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Biomedical subjects

B Stein

Publications and source records attributed to B Stein.

At least 91 records · Page 5Linked to original sources
PubMed1994

The bZIP transactivator of Epstein-Barr virus, BZLF1, functionally and physically interacts with the p65 subunit of NF-kappa B.

The Epstein-Barr virus (EBV) BZLF1 (Z) immediate-early transactivator initiates the switch between latent and productive infection in B cells. The Z protein, which has homology to the basic leucine zipper protein c-Fos, transactivates the promoters of several replicative cycle proteins. Transactivation efficiency of the EBV BMRF1 promoter by Z is cell type dependent. In B cells, in which EBV typically exists in a latent form, Z activates the BMRF1 promoter inefficiently. We have discovered that the p65 component of the cellular factor NF-kappa B inhibits transactivation of several EBV promoters by Z. Furthermore, the inhibitor of NF-kappa B, I kappa B alpha, can augment Z-induced transactivation in the B-cell line Raji. Using glutathione S-transferase fusion proteins and coimmunoprecipitation studies, we demonstrate a direct interaction between Z and p65. This physical interaction, which requires the dimerization domain of Z and the Rel homology domain of p65, can be demonstrated both in vitro and in vivo. Inhibition of Z transactivation function by NF-kappa B p65, or possibly by other Rel family proteins, may contribute to the inefficiency of Z transactivator function in B cells and may be a mechanism of maintaining B-cell-specific viral latency.

Cell Line↗
PubMed1994

Function of NF-kappa B/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different NF-kappa B/Rel subunits.

The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-kappa B/Rel binding sites located at -109 to -118 (Ii kappa B-1) and -163 to -172 (Ii kappa B-2) from the transcription start site. We report here that the differential function of each of these NF-kappa B/Rel sites in several distinct cell types depends on cell-specific binding of NF-kappa B/Rel transcription factors. Ii kappa B-1 is a positive regulatory element in B-cell lines and in the Ii-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glia cell lines. In vivo protein-DNA contacts are detectable at Ii kappa B-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-kappa B/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii kappa B-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again, in vitro supershift studies indicate that the presence of p50, p52, p65, and cRel correlates with positive function whereas the presence of only p50 and p52 correlates with negative function. This differential binding of specific NF-kappa B/Rel subunits is likely to mediate the disparate functions of these two NF-kappa B/Rel binding sites.

B-Lymphocytes↗
PubMed1994

Organelle sedimentation in gravitropic roots of Limnobium is restricted to the elongation zone.

Roots of the aquatic angiosperm Limnobium spongia (Bosc) Steud. were evaluated by light and electron microscopy to determine the distribution of organelle sedimentation towards gravity. Roots of Limnobium are strongly gravitropic. The rootcap consists of only two layers of cells. Although small amyloplasts are present in the central cap cells, no sedimentation of any organelle, including amyloplasts, was found. In contrast, both amyloplasts and nuclei sediment consistently and completely in cells of the elongation zone. Sedimentation occurs in one cell layer of the cortex just outside the endodermis. Sedimentation of both amyloplasts and nuclei begins in cells that are in their initial stages of elongation and persists at least to the level of the root where root hairs emerge. This is the first modern report of the presence of sedimentation away from, but not in, the rootcap. It shows that sedimentation in the rootcap is not necessary for gravitropic sensing in at least one angiosperm. If amyloplast sedimentation is responsible for gravitropic sensing, then the site of sensing in Limnobium roots is the elongation zone and not the rootcap. These data do not necessarily conflict with the hypothesis that sensing occurs in the cap in other roots, since Limnobium roots are exceptional in rootcap origin and structure, as well as in the distribution of organelle sedimentation. Similarly, if nuclear sedimentation is involved in gravitropic sensing, then nuclear mass would function in addition to, not instead of, that of amyloplasts.

Cell Nucleus↗
PubMed1994

Incontinence: defining the problem.

As this issue of Rhode Island Medicine demonstrates, incontinence is felt within all segments of society. No practitioners are insulated from patients suffering from this condition. Often, the suffering is silent for many reasons. Patients may have been conditioned to believe that theirs is a situation that is best left alone, and one that they should accept. If they overcome this hurdle, then they must be willing to address the problem with their physicians. This is obviously assisted if the subject matter is broached by the caregiver rather than relying on the patient to come forth regarding a socially compromising matter. Therefore, physicians need to be aware of the problems of incontinence, familiar with the various modes of therapy, and willing to take on these issues with their patients with compassion and understanding. In this way can we address the full needs of our patients regarding a problem long ignored but easily diagnosed and treatable.

Adolescent↗
PubMed1993

Interleukin 1 induction of the c-jun promoter.

Interleukin 1 (IL-1) induces pleiotropic effects in many cell types during inflammation and immunity. We have recently shown how the IL-1 signal is transmitted to the nucleus: In T cells and in pituitary cells, IL-1 induced genes via activation of the nuclear factor AP-1. We now demonstrate how IL-1 activates the AP-1 factor in liver cells, which are a major target for IL-1 during the acute phase response in vivo. IL-1 induced gene transcription of both AP-1 components, c-jun and c-fos. IL-1 also increased the stability of c-jun mRNA. We define two enhancer sites in the jun promoter that are required for induction by IL-1. Although the binding sites share some similarity with the AP-1 binding site, the nuclear factors binding the jun motifs are not composed of Jun or Fos proteins. Thus these data identify two binding proteins that serve as one of the first nuclear targets for IL-1 signal transduction.

Base Sequence↗
PubMed1993

Cross-coupling of the NF-kappa B p65 and Fos/Jun transcription factors produces potentiated biological function.

NF-kappa B and AP-1 represent distinct mammalian transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is typically composed of two DNA binding subunits, NF-kappa B p50 and NF-kappa B p65, which share structural homology with the c-rel proto-oncogene product. Similarly, the AP-1 transcription factor complex is comprised of dimers of the c-fos and c-jun proto-oncogene products or of closely related proteins. We now demonstrate that the bZIP regions of c-Fos and c-Jun are capable of physically interacting with NF-kappa B p65 through the Rel homology domain. This complex of NF-kappa B p65 and Jun or Fos exhibits enhanced DNA binding and biological function via both the kappa B and AP-1 response elements including synergistic activation of the 5' long terminal repeat of the human immunodeficiency virus type 1. These findings support a combinatorial mechanism of gene regulation involving the unexpected cross-coupling of two different classes of transcription factors to form novel protein complexes exhibiting potentiated biological activity.

3T3 Cells↗
PubMed1993

Pertussis toxin unmasks stimulatory myocardial A2-adenosine receptors on ventricular cardiomyocytes.

Pertussis toxin-pretreatment abolished the contractility- and cAMP-decreasing effects of the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (R-PIA) in the presence of isoprenaline in isolated ventricular cardiomyocytes from guinea-pigs, indicating that these stimulatory effects of A1-adenosine receptors are mediated via pertussis toxin-sensitive G-proteins. Furthermore, the decrease in contractile response by the A1/A2-adenosine receptor agonist 5'-N-ethylcarboxamidadenosine (NECA) was abolished. Moreover, NECA increased cAMP content in pertussis toxin-pretreated cells. Thus, pertussis toxin unmasked cAMP-augmenting effects of NECA, indicating that NECA can stimulate A2-adenosine receptors on cardiomyocytes. Thereby, the present study provides evidence that besides cAMP- and contractility-decreasing A1-adenosine receptors, cAMP-increasing A2-adenosine receptors coexist on ventricular cardiomyocytes, which do not influence contractile response.

Adenosine↗
PubMed1993

Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant.

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the caMV-35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes. Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing beta-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.

Base Sequence↗
PubMed1993

Evidence for possible involvement of an elastolytic serine protease in aspergillosis.

A number of isolates of Aspergillus fumigatus obtained from the hospital environment produced extracellular elastolytic activity. This activity was found to be catalyzed by a single 33-kDa protein which was purified and characterized to be a serine protease. A. fumigatus, when grown on the insoluble structural material obtained from murine and bovine lung, produced the same extracellular 33-kDa elastolytic protease, indicating that this enzyme is likely to be produced when the organism infects the lung. Polymerase chain reaction with an oligonucleotide primer based on the N-terminal amino acid sequence of the elastolytic enzyme yielded a cDNA which was cloned and sequenced. The active serine motif showed more similarity to subtilisin than to mammalian elastase. The amino acid sequence showed 80% identity to the alkaline protease from Aspergillus oryzae. Screening of hospital isolates of Aspergillus flavus showed great variation in the production of elastolytic activity and a much lower level of activity than that produced by A. fumigatus. The elastolytic protease from A. flavus was shown to be a serine protease susceptible to modification and inactivation by active serine and histidine-directed reagents. This protease cross-reacted with the antibodies prepared against the elastolytic protease from A. fumigatus. Immunogold localization of the elastolytic enzyme showed that A. fumigatus germinating and penetrating into the lungs of neutropenic mice secreted the elastolytic protease. An elastase-deficient mutant generated from a highly virulent isolate of A. fumigatus caused drastically reduced mortality when nasally introduced into the lung of neutropenic mice. All of the evidence suggests that extracellular elastolytic protease is a significant virulence factor in invasive aspergillosis.

Amino Acid Sequence↗
PubMed1993

Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B.

The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65, p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter. Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B. Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.

Animals↗
PubMed1993

Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction.

NF-kappa B and C/EBP represent distinct families of transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is composed of two subunits, a 50- and a 65-kDa protein. All members of the NF-kappa B family, including the product of the proto-oncogene c-rel, are characterized by their highly homologous approximately 300-amino-acid N-terminal region. This Rel homology domain mediates DNA binding, dimerization, and nuclear targeting of these proteins. C/EBP contains the bZIP region, which is characterized by two motifs in the C-terminal half of the protein: a basic region involved in DNA binding and a leucine zipper motif involved in dimerization. The C/EBP family consist of several related proteins, C/EBP alpha, C/EBP beta, C/EBP gamma, and C/EBP delta, that form homodimers and that form heterodimers with each other. We now demonstrated the unexpected cross-coupling of members of the NF-kappa B family three members of the C/EBP family. NF-kappa B p65, p50, and Rel functionally synergize with C/EBP alpha, C/EBP beta, and C/EBP delta. This cross-coupling results in the inhibition of promoters with kappa B enhancer motifs and in the synergistic stimulation of promoters with C/EBP binding sites. These studies demonstrate that NF-kappa B augments gene expression mediated by a multimerized c-fos serum response element in the presence of C/EBP. We show a direct physical association of the bZIP region of C/EBP with the Rel homology domain of NF-kappa B. The cross-coupling of NF-kappa B with C/EBP highlights a mechanism of gene regulation involving an interaction between distinct transcription factor families.

Amino Acid Sequence↗
PubMed1993

Ca(++)-dependent constitutive nitric oxide synthase is not involved in the cyclic GMP-increasing effects of carbachol in ventricular cardiomyocytes.

We investigated the effects of the M-cholinoceptor agonist carbachol on cyclic GMP (cGMP) content and contractile response in the absence and presence of the nitric oxide synthase inhibitor NG-nitro-L-arginine in guinea-pig isolated ventricular cardiomyocytes. Carbachol (10 mumol/l, 10 min) increased basal cGMP content to approximately 200% and contractile response to 118%. Preincubation of the cardiomyocytes with NG-nitro-L-arginine (0.1 mumol/l, 60 min) did not alter the effects of carbachol on neither cGMP content or contractile response. Moreover, nitric oxide synthase activity was undetectable in crude or ADP-agarose purified cytosolic and particulate fractions of homogenized isolated ventricular cardiomyocytes. Pretreatment with pertussis toxin did not affect the carbachol-mediated increase in cGMP content or contractile response. However, methylene blue abolished the elevation in cGMP content by carbachol, without changing contractile response. It is concluded that the carbachol-mediated increase in cGMP content and contractile response in ventricular cardiomyocytes is neither mediated via a nitric oxidebiosynthesis pathway nor via a pertussis toxin-sensitive GTP-binding protein. Furthermore, the cGMP increase by carbachol is due to an activation of soluble guanylyl cyclase and is dissociated from the contractile response. We therefore assume that carbachol activates two independent effector cascades, one leading to an elevation in cGMP content and the other to an increase in contractile response and that none of the effects are mediated via endogenous nitric oxide formation.

Amino Acid Oxidoreductases↗
PubMed1992

The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression.

Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-kappa B. Functional analyses of chimeric proteins composed of NF-kappa B p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-kappa B p65 comprise a potent transcriptional activation domain. Transient transfection of human T cells with an expression vector encoding NF-kappa B p65, but not NF-kappa B p50, produced marked transcriptional activation of a basal promoter containing duplicated kappa B enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus. These stimulatory effects of NF-kappa B p65 were synergistically enhanced by coexpression of NF-kappa B p50 but were completely inhibited by coexpression of the v-rel oncogene product. Together, these functional studies demonstrate that NF-kappa B p65 is a transactivating subunit of the heterodimeric NF-kappa B complex and serves as one cellular target for v-Rel-mediated transcriptional repression.

Amino Acid Sequence↗
PubMed1992

Prostaglandin E2 alleviates cyclosporin A-induced bone loss in the rat.

Cyclosporine A (CsA) administered to the male and female rat produces high-turnover osteopenia. Prostaglandins have both bone-resorbing and bone-forming properties, but administration of prostaglandin E2 (PGE2) to the rat in vivo produces a net increase in cancellous bone. To investigate the effects of PGE2 on CsA-induced alteration in bone mass, 43 male Sprague-Dawley rats (9 weeks old) were administered 15 mg/kg of CsA by oral gavage and/or 6 mg/kg of PGE2 by subcutaneous injection daily for 21 days according to the following protocol: group A was an age-matched control; group B received CsA only; group C received PGE2 only; and group D received CsA and PGE2. Serum was assayed on days 0, 7, 14, and 21 for bone gla protein (BGP), PTH, and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. A computerized image analysis system was used for bone histomorphometry of the proximal tibial metaphysis after double tetracycline labeling. Compared to control animals (group A), treatment with CsA alone (group B) and PGE2 alone (group C) significantly elevated BGP levels. Combination therapy (group D) resulted in BGP levels that were significantly higher on days 7 and 14 than with either agent alone. 1,25-(OH)2D was significantly elevated in the CsA group only (group B). Therapy with CsA alone (group B) resulted in a significant osteopenia. The concurrent administration of PGE2 with CsA (group D) alleviated the altered bone mass induced by CsA alone by adding a significant amount of additional bone. This report confirms and extends the current knowledge of the different effects of CsA and PGE2 on bone mineral metabolism and demonstrates that PGE2 can alleviate the deleterious effects of CsA on bone.

Administration, Oral↗
PubMed1992

The effect of a new vitamin D analog, 22-oxa-1 alpha,25(OH)2D3, on bone mineral metabolism in normal male rats.

The in vivo effects of 22-oxa-1 alpha,25 dihydroxyvitamin D3 (OCT), on bone mineral metabolism were investigated in normal male Sprague-Dawley rats. The rats were administered either vehicle (control), low-dose OCT (25 ng/100 g body weight), or high-dose OCT (250 ng/100 g body wt). High-dose OCT increased serum ionized calcium (P less than 0.05) and decreased serum parathyroid hormone (PTH) (P less than 0.05) at all time points and increased serum bone Gla protein on days 7 and 28 (P less than 0.05) compared with controls. Low-dose OCT decreased serum PTH at all the time points (P less than 0.05) compared with controls. Tibial bone histomorphometry showed no significant differences between the two doses of OCT and controls. We found that OCT has minimal direct effects on bone metabolism in normal male rats in contrast to 1,25 dihydroxyvitamin D3. This property may be advantageous in the treatment with OCT of cell-proliferative diseases.

Analysis of Variance↗
PubMed1992

Surgical considerations in bypassing coronary arteries with 100% proximal occlusion.

Objective data on the ability of cineangiography to predict the size of reconstituted totally occluded coronary arteries, as well as the clinical outcome of such revascularization, are sparse. Accordingly, we reviewed 200 consecutive cases of coronary revascularization to determine the answers to these questions. Group I patients (n = 57, with 86 totally occluded coronary arteries) had at least one coronary artery with a 100% proximal occlusion that reconstituted distally. Group II patients (n = 143, with 205 subtotally occluded coronary arteries) had 50% to 99% proximal stenosis of at least one coronary artery. Cineangiograms were blindly reviewed to measure the size of the coronary arteries, which were compared with the actual vessel size at operation. In group I, the totally occluded coronary arteries had a cineangiographic size of 1.9 +/- 0.7 mm and an actual size of 1.6 +/- 0.4 mm (p = 0.00004). In group II, the subtotally occluded coronary arteries had a cineangiographic size of 1.8 +/- 0.4 mm compared with an actual size of 1.8 +/- 0.3 mm (p = not significant). The site of bypass grafting was significantly smaller in group I (1.6 +/- 0.4 mm versus 1.8 +/- 0.3 mm; p = 0.00008). The two groups were similar with respect to preoperative and intraoperative parameters. Operative mortalities were similar (group I, 1.8%; group II, 3.5%; p = 0.68). Creatine kinase isoenzyme profiles and electrocardiographic changes were similar, except for a significant late rise of creatine kinase-MB in group I (56.1 +/- 14.7 versus 30.7 +/- 33.7 MIU/mL; p < 0.001). In conclusion, cineangiography significantly overestimates the size of totally occluded coronary arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗