Biomedical subjects

S C Baker

Publications and source records attributed to S C Baker.

At least 19 recordsLinked to original sources

PubMed1998

Processing of the coronavirus MHV-JHM polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of ORF1a.

The replicase of mouse hepatitis virus strain JHM (MHV-JHM) is encoded by two overlapping open reading frames, ORF1a and ORF1b, which are translated to produce a 750-kDa precursor polyprotein. The polyprotein is proposed to be processed by viral proteinases to generate the functional replicase complex. To date, only the MHV-JHM amino-terminal proteins p28 and p72, which is processed to p65, have been identified. To further elucidate the biogenesis of the MHV-JHM replicase, we cloned and expressed five regions of ORF1a in bacteria and prepared rabbit antisera to each region. Using the immune sera to immunoprecipitate radiolabeled proteins from MHV-JHM infected cells, we determined that the MHV-JHM ORF1a is initially processed to generate p28, p72, p250, and p150. Pulse-chase analysis revealed that these intermediates are further processed to generate p65, p210, p40, p27, the MHV 3C-like proteinase, and p15. A putative replicase complex consisting of p250, p210, p40, p150, and a large protein (> 300 kDa) coprecipitate from infected cells disrupted with NP-40, indicating that these proteins are closely associated even after initial proteolytic processing. Immunofluorescence studies revealed punctate labeling of ORF1a proteins in the perinuclear region of infected cells, consistent with a membrane-association of the replicase complex. Furthermore, in vitro transcription/translation studies of the MHV-JHM 3Cpro and flanking hydrophobic domains confirm that 3C protease activity is significantly enhanced in the presence of canine microsomal membranes. Overall, our results demonstrate that the MHV-JHM ORF1a polyprotein: (1) is processed into more than 10 protein intermediates and products, (2) requires membranes for efficient biogenesis, and (3) is detected in discrete membranous regions in the cytoplasm of infected cells.

Animals↗

PubMed1998

Maturation of the polymerase polyprotein of the coronavirus MHV strain JHM involves a cascade of proteolytic processing events.

The RNA polymerase gene of the murine coronavirus mouse hepatitis virus (MHV) encodes a polyprotein of greater than 750 kDa. The amino-terminal cleavage product of the MHV polymerase polyprotein, p28, has been shown to be cleaved from the polyprotein by the virus-encoded protease PCP-1. We aim to identify the MHV-JHM proteolytic products downstream of p28 and to determine which viral proteinase domains are responsible for generating each of them. To this end, we have generated antisera directed at specific MHV-JHM ORF1a regions and have used these antisera to identify six viral proteins, representing a large portion of ORF1a, from MHV-JHM-infected cells. These proteins include p28, p72, p65, p250, p210, and p27.

Animals↗

PubMed1998

Quasispecies development by high frequency RNA recombination during MHV persistence.

Recent studies suggest that infectious viruses and particularly persisting viral RNAs often exist as diverse populations or "quasispecies". We have developed an approach to characterize populations of the murine coronavirus mouse hepatitis virus (MHV) generated during persistent infection which has allowed us to begin to address the role of the viral quasispecies in MHV pathogenesis. We analyzed the population of persisting viral RNAs using reverse-transcription polymerase chain reaction amplification (RT-PCR) of the S1 "hypervariable" region of the spike gene followed by differential colony hybridization to identify spike deletion variants (SDVs) from acute and persistently infected mice. Sequence analysis revealed that mice with the most severe chronic paralysis harbored the most complex quasispecies. Mapping of the SDVs to the predicted RNA secondary structure of the spike RNA revealed that an isolated stem loop structure is frequently deleted. Overall, these results are consistent with high frequency recombination at sites of RNA secondary structure contributing to expansion of the viral quasispecies and persisting viral pathogenesis.

Animals↗

PubMed1998

Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility.

Paracoccus denitrificans and its near relative Paracoccus versutus (formerly known as Thiobacilllus versutus) have been attracting increasing attention because the aerobic respiratory system of P. denitrificans has long been regarded as a model for that of the mitochondrion, with which there are many components (e.g., cytochrome aa3 oxidase) in common. Members of the genus exhibit a great range of metabolic flexibility, particularly with respect to processes involving respiration. Prominent examples of flexibility are the use in denitrification of nitrate, nitrite, nitrous oxide, and nitric oxide as alternative electron acceptors to oxygen and the ability to use C1 compounds (e.g., methanol and methylamine) as electron donors to the respiratory chains. The proteins required for these respiratory processes are not constitutive, and the underlying complex regulatory systems that regulate their expression are beginning to be unraveled. There has been uncertainty about whether transcription in a member of the alpha-3 Proteobacteria such as P. denitrificans involves a conventional sigma70-type RNA polymerase, especially since canonical -35 and -10 DNA binding sites have not been readily identified. In this review, we argue that many genes, in particular those encoding constitutive proteins, may be under the control of a sigma70 RNA polymerase very closely related to that of Rhodobacter capsulatus. While the main focus is on the structure and regulation of genes coding for products involved in respiratory processes in Paracoccus, the current state of knowledge of the components of such respiratory pathways, and their biogenesis, is also reviewed.

Base Sequence↗

PubMed1998

Pattern formation and growth during floral organogenesis: HUELLENLOS and AINTEGUMENTA are required for the formation of the proximal region of the ovule primordium in Arabidopsis thaliana.

Our understanding of the molecular mechanisms that regulate and integrate the temporal and spatial control of cell proliferation during organ ontogenesis, particularly of floral organs, continues to be primitive. The ovule, the progenitor of the seed, of Arabidopsis thaliana has been used to develop an effective model system for the analysis of plant organogenesis. A typical feature of a generalized ovule is the linear arrangement of at least three distinct elements, the funiculus, chalaza and nucellus, along a proximal-distal axis. This pattern is supposed to be established during the early proliferative phase of ovule development. We provide genetic evidence that the young ovule primordium indeed is a composite structure. Two genes, HUELLENLOS and AINTEGUMENTA have overlapping functions in the ovule and differentially control the formation of the central and proximal elements of the primordium. The results indicate that proximal-distal pattern formation in the Arabidopsis ovule takes place in a sequential fashion, starting from the distal end. Furthermore, we show that HUELLENLOS also regulates the initiation and/or maintenance of integument and embryo sac ontogenesis and interestingly prevents inappropriate cell death in the young ovule.

Arabidopsis↗

PubMed1997

IgA plasma cells in vascular tissue of patients with Kawasaki syndrome.

The etiology and pathogenesis of Kawasaki syndrome (KS) remain unknown. Clinical and epidemiologic features of KS are consistent with an infectious cause. To search for an etiologic agent of KS, a phage cDNA expression library was constructed from the aorto-iliac junction of a patient with fatal acute KS and screened with convalescent KS serum followed by anti-human Ig. Unexpectedly, 0.1% of the clones in the library react with anti-human Ig, indicating the presence of many Ig-producing B lymphocytes in the vasculitic tissue. To confirm this finding and to determine the isotypes produced, frozen vascular tissue sections from the patient and paraffin sections from coronary arteries from six additional patients with fatal acute or subacute KS were incubated with Abs to Ig isotypes. Histopathology of the tissues revealed the presence of many plasma cells in the inflammatory infiltrate. IgA was the predominant isotype produced in vascular tissue in all seven KS patients. IgM- and IgG-producing cells were less often detected. We conclude that there is a marked plasma cell response within the vasculitic tissue in KS, with unusual IgA production locally in this nonlymphoid, nonmucosal tissue. We suggest that the prominence of IgA plasma cells in the vascular infiltrate in the early, acute, and subacute stages of KS indicates an Ag-driven immune response to an etiologic agent with a respiratory or gastrointestinal portal of entry and speculate that this unusual immune response is integral to the pathogenesis of the illness.

Adolescent↗

PubMed1997

Cytochrome cd1 structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to beta-propeller folds.

The central tunnel of the eight-bladed beta-propeller domain of cytochrome cd1 (nitrite reductase) is seen, from a 1.28 A resolution structure, to contain hydrogen donors and acceptors that are satisfied by interaction either with water or the d1 haem. The d1 haem, although bound by an extensive network of hydrogen bonds, is not distorted in its binding pocket and is confirmed to have exactly the dioxoisobacteriochlorin structure proposed from chemical studies. A biological rationale is advanced for the undistorted structure of the d1 haem and the large number of hydrogen bonds it makes. The beta-propeller domain can be closely superimposed on that of methanol dehydrogenase despite the enzymes sharing no common sequence motifs and using a different set of interactions to "Velcro" close the propeller. The sequence and likely structural relationships between cytochrome cd1 or methanol dehydrogenase and other predicted eight-bladed beta-propeller domains in proteins, such as the pyrolloquinoline quinone-dependent alcohol dehydrogenase, are discussed and compared with other propeller proteins. From sequencing the nirS gene of Thiosphaera pantotropha, it is established that the amino acid sequence deduced previously in part from X-ray diffraction data at lower resolution was largely correct, as was the proposal that eight N-terminal amino acid residues were not seen in the structure. The unusual haem iron environments in both the c-type cytochrome domain, with His/His coordination, and the d1-type cytochrome domain with Tyr/His coordination are related to the functions of the redox centres.

Amino Acid Sequence↗

PubMed1997

Dopamine efflux in the rat nucleus accumbens evoked by dopamine receptor stimulation in the entorhinal cortex is modulated by oestradiol and progesterone.

This study compared the effects of dopamine receptor stimulation in the entorhinal cortex on dopamine release in the nucleus accumbens, measured by in vivo microdialysis in conscious Sprague-Dawley rats, with and without oestradiol and progesterone priming. Nonselective dopamine receptor stimulation with apomorphine reduced dopamine release in the nucleus accumbens, an effect which was prevented by injection of cis-flupenthixol into the entorhinal cortex. Selective D1 receptor stimulation with SKF38393 increased dopamine release, whereas selective D2 receptor stimulation with quinpirole did not affect dopamine release. Combined administration of oestradiol and progesterone potentiated the response to apomorphine and prevented the response to SKF38393. The effects of single hormone administration on the response to apomorphine suggested that the modulation was primarily due to oestradiol enhancing effects of progesterone. Experiments with high [K+] suggested these hormonal effects were exerted predominantly in the entorhinal cortex. The present experiments have demonstrated that dopaminergic modulation of transmission in a cortico-striatal loop linking temporal and prefrontal cortex is regulated by oestradiol and progesterone. Dysfunction in this system in humans may give rise to affective and cognitive symptoms which may, if initiated by a postpartum fall in oestrogen and progesterone concentrations, constitute the core pathophysiology of puerperal psychosis.

Animals↗

PubMed1997

Methylosulfonomonas methylovora gen. nov., sp. nov., and Marinosulfonomonas methylotropha gen. nov., sp. nov.: novel methylotrophs able to grow on methanesulfonic acid.

Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the alpha-Proteobacteria. Methylosulfonomonas is a discrete lineage within the alpha-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the alpha-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera.

Bacteria↗

PubMed1997

The interaction between mood and cognitive function studied with PET.

BACKGROUND: Experimentally induced depressed mood is a suggested model for retarded depression. We describe the neural response associated with induced mood and the locus of the interaction between systems mediating mood and cognitive function. METHODS: Normal subjects performed a verbal fluency task during induced elated and depressed mood states. Regional cerebral blood flow (rCBF) was measured as an index of neural activity using Positron Emission Tomography (PET). RESULTS: In both elated and depressed mood state rCBF was increased in lateral orbitofrontal cortex, rCBF was also increased in the midbrain in elated mood. In the depressed condition rCBF was decreased in rostral medial prefrontal cortex. Verbal fluency produced an expected increase of rCBF in left dorsolateral prefrontal, inferior frontal and premotor cortex, anterior cingulate and insula cortex bilaterally, the left supramarginal gyrus posteriorly and the thalamus. Activation in the verbal fluency task was attenuated throughout the left prefrontal, premotor and cingulate cortex and thalamus in both elated and depressed mood conditions. An attenuation of anterior cingulate activation was specific to depressed mood. CONCLUSIONS: Alteration of mood is associated with activation of orbitofrontal cortex which may be critical to the experience of emotion. The mood induced modulation of verbal fluency induced activations is consistent with resting state findings of decreased function in these regions in depressed patients. The present data suggest that resting state rCBF profile may represent the modulation of spontaneous activity in this network by a core system that is dysfunctional in depression.

Adolescent↗

PubMed1997

Prefrontal dysfunction in depressed patients performing a complex planning task: a study using positron emission tomography.

INTRODUCTION: Patients with unipolar depression show impaired performance on the Tower of London planning task. Positron emission tomography, which has previously identified resting state blood flow abnormalities in depression, was used to investigate neural activity associated with performance of this task in depressed patients and normal controls. METHODS: Six patients with unipolar depression and six matched controls were scanned while performing easy and hard Tower of London problems in a one-touch computerized paradigm and while performing a perceptuomotor control task. RESULTS: The patients in this study showed an expected task-related performance deficit compared with normal subjects. In normal subjects, the task engaged a network of prefrontal cortex, anterior cingulate, posterior cortical areas and subcortical structures including the striatum, thalamus and cerebellum. Depressed patients failed to show significant activation in the cingulate and striatum; activation in the other prefrontal and posterior cortical regions was significantly attenuated relative to controls. Crucially, patients also failed to show the normal augmentation of activation in the caudate nucleus, anterior cingulate and right prefrontal cortex associated with increasing task difficulty. CONCLUSIONS: These findings provide evidence for cingulate, prefrontal and striatal dysfunction associated with impaired task performance in depression. The present results are consistent with a central role of cingulate dysfunction in depression as well as suggesting impaired frontostriatal function.

Adult↗

PubMed1997

Interactions among genes regulating ovule development in Arabidopsis thaliana.

The INNER NO OUTER (INO) and AINTEGUMENTA (ANT) genes are essential for ovule integument development in Arabidopsis thaliana. Ovules of ino mutants initiate two integument primordia, but the outer integument primordium forms on the opposite side of the ovule from the normal location and undergoes no further development. The inner integument appears to develop normally, resulting in erect, unitegmic ovules that resemble those of gymnosperms. ino plants are partially fertile and produce seeds with altered surface topography, demonstrating a lineage dependence in development of the testa. ant mutations affect initiation of both integuments. The strongest of five new ant alleles we have isolated produces ovules that lack integuments and fail to complete megasporogenesis. ant mutations also affect flower development, resulting in narrow petals and the absence of one or both lateral stamens. Characterization of double mutants between ant, ino and other mutations affecting ovule development has enabled the construction of a model for genetic control of ovule development. This model proposes parallel independent regulatory pathways for a number of aspects of this process, a dependence on the presence of an inner integument for development of the embryo sac, and the existence of additional genes regulating ovule development.

Arabidopsis↗

PubMed1997

Evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection.

High-frequency RNA recombination has been proposed as an important mechanism for generating viral deletion variants of murine coronavirus. Indeed, a number of variants with deletions in the spike glycoprotein have been isolated from persistently infected animals. However, the significance of generating and potentially accumulating deletion variants in the persisting viral RNA population is unclear. To study this issue, we evaluated the evolution of spike variants by examining the population of spike RNA sequences detected in the brains and spinal cords of mice inoculated with coronavirus and sacrificed at 4, 42, or 100 days postinoculation. We focused on the S1 hypervariable region since previous investigators had shown that this region is subject to recombination and deletion. RNA isolated from the brains or spinal cords of infected mice was rescued by reverse transcription-PCR, and the amplified products were cloned and used in differential colony hybridizations to identify individual isolates with deletions. We found that 11 of 20 persistently infected mice harbored spike deletion variants (SDVs), indicating that deletions are common but not required for persistent infection. To determine if a specific type of SDV accumulated during persistence, we sequenced 106 of the deletion isolates. We identified 23 distinct patterns of SDVs, including 5 double-deletion variants. Furthermore, we found that each mouse harbored distinct variants in its central nervous system (CNS), suggesting that SDVs are generated during viral replication in the CNS. Interestingly, mice with the most severe and persisting neurological disease harbored the most prevalent and diverse quasispecies of SDVs. Overall, these findings illustrate the complexity of the population of persisting viral RNAs which may contribute to chronic disease.

Animals↗

PubMed1997

Generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted RNA secondary structure.

Coronavirus RNA evolves in the central nervous systems (CNS) of mice during persistent infection. This evolution can be monitored by detection of a viral quasispecies of spike deletion variants (SDVs) (C. L. Rowe, S. C. Baker, M. J. Nathan, and J. O. Fleming, J. Virol. 71:2959-2969, 1997). We and others have found that the deletions cluster in the region from 1,200 to 1,800 nucleotides from the 5' end of the spike gene sequence, termed the "hypervariable" region. To address how SDVs might arise, we generated the predicted folding structures of the positive- and negative-strand senses of the entire 4,139-nt spike RNA sequence. We found that a prominent, isolated stem-loop structure is coincident with the hypervariable region in each structure. To determine if this predicted stem-loop is a "hot spot" for RNA recombination, we assessed whether this region of the spike is more frequently deleted than three other selected regions of the spike sequence in a population of viral sequences isolated from the CNS of acutely and persistently infected mice. Using differential colony hybridization of cloned spike reverse transcription-PCR products, we detected SDVs in which the hot spot was deleted but did not detect SDVs in which other regions of the spike sequence were exclusively deleted. Furthermore, sequence analysis and mapping of the crossover sites of 25 distinct patterns of SDVs showed that the majority of crossover sites clustered to two regions at the base of the isolated stem-loop, which we designated as high-frequency recombination sites 1 and 2. Interestingly, the majority of the left and right crossover sites of the SDVs were directly across from or proximal to one another, suggesting that these SDVs are likely generated by intramolecular recombination. Overall, our results are consistent with there being an important role for the spike RNA secondary structure as a contributing factor in the generation of SDVs during persistent infection.

Base Sequence↗

PubMed1996

Neural systems engaged by planning: a PET study of the Tower of London task.

The functional anatomy of planning was investigated using the Tower of London task. Activation was observed in a distributed network of cortical areas incorporating prefrontal, cingulate, premotor, parietal and occipital cortices. Activation in corresponding areas has been observed in visuospatial working memory tasks with the exception of the rostral prefrontal cortex. This area may be identified with the executive components of planning comprising response selection and evaluation. Enhanced neural activity in both this rostral prefrontal area and the visuospatial working memory system was associated with increased task difficulty.

Adolescent↗

PubMed1996

Identification of the polymerase polyprotein products p72 and p65 of the murine coronavirus MHV-JHM.

The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. The amino-terminal product of the MHV polymerase polyprotein, p28, is generated by cleavage of the polyprotein by PCP-1. To identify the viral products downstream of p28, we generated a fusion-protein specific antiserum directed against the region adjacent to p28 and used the antiserum to detect virus-specific proteins from MHV-JHM infected cells. When this antiserum was used to immunoprecipitate radiolabeled proteins from MHV-JHM infected cell lysates, virus-specific proteins of 72 and 65 kDa were detected. Furthermore, pulse and chase experiments demonstrated that p72 is likely a precursor to the mature protein product, p65. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5'-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. We also cloned and expressed the 72 kDa region immediately downstream from p28, and tested the ability of in vitro translated PCP-1 and PCP-2 to cleave p72 to p65 in trans. Our results indicate that neither viral proteinase domain PCP-1 nor PCP-2 is capable of cleavage of p72 to produce p65 in vitro. The role of MHV proteinases in the processing of p72 and p65 is discussed.

Animals↗

PubMed1996

Primary structure and phylogeny of the Calvin cycle enzymes transketolase and fructosebisphosphate aldolase of Xanthobacter flavus.

Xanthobacter flavus, a gram-negative facultatively autotrophic bacterium, employs the Calvin cycle for the fixation of carbon dioxide. Cells grown under autotrophic growth conditions possess an Fe(2+)-dependent fructosebisphosphate (FBP) aldolase (class II) in addition to a class I FBP aldolase. By nucleotide sequencing and heterologous expression in Escherichia coli, genes encoding transketolase (EC 2.2.1.1.; CbbT) and class II FBP aldolase (EC 4.1.2.13; CbbA) were identified. A partial open reading frame encoding a protein similar to pentose-5-phosphate 3-epimerase was identified downstream from cbbA. A phylogenetic tree of transketolase proteins displays a conventional branching order. However, the class II FBP aldolase protein from X. flavus is only distantly related to that of E. coli. The autotrophic FBP aldolase proteins from X. flavus, Alcaligenes eutrophus, and Rhodobacter sphaeroides form a tight cluster, with the proteins from gram-positive bacteria as the closest relatives.

Amino Acid Sequence↗

PubMed1995

Evolution of mouse hepatitis virus (MHV) during chronic infection: quasispecies nature of the persisting MHV RNA.

Coronavirus infection of mice has been used extensively as a model for the study of acute encephalitis and chronic demyelination. To examine the evolution of coronavirus RNA during chronic demyelinating infection, we isolated RNA from intracerebrally inoculated mice at 4, 6, 8, 13, 20, and 42 days postinfection and used reverse transcription-polymerase chain reaction amplification methods (RT-PCR) to detect viral sequences. RNA sequences from two viral structural genes, the spike gene and the nucleocapsid gene, were detected throughout the chronic infection. In contrast, infectious virus was not detectable from brain homongenates beyond 13 days postinfection. These results indicate that coronavirus RNA persists in the brain at times when infectious virus is not detected. To determine if genetic changes were occurring during viral replication in the host, we cloned and sequenced the RT-PCR products from the spike and nucleocapsid regions and analyzed the sequences for mutations. Sequencing of the cloned products revealed that a variety of mutant forms of viral RNA persisted in the CNS, including point mutants, deletion mutants, and termination mutants. The mutations accumulated during persistent infection in both the spike and the nucleocapsid sequences, with greater than 65% of the mutations encoding amino acid changes. These results show that a diverse population or quasispecies consisting of mutant and deletion variant viral RNAs (which may not be capable of producing infectious virus particles) persists in the central nervous system of mice during chronic demyelinating infection. The implications of these results for the role of persistent viral genetic information in the pathogenesis of chronic demyelination are discussed.

Amino Acid Sequence↗