Transcriptional profile of glucose-shocked and acid-adapted strains of Streptococcus mutans.

The aciduricity of Streptococcus mutans is an important virulence factor of the organism, required to both out-compete commensal oral microorganisms and cause dental caries. In this study, we monitored transcriptional changes that occurred as a continuous culture of either an acid-tolerant strain (UA159) or an acid-sensitive strain (fabM::Erm) moved from steady-state growth at neutral pH, experienced glucose-shock and acidification of the culture, and transitioned to steady-state growth at low pH. Hence, the timing of elements of the acid tolerance response (ATR) could be observed and categorized as acute vs. adaptive ATR mechanisms. Modulation of branched chain amino acid biosynthesis, DNA/protein repair mechanisms, reactive oxygen species metabolizers and phosphoenolpyruvate:phosphotransferase systems occurred in the initial acute phase, immediately following glucose-shock, while upregulation of F1 F0 -ATPase did not occur until the adaptive phase, after steady-state growth had been re-established. In addition to the archetypal ATR pathways mentioned above, glucose-shock led to differential expression of genes suggesting a re-routing of resources away from the synthesis of fatty acids and proteins, and towards synthesis of purines, pyrimidines and amino acids. These adjustments were largely transient, as upon establishment of steady-state growth at acidic pH, transcripts returned to basal expression levels. During growth at steady-state pH 7, fabM::Erm had a transcriptional profile analogous to that of UA159 during glucose-shock, indicating that even during growth in rich media at neutral pH, the cells were stressed. These results, coupled with a recently established collection of deletion strains, provide a starting point for elucidation of the acid tolerance response in S. mutans.

CD45RO: a marker for BCR-mediated selection.

We previously showed that IgH sequence alone minimally influenced germinal centre (GC) B-cell survival fate. As end-stage effector B cells are typically more mutated than founder GC B cells, we worked to develop an assay that would enrich for populations of GC B cells with progressively increasing numbers of somatic mutations, which could potentially be used as an indicator of positive selection. We targeted CD45 as it has been shown to influence activation-induced cytidine deaminase (AID) expression. In this study, anti-CD77 and anti-CD45RO (RO) were used to subdivide CD19(+)IgD(-)CD38(+)CD77(+) centroblasts (CB) and CD19(+)IgD(-)CD38(+)CD77(-) centrocytes (CC) into three contiguous RO fractions (RO(-), RO(+/-) and RO(+)) and assessed whether mutation frequency and characteristics associated with selection varied with respect to increasing RO expression. Here, we show that the average number of mutations per IgV(H)4 transcript increased concordantly with RO for CC, but not for CB. CC also exhibited an RO-associated increase in replacement mutations. Comparative analysis of clonally related sequences revealed that increased mutations were not due to the exclusive persistence of surface RO on highly mutated cells. RO-expressing CC and CB pools showed increased signs of activation (CD69(+)) and were enriched for surface Ig(+) cells. BCR-crosslinking induced a significant increase in surface RO on total tonsillar and GC B cells, which collectively suggests that the RO-associated increase in mutations is attributable, at least in part, to the cycling of cells that may have recently undergone BCR-mediated selection, or are potentially in developmental transition between CC and CB stages.

Induction of fibrinogen expression in the lung epithelium during Pneumocystis carinii pneumonia.

Pneumocystis carinii is an important pulmonary pathogen responsible for morbidity and mortality in patients with AIDS. The acute-phase response (APR), the primary mechanism used by the body to restore homeostasis following infection, is characterized by increased levels of circulating fibrinogen (FBG). Although the liver is the primary site of increased FBG synthesis during the APR, we unexpectedly discovered that FBG is synthesized and secreted by lung alveolar epithelial cells in vitro during an inflammatory stimulus. Therefore, we sought to determine whether lung epithelial cells produce FBG in vivo using animal models of P. carinii pneumonia (PCP). Inflammation was noted by an influx of macrophages to P. carinii-infected alveoli. Northern hybridization revealed that gamma-FBG mRNA increased two- to fivefold in P. carinii-infected lung tissue, while RNA in situ hybridization demonstrated increased levels of gamma-FBG mRNA in the lung epithelium. Immunoelectron microscopy detected lung epithelial cell-specific production of FBG, suggesting induction of a localized inflammatory response resembling the APR. A systemic APR was confirmed by a two- to fivefold upregulation of the levels of hepatic gamma-FBG mRNA in animals with PCP, resulting in a corresponding increase in levels of FBG in plasma. Furthermore, immunoelectron microscopy revealed the presence of FBG at the junction of cell membranes of trophic forms of P. carinii organisms aggregated along the alveolar epithelium. These results implicate FBG in the pathogenesis of PCP in a manner similar to that of the adhesive glycoproteins fibronectin and vitronectin, which are known to participate in intra-alveolar aggregation of organisms and adherence of P. carinii to the lung epithelium.

Tissue factor mRNA expression in the endothelium of an intact umbilical vein.

Tissue factor (TF) mRNA expression was measured by in situ hybridization in the endothelium of the intact human umbilical vein after infection with Rickettsia rickettsii. At 4 hours, R rickettsii organisms were clearly visible within approximately 70% of endothelial cells by immunocytochemical staining. Quantitation of TF mRNA expression revealed that the level within endothelial cells of the infected vein was significantly greater (3.7-fold, P < .0001) than that detected in uninfected endothelial cells. Serial sections of the umbilical cord vein were processed for in situ hybridization, and immunocytochemical staining and showed TF expression in those endothelial cells that contained R rickettsii organisms. Immunocytochemical staining for TF antigen at 6 hours was negative, but TF was clearly demonstrated within macrophages and fibroblasts of both control and infected umbilical cords. These studies demonstrate that the vascular endothelial cell, ex vivo, can be directly induced to express TF mRNA. This observation has not heretofore been clearly demonstrated except for in cultured endothelial cells. Since R rickettsii infection induces thrombotic vascular occlusions in patients with Rocky Mountain Spotted Fever, the results imply a potential role for endothelial cell TF in the pathogenesis of thrombotic disease.

Cloning and characterization of a lung-specific cDNA corresponding to the gamma chain of hepatic fibrinogen.

The gene (gamma FBG) encoding the fibrinogen gamma chain (gamma FBG) has been shown to exhibit both tissue-specific and ubiquitous expression. To confirm the identity of the gamma FBG transcripts expressed in extrahepatic tissue, lung tissue was chosen as a model of extrahepatic gamma FBG gene expression. A ferret lung cDNA clone bank was constructed in lambda gt11 and several positive plaques were isolated using cross-species hybridization with the rat gamma FBG cDNA. Sequence data of the longest clone, designated pFLG gamma 3, was compared at the nucleotide and deduced amino acid (aa) levels with sequences of gamma FBG from other species. The results indicated that the identity of the ferret lung-specific gamma FBG cDNA to pig, rat, bovine and human gamma FBG cDNAs ranged from 78-88%; the similarity of the ferret lung-specific gamma FBG deduced aa sequence ranged from 84-88% across species. Cysteine aa involved in intra- and inter-chain disulfide-bonded secondary and tertiary structure are absolutely conserved in ferret gamma FBG. The putative cell-cell adhesion sites for both platelet alpha IIb beta 3 and intercellular adhesion molecule-1 receptor binding to ferret gamma FBG are > 90% similar to the corresponding sites in the human gamma FBG. The results of Northern hybridization indicated that the ferret lung gamma FBG mRNA was equivalent in size to the liver gamma FBG mRNA; Southern hybridization suggested that ferret gamma FBG is a single-copy gene, as is the gamma FBG of other species. Lung-specific gamma FBG expression was localized to epithelial cells of the large and small airways and chondrocytes by in situ RNA:RNA hybridization. The functional significance of gamma FBG expression in lung is not presently known. Since expression of FBG is up-regulated 2-10-fold in the liver during an inflammatory event, it is possible that lung-specific gamma FBG expression occurs predominantly during lung disease or injury.

Cloning of the complete coding sequence of rat fibrinogen B beta chain cDNA: interspecies conservation of fibrin beta 15-42 primary structure.

After thrombin cleavage, the newly exposed NH2-termini of the beta chains play a role in both fibrin polymerization and fibrin interactions with cells in the process of wound healing. These physiological responses have been shown to be mediated, at least in part, by beta 15-42. To compare the sequence of the beta chain of fibrin across species, the complete coding sequence of the rat fibrinogen B beta chain cDNA was cloned (designated pRB beta 3) and characterized. The sequence newly determined from pRB beta 3 encompassed nucleotides 121-589, encoding residues 9-165 of the mature polypeptide. Significant homology of pRB beta 3 cDNA and deduced amino acid sequences was found when compared with other species' B beta chains. The rat B beta-Arg14-Gly15 thrombin cleavage site is conserved; however, the fibrinopeptide B sequences are only 50% similar when rat is compared with human. In contrast, the beta 15-42 region is 100% similar when allowing for conservative amino acid substitutions. Monoclonal antibodies (MAbs) specific for human fibrinogen B beta 1-21 (1-8C6) and human fibrin beta 15-21 (59D8 and T2G1) failed to cross-react with rat fibrinogen or fibrin by ELISA, respectively, even though thrombin conversion of rat fibrinogen to fibrin was confirmed. MAb 1-8C6 reacted with reduced and denatured human fibrinogen B beta chain by Western blotting, whereas, MAb T2G1 did not blot with reduced and denatured human fibrin beta chain. A comparative analysis of the binding affinity of the human B beta fibrin(ogen) specific MAbs with B beta fibrin(ogen) from several species suggested that amino acid residues preceding and including Arg14-Gly15 are important in the epitope of the B beta 1-21 specific MAb 1-8C6, and that residues Gly15, Leu19 and/or Lys21 play an important role in the epitope shared by the beta 15-21-specific MAbs T2G1 and 59D8.

Perioperative analgesia with subarachnoid sufentanil administration.

BACKGROUND AND OBJECTIVES: Thirty-seven ASA Physical Status I parturients undergoing elective cesarean delivery were evaluated to determine the effects of subarachnoid sufentanil administration. METHODS: This study was carried out in a randomized, double-blind fashion. All patients received 1.4 ml 0.75% bupivacaine with 8.25% dextrose following 10 micrograms, 15 micrograms, or 20 micrograms sufentanil, or 1 ml of normal saline containing no sufentanil. Onset and duration of sensory and motor anesthesia, duration of complete analgesia, duration of effective analgesia, and intraoperative parenteral opioid requirements were evaluated. Incidence of side effects such as respiratory depression, pruritus, nausea, and vomiting were evaluated. RESULTS: Duration of complete analgesia and duration of effective analgesia were prolonged significantly in all patient groups receiving sufentanil as compared to control groups receiving no narcotic. Pruritus was significantly increased in patient groups receiving subarachnoid sufentanil. Respiratory depression was not observed in any patient studied. One- and five-minute Apgar scores; umbilical, venous, and arterial blood gas results; and Early Neonatal Neurobehavioral Scale results were all within normal limits and were not significantly different among the groups.

Liver-specific RNA processing of the ubiquitously transcribed rat fibrinogen gamma-chain gene.

Fibrinogen gamma-chains differ in amino acid sequence at the carboxyterminus due to alternative 3' RNA processing. Previous studies reported differences between humans and rats in the mechanism of gamma-chain RNA processing and that it was a nonregulated event. To test the hypothesis that rat gamma-chain RNA processing involves both alternative splicing and poly(A) site selection and that it is regulated in a tissue-specific manner, we determined the tissue distribution of gamma-chain mRNA expression and the pattern of gamma-chain pre-mRNA processing. The results of in situ hybridization demonstrated that gamma A and gamma B transcripts were localized to and codistributed in liver hepatocytes, indicating that no subset of cells process gamma B mRNA. The ubiquitous expression of the fibrinogen gamma-chain promoter was demonstrated in marrow, lung, brain, and liver by RNase protection using a 5'-specific gamma-chain probe. RNase protection studies to map 3' RNA processing sites suggested that, in addition to the distal poly(A) signal previously identified, two alternative poly(A) signals within the last intron (ATTAAA and AATAAA) were used only in liver to produce gamma B transcripts. Approximately equal usage of the three poly(A) signals (27%, 37%, and 36%, respectively) to form the 3' end of mature gamma B transcripts suggested that poly(A) site selection is random. These results indicate that splicing of the last intron to produce gamma A mRNA is the ubiquitous and constitutive pattern of gamma-chain RNA processing, while retention of the last intron to produce gamma B mRNAs is the tissue-specific and regulated pattern of gamma-chain RNA processing. The pattern of rat gamma-chain RNA processing is similar to human, implying that the mechanism is conserved. These data support a mechanism of tissue-specific splice site selection predominating over poly(A) site selection in gamma-chain pre-mRNA processing. The expression of both fibrinogen gamma-chain transcripts in liver, rather than mutually exclusive expression in liver and other tissues, provides a new model for studying tissue-specific alternative 3' end formation regulatory mechanisms.

Developmental expression of mRNAs encoding platelet proteins in rat megakaryocytes.

The committed bone marrow megakaryocyte (MK) progenitor undergoes a series of highly regulated stages of development resulting in a large multi-nucleated platelet-producing cell. We studied the developmental expression of the mRNA for two alpha granule proteins, fibronectin (FN) and fibrinogen gamma chain (gamma-FIB), and a cytoskeletal protein, actin, in MKs from marrow of Sprague-Dawley rats. By the method of in situ RNA:RNA hybridization, we showed that mRNAs for the alpha granule proteins were expressed most abundantly in a population of 15-microns diameter promegakaryocytes and in cells as small as 10 microns whose identity as immature MKs was inferred by positive staining for platelet- and MK-specific markers. gamma-FIB and FN mRNAs were present in reduced abundance in a small proportion of intermediate MKs; however, little or no expression was seen in mature platelet-producing MKs. In contrast, high levels of actin mRNA were expressed predominantly in mature, multi-nucleated MKs, and less abundantly in the immature forms. These results suggest that FN and gamma-FIB are transcribed early in MK development to permit translation and packaging of the protein into alpha granules, after which transcription ceases. On the other hand, transcription of actin occurs continuously throughout development, with highest levels in mature platelet-producing MKs, in which actin is needed for shape changes and intracellular movement of organelles. Our data suggest that in situ RNA:RNA hybridization for platelet-specific markers will provide additional criteria by which to establish MK lineage in immature marrow progenitors.

Tissue-specific and ubiquitous expression of fibrinogen gamma-chain mRNA.

Fibrinogen gamma-chains are derived from differential mRNA splicing resulting in polypeptide that differ in their carboxyterminal sequences as well as tissue distribution. We examined the expression of the rat fibrinogen gamma-chain mRNAs to determine the nature of the differential tissue expression of the gamma-fibrinogens. Here we demonstrate that the expression of the rat gamma B mRNA is tissue-specific. Northern blot analysis indicated that gamma A mRNA was expressed in liver, lung, brain and marrow, while the alternatively spliced gamma B mRNA was expressed only in liver. Three distinct full-length species of gamma B mRNA were identified in liver, indicative of multiple sites for polyadenylation that is suggestive of regulation at the level of 3' RNA processing. The restricted expression of gamma B mRNA in liver was contrasted by the ubiquitous expression of the gamma-chain promoter in brain and lung tissues that are not known for production of plasma coagulation proteins. The results of in situ hybridization showed that only gamma A mRNA was found in lung, localized to bronchiolar epithelial cells and chondrocytes but not smooth muscle or endothelial cells. Tissue-specific regulation of the gamma-chain gene results in compartmentalization of gamma B-fibrinogen to the circulation.

Temperature-sensitive mutants in herpes simplex virus type 1 ICP4 permissive for early gene expression.

A large number of temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1) in the gene encoding the immediate-early transcriptional regulatory protein, ICP4, have been isolated and characterized with respect to expression of the immediate-early, early, and late viral gene products. The hallmark of these mutants is the overproduction of immediate-early gene products and the underproduction of early and late gene products. The present study involves the preliminary genetic and molecular characterization of two unique regulatory mutants of HSV-1, ts48 and ts303. Genetically, both mutants exhibit inefficient complementation with eight ts mutants in complementation group 1-2, which defines the gene for ICP4, and marker rescue experiments place the mutations in both mutants in the 3' portion of the coding sequence for ICP4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of ts48- and ts303-infected cell polypeptides synthesized at the nonpermissive temperature demonstrates that immediate-early polypeptides ICP4 and ICP27 are overproduced, with the simultaneous production of early polypeptides ICP6, ICP8, gB, and others. Immediate-early polypeptides are resynthesized upon temperature shift-up early in infection; however, shift-up late in infection does not result in the resynthesis of immediate-early polypeptides. Late gene products are either absent or underrepresented under long-term labeling conditions. To examine the effects of the mutations in ts48, ts303, and other ICP4 mutants specifically on early gene expression, trans-induction experiments were performed in cells transfected with the gene for chloramphenicol acetyltransferase under early gene control (tk) and superinfected with KOS, tsB32, ts48, and ts303. Mutant tsB32 did not induce chloramphenicol acetyltransferase activity above the basal level; however, ts48 and ts303 induced chloramphenicol acetyltransferase activity nearly equal to wild-type levels. Fifteen to fifty percent of wild-type levels of viral DNA are synthesized at the nonpermissive temperature in ts48- and ts303-infected cells, indicating that immediate-early and early gene functions are intact (or nearly so) and that the block in ts48 and ts303 is in a regulatory event subsequent to that exhibited by other mutants in complementation group 1-2 which are DNA-.

R-factor responsible for an outbreak of multiply antibiotic-resistant Klebsiella pneumoniae.

Seven serotypes of Klebsiella pneumoniae isolated from different patients demonstrated resistance to the same eight antibiotics. A plasmid carrying resistance determinants to these antibiotics and mercury salts could be transferred in toto to a plasmidless strain of Escherichia coli. All E. coli transconjugants showed the same antibiotic resistance pattern. Digestion with restriction endonucleases yielded patterns that were identical for each of the R-factor transferred from the multiply resistant serotypes. Moreover, deoxyribonucleic acid-deoxyribonucleic acid hybridization demonstrated identity between the probe, pMAC20 (an R-factor from one serotype), and all R-factors isolated from the multiply resistant strains of K. pneumoniae and the E. coli transconjugants tested.