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M G Schmidt

Publications and source records attributed to M G Schmidt.

At least 19 recordsLinked to original sources
PubMed1999

Effect of rev on the cytoplasmic localization of intron-containing human immunodeficiency virus type 1 RNA.

Human immunodeficiency virus type 1 (HIV-1) proteins are expressed from both intron-containing and completely spliced RNAs. Rev, an HIV-1 regulatory protein, is necessary for the expression of intron-containing RNAs. The effect of Rev on the subcellular localization of intron-containing HIV-1 RNA was examined by in situ RNA hybridization. In the presence of Rev, intron-containing HIV-1 RNA accumulated at the nuclear membrane and within the cytoplasm of transfected cells. In the absence of Rev, intron-containing HIV-1 RNA accumulated within the nucleus. In approximately 20% of the cells transfected in the absence of Rev, intron-containing HIV-1 RNA was also found in the cytoplasm. Differences in the subcytoplasmic localization of intron-containing HIV-1 RNA in the presence and absence of Rev were not observed using in situ RNA hybridization. To determine the effect of Rev on RNA localization within the cytoplasm, an extensive fractionation protocol involving both hypotonic and detergent lysis was used. In the presence of Rev, 40.9 +/- 4.6% of the cytoplasmic intron-containing HIV-1 RNA was released by hypotonic lysis. A similar fractionation profile was seen for several other translated viral and cellular RNAs. However, in the absence of Rev, only 16.5 +/- 5.1% of the cytoplasmic intron-containing HIV-1 RNA was released on hypotonic lysis (P < 0. 005). Thus the cytoplasmic fractionation pattern of this RNA was altered in the absence of Rev.

Cell Line↗
PubMed1999

Regulation of the Escherichia coli secA gene is mediated by two distinct RNA structural conformations.

Expression from the secA gene, encoding a key component of the general secretory pathway of Escherichia coli, is influenced by the secretion status of the cell, autogenous translational repression, and translational coupling to the upstream gene, X. SecA binds to its mRNA in a region overlapping its ribosome binding site, thus competing with ribosomes that would initiate secA translation. Mapping of the geneX-secA mRNA secondary structure has demonstrated that the RNA can adopt two distinct conformations in solution. The first conformation arises from the base-pairing of the secA Shine-Dalgarno (SD) sequence with the geneX terminus. The second conformation, in which the secA SD sequence is no longer paired with the geneX terminus, contains a GC-rich stem upstream of the secA SD sequence. The presence of this GC-rich stem is supported by structure mapping of a mutant RNA containing a deletion in the geneX terminus. The former structure appears to be involved in translational coupling by directly linking the geneX and secA sequences, where geneX translation activates secA translational initiation through the unpairing and unmasking of the secA SD sequence. As indicated by SecA-RNA binding assays, the latter structure is probably involved in SecA binding and translational repression of the secA gene. The stabilizing effect of magnesium ions toward occlusion of the secA SD sequence supports the presence of RNA tertiary structure in this regulatory domain.

Adenosine Triphosphatases↗
PubMed1999

SecA: the ubiquitous component of preprotein translocase in prokaryotes.

SecA is an obligatory component of the complex hetero-septameric translocase of prokaryotes. It is unique in that it exists as two forms within the holoenzyme; first, as a structural component of the preprotein channel and second, as an ATP-dependent membrane cycling factor facilitating the translocation of a broad class of proteins across the cytoplasmic membrane. While the translocase activity of SecA appears to be functionally conserved, it is not clear whether the mechanisms of regulation of the secA gene are similarly maintained. The recent characterization of an ATP-dependent RNA helicase activity of SecA offers a unique mechanism for SecA to communicate the secretion status of the cell to the appropriate regulatory circuits simply by the unwinding of an appropriate RNA target. Resolution of these two activities through combined biochemical, genetic, and biophysical studies should lead to a better understanding of the role of SecA in bacterial secretion.

Adenosine Triphosphatases↗
PubMed1998

Effect of Rev on the intranuclear localization of HIV-1 unspliced RNA.

Human immunodeficiency virus type 1 (HIV-1) Rev is a 19-kDa regulatory protein which binds to unspliced and partially spliced HIV-1 RNAs. Export, splicing, stability, and translation of HIV-1 RNAs are influenced by Rev. To further understand the effect of Rev on HIV-1 RNA splicing, the intranuclear localization of unspliced HIV-1 RNA and a cellular splicing factor was examined in the presence and absence of Rev. Splicing component-35 (SC-35) is an essential SR protein splicing factor which localizes into 20-40 nuclear granules (Fu, X. D., and Maniatis, T. Nature 343 (6257), 437-441, 1990). Laser scanning confocal microscopy was utilized to examine the colocalization of unspliced HIV-1 RNA and SC-35-containing granules. In the presence of Rev, many of the SC-35-containing granules were colocalized on their edges or completely colocalized with HIV-1 unspliced RNA speckles. In the absence of Rev, however, little colocalization of the unspliced HIV-1 RNA speckles and the SC-35-containing granules was observed. Quantitative RT-PCR was utilized to examine the effect of Rev on the level of fully spliced HIV-1 RNA. In the presence of Rev, a decrease in the level of fully spliced HIV-1 RNA was observed. Thus both the intranuclear localization and posttranscriptional processing of HIV-1 unspliced RNA are affected by Rev.

Cell Line↗
PubMed1998

Regulation of intracellular human immunodeficiency virus type-1 protease activity.

The maturation of HIV-1 virions is accomplished through the proteolytic cleavage of Gag and GagPol precursor polyproteins by the viral-encoded protease (PR). Since virions are assembled from unprocessed polyproteins, the intracellular activation of PR must be limited. An experimental system was established that allows the investigation of the intracellular regulation of PR activity. By expressing Gag in trans with the GagPol precursor, downregulation of the intracellular PR activity associated with GagPol was demonstrated. Inhibition of PR activity was dependent upon the context of PR expression. Sequences capable of mediating this inhibition were localized to capsid. A mechanism through which Gag regulates PR activity is proposed whereby the disproportionate synthesis of Gag inhibits the activation of PR in the cytoplasm. Further elucidation of the mechanism of intracellular inhibition of PR activity may facilitate the development of novel PR inhibitors capable of inhibiting viral replication in vivo.

Amino Acid Sequence↗
PubMed1994

Rapid purification of native SecA from Escherichia coli: development of a new affinity chromatography procedure.

The SecA protein occupies a pivotal position in the public protein export pathway in Escherichia coli. The multifunctional SecA protein recognizes cytoplasmic factors associated with export including the presecretory protein and targets the complex to the inner membrane, where it acts in the early stages of protein translocation. The ability of SecA to bind ATP was the basis for the development of a novel, rapid purification scheme involving a single chromatographic step. Affinity chromatography was carried out on Red Sepharose CL-6B. The SecA present in crude extracts of E. coli binds strongly to this dye-ligand matrix, and active protein was purified to greater than 90% homogeneity. The protein isolated by this procedure retained the previously described ATPase and RNA-binding activities of SecA. This approach should permit the rapid purification of SecA homologs from a variety microorganisms.

Adenosine Triphosphatases↗
PubMed1991

Regulation of Escherichia coli secA mRNA translation by a secretion-responsive element.

The Escherichia coli secA gene, whose translation is responsive to the proficiency of protein export within the cell, is the second gene in a three-gene operon and is flanked by gene X and mutT. By using gene fusion and oligonucleotide-directed mutagenesis techniques, we have localized this translationally regulated site to a region at the end of gene X and the beginning of secA. This region has been shown to bind SecA protein in vitro. These studies open the way for a direct investigation of the mechanism of secA regulation and its coupling to the protein secretion capability of the cell.

Adenosine Triphosphatases↗
PubMed1989

SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli.

The Escherichia coli secA gene, whose expression is responsive to the protein secretion status of the cell, is the second gene in an operon. We found that both the basal and induced levels of SecA biosynthesis are dependent on prior translation of the upstream gene, gene X, and identified two large gene X-secA transcripts. The 10-fold derepression of secA expression by protein export defects was at the translational level since no further increases in gene X or secA mRNA levels were detected during this period, and a secA-lacZ protein fusion but not an operon fusion was appropriately derepressed. Furthermore, overexpression of the SecA protein severely reduced expression of only the secA-lacZ protein fusion, indicating that SecA autogenously represses its own translation.

Bacterial Proteins↗
PubMed1988

Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coli.

The DNA sequence of the secA gene, essential for protein export in Escherichia coli, was determined and found to encode a hydrophilic protein of 901 amino acid residues with a predicted molecular weight of 101,902, consistent with its previously determined size and subcellular location. Sequence analysis of 9 secA(Ts) mutations conferring general protein export and secA regulatory defects revealed that these mutations were clustered in three specific regions within the first 170 amino acid residues of the SecA protein and were the result of single amino acid changes predicted to be severely disruptive of protein structure and function. The DNA sequence immediately upstream of secA was shown to encode a previously inferred gene, gene X. Sequence analysis of a conditionally lethal amber mutation, am109, previously inferred to be located proximally in the secA gene, revealed that it was located distally in gene X and was conditionally lethal due to its polar effect on secA expression. This and additional evidence are presented indicating that gene X and secA are cotranscribed.

Amino Acid Sequence↗
PubMed1987

The patient's partner: the spouse in residential care.

When an elderly patient enters residential care, tensions often arise because the patient's partner attempts to defend a threatened spousal role that is incompatible with the new setting. Patterns of patient entry into the facility also influence the interaction between the patient's spouse and nursing staff. The social worker's task is to reduce friction between the spouse and staff members by sustaining the husband or wife in an acceptable caring role.

Aged↗
PubMed1979

Patient as volunteer: an assault on chronicity.

By integrating selected former patients into its regular volunteer program, a South Australian state hospital reduced the hospital stays and the readmissions of a large number of the participants who had previously been seen as failures of the community mental health movement. The author describes the program, its benefits, and some limitations and problems, such as an occasional extra burden on ward personnel. The former patients' new roles as volunteers enabled them to get the support they needed from the hospital but through means more acceptable to them. The program also improved their self-esteem and enabled them to engage in more normalizing social relationships.

Adult↗
PubMed1976

Strain-related differences in immunosuppressive effects of Enterobacteriaceae and their lipopolysaccharides on production in rabbits of antibody to enterobacterial common antigen.

Certain polysaccharides have been shown to inhibit the antibody response of rabbits to the common enterobacterial antigen (CA). The present investigation revealed that striking differences exist in the immunosuppressive effects of enteric bacteria and their lipolysaccharides (lps), depending upon CA production by the strains. Mixtures of immunogenic strains (Escherichia coli F2378 [R4], E. coli F470 [R1], or Shigella boydii F3140 [R]) and non-immunogenic CA-producing strains, such as E. coli O1, E. coli O113, Salmonella montevideo, and S. minnesota, as well as the R mutants E. coli F614 (R1), E. coli F757 (R1), and S. typhimurium his 642 (Ra), failed to elicit CA antibodies. In contrast, mixtures of the immunogen and CA-negative strains S. typhimurium his 386 (Ra) and S. minnesota P595 (Re) or R555 (Ra) yielded antibodies in titers similar to those elicited by the immunogen alone. Further, LPS of CA-positive but not of CA-negative strains exerted this immunosuppressive effect. Quantitative studies revealed that LPS of S. minnesota in amounts of 100 mug/ml was strongly immunosuppressive, in amounts of 20 mug/ml slightly effective, and in amounts of 4 mug/ml ineffective. It is postulated that hitherto unknown differences exist, either in composition or in configuration, between LPS obtained from different microorganisms to account for the strain-related differences in immunosuppressive effects and, further, that the immunosuppressive LPS interacts with immunogenic CA.

Animals↗