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Biomedical subjects

R Gilmore

Publications and source records attributed to R Gilmore.

At least 19 recordsLinked to original sources
PubMed2001

Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase.

The oligosaccharyltransferase (OST) preferentially utilizes the fully assembled dolichol-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-PP-Dol as the donor for N-linked glycosylation of asparagine residues in N-X-T/S consensus sites in newly synthesized proteins. A wide variety of assembly intermediates (Glc(0-2)Man(0-9)GlcNAc(2)-PP-Dol) can serve as the donor substrate for N-linked glycosylation of peptide acceptor substrates in vitro or of nascent glycoproteins in mutant cells that are defective in donor substrate assembly. A kinetic mechanism that can account for the selection of the fully assembled donor substrate from a complex mixture of dolichol-linked oligosaccharides (OS-PP-Dol) has not been elucidated. Here, the steady-state kinetic properties of the OST were reinvestigated using a proteoliposome assay system consisting of the purified yeast enzyme, near-homogeneous preparations of a dolichol-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol) and an (125)I-labeled tripeptide as the acceptor substrate. The K(m) of the OST for the acceptor tripeptide was only slightly enhanced when Glc(3)Man(9)GlcNAc(2)-PP-Dol was the donor substrate relative to when Man(9)GlcNAc(2)-PP-Dol was the donor substrate. Evaluation of the kinetic data for both donor substrates showed deviations from typical Michaelis-Menten kinetics. Sigmoidal saturation curves, Lineweaver-Burk plots with upward curvature, and apparent Hill coefficients of about 1.4 suggested a substrate activation mechanism involving distinct regulatory (activator) and catalytic binding sites for OS-PP-Dol. Results of competition experiments using either oligosaccharide donor as an alternative substrate were also consistent with this hypothesis. We propose that binding of either donor substrate to the activator site substantially enhances Glc(3)Man(9)GlcNAc(2)-PP-Dol occupancy of the enzyme catalytic site via allosteric activation.

Allosteric Regulation↗
PubMed2001

Hsp90 phosphorylation is linked to its chaperoning function. Assembly of the reovirus cell attachment protein.

Studies on Hsp90 have mainly focused on its involvement in the activation of several families of protein kinases and of steroid hormone receptors. Little is known regarding the role of Hsp90 in the folding of nascent proteins. We previously reported that Hsp90 plays an active role in the posttranslational assembly of the C-terminal globular head of the reovirus attachment protein final sigma1. We show here that Hsp90 becomes phosphorylated in this process. However, only the unphosphorylated form of Hsp90 is complexed with final sigma1, suggesting that Hsp90 phosphorylation is coupled to the release of the chaperone from the target protein. Geldanamycin, which blocks final sigma1 maturation by preventing the release of Hsp90 from final sigma1, also inhibits Hsp90 phosphorylation. Taken together, these results demonstrate that Hsp90 phosphorylation is linked to its chaperoning function.

Animals↗
PubMed2001

Large-scale isolation of dolichol-linked oligosaccharides with homogeneous oligosaccharide structures: determination of steady-state dolichol-linked oligosaccharide compositions.

The dolichol-linked oligosaccharide donor (Glc(3)Man(9)GlcNAc(2)-PP-Dol) for N-linked glycosylation of proteins is assembled in a series of reactions that initiate on the cytoplasmic face of the rough endoplasmic reticulum and terminate within the lumen. The biochemical analysis of the oligosaccharyltransferase and the glycosyltransferases that mediate assembly of dolichol-linked oligosaccharides (OS-PP-Dol) has been hindered by the lack of structurally homogeneous substrate preparations. We have developed an improved method for the preparative-scale isolation of dolichol-linked oligosaccharides from vertebrate tissues and yeast cells. Preparations that were highly enriched in either Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol were obtained from porcine pancreas and a Man(5)GlcNAc(2)-PP-Dol preparation was obtained from an alg3 yeast culture. Chromatography of the OS-PP-Dol preparations on an aminopropyl silica column was used to obtain dolichol-linked oligosaccharides with defined structures. A single chromatography step could achieve near-baseline resolution of dolichol-linked oligosaccharides that differed by one sugar residue. A sensitive oligosaccharyltransferase endpoint assay was used to determine the concentration and composition of the OS-PP-Dol preparations. Typical yields of Glc(3)Man(9)GlcNAc(2)-PP-Dol, Man(9)GlcNAc(2)-PP-Dol, and Man(5)GlcNAc(2)-PP-Dol ranged between 5 and 15 nmol per chromatographic run. The homogeneity of these preparations ranged between 85 and 98% with respect to oligosaccharide composition. Purification of dolichol-linked oligosaccharides from cultures of alg mutant yeast strains provides a general method to obtain authentic OS-PP-Dol assembly intermediates of high purity. The analytical methods described here can be used to accurately evaluate the steady-state dolichol-linked oligosaccharide compositions of wild-type and mutant cell lines.

Animals↗
PubMed2001

Intramural leiomyoma during pregnancy becoming pedunculated postpartally. A case report.

BACKGROUND: The natural history of uterine leiomyomas during pregnancy has been reported. CASE: A 39-year-old primigravida presented with vaginal spotting in the 10th week of pregnancy. Ultrasonic evaluation revealed a large intramural leiomyoma. Unsuccessful tocolysis at 25 weeks' gestation resulted in a cesarean section for breech presentation. At hysterotomy a 10-cm intramural leiomyoma was found in the right fundus and was left in situ. Six months later, at open laparotomy for myomectomy, the 10-cm leiomyoma was pedunculated, on a 4-cm stalk. CONCLUSION: Large intramural leiomyomas found at cesarean section may become pedunculated postpartally, thus making myomectomy easier and safer at a postpartum intervention than at the time of cesarean section. Also, prior knowledge of the possibility of myoma transformation from intramural to pedunculated postpartally may help in planning a later myomectomy. Myomectomy prior to pregnancy should be considered when it has caused a prior pregnancy complication.

Adult↗
PubMed2000

Covering dynamical systems: twofold covers.

We study the relation between a dynamical system, which is unchanged (equivariant) under a discrete symmetry group G and another locally identical dynamical system with no residual symmetry. We also study the converse mapping: lifting a dynamical system without symmetry to a multiple cover, which is equivariant under G. This is done in R3 for the two element rotation and inversion groups. Comparisons are done for the equations of motion, the strange attractors that they generate, and the branched manifolds that classify these strange attractors. A dynamical system can have many inequivalent multiple covers, all equivariant under the same symmetry group G. These are distinguished by the value of a certain topological index. Many examples are presented. A new global bifurcation, the "peeling bifurcation," is described.

Journal Article↗
PubMed2000

Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex.

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH(2) and COOH terminus of Sec61alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61alpha. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.

Animals↗
PubMed2000

Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel.

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.

Animals↗
PubMed2000

Role of ubiquitin in proteasomal degradation of mutant alpha(1)-antitrypsin Z in the endoplasmic reticulum.

A delay in intracellular degradation of the mutant alpha(1)-antitrypsin (alpha(1)AT)Z molecule is associated with greater retention within the endoplasmic reticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha(1)AT deficiency. Recent studies have shown that alpha(1)ATZ is ordinarily degraded in the ER by a mechanism that involves the proteasome, as demonstrated in intact cells using human fibroblast cell lines engineered for expression of alpha(1)ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha(1)ATZ mRNA. To determine whether the ubiquitin system is required for proteasomal degradation of alpha(1)ATZ and whether specific components of the ubiquitin system can be implicated, we have now used two approaches. First, we overexpressed a dominant-negative ubiquitin mutant (UbK48R-G76A) by transient transfection in the human fibroblast cell lines expressing alpha(1)ATZ. The results showed that there was marked, specific, and selective inhibition of alpha(1)ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha(1)ATZ. Second, we subjected reticulocyte lysate to DE52 chromatography and tested the resulting well-characterized fractions in the cell-free system. The results showed that there were both ubiquitin-dependent and -independent proteasomal mechanisms for degradation of alpha(1)ATZ and that the ubiquitin-conjugating enzyme E2-F1 may play a role in the ubiquitin-dependent proteasomal mechanism.

Animals↗
PubMed1999

Conduction aphasia and the arcuate fasciculus: A reexamination of the Wernicke-Geschwind model.

Wernicke, and later Geschwind, posited that the critical lesion in conduction aphasia is in the dominant hemisphere's arcuate fasciculus. This white matter pathway was thought to connect the anterior language production areas with the posterior language areas that contain auditory memories of words (a phonological lexicon). Alternatively, conduction aphasia might be induced by cortical dysfunction, which impairs the phonological output lexicon. We observed an epileptic patient who, during cortical stimulation of her posterior superior temporal gyrus, demonstrated frequent phonemic paraphasias, decreased repetition of words, and yet had intact semantic knowledge, a pattern consistent with conduction aphasia. These findings suggest that cortical dysfunction alone may induce conduction aphasia.

Aphasia, Conduction↗
PubMed1999

Adult-onset seizures: clinical solutions to a challenging patient work-up.

The age-related increase in the incidence of seizures in older persons is directly related to the increase in prevalence of causative factors such as cardiovascular disease and stroke, primary and metastatic brain tumors, toxic-metabolic disturbances, and medications. Because the treatment plan depends on seizure etiology, comprehensive evaluation of each of these causes is imperative. Reliable history and thorough physical examination remain the most important steps for diagnosis and effective treatment. Nevertheless, assessment and treatment of new-onset paroxysmal events can be problematic, because numerous morbidities and syndromes--including transient ischemic attack, syncope, drug intoxication, amnesia, movement disorders, and psychiatric disorders--can present with similar symptomology.

Age Factors↗
PubMed1999

Strategies for successful management of older patients with seizures.

The incidence of seizures increases dramatically with age, making epilepsy in the older patient a common clinical presentation in the primary care practice. In the case of a single seizure or when the underlying cause can be corrected, antiepileptic drug (AED) therapy may not be warranted. For recurrent seizures, single AED therapy should be initiated at a low dose and gradually titrated upward. Control of seizure frequency is dependent on appropriate AED selection and compliance, drug-drug interactions, and minimization of side effects. Monitoring of AED serum levels is imperative for effective AED therapy. Conventional AEDs remain the standard initial anticonvulsants for epilepsy in older patients. The newer AEDs have demonstrated efficacy as adjunctive therapy and may offer reduced side-effect profiles and fewer drug-drug interactions.

Age Distribution↗
PubMed1998

A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum.

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Deltascj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Deltascj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.

Alleles↗
PubMed1998

Chemokines are present in the genital tract of HIV-seropositive and HIV-seronegative women: correlation with other immune mediators.

In this cross-sectional study, 53 cervicovaginal lavage samples (CVL) from 41 women were analyzed for the chemokines interleukin-8 (IL-8), regulated-on-activation normal T-expressed and secreted (RANTES) factor, and macrophage inflammatory protein-1alpha (MIP-1alpha) by enzyme-linked immunosorbent assay (ELISA). IL-8 was detected in 81% of CVL, whereas RANTES was detected in 32%, and MIP-1alpha in 15% of the CVL. The mean levels of IL-8, RANTES, and MIP-1alpha in positive samples were 396 pg/ml, 102 pg/ml, and 34 pg/ml, respectively. IL-8 levels correlated positively with IL-1beta and IgG in a subset of CVL samples. RANTES levels correlated positively with complement protein levels. Additionally, the levels of RANTES, but not MIP-1alpha, reached levels reported in previous studies of the effects of beta chemokines to inhibit HIV replication. These results suggest that measuring chemokines in CVL specimens can provide important information regarding immune responses in the genital tract.

Cervix Uteri↗
PubMed1998

Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein sigma1.

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

Adenosine Triphosphate↗
PubMed1998

An efficient motion estimator with application to medical image registration.

Image registration is a very important problem in computer vision and medical image processing. Numerous algorithms for registering single and multi-modal image data have been reported in these areas. Robustness as well as computational efficiency are prime factors of importance in image data registration. In this paper, a robust/reliable and efficient algorithm for estimating the transformation between two image data sets of a patient taken from the same modality over time is presented. Estimating the registration between two image data sets is formulated as a motion-estimation problem. We use a hierarchical optical flow motion model which allows for both global as well as local motion between the data sets. In this hierarchical motion model, we represent the flow field with a B-spline basis which implicitly incorporates smoothness constraints on the field. In computing the motion, we minimize the expectation of the squared differences energy function numerically via a modified Newton iteration scheme. The main idea in the modified Newton method is that we precompute the Hessian of the energy function at the optimum without explicitly knowing the optimum. This idea is used for both global and local motion estimation in the hierarchical motion model. We present examples of motion estimation on synthetic and real data (from a patient acquired during pre- and post-operative stages) and compare the performance of our algorithm with that of competing ones.

Algorithms↗
PubMed1998

Signal recognition particle-dependent targeting of ribosomes to the rough endoplasmic reticulum in the absence and presence of the nascent polypeptide-associated complex.

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.

Animals↗