Biomedical subjects

M J Wick

Publications and source records attributed to M J Wick.

At least 19 recordsLinked to original sources

PubMed2005

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia.

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.

Animals↗

PubMed2002

Transgenic expression of a mutant glycine receptor decreases alcohol sensitivity of mice.

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that inhibit neurotransmission in the adult brainstem and spinal cord. GlyR function is potentiated by ethanol in vitro, and a mutant GlyR subunit alpha(1)(S267Q) is insensitive to the potentiating effects of ethanol. To test the importance of GlyR for the actions of ethanol in vivo, we constructed transgenic mice with this mutation. Under the control of synapsin I regulatory sequences, transgenic expression of S267Q mutant GlyR alpha(1) subunits in the nervous system was demonstrated using [(3)H]strychnine binding and immunoblotting. These mice showed decreased sensitivity to ethanol in three behavioral tests: ethanol inhibition of strychnine seizures, motor incoordination (rotarod), and loss of righting reflex. There was no change in ethanol sensitivity in tests of acute functional tolerance or body temperature, and there was no change in ethanol metabolism. Transgene effects were pharmacologically specific for ethanol, compared with pentobarbital, flurazepam, and ketamine. These results support the idea that glycine receptors contribute to some behavioral actions of ethanol and that ethanol sensitivity can be changed in vivo by transgenic expression of a single receptor subunit.

Animals↗

PubMed2001

Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt.

A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Differential involvement of dendritic cell subsets during acute Salmonella infection.

Within murine CD11c(+) dendritic cells (DC), CD8alpha+, CD8alpha-CD4+, and CD8alpha-CD4- subsets are defined. This study characterized the localization, number, and function of these subsets during acute Salmonella typhimurium infection. Immunohistochemical and flow cytometric analyses of spleens from mice orally infected with virulent S. typhimurium revealed that in situ redistribution and alteration in the absolute number and function of DC occurred in a subset-specific manner during infection. CD8alpha-CD4+ DC present at B cell follicle borders in the spleen of naive mice were absent 5 days post-Salmonella infection, despite no overall change in the absolute number of CD8alpha-CD4+ splenic DC. CD8alpha+ and CD8alpha-CD4- DC were prominently associated with the red pulp, and the frequency of these cells increased strikingly 5 days post-Salmonella infection. Significant quantitative increases in both CD8alpha+ and CD8alpha-CD4- subsets were associated with the in situ redistribution. Examination of Salmonella-infected TAP1(-/-)/beta(2)-microglobulin(-/-) mice, which lack CD8alpha+ T cells, confirmed the differential subset-specific modulations in the DC populations both in situ and quantitatively. Ex vivo intracellular cytokine analysis showed significantly increased frequencies of CD8alpha(+) DC producing TNF-alpha at days 2 and 5 postinfection. In contrast, CD4+ DC producing TNF-alpha were transiently increased followed by a significant reduction. No significant increase in IL-12p40 or IL-10 production by splenic DC was detected during the first 5 days post-S. typhimurium infection. Together these data reveal differential modulation of splenic DC subsets with regard to organization, number, and cytokine production during the course of acute Salmonella infection.

Acute Disease↗

PubMed2001

Salmonella typhimurium-induced cytokine production and surface molecule expression by murine macrophages.

The influence of Salmonella enterica serovar Typhimurium (S. typhimurium) on co-stimulatory molecule expression, cytokine production and induction of nitric oxide synthase (iNOS) by murine macrophages (Mphi), as well as the influence of the phoP locus on these aspects of S. typhimurium-Mphi interaction, was characterized. Pulsing Mphi with S. typhimurium resulted in increased surface expression of MHC-I, MHC-II, CD86 and CD54. Furthermore, co-incubating S. typhimurium with Mphi resulted in interleukin (IL)-12p40, IL-6 and tumor necrosis factor-alpha production as well as iNOS induction while IL-12p70 was not detectable. Finally, although phoP did not influence the level of surface molecule expression or cytokine production by S. typhimurium-pulsed Mphi phoP did influence the level of iNOS induction. Together these data show that S. typhimurium interaction with Mphi activates these cells in ways that may enhance their ability to productively stimulate Salmonella-specific T cells following phagocytic processing and presentation of Salmonella antigens.

Animals↗

PubMed2001

The influence of curli, a MHC-I-binding bacterial surface structure, on macrophage-T cell interactions.

Escherichia coli express thin surface fimbriae called curli which bind soluble matrix proteins and major histocompatibility complex (MHC)-I molecules. The present study addressed the ability of purified curli or curliated E. coli to influence peptide presentation on MHC-I, T cell proliferation and bacterial uptake by macrophages. In vitro studies with curli-proficient E. coli YMel and the isogenic curli-deficient strain YMel-1, both expressing the model antigen Crl-OVA, showed that curli expression by E. coli does not appear to influence the efficiency by which the bacteria are processed by murine macrophages for OVA(257-264) presentation on K(b). Furthermore, curli expression by E. coli did not influence the binding of exogenously added OVA(257-264) peptide to K(b) on the surface of prefixed macrophages. In addition, neither curliated nor non-curliated heat-killed bacteria influenced proliferation of either murine or human T cells stimulated with anti-CD3. Finally, curliated E. coli adhered to and were internalized by macrophages from C57BL/6 and MHC-I-deficient TAP1(-/-) mice equally well. Together these studies show that curli expression by E. coli does not appear to influence phagocytic processing of bacteria expressing Crl-OVA for OVA(257-264)/K(b) presentation, the binding of exogenously added OVA(257-264) to K(b) or T cell proliferation. In addition, although curli expression by E. coli enhances bacterial interaction with macrophages, curli interaction with MHC-I does not significantly contribute to this adherence.

Animals↗

PubMed2001

In vivo activation of dendritic cells and T cells during Salmonella enterica serovar Typhimurium infection.

The present study was initiated to gain insight into the interaction between splenic dendritic cells (DC) and Salmonella enterica serovar Typhimurium in vivo. Splenic phagocytic cell populations associated with green fluorescent protein (GFP)-expressing bacteria and the bacterium-specific T-cell response were evaluated in mice given S. enterica serovar Typhimurium expressing GFP and ovalbumin. Flow cytometry analysis revealed that GFP-positive splenic DC (CD11c+ major histocompatibility complex class II-positive [MHC-II+] cells) were present following bacterial administration, and confocal microscopy showed that GFP-expressing bacteria were contained within CD11c+ MHC-II+ splenocytes. Furthermore, splenic DC and T cells were activated following Salmonella infection. This was shown by increased surface expression of CD86 and CD40 on CD11c+ MHC-II+ cells and increased CD44 and CD69 expression on CD4+ and CD8+ T cells. Salmonella-specific gamma interferon (IFN-gamma)-producing cells in both of these T-cell subsets, as well as cytolytic effector cells, were also generated in mice given live bacteria. The frequency of Salmonella-specific CD4+ T cells producing IFN-gamma was greater than that of specific CD8+ T cells producing IFN-gamma in the same infected animal. This supports the argument that the predominant source of IFN-gamma production by cells of the specific immune response is CD4+ T cells. Finally, DC that phagocytosed live or heat-killed Salmonella in vitro primed bacterium-specific IFN-gamma-producing CD4+ and CD8+ T cells as well as cytolytic effector cells following administration into naïve mice. Together these data suggest that DC are involved in priming naïve T cells to Salmonella in vivo.

Animals↗

PubMed2000

Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1.

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2000

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton.

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr(774) in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Czeta, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2000

Salmonella-induced apoptosis of infected macrophages results in presentation of a bacteria-encoded antigen after uptake by bystander dendritic cells.

Salmonella typhimurium is a gram-negative bacterium that survives and replicates inside vacuolar compartments of macrophages. Infection of macrophages with S. typhimurium grown under conditions allowing expression of the type III secretion system results in apoptotic death of the infected cells. Here, we show that infection of bone marrow-derived macrophages (MPhi) with wild-type S. typhimurium 14028 results in presentation of epitopes derived from a bacteria-encoded antigen on major histocompatibility complex (MHC) class I and MHC class II molecules after internalization of apoptotic MPhi by bystander dendritic cells (DCs). In contrast, infection of MPhi with the phoP constitutive mutant strain CS022, which does not induce apoptosis in infected MPhi, does not result in presentation of a bacteria-derived antigen by bystander DCs unless the infected MPhi are induced to undergo apoptosis by treatment with lipopolysaccharide and ATP. DCs appear to be unique in their ability to present antigens derived from MPhi induced to undergo apoptosis by Salmonella, as bystander MPhi are not capable of presenting the bacteria-derived antigen despite the fact that they efficiently internalize the apoptotic cells. These data suggest that apoptosis induction by bacterial infection of MPhi may not be a quiescent death that allows the bacteria to escape recognition by the immune system, but rather may contribute to an antimicrobial immune response upon engulfment by bystander DCs.

Animals↗

PubMed2000

Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

Age Factors↗

PubMed2000

Behavioural changes produced by transgenic overexpression of gamma2L and gamma2S subunits of the GABAA receptor.

Transgenic mice overexpressing either the mouse gamma2L or gamma2S subunit of the GABAA receptor were generated in a C57BL/6 J x DBA/2 J mixed background and expanded into transgenic lines. Transgenic mice and littermate controls were analysed with respect to altered behaviour indicative of anxiety, motor activity and acute effects of benzodiazepines and alcohol, as well as with regard to altered responses to alcohol withdrawal and acute functional tolerance to alcohol. Biochemical tests assessed flunitrazepam- and ethanol-enhanced 36Cl- flux stimulated by muscimol in cerebellar and cortical microsacs and [3H]-flunitrazepam binding to cerebellar membranes. There were no significant differences in any of these measures between the transgenic and control mice, except in tests of acute functional tolerance to acute injection of ethanol. Compared to controls, mice carrying either the gamma2L or gamma2S transgene developed significantly less tolerance to the ataxic effects of ethanol. We conclude that acute functional tolerance to ethanol is very sensitive to the amount of GABAA receptor gamma2 subunit available (regardless of whether it is gamma2L or gamma2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.

Animals↗

PubMed2000

Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response.

Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMPhi. These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes naïve CD4(+) and CD8(+) T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.

Animals↗

PubMed2000

Salmonella enterica serovar typhimurium-induced maturation of bone marrow-derived dendritic cells.

Murine bone marrow-derived dendritic cells (DC) can phagocytose and process Salmonella enterica serovar Typhimurium for peptide presentation on major histocompatibility complex class I (MHC-I) and MHC-II molecules. To investigate if a serovar Typhimurium encounter with DC induces maturation and downregulates their ability to present antigens from subsequently encountered bacteria, DC were pulsed with serovar Typhimurium 24 h prior to coincubating with Escherichia coli expressing the model antigen Crl-OVA. Quantitating presentation of OVA epitopes contained within Crl-OVA showed that Salmonella-pulsed DC had a reduced capacity to process Crl-OVA-expressing E. coli for OVA(257-264)/K(b) and OVA(265-277)/I-A(b) presentation. In addition, time course studies of DC pulsed with Crl-OVA-expressing serovar Typhimurium showed that OVA(257-264)/K(b) complexes could stimulate CD8OVA T-hybridoma cells for <24 h following a bacterial pulse, while OVA(265-277)/I-A(b) complexes could stimulate OT4H T-hybridoma cells for >24 but <48 h. The phoP-phoQ virulence locus of serovar Typhimurium also influenced the ability of DC to process Crl-OVA-expressing serovar Typhimurium for OVA(265-277)/I-A(b) presentation but not for OVA(257-264)/K(b) presentation. Furthermore, pulsing of DC with serovar Typhimurium followed by incubation for 24 or 48 h altered surface expression of MHC-I, MHC-II, CD40, CD54, CD80, and CD86, generating a DC population with a uniform, high expression level of these molecules. Finally, neither the serovar Typhimurium phoP-phoQ locus nor lipopolysaccharides (LPS) containing lipid A modifications purified from phoP mutant strains had a different effect on DC maturation from that of wild-type serovar Typhimurium or purified wild-type LPS. Thus, these data show that Salmonella or Salmonella LPS induces maturation of DC and that this process is not altered by the Salmonella phoP virulence locus. However, phoP did influence OVA(265-277)/I-A(b) presentation by DC infected with Crl-OVA-expressing serovar Typhimurium when quantitated after 2 h of bacterial infection.

Animals↗

PubMed2000

Diagnosis of human papillomavirus gynecologic infections.

The identification in the early 1980s of human papillomavirus (HPV) DNA in cervical carcinoma generated interest in molecular classification of the virus, and prompted studies regarding the oncogenic potential of genital HPVs. Subsequent studies confirming the presence of HPV in greater than 90% of precancerous cervical lesions and close to 100% of cervical cancers has raised concerns regarding the adequacy of Papanicolaou (Pap) smear testing for the detection of precancerous lesions/HPV infection. A variety of detection methods adjunctive to cytologic testing have been described, including detection at the macroscopic level, cerviography, colposcopy, and serologic and molecular-based HPV testing. Recently, there has been intense interest in molecular-based detection and typing of HPV-induced genital lesions. This has resulted in the development of a variety of molecular-based detection methods including Southern transfer, dot blotting, in situ hybridization, hybrid capture, and PCR-based assays. This article provides an overview of each of the molecular methods, and addresses the potential future role of molecular-based HPV testing.

Antibodies, Viral↗

PubMed1999

Stable expression of human glycine alpha1 and alpha2 homomeric receptors in mouse L(tk-) cells.

We report the development of two mouse fibroblast-like stably-transfected cell lines (alpha1-62-4 and alpha2-B36-1) that express human alpha1 or alpha2 glycine receptor subunits, respectively. Transfected cDNAs were cloned into the pMSGneo expression vector, for which transcription is controlled by the dexamethasone-inducible MMTV promoter. Patch-clamp electrophysiological recordings revealed that the alpha1 or alpha2 glycine receptor subunits expressed in these cells form functional glycine receptors that are inhibited by strychnine and picrotoxin. Glycine activated currents in these cells with EC50s of 101+/-7 or 112+/-23 microM for cells stably expressing alpha1 or alpha2 receptors, respectively. As indicated by assays of glycine-stimulated 36Cl-- uptake, these cells express glycine receptors only after treatment with dexamethasone. In order to measure expression of the glycine alpha1 or alpha2 receptor protein, we produced a new anti-alpha1/alpha2 glycine receptor antibody (anti-alpha GR). Western blot analysis with this antibody showed a band of approximately 48 kDa only in homogenates from cells which had been transfected with the glycine alpha1 or alpha2 receptor cDNAs. Thus, through use of this stable expression system, we successfully produced cell lines expressing strychnine-sensitive glycine receptors that display similar functional characteristics to homomeric glycine receptors expressed in other systems. These stably transfected cells should provide a useful in vitro system for the study of the physiology and pharmacology of strychnine-sensitive glycine receptors.

Animals↗

PubMed1999

Analysis of a very large trinucleotide repeat in a patient with juvenile Huntington's disease.

A patient with juvenile Huntington's disease (HD) of probable maternal inheritance is reported. The expanded IT-15 allele was only detected with the use of modified PCR and Southern transfer techniques, which showed a CAG trinucleotide repeat expansion of approximately 250 repeats-the largest CAG expansion reported within the huntingtin gene. This case emphasizes the need for communication between the diagnostic laboratory and the clinician to define the molecular genetics of unusual cases.

Adolescent↗

PubMed1999

Classical MHC class I peptide presentation of a bacterial fusion protein by bone marrow-derived dendritic cells.

Dendritic cells (DC) expanded in the presence of GM-CSF from the bone marrow of C57BL/6 mice process Gram-negative bacteria expressing the model antigen Crl-OVA for peptide presentation on MHC class I molecules. Here we show that presentation of OVA(257-264) processed by DC co-incubated with E. coli expressing Crl-OVA, which contains the Kb-binding OVA(257-264) epitope, occurs by a cytosolic MHC-I presentation pathway. First, we demonstrate the requirement for the transporter associated with antigen processing (TAP) by showing that DC from TAP1-/- mice co-incubated with E. coli expressing Crl-OVA did not result in Kb presentation of OVA(257-264). Second, the proteasome inhibitor MG132 abrogated presentation of OVA(257-264) on Kb when C57BL/6 DC phagocytosed and processed E. coli expressing Crl-OVA. Third, inhibiting protein synthesis using cycloheximide or blocking exocytosis of newly synthesized proteins from the endoplasmic reticulum using brefeldin A abrogated presentation of OVA(257-264) processed from bacteria expressing Crl-OVA by C57BL/6 DC. Finally, peptide regurgitation and loading of OVA(257-264) on neighboring bystander Kb-expressing antigen-presenting cells after BALB/c (H-2d) DC phagocytosed E. coli expressing Crl-OVA could not be detected. Together, these data support a cytosolic MHC-I presentation pathway for OVA(257-264) processed from E. coli expressing Crl-OVA by bone marrow-derived DC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗