Biomedical subjects

K S Harris

Publications and source records attributed to K S Harris.

At least 19 recordsLinked to original sources

PubMed2006

The most polymorphic residue on Plasmodium falciparum apical membrane antigen 1 determines binding of an invasion-inhibitory antibody.

Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed alpha-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.

Amino Acid Sequence↗

PubMed1999

A comparison of intergestural patterns in deaf and hearing adult speakers: implications from an acoustic analysis of disyllables.

Coarticulation studies in speech of deaf individuals have so far focused on intrasyllabic patterning of various consonant-vowel sequences. In this study, both inter- and intrasyllabic patterning were examined in disyllables /symbol see text #CVC/ and the effects of phonetic context, speaking rate, and segment type were explored. Systematic observation of F2 and durational measurements in disyllables minimally contrasting in vocalic ([i], [u,][a]) and in consonant ([b], [d]) context, respectively, was made at selected locations in the disyllable, in order to relate inferences about articulatory adjustments with their temporal coordinates. Results indicated that intervocalic coarticulation across hearing and deaf speakers varied as a function of the phonetic composition of disyllables (b_b or d_d). The deaf speakers showed reduced intervocalic coarticulation for bilabial but not for alveolar disyllables compared to the hearing speakers. Furthermore, they showed less marked consonant influences on the schwa and stressed vowel of disyllables compared to the hearing controls. Rate effects were minimal and did not alter the coarticulatory patterns observed across hearing status. The above findings modify the conclusions drawn from previous studies and suggest that the speech of deaf and hearing speakers is guided by different gestural organization.

Adult↗

PubMed1997

smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans.

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal gene expression by modulating the levels of alternatively spliced SRp20 and SRp30b mRNAs. These SR genes contain alternatively spliced exons that introduce upstream stop codons. The effect of smg genes on SR transcripts is specific, because the gene encoding the catalytic subunit of the cAMP-dependent protein kinase, which also contains an alternatively spliced exon that introduces upstream stop codon, is not effected in a smg background. These results suggest that the levels of alternatively spliced mRNAs may, in part, be regulated by alternative mRNA stability.

Alternative Splicing↗

PubMed1997

Dual processing of open- and closed-class words.

A series of articles in the past two decades has suggested differential processing of open- and closed-class lexical items by normal adults. Difficulties in replicating a crucial study (Bradley, 1978), however, have weakened the dual route hypothesis. We matched 16 French open-class items to 16 closed-class items for phonological structure, world length, and relative word frequency. Three agrammatic aphasics revealed strikingly more phonological errors on closed-class than open-class items. Dysfluencies were greater on closed-class items and contributed to greater overall reading time for the closed-class words, consistent with a two-route model for the production of closed- and open-class lexical items in Broca's aphasics and, thus, normals.

Aged↗

PubMed1996

Pre- and posttreatment comparison of the kinematics of the fluent speech of persons who stutter.

This study reports changes in acoustic, respiratory, laryngeal, and articulatory kinematics of 3 males who stutter, following participation in a version of the Hollins Precision Fluency Shaping Program. Two nonstuttering controls received no treatment. Subjects repeated phrases of the form "He see CVC again" at self-selected slow, normal, and fast speaking rates. For experimental subjects, acoustic duration of the phrases increased significantly in 7 out of 9 comparisons of before- and after-treatment conditions, whereas controls decreased the duration of the phrases in 4 out of 6 comparisons of measurements made over approximately the time interval during which the experimental group received treatment. The experimental group increased inspiratory volume for 7 out of 9 conditions and average expiratory flow significantly for all conditions, whereas the controls decreased both. The experimental group prolonged laryngeal opening in 6 of 7 comparisons, but only 3 of the increases were significant. Lip and jaw movements for consonants were significantly reduced in amplitude for the experimental group for 30 of 36 measures. The direction of change for laryngeal and upper articulator measures was mixed for controls. These results show that behavioral treatment can produce significant changes in the fluent speech of persons who stutter with respect to respiration, laryngeal valving, and articulation. Possible relationships between the observed changes in speech production and the increased fluency of the subjects are discussed.

Adult↗

PubMed1995

Poliovirus protease 3C mediates cleavage of microtubule-associated protein 4.

Poliovirus infection results in a number of host cell changes, including specific alterations in cellular proteins. This study further characterizes the cleavage of a cytoskeletal protein, microtubule-associated protein 4 (MAP-4) and investigates the identity of the viral protease which mediates its cleavage. MAP-4 cleavage by poliovirus was previously identified using a monoclonal antibody (M. Joachims and D. Etchison, 1992, J. Virol. 66, 5997-5804). In this study, MAP-4 cleavage was found to occur in cells infected by only some picornaviruses, poliovirus and human rhinovirus 14. Infection by other types of viruses, vesicular stomatitis virus and adenovirus, or by other types of picornaviruses, encephalomyocarditis virus, did not result in MAP-4 cleavage. To determine the viral mediator of MAP-4 cleavage, the effects of purified poliovirus proteases on MAP-4 integrity were examined by immunoblot. When MAP-4 substrates were incubated with concentrations of poliovirus 2A that were more than sufficient to induce p220 cleavage, there was no effect on MAP-4. However, when MAP-4 substrates were incubated with purified 3C protease (3Cpro), cleavage products were detected that were identical in size to those generated in vivo in poliovirus-infected cells; the use of a mutant 3C protease did not result in MAP-4 cleavage. Cleavage of MAP-4 was also demonstrated with purified 3CDpro, and the in vitro cleavage kinetics were examined. Indirect immunofluorescence revealed that MAP-4 cleavage also correlated with a marked "collapse" of microtubules during late infection, indicating a possible relationship between 3Cpro-mediated MAP-4 cleavage and changes in the microtubule system of infected cells.

3C Viral Proteases↗

PubMed1995

Bioavailability of soybean isoflavones depends upon gut microflora in women.

Soybean isoflavones have been proposed to be anticarcinogenic, but their effective doses have not been established. To study their bioavailability, seven women consumed 3.4, 6.9, or 10.3 mumol isoflavones/kg body wt in soymilk in each of three meals of a liquid diet on one of three feeding days that were separated by 2-wk washout periods. Subjects were randomly assigned to doses in a cross-over design. Plasma, urine and fecal isoflavones were measured by reverse phase HPLC. In two subjects, fecal isoflavone recovery was 10-20 times that in the other five subjects. Average 48-h urinary recoveries of ingested daidzein and genistein were 16 +/- 4 and 10 +/- 4%, respectively, at all three doses among the five subjects excreting only small amounts of isoflavones in feces, whereas urinary recoveries of daidzein and genistein in the two subjects who excreted large amounts of fecal isoflavones were 32 +/- 5 and 37 +/- 6%, respectively. Urinary isoflavone excretion was nearly zero in all subjects at 48 h after dosing. Average plasma concentration of genistein at 24 h after the breakfast isoflavone dose in subjects excreting large amounts of fecal isoflavones was significantly greater by 2.5-fold than in subjects who excreted small amounts of fecal isoflavones (P < 0.05). In vitro anaerobic incubation of isoflavones with human feces showed that intestinal half-life of daidzein and genistein may be as little as 7.5 and 3.3 h, respectively. These data suggest that human isoflavone bioavailability depends upon the relative ability of gut microflora to degrade these compounds.

Adult↗

PubMed1995

Articulatory organization of mandibular, labial, and velar movements during speech.

It has been shown that articulator movements during speech are adjusted along a number of spatiotemporal dimensions. For example, variations in the extent of lip, jaw, or tongue motion are associated with proportional changes in the respective articulators' peak velocity. Modifications in the timing of lip and jaw actions are apparently constrained, exhibiting relative timing covariation. Syllable prominence systematically affects some combination of the articular motion parameters, i.e., extent, speed, and duration. The present investigation is an attempt to extend observations of the spatiotemporal properties of articulator movement to include the velum. Lip, jaw, and velar kinematics were recorded optoelectronically and simultaneously with the acoustic signal during productions of the utterance/mabnab/. The spatial and temporal relations between the lips, the jaw, and the velum were examined and compared across articulators. For movements associated with each syllable, the velum displayed scaling pattern qualitatively similar to those of the lips and jaw. Moreover, velocity-displacement relations were more robust for the lowering than for the raising movements of the velum. There was evidence of interarticulator coupling between the velum and the jaw, and between the velum and the upper lip, although this coupling was not as strong as that observed among the oral articulators. Articulator specific differences in velocity-displacement correlations and degree of interarticulator cohesion for the various movement phases may be related to a combination of aerodynamic and phonetic factors, such as the phonologically noncontrastive nature of nasalization in English.

Adult↗

PubMed1995

Interaction between the 5'-terminal cloverleaf and 3AB/3CDpro of poliovirus is essential for RNA replication.

On the basis of sequence alignments and secondary structure comparisons of the first 100 nucleotides of enterovirus and rhinovirus RNAs, chimeric constructs in which this region of poliovirus type 1 Mahoney [PV1(M)] is replaced with that of human rhinovirus type 2 (HRV2) or HRV14 have been engineered. These chimeric constructs contain the internal ribosomal entry site of either poliovirus or encephalomyocarditis virus. Independent of the internal ribosomal entry site elements, only the constructs containing either the PV1(M) or HRV2 cloverleaf sequences yielded viable viruses. The secondary structures of all three cloverleaves are quite similar. However, highly purified polioviral proteins 3CDpro and 3AB together bound to the PV1(M) and HRV2 cloverleaves, albeit with different affinities, whereas the HRV14 homolog did not interact with these proteins to any appreciable extent. These results support a mechanism of poliovirus genomic replication in which the formation of a complex between the cloverleaf structure and the 3CDpro/3AB proteins of poliovirus plays an essential role.

3C Viral Proteases↗

PubMed1994

Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB.

The synthesis in vitro of poly(U) on a poly(A) template with oligo(dT)15 primer by poliovirus RNA polymerase 3Dpol (280 ng/ml) is strongly stimulated (50-100 fold) by the addition of purified poliovirus polypeptide 3AB. The synthesis of product continues linearly with time for up to 90 min. The reaction with 3Dpol alone can be reactivated and similarly enhanced by the addition of 3AB at 30 min of incubation. Optimal stimulation is achieved under conditions where the concentration of 3Dpol and of template is low, when the molar ratio of 3AB to 3Dpol is about 100:1 and that of 3AB to poly(A) is about 25:1. In the presence of 3AB, the yield of product made by 3Dpol is much increased but its size is unchanged. From a number of basic proteins and peptides tested, a few were found which also exhibited limited enhancement of polymerase activity. The stimulatory effect of 3AB is probably related to its ability to bind both the template-primer, poly(A).oligo(dT)15, and 3Dpol (Molla, A., Harris, K. S., Paul, A. V., Shin, S. H., Mugavero, J., and Wimmer, E. J. (1994) J. Biol. Chem. 269, 27015-27020). RNA synthesis on purified poliovirus RNA with oligo(dT)15 primer is enhanced by 3AB about 5-10 fold, and this reaction is highly sensitive to detergent.

Amino Acid Sequence↗

PubMed1994

Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding.

Poliovirus proteinase 3CDpro by itself is not an RNA-binding protein. Two cellular proteins have been purified from HeLa cells (p50 and p36) which interact with purified 3CDpro but only p36-3CDpro bind to the 5'-terminal 110 nucleotides of polioviral RNA genome, an RNA segment whose secondary structure resembles a cloverleaf. The identity of these factors was determined by microsequencing tryptic digests of the purified proteins. Host protein p50 is the eukaryotic elongation factor EF-1 alpha, and p36 an N-terminal fragment thereof. p36, referred to as host factor, did not appear to interact with purified 3Cpro or 3Dpol. Significantly, the formation of a 3CDpro-cloverleaf complex was also observed in the presence of purified poliovirus polypeptide 3AB, the precursor of VPg. 3AB by itself does not stably bind to the cloverleaf. Competition experiments have demonstrated that the RNA-protein interactions are specific for the full-length cloverleaf. UV cross-linking studies were employed to examine the protein components of the cloverleaf ribonucleoproteins. RNA footprinting was used to determine the site on the cloverleaf where the viral and cellular factors bind. Finally, we have discovered that 3AB-3CDpro also interacts with the 3'-terminal sequence of poliovirus RNA. In contrast to the 5'-terminal cloverleaf, the 3'-terminal RNA can bind 3AB in the absence of other proteins. A model for initiation of poliovirus RNA synthesis is presented.

3C Viral Proteases↗

PubMed1994

Stimulation of poliovirus proteinase 3Cpro-related proteolysis by the genome-linked protein VPg and its precursor 3AB.

Purified recombinant poliovirus polypeptide 3AB interacts with 3CDpro and 3Dpol as shown by coimmunoprecipitation with anti-3Dpol antibodies. A consequence of this interaction is an accelerated autoprocessing of 3CDpro to produce 3Cpro and 3Dpol. The activation of 3Dpol polymerase activity by cleavage of 3CDpro, a polypeptide that has no polymerase activity, can be shown by template- and primer-dependent poly(U) synthesis. Anti-VPg antibodies (VPg = 3B) added to HeLa translation extracts programmed with poliovirion RNA inhibit cleavage of 3CDpro whereas addition of purified 3AB or VPg to these translation reactions increases 3CDpro processing. 3AB stimulates also 3Cpro-related proteolysis of 2BC, a poliovirus-specific, nonstructural processing intermediate. In contrast, 3CDpro-specific cleavage of the structural precursor P1 is inhibited by the addition of 3AB as shown by a decrease in the production of VP0 and VP3. These data shed new light on a phenomenon in the regulation of expression of poliovirus genetic information: whereas the proteinase 3CDpro is needed for processing of the capsid precursor, the cleavage product of this relatively stable precursor is required for RNA replication.

3C Viral Proteases↗

PubMed1994

Properties of purified recombinant poliovirus protein 3aB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol.

The poliovirus-specific polypeptide 3AB (B = VPg) was expressed in Escherichia coli and purified to near homogeneity. Corresponding to its known association with membranes in poliovirus-infected HeLa cells, 3AB expressed in E. coli was also membrane-associated, and it could be solubilized only in detergent-containing buffers. In soluble form, 3AB was resistant to digestion with the virus-specific proteinases 3Cpro and 3CDpro. However, it was cleaved by these enzymes to 3A and VPg when bound to the bacterial membranes, an observation suggesting that 3AB may deliver the genome-linked protein VPg to the membrane-associated poliovirus replication complex. The specific activity of 3CDpro in processing 3AB was significantly higher than that of 3Cpro. Soluble 3AB was found to stimulate nearly 100-fold poly (A)-dependent, primer-dependent poly(U) synthesis, catalyzed by purified poliovirus RNA polymerase 3Dpol. We propose that 3AB has a dual function in poliovirus genome replication: as a precursor for VPg, and as a co-factor for 3Dpol.

3C Viral Proteases↗

PubMed1992

Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase.

A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.

3C Viral Proteases↗

PubMed1990

Rotation and translation of the jaw during speech.

A two-dimensional rigid-body model of jaw movement was used to describe jaw opening and closing gestures for vowels and for bilabial and alveolar consonants. Jaw movements were decomposed into three components: (a) rotation about the terminal hinge axis, (b) the horizontal translation of that axis, and (c) the vertical translation of that axis. Data were collected for 3 subjects in two separate recording sessions. Multiple regression analysis was used to examine the relationships among the three jaw movement components. For 2 subjects, but not for the third, an interdependence between jaw rotation and the first principal component of jaw translation, horizontal translation, was observed. For these 2 subjects, the first degree of freedom of jaw movement corresponded to a combination of rotation and the first principal component of jaw translation. For the third subject, the first degree of freedom of jaw movement corresponded to rotation alone. The results of this study, like those of Westbury (1988), indicate that an accurate description of jaw movement during speech requires the recording of two points of jaw movement.

Adult↗

PubMed1989

Cleavage of synthetic peptides by purified poliovirus 3C proteinase.

Synthetic peptides, 14-16 residues in length, were used as substrates for purified recombinant poliovirus proteinase 3C. The sequences of the substrates correspond to the sequences of authentic cleavage sites in the poliovirus polyprotein, all of which contain Gln-Gly at the scissile bond. Specificity of cleavages was demonstrated by analysis of 3C digests of synthetic peptides. Relative rate constants for the cleavages were derived by competition experiments. The rate constants roughly correlated with the estimated half-life of the homologous precursor proteins detected in poliovirus-infected cells. The peptide most resistant to cleavage corresponded to the 3C/3D junction, a site known to be cleaved very slowly by 3C in vivo. Substitution of threonine for alanine in P4 position of this peptide, however, resulted in significant cleavage. This observation supports the hypothesis that the residue in P4 position, in addition to the Gln-Gly in P1 and P1', respectively, contributes to substrate recognition. Ac-Gln-Gly-NH2 was not a substrate for 3C.

3C Viral Proteases↗

PubMed1989

Determining the extent of coarticulation: effects of experimental design.

The purpose of this letter is to explore some reasons for what appear to be conflicting reports regarding the nature and extent of anticipatory coarticulation, in general, and anticipatory lip rounding, in particular. Analyses of labial electromyographic and kinematic data using a minimal-pair paradigm allowed for the differentiation of consonantal and vocalic effects, supporting a frame versus a feature-spreading model of coarticulation. It is believed that the apparent conflicts of previous studies of anticipatory coarticulation might be resolved if experimental design made more use of contrastive minimal pairs and relied less on assumptions about feature specifications of phones.

Electromyography↗

PubMed1988

Patterns of interarticulator phasing and their relation to linguistic structure.

Work by Tuller and Kelso [J. Acoust. Soc. Am. 76, 1030-1036 (1984)] and Kelso et al. [J. Phon. 14, 29-59 (1986)] has demonstrated stable relations between jaw and lip movements in (bV#CVb) utterances across rate and stress conditions. Specifically, the onset of lip movement toward the intervocalic consonant was found to be constant with respect to the vowel-to-vowel jaw cycle in both time and relative phasing. An attempt was made to replicate and extend this work by investigating interarticulator phase relations for utterances having a broader range of linguistic organization: In addition to rate and stress, syllable structure (open versus closed syllables) and identity of the intervocalic consonant (/p/ vs /m/) were manipulated. Results showed that the upper lip's lowering onset varied systematically with respect to the jaw vowel cycle as a function of both rate and stress. In addition, syllable structure and consonant identity influenced the relation of lip and jaw gestures. There was a general tendency for any condition that shortened the first vowel to produce earlier onsets of the upper lip relative to the jaw. However, the within-condition jaw cycle duration variability did not correlate with the within-condition variability in phase. Thus it seems that stable interarticulator phase relations maintain not only the integrity of phonological structure, as suggested by Kelso et al., but structural integrity at other levels of linguistic organization as well.

Female↗