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Biomedical subjects

J R Klein

Publications and source records attributed to J R Klein.

At least 37 records · Page 2Linked to original sources
PubMed2000

Transcriptional organization and function of invasion genes within Salmonella enterica serovar Typhimurium pathogenicity island 1, including the prgH, prgI, prgJ, prgK, orgA, orgB, and orgC genes.

Salmonella enterica serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of Salmonella into intestinal epithelial cells is dependent upon invasion genes that are clustered together in Salmonella pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the prgH, -I, -J, and -K and orgA genes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published orgA DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designated orgA and orgB. Additionally, an opening reading frame downstream of orgB, which we have designated orgC, has been identified and partially characterized. Previously published work has indicated that the prgH, -I, -J, and -K genes are transcribed from a promoter distinct from that used by the gene immediately downstream, orgA. Here, we present experiments indicating that orgA expression is driven by the prgH promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of prgH to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that the prgH, prgI, prgJ, prgK, orgA, and orgB genes are each required for invasion and secretion, while orgC is not essential for the invasive phenotype.

Bacterial Proteins↗
PubMed1999

Primary structure and features of the genome of the Lactobacillus gasseri temperate bacteriophage (phi)adh.

The complete DNA sequence of the Lactobacillus (Lb.) gasseri temperate phage (phi)adh was determined. The linear and double-stranded genome consists of 43.785bp with a G+C content of 35. 3% and 3' protruding cohesive ends of 12nt. Sixty-two possible ORFs were identified. On the basis of homology comparisons, some of them could be assigned to possible functions, such as a helicase, a nucleic acid polymerase and a protease. In a non-coding area of the (phi)adh genome, structural features of a potential replication origin were detected. After subcloning, this region was functional as a replicon in Lb. gasseri and Lactococcus lactis. N-terminal aa sequencing and electron microscopic analysis of intact and defective phage particles enabled the identification of two capsid protein genes. One of their products, the major head protein, seems to be processed on the posttranslational level.

Amino Acid Sequence↗
PubMed1999

Neuroendocrine-induced synthesis of bone marrow-derived cytokines with inflammatory immunomodulating properties.

Although cytokines and other soluble regulators of immunity are known to be involved in hematopoiesis, little is known about the signals that induce the synthesis of those mediators locally. Based on recent studies linking the neuroendocrine hormone thyrotropin [thyroid-stimulating hormone (TSH)] to immune cell function in other tissues, we investigated the capacity of TSH to activate cytokine responses from bone marrow cells. These studies reveal that stimulation of the TSH receptor on bone marrow cells-using highly purified or recombinant TSH or by direct stimulation with anti-TSH receptor antibodies-rapidly induces the synthesis of cytokines from bone marrow cells that are classically used in the regulation of inflammatory responses. Of 13 cytokines screened for activity by ELISA or by RNase protection assays for gene expression, IL-6, IFN-beta, TNFalpha, TNFbeta, TGFbeta2, and lymphotoxin-beta responses were reproducibly induced by TSH within 2-3 h of stimulation. Intracellularly, TSH stimulation of bone marrow cells caused rapid increases in cAMP levels and induced the phosphorylation of the Jak2 protein kinase, thereby defining a novel G-protein-coupled receptor/cytokine synthesis pathway. These findings demonstrate that TSH can serve as a primary inductive signal of cytokine production by bone marrow cells.

Animals↗
PubMed1999

Differential expression of CD43 isoforms on murine T cells and their relationship to acute intestinal graft versus host disease: studies using enhanced-green fluorescent protein transgenic mice.

Three mAb (R2/60, S7 and 1B11) were used to study the expression of murine CD43 on peripheral T cells and intestinal intraepithelial lymphocytes (IEL) from normal mice, and from mice during acute graft versus host disease (GVHD). In the spleen, essentially all T cells expressed the R2/60 and S7 antigens, whereas the 1B11 antigen was expressed on about half of the CD8(+) cells and approximately 15% of CD4(+) T cells. Interestingly, a significant proportion of resting splenic B cells expressed the 1B11 and R2/60 antigens, but not the S7 antigen. The majority of IEL expressed R2/60 antigen; however, the S7 and 1B11 markers were differentially expressed on CD8alpha, CD8beta, TCRalphabeta and TCRgammadelta cells. Immunoprecipitation and Western blotting analyses identified characteristic 115 and 130 kDa reactive components from IEL lysates with mAb S7 and 1B11 respectively, and reactivity to both molecular entities by mAb R2/60. During acute intestinal GVHD induced by injecting CB6F(1) athymic nude mice with spleen cells from C57BL/6 enhanced-green fluorescent protein transgenic mice, 80-90% of donor T cells in the intestine epithelium expressed all CD43 isoforms; however, the level of expression of the 130 kDa CD43 antigen increased significantly and the level of the 115 kDa antigen decreased on GVHD donor T cells compared to cells at the time of transfer. Using EL4 cells, a similar shift in the expression of CD43 isoforms occurred experimentally following treatment with neuraminidase, suggesting that the type of CD43 isoform expressed on T cells is strongly influenced by conditions which affect membrane charge. The significance of these findings for intestinal immunopathology is discussed.

Animals↗
PubMed1999

Introduction of peptidase genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and controlled expression.

Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene (P(nisA)). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of P(nisA)::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools.

Aminopeptidases↗
PubMed1999

Synthesis and automated detection of fluorescently labeled primer extension products.

Common methods for determination of transcription initiation sites are based on the extension of radioactively labeled primers or internal labeling of extension products with reverse transcriptase. Here, we describe a modification of this procedure, adapted for automated detection of extension products by using fluorescent primers and an optimized reaction protocol. This facilitates high-throughput screening to detect mRNA start-points of large DNA regions and significantly reduces the time required to determine sites of transcription initiation.

Bacteriophages↗
PubMed1999

Simultaneous anterior and posterior approaches to the spine for revision surgery: current indications and techniques.

Revision spinal surgery often requires attention to both the anterior and the posterior portions of the spine. Staged, sequential, and more recently simultaneous anterior and posterior approaches have been proposed. A simultaneous approach has the distinct advantage of allowing complete and constant control of the anterior and posterior portions of the spine during surgery. The simultaneous approach has been shown to offer decreased operating time, blood loss, complication rate, and hospital length of stay as compared with staged procedures. The evolution in spinal instrumentation and ancillary equipment has greatly advanced the simultaneous technique. The development of a special operating table has facilitated patient positioning and intraoperative patient adjustments, optimizing operative exposure for the anterior and posterior surgical teams. The two-rod and four-rod techniques offer the surgeon the possibility to safely address complex deformities, particularly in kyphosis.

Female↗
PubMed1998

Hormone regulation of immune homeostasis: local or long distance?

The endocrine system and the immune system consist of dynamic biological processes involved on the one hand in the regulation of a complex array of metabolic and physiologic activities, and on the other hand in protection against infection and disease. Evidence for bidirectional functional involvement of immune-endocrine interactions can be seen at many levels, including codependence during critical stages of development, the complementary use of shared molecular mediators and receptors by both systems, and the integrated participation of the immune system and the endocrine system in resistance or susceptibility to disease. Moreover, recent findings-principally derived from studies of intestinal intraepithelial lymphocytes in mice--suggest that immune-endocrine interactions are essential for the proper development of intestinal T cells, and indicate that this most likely occurs via a local network of hormone synthesis and utilization. In the present article, these findings will be discussed in the context of immune-endocrine collaboration, with particular attention given to the involvement of the thymus in this process, and a hypothesis will be proposed which suggests that the homeostatic balance between health and disease is largely driven by local rather than systemic hormonal regulatory events.

Animals↗
PubMed1998

The arbZ gene from Lactobacillus delbrueckii subsp. lactis confers to Escherichia coli the ability to utilize the beta-glucoside arbutin.

From a genomic library of the industrially used strain Lactobacillus delbrueckii subsp. lactis DSM7290, a gene designated arbZ (869bp; encoding a 33.5kDa protein) was isolated by screening E. coli transformants for the ability to utilize the beta-glucoside arbutin. Out of 9000 transformants nine were able to ferment arbutin, whereas no utilization of the beta-glucosides salicin, esculin or cellobiose could be detected. Overexpression of arbZ using the T7-polymerase-T7-promoter-system resulted in the formation of insoluble, catalytically inactive protein aggregates (inclusion bodies). Accordingly, overexpression was not accompanied by an increase in ArbZ activity. Induction of arbZ controlled by the lac promoter under conditions that reduce protein aggregation resulted in a 12-fold increase in arbutin hydrolyzing activity of intact cells and a 13-fold increase in phospho-beta-glycosidase activity in cell-free extracts of the respective transformants. Nucleotide sequence analysis revealed a second gene upstream of arbZ that was designated arbX (830bp). ArbX (32.6kDa) shared similarity with several glycosyltransferases involved in the biosynthesis of lipopolysaccharides in Gram-negative bacteria. In Lb. delbrueckii subsp. lactis DSM7290 two transcripts, one covering arbX together with arbZ and one covering arbZ alone were detected by Northern blot analysis.

Amino Acid Sequence↗
PubMed1998

Structure of a genome region of the Lactobacillus gasseri temperate phage phiadh covering a repressor gene and cognate promoters.

By sequencing the DNA regions which flank the intG gene encoding integrase of the temperate Lactobacillus (Lb.) gasseri bacteriophage phiadh, a continuous sequence of 6590 bp was established. It encompasses five newly identified ORFs, of which four are located upstream, and one (orfC) downstream of intG. Proteins corresponding to the expected products of the intG upstream coding regions, orfA (33 kDa), orf2 (14 kDa), rad (12.1 kDa), and tec (7.9 kDa), were identified by in vitro expression of subcloned DNA fragments. Rad shares homology with transcription regulators, including SinR of Bacillus species and the repressor of phage phi105. The gporf2 is similar to predicted products of topologically equivalent coding regions of the Lactococcus lactis phage TP901-1 and the B. subtilis phage phi105. Promoters for the divergently oriented rad and tec genes were mapped within the 435-bp region between them and specify overlapping transcripts with extended 5'-untranslated sequences. As shown with lacZ fusions, Rad repressed transcription from the tec and rad promoters 20- and 5-fold, respectively. In Lb. gasseri, weak expression of cloned rad ws sufficient to mediate immunity towards phiadh.

Amino Acid Sequence↗
PubMed1998

Dynamic regulation of gastric autoimmunity by thyroid hormone.

Neonatal thymectomy (NTX) of BALB/c mice between days 1 and 3 post-birth leads to a high incidence of autoimmune gastritis involving T cells and autoantibodies. Although progress has been made in understanding various immunological events associated with that disease process, much remains to be learned about the regulatory factors which control autoimmune gastritis. In this study we have examined the potential for neuroendocrine hormones of the hypothalamus-pituitary-thyroid (HPT) axis to alter the outcome of experimental autoimmune gastritis in NTX mice. As reported here, thyroxine administered to young adult NTX mice during an active phase of disease development dramatically reduced the incidence of gastritis and the overall severity of disease, and lowered the levels of anti-parietal cell antibodies. In contrast, treatment of young NTX mice with thyroxine or other HPT hormones prior to the onset of disease had no beneficial effect on gastritis, but instead resulted in significantly higher anti-parietal cell antibody levels compared to non-hormone-treated NTX mice or to NTX mice treated as adults. Reverse transcriptase spectratype analyses of 22 Vbeta gene junctional sites revealed pronounced oligoclonality and limited junctional diversity in stomach lymphocytes from untreated NTX mice compared to normal mice or thyroxine-treated NTX mice. These findings identify a set of dynamic immune-endocrine interactions, linked to critical development-dependent events, that are involved in the expression and regulation of peripheral autoimmunity.

Age Factors↗
PubMed1998

The biomechanics of the patellofemoral joint.

Physical therapy for patella malalignment differs from therapy for other knee conditions. In fact, accepted forms of knee therapy can be counterproductive when one is dealing with patella malalignment. This article reviews some of the biomechanical foundations of patella-specific therapy and addresses common controversies. We emphasize the concept of articular cartilage stress over that of joint reaction force. The rationale for strengthening the vastus medialis obliquus and for avoiding certain forms of open chain strengthening are discussed, and we outline some of the exciting work being done in our laboratory concerning patellofemoral tracking, contact area, and cartilage properties.

Biomechanical Phenomena↗
PubMed1997

Hemodynamic effects of chronic prenatal ventricular pacing for the treatment of complete atrioventricular block.

BACKGROUND: Increasing the heart rate of the fetus with cardiac failure caused by complete AV block (CAVB) may allow delivery of a full-term, stable neonate with preserved ventricular function. Direct fetal pacing may be a feasible method to achieve this, but the effect of pacing on the structure and function of the rapidly developing fetal heart is unknown. METHODS AND RESULTS: CAVB was created in fetal lambs at 80% gestation by cryoablating the AV node. Epicardial ventricular pacing at 130 bpm was achieved by use of a pacemaker placed under the pectoral muscles. The fetus was returned to the uterus and allowed to continue to term. Ventricular function was assessed 1 week after birth in 7 lambs with CAVB and 10 control lambs. By use of the conductance catheter technique, the end-systolic pressure-volume relationship was determined at different heart rates, pacing conditions, and inotropic states. The contractility was not different between the two groups at their baseline heart rates and rhythms or when they were paced synchronously compared with asynchronously. Also, both groups responded significantly and similarly to inotropic manipulation, indicating preserved contractile reserve. Finally, in both groups, increased heart rates were associated with increased contractility, indicating an intact force-frequency relationship. CONCLUSIONS: We conclude that chronic epicardial ventricular pacing is well tolerated by the fetus, can be successfully applied as a treatment for CAVB, and does not adversely affect myocardial function in the rapidly developing, immature heart.

Animals↗
PubMed1997

Local hormone networks and intestinal T cell homeostasis.

Neuroendocrine hormones of the hypothalamus-pituitary-thyroid axis can exert positive or negative immunoregulatory effects on intestinal lymphocytes. Small intestine epithelial cells were found to express receptors for thyrotropin-releasing hormone (TRH) and to be a primary source of intestine-derived thyroid-stimulating hormone (TSH). The gene for the TSH receptor (TSH-R) was expressed in intestinal T cells but not in epithelial cells, which suggested a hormone-mediated link between lymphoid and nonhematopoietic components of the intestine. Because mice with congenitally mutant TSH-R (hyt/hyt mice) have a selectively impaired intestinal T cell repertoire, TSH may be a key immunoregulatory mediator in the intestine.

Animals↗
PubMed1996

Characterization of pepR1, a gene coding for a potential transcriptional regulator of Lactobacillus delbrueckii subsp. lactis DSM7290.

The gene designated pepR1, encoding a potential transcription regulator of Lactobacillus delbrueckii subsp. lactis DSM7290, was identified by sequence similarity of an open reading frame located upstream of the prolidase pepQ orientated in opposite direction. pepQ and pepR1 coding regions are spaced by 152 nucleotides. Upstream of the -35 region of pepQ, a 14-bp palindromic sequence, homologous to the catabolite responsive element, could be identified. The pepRl gene has the potential to encode a protein of 333 amino acids with a calculated molecular mass of 36955 Da and a calculated pl of 5.5. The deduced protein sequence shows significant identity to the catabolite control protein of Bacillus. Co-expression in Escherichia coli was studied with the pepR1-pepQ intergenic region fused to the promoterless beta-galactosidase reporter gene. The pepQ-beta-galactosidase hybrid displayed an enhanced expression in the presence of cloned pepR1.

Amino Acid Sequence↗
PubMed1996

Fetal model of single ventricle physiology: hemodynamic effects of oxygen, nitric oxide, carbon dioxide, and hypoxia in the early postnatal period.

BACKGROUND: In patients with single ventricle physiology, the distribution of flow to the systemic and pulmonary circulations, which are in parallel, largely depends on the relative resistances in the respective vascular beds. Although neonatal palliation in patients with single ventricle physiology has become more common, medical management during the perinatal and perioperative periods is often based primarily on personal and institutional experience and is complicated by the transition to postnatal life and the effects of cardiopulmonary bypass. The lack of an animal model that suitably mimics single ventricle physiology has impeded progress in this area. OBJECTIVE: The purpose of the current study was to investigate the effects of respiratory manipulations on pulmonary hemodynamics in the early neonatal period in a reproducible model of single ventricle physiology created in utero. METHODS: A 10 mm Damus-Kaye-Stansel aortopulmonary anastomosis was created in fetal sheep (n = 14) at 140 +/- 1.2 days' gestation, with pulmonary blood flow provided through a 5 mm aortopulmonary shunt after ligation of the main pulmonary artery distally. Two to 3 days after delivery at term, lambs (n = 11) underwent an open sternotomy and 30 minutes of deep hypothermic circulatory arrest. Both before and after bypass, respiratory manipulations, including administration of nitric oxide (80 ppm), 100% oxygen, 10% oxygen, and 5% carbon dioxide, were performed and hemodynamic variables were measured. RESULTS: Nitric oxide and oxygen caused a decrease in pulmonary vascular resistance and an increase in the pulmonary/systemic blood flow ratio, both before and after bypass. Hypoxia and carbon dioxide produced a significant rise in pulmonary vascular resistance and a significant drop in the ratio of pulmonary to systemic blood flow. CONCLUSIONS: Oxygen, nitric oxide, and carbon dioxide all appear to be useful means of manipulating pulmonary vascular resistance and pulmonary/systemic blood flow ratio in neonatal lambs with single ventricle physiology, but further investigation is necessary to understand their dose responsive effects and the effects of prolonged administration.

Animals↗