling technique, we demonstrate that hexa-acylated species of lipid A predominate in the outer membranes of wild type E. coli labeled for several generations at 42 degreesC. In contrast, in htrB mutants shifted to 42 degreesC for 3 h, tetra-acylated lipid A species and glycerophospholipids accumulate in the inner membrane. Extra copies of the cloned msbA gene restore the ability of htrB mutants to grow at 42 degreesC, but they do not increase the extent of lipid A acylation. However, a significant fraction of the tetra-acylated lipid A species that accumulate in htrB mutants are transported to the outer membrane in the presence of extra copies of msbA. E. coli strains in which msbA synthesis is selectively shut off at 42 degreesC accumulate hexa-acylated lipid A and glycerophospholipids in their inner membranes. Our results support the view that MsbA plays a role in lipid A and possibly glycerophospholipid transport. The tetra-acylated lipid A precursors that accumulate in htrB mutants may not be transported as efficiently by MsbA as are penta- or hexa-acylated lipid A species.

ATP-Binding Cassette Transporters↗

Formation and amplification of a novel tombusvirus defective RNA which lacks the 5' nontranslated region of the viral genome.

Defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) are small, subgenomic, helper-dependent replicons that are believed to be generated primarily by aberrant events during replication of the plus-sense RNA genome. Prototypical TBSV DI RNAs contain four noncontiguous segments (regions I through IV) derived from the 5' nontranslated region (NTR) (I), an internal section (II), and the 3'-terminal portion (III and IV) of the viral genome. We have studied the formation of these molecules by using engineered precursor DI RNA transcripts and report here the consistent accumulation of a novel defective RNA species, designated RNA B. Northern blot, primer extension, and sequence analyses indicated that, unlike prototypical DI RNAs, RNA B lacks region I. In vitro transcripts corresponding to the region II-III-IV structure of RNA B were amplified when coinoculated with helper, indicating that the 5' NTR of the genome does not harbor cis-acting replication elements essential for viral RNA replication. Region I is, however, important for DI RNA fitness, since molecules lacking it accumulated to significantly lower levels ( approximately 10-fold reduction). Analysis of the minus-strand sequence of region I led to the identification of an RNA undecamer sequence, arranged in tandem, at its very 3' terminus. Additional variants of the undecamer motif were also identified at internal positions in region I and in the negative strands of regions II, III, and IV. Features of the undecamer motif, the consensus of which is (-)3'-CCCAAAGAGAG, are consistent with a role as a cis-acting replication element. It is proposed that the ability of RNA B to be amplified is due, in part, to compensatory effects of a strategically positioned undecamer motif in region II. Possible replicase-mediated mechanisms for the generation of this novel viral RNA are also presented.

5' Untranslated Regions↗

Uncoupled expression of p33 and p92 permits amplification of tomato bushy stunt virus RNAs.

Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5'-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal approximately 6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3'-located segment, region 3.5, was tested. However, when 5'-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5' cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.

Gene Amplification↗

A mono-functional 3-deoxy-D-manno-octulosonic acid (Kdo) transferase and a Kdo kinase in extracts of Haemophilus influenzae.

Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. coli Kdo transferase and demonstrated that homogeneous preparations of KdtA polypeptide catalyzed the attachment of both Kdo groups to the precursor, lipid IVA. E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IVA in extracts of H. influenzae. The mono-functional Kdo transferase of H. influenzae is membrane-bound, and the reaction is dependent upon a CMP-Kdo-generating system, as in E. coli. The specific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IVA were prepared for analysis. Matrix-assisted laser desorption/ionization mass spectrometry revealed a parent ion (M - H)- at m/z 1626.0, consistent with the addition of a single Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IVA with a parent ion (M - H)- at m/z 1703.9. The identification of the mono-functional H. influenzae Kdo transferase, which is encoded by a KdtA homologue that displays 50% identity to its E. coli counterpart, should facilitate the mechanistic dissection of more complex multi-functional Kdo transferases, like those of E. coli and Chlamydia trachomatis.

Escherichia coli↗

Regulation of complex host dynamics by a macroparasite.

The impact of a macroparasite on a host population which can exhibit highly complex dynamics is considered. We focus on the case where the host reproduces annually, in which case we couple within-season density dependent interactions with a discrete-time pulse to reflect seasonal reproduction. Results from analytic arguments, supported by extensive numerical simulations, indicate that within season parasite-induced host mortality is potentially capable of stabilising and simplifying host dynamics. Moreover, the interaction of demographic processes occurring on different time-scales is, in some cases, stabilising. In particular, parasite reduction in host frequency, which is a destabilising process in the May & Anderson continuous time model, is found to stabilise host macroparasite interactions under certain conditions. These results are discussed in an ecological context.

Animals↗