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PubMed1994

Constitutive and regulated prolactin secretion: effects of estradiol.

We have utilized a combined reverse hemolytic plaque/in situ hybridization assay to assess the relationship between individual cell secretion of prolactin and the level of prolactin gene expression within the same cell. It is thought that cells utilizing predominantly a constitutive pathway of protein secretion will exhibit a direct relationship between the level of secretion and that of gene expression. In contrast, cells that are secreting protein predominantly from stored pools exhibit no relationship between these two parameters since secretion does not depend upon synthesis of new hormone. In addition, regulated protein secretion depends upon calcium; therefore, regulated secretion is inhibited by calcium channel blockers such as nifedipine. We tested whether the presence of estradiol influences the proportion of hormone secreted by the constitutive and regulated pathway by 1) assessing whether estradiol changes the relationship between gene expression and secretion in individual cells and 2) measuring the effects of nifedipine on prolactin secretion. Our data demonstrate that estradiol priming results in a direct relationship between the amount of hormone secreted by an individual lactotroph and the level of prolactin mRNA in the same cell; the findings also show that prolactin secretion from lactotrophs of estradiol-treated rats is not suppressed by nifedipine. Therefore, we conclude that estradiol enhances the proportion of hormone that is secreted via a constitutive pathway.

Animals↗
PubMed2001

Sepsis is associated with reciprocal expressional modifications of constitutive nitric oxide synthase (NOS) in human skeletal muscle: down-regulation of NOS1 and up-regulation of NOS3.

OBJECTIVE: To study the expression (mRNA and protein) and activity of the constitutive isoforms of nitric oxide synthase (NOS1 and NOS3) in a skeletal muscle of septic patients. DESIGN: Prospective study. SETTING: An adult trauma/surgical intensive care unit in an urban teaching hospital. PATIENTS: Sixteen septic patients and 21 controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Samples of the rectus abdominis muscle were obtained during surgical procedure. NOS mRNA, protein, and activity were detected by reverse-transcriptase polymerase chain reaction, Western blot, and the conversion of [3H]L-arginine to [3H]L-citrulline, respectively. The main results of this study are as follows: a) Levels of NOS1 mRNA and protein were significantly higher than those of NOS3 in the rectus abdominis muscle of control patients; b) NOS1 expression was down-regulated in septic patients, whereas NOS3 was up-regulated; c) these modulations were associated with a reduction in constitutive NOS activity; and d) modifications of NOS1 and NOS3 protein expression were correlated significantly with the severity of sepsis, assessed by the Simplified Acute Physiology Score II. CONCLUSIONS: Sepsis induces reciprocal expressional modifications of NOS1 and NOS3 in human skeletal muscle, which decreases muscular constitutive NOS activity. These modifications may have implications for muscle impairment in septic patients.

Caenorhabditis elegans Proteins↗
PubMed1989

Circulatory changes in muscle and skin arteries in orthostatic hypotension and constitutional hypotension.

It is not known whether there are vascular changes in the peripheral circulation of patients with orthostatic hypotension and patients with constitutional hypotension. Therefore, blood flow was studied simultaneously at the calf (mainly muscle circulation) and at the finger (mainly finger circulation) with ECG-triggered venous occlusion plethysmography. Calf and finger blood flow were measured for 20 min at rest and during reactive hyperaemia due to an arterial occlusion for 3 min. Vascular resistance was calculated from the blood flow and arterial blood pressure. In orthostatic hypotension there is a decreased vasodilator capacity of the calf resistance vessels and not of the finger resistance vessels. In constitutional hypotension there is a decreased vasodilator capacity of the calf and finger resistance vessels. These results suggest that there are structural or functional changes, or both, in the muscle and skin circulation of patients with constitutional hypotension, whereas there are only structural or functional changes, or both, in the muscle circulation of patients with orthostatic hypotension.

Adult↗
PubMed1993

The constitutive exocytotic pathway in microvillous atrophy.

Microvillous atrophy is a disorder within the intractable diarrhea of infancy syndrome. The disease is believed to stem from a transport defect that prevents exocytosis of brush border-related material. We investigated this hypothesis by examining the direct constitutive exocytotic pathway using sucrase-isomaltase as a representative protein. We also studied various other brush border and lysosomal marker enzymes. The biosynthesis and localization of selected intestinal epithelial enzymes were studied in small-intestinal mucosal biopsy specimens from a total of nine children with microvillous atrophy by: (a) metabolic labeling in organ culture, (b) radioiodination and immunoprecipitation, (c) indirect immunoperoxidase immunocytochemistry, and (d) immunogold electron microscopy. The results demonstrated that brush border enzymes were synthesized normally and could be located in the apical brush border membrane and on microvillous membrane within microvillous inclusions. Brush border enzymes were not detected in the "secretory granules" that accumulated within the apical cytoplasm of epithelial cells. Lysosomal enzymes were only detected within lysosomal bodies. Thus, the direct constitutive pathway is not involved in microvillous atrophy, and a disturbance of endocytosis or the indirect constitutive pathway is unlikely. Any transport defect in the disease probably involves a different, unidentified exocytotic pathway.

Atrophy↗
PubMed2002

Constitutive activation of Jak/STAT proteins in Epstein-Barr virus-infected B-cell lines from patients with posttransplant lymphoproliferative disorder.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a major complication after bone marrow and solid organ transplantation. The disease encompasses a spectrum of abnormal, Epstein-Barr virus (EBV)-associated B-cell proliferations. We have previously shown that EBV-infected, spontaneous lymphoblastoid cell lines (SLCL) derived from PTLD patients require autocrine interleukin (IL)-10 to proliferate. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of the Jak/STAT signaling pathway proteins were analyzed in three different SLCL, termed JB7, MF4, and VB4. METHODS: The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblot. Activated STAT dimer formation was determined by electromobility shift assays. RESULTS: All three SLCL, but not the Daudi Burkitt's lymphoma B-cell line, expressed the four known Jak kinases constitutively tyrosine phosphorylated, with particularly high levels of P-tyr Jak1 in the JB7 line. STAT1 and STAT3, but not STAT2 or STAT5, are also constitutively activated in all SLCL. The ability of the activated STAT proteins to form DNA-binding dimers was confirmed by electromobility shift assay. The SLCL, but not the Daudi line, express activated STAT complexes composed of STAT1 and STAT3. Another EBV-infected B-cell line, isolated from a lymph node biopsy after kidney transplantation, is phenotypically similar to the other SLCL in both surface antigen and activated STAT1 and STAT3 expression. CONCLUSION: These data support the presence of a constitutively active autocrine signaling pathway consistent with IL-10 in the SLCL.

Antigens, CD↗
PubMed2001

Constitutional balanced chromosomal rearrangements and neoplasm in children.

A predisposition to tumor development is currently associated with some, but not all, constitutional chromosomal abnormalities. In a series of 578 children, in which conventional cytogenetic investigation was performed on material from various benign and malignant tumors, four boys and one girl were also found to have constitutional balanced chromosomal rearrangements. The figure of 5 in 578 is notable because the reported incidence of balanced rearrangements in newborns is approximately 1 in 450. Thereby suggesting that some, if not all, children with balanced constitutional chromosomal rearrangements have an increased predisposition for neoplasms developing.

Abnormalities, Multiple↗
PubMed2004

Chronic low back pain-associated paraspinal muscle dysfunction is not the result of a constitutionally determined "adverse" fiber-type composition.

STUDY DESIGN: Investigative case control study. OBJECTIVES: To determine whether excessive paraspinal muscle fatigue in chronic low back pain results from a paucity of muscle type I fiber content. SUMMARY OF BACKGROUND DATA: Paraspinal muscle function is vital for spinal protection. Prospective studies suggest that excessive paraspinal muscle fatigability may increase risk of first-time low back pain. As contractile performance of the paraspinal muscles is governed by their constitutionally determined fiber composition, the question arises whether a constitutionally determined "adverse" composition could predispose to low back pain through impaired spinal protection. METHODS: Thirty-five male patients with chronic low back pain were compared with 32 male control patients of similar age and anthropometry. During Sorensen and 60% of maximum voluntary isometric contraction fatigue tests, median frequency declines in the paraspinal muscle surface electromyograph signal were monitored and correlated with muscle histomorphometry. RESULTS: Patients were weaker than controls during maximum voluntary isometric contractions (84.47 [28.44]vs. 98.74 [18.11] kg, respectively; P = 0.02) and more fatigable during their Sorensen tests (endurance time 105.29 [28.53]vs. 137.50 [40.38] sec, respectively; P < 0.01). There were no between-group differences in median frequency declines during the Sorensen (-0.37 [0.16]vs. -0.36 [0.12]%.sec) or 60% maximum voluntary isometric contraction (-0.42 [0.31]vs. -0.51 [0.29]%.sec) tests, for patients and controls, respectively. There were no between-group differences in the percent number of paraspinal muscle type I fibers (64 [11]vs. 64 [9]%) or the percent area occupied by type I fibers (67 [11]vs. 69 [9]%), for patients and controls, respectively. Type I and II muscle fiber narrow diameters were similar for both groups. CONCLUSION: In the patients with chronic low back pain tested, their associated paraspinal muscle dysfunction was not the result of a constitutionally determined "adverse" fiber type composition.

Adult↗
PubMed2005

Constitutive innate immunity is a component of the pace-of-life syndrome in tropical birds.

We studied the relationship between one component of immune function and basal metabolic rate (BMR), an indicator of the 'pace-of-life syndrome', among 12 tropical bird species and among individuals of the tropical house wren (Troglodytes aedon), to gain insights into functional connections between life history and physiology. To assess constitutive innate immunity we introduced a new technique in the field of ecological and evolutionary immunology that quantifies the bactericidal activity of whole blood. This in vitro assay utilises a single blood sample to provide a functional, integrated measure of constitutive innate immunity. We found that the bactericidal activity of whole blood varied considerably among species and among individuals within a species. This variation was not correlated with body mass or whole-organism BMR. However, among species, bacteria killing activity was negatively correlated with mass-adjusted BMR, suggesting that species with a slower pace-of-life have evolved a more robust constitutive innate immune capability. Among individuals of a single species, the house wren, bacteria killing activity was positively correlated with mass-adjusted BMR, pointing to physiological differences in individual quality on which natural selection potentially could act.

Animals↗
PubMed1993

pRJ5: a naturally occurring Staphylococcus aureus plasmid expressing constitutive macrolide-lincosamide-streptogramin B resistance contains a tandem duplication in the leader region of the ermC gene.

The 2.55 kb Staphylococcus aureus plasmid, pRJ5, confers constitutive resistance to macrolide-lincosamide-streptogramin B (MLS) antibiotics. pRJ5 is nearly identical to the inducible MLS resistance plasmid pT48, and has homology with the S. aureus plasmids pE194 and pSN2. The HindIII-C and/or Hind-B fragments were required for stable maintenance of the plasmid and probably carry palA. Plasmids pRJ5 and pT48 were shown to belong to the same incompatibility group, Inc12 (L). DNA sequencing showed that pRJ5 contains a 28 bp direct tandem duplication in the leader/attenuator region of ermC. This is likely to change the secondary structure of the methylase mRNA, allowing constitutive expression of ermC. The type of mutation found on plasmid pRJ5 is different from those observed in similar 2.5 kb constitutive MLS-resistance plasmids isolated from other Gram-positive bacteria, including staphylococci.

Anti-Bacterial Agents↗
PubMed2001

Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.

Genes placed under the control of the arabinose-inducible araBAD promoter (P(BAD)) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from P(BAD) was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P(BAD) in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of P(BAD). In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne P(BAD)-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.

Arabinose↗
PubMed1975

The synthesis of beta-galactosidase by constitutive and other regulatory mutants of Escherichia coli in chemostat culture.

The synthesis of beta-galactosidase by an E. coli constitutive mutant was examined in a chemostat using glucose-, glycerol-, succinate- or N-limited growth media. Except for glucose-grown bacteria, the steady-state intracellular level of beta-galactosidase was maximal at dilution rates between 0-2 and 0-3 h-1. At higher dilution rates enzyme synthesis was reduced by catabolite repression, which could be relieved by the addition of cyclic AMP. With a catabolite-resistant mutant (UV5c), no decrease in enzyme level at high dilution rates were observed. All mutants examined were constitutive and gave decreased enzyme levels at low dilution rates, with the exception of lac-/F'lac UV5c mutants where the enzyme levels rose at low dilution rates. Hyper-producing mutants were isolated but were unstable. A constitutive mutant growing on glycerol-limited media was considered the most suitable for large-scale production of beta-galactosidase in a chemostat.

Bacteriological Techniques↗
PubMed2003

Integrative, multifunctional plasmids for hypha-specific or constitutive expression of green fluorescent protein in Candida albicans.

The authors have engineered plasmid constructs for developmental and constitutive expression of yeast-enhanced green fluorescent protein (yEGFP3) in Candida albicans. The promoter for the hyphae-specific gene Hyphal Wall Protein 1 (HWP1) conferred developmental expression of yEGFP3 in germ tubes and hyphae but not in yeasts or pseudohyphae when targeted to the ENO1 (enolase) locus in single copy. The pHWP1GFP3 construct allows for the easy visualization of HWP1 promoter activity in individual cells expressing true hyphae without having to prepare RNA for analysis. Constitutive expression of yEGFP was seen in all cell morphologies when the HWP1 promoter was replaced with the ENO1 promoter region. The use of the plasmids for expression of genes other than yEGFP3 was examined by substituting the putative C. albicans BCY1 (SRA1) gene, a component of the cAMP signalling pathway involved in yeast to hyphae transitions, for yEGFP3. Strains overexpressing BCY1 from the ENO1 promoter were inhibited in germ tube formation and filamentation in both liquid and solid media, a phenotype consistent with keeping protein kinase A in its inactive form by association with Bcy1p. The plasmids are suitable for studies of germ tube induction or assessing germ tube formation by measuring yEGFP3 expression, for inducible expression of genes concomitant with germ tube formation by the HWP1 promoter, for constitutive expression of genes by the ENO1 promoter, and for expressing yEGFP3 using a promoter of choice.

Candida albicans↗
PubMed2005

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system.

Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive rcsC alleles were also found, in agreement with the postulated double role (positive and negative) of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading to avirulence in constitutively activated rcsC mutants.

Animals↗
PubMed1996

The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing.

The splicing factor U2AF (U2 snRNP auxiliary factor) is a heterodimer with subunits of 65 and 35 kD (U2AF65 and U2AF35). U2AF65 binds specifically to 3' splice sites, but previous studies failed to demonstrate a function for U2AF35. Here, we report that U2AF35 is required for constitutive splicing and also functions as a mediator of enhancer-dependent splicing. Nuclear extracts deficient in U2AF35 were inactive; however, both constitutive and enhancer-dependent splicing could be restored by the addition of purified recombinant U2AF35. In vitro protein-RNA interaction studies with pre-mRNAs containing either a constitutive or regulated splicing enhancer revealed that U2AF35 directly mediates interactions between U2AF65 and proteins bound to the enhancers. Thus, U2AF35 functions as a bridge between U2AF65 and the enhancer complex to recruit U2AF65 to the adjacent intron.

Enhancer Elements, Genetic↗
PubMed2001

Dynamic constitutive relations for polarization and magnetization.

In this paper we develop constitutive relations for materials where the magnetization and polarization may depend on both the electric and magnetic fields. The approach is general, and is based on a previously developed statistical-mechanical theory. We include the quadrupole-moment density as well as the dipole-moment density in the microscopic displacement field. This yields an electric gradient term in the constitutive equations. This leads to origin invariance in the multipole moments from which Maxwell's equations are defined. We present generalizations of Debye and Landau-Lifshitz equations of motion which are valid for nonequilibrium and contain memory. The reversible and relaxation terms in the polarization and magnetization evolution equations include the possibility of magnetoelectric coupling. Using constitutive relationship, we derive evolution equations for the displacement and induction fields from a Hamiltonian approach.

Journal Article↗
PubMed2003

Structural and biochemical dissection of photorespiration in hybrids differing in genome constitution between Diplotaxis tenuifolia (C3-C4) and radish (C3).

We compared the structural, biochemical, and physiological characteristics involved in photorespiration of intergeneric hybrids differing in genome constitution (DtDtR, DtDtRR, and DtRR) between the C(3)-C(4) intermediate species Diplotaxis tenuifolia (DtDt) and the C(3) species radish (Raphanus sativus; RR). The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of radish had only a few chloroplasts and mitochondria. In the hybrids, the numbers of chloroplasts and mitochondria, the ratio of centripetally located organelles to total organelles, and the mitochondrial size in the BS cells increased with an increase in the constitution ratio of the Dt:R genome. The P-protein of glycine decarboxylase (GDC) was confined to the BS mitochondria in D. tenuifolia, whereas in radish, it accumulated more densely in the mesophyll than in the BS mitochondria. In the hybrids, more intense accumulation of GDC in the BS relative to the mesophyll mitochondria occurred with an increase in the Dt:R ratio. These structural and biochemical features in the hybrids were reflected in the gas exchange characteristics of leaves, such as the CO(2) compensation point. Our data indicate that the leaf structure, the intercellular pattern of GDC expression, and the gas exchange characteristics of C(3)-C(4) intermediate photosynthesis are inherited in the hybrids depending on the constitution ratio of the parent genomes. Our findings also demonstrate that the apparent reduced photorespiration in C(3)-C(4) intermediate plants is mainly due to the structural differentiation of mitochondria and chloroplasts in the BS cells combined with the BS-dominant expression of GDC.

Amino Acid Oxidoreductases↗
PubMed2004

Overexpression of constitutive differential growth 1 gene, which encodes a RLCKVII-subfamily protein kinase, causes abnormal differential and elongation growth after organ differentiation in Arabidopsis.

To better understand genetic regulation of differential growth of plant organs, a dominant and semidwarf mutant, constitutive differential growth 1-Dominant (cdg1-D), was isolated utilizing the technique of activation tagging. cdg1-D showed pleiotropic phenotype including dwarfism, exaggerated leaf epinasty, and twisted or spiral growth in hypocotyl, inflorescence stem, and petiole. Hypocotyls of cdg1-D were longer than those of wild type under light conditions. The phenotype was caused by activation tagging of CDG1 gene that encodes a receptor-like cytoplasmic kinase of RLCKVII subfamily. When treated with high concentrations of brassinolide, light-grown wild-type seedlings showed long hypocotyls and strong leaf epinasty as observed in cdg1-D seedlings. Treatment of cdg1-D with brassinazole, a specific inhibitor of brassinosteroid (BR) biosynthesis, did not rescue the mutant phenotype. Gene expression of CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM involved in BR biosynthesis and phyB ACTIVATION-TAGGED SUPPRESSOR1 that inactivates BR was repressed and induced, respectively, in cdg1-D plants, suggesting constitutive activation of BR signaling in the mutant. CDG1 was expressed at a very low level in all the organs of the wild type tested. We isolated two independent intragenic suppressors of cdg1-D. However, they showed normal morphology and responded to BR in a similar manner to wild type. Taken together, CDG1 gene may interfere with signal transduction of BR when overexpressed, but is not an essential factor for it in the wild type.

Amino Acid Sequence↗
PubMed1997

Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat.

Oxidant stress resistance in Conyza bonariensis and wheat (Triticum aestivum) has been correlated with high levels of antioxidant enzyme activities. Additionally, external oxidant stresses can increase a plant's levels of the enzymes of polyamine biosynthesis and polyamines, especially putrescine. We investigated the constitutive relationships between putrescine, putrescine-generating enzymes, and oxidant stress resistance in wheat and C. bonariensis. Putrescine was Constitutively elevated (2.5- to 5.7-fold) in 2-week-old-resistant wheat and C. bonariensis biotypes, which correlated with a 10- to 15-fold increase in paraquat oxidant resistance. Arginine and ornithine decarboxylase activities doubled, along with higher putrescine levels in resistant C. bonariensis. The variations in levels of putrescine and arginine and ornithine decarboxylase activities paralleled the constitutive variation of antioxidant enzymes, as well as oxidant resistance. Higher levels of both putrescine and antioxidant enzyme activities occurred during a peak of oxidant resistance at 10 weeks, when paraquat resistance in C. bonariensis plants is >50-fold greater than in the sensitive biotype. Application of 100 [mu]M putrescine can double oxidant-stress resistance in the resistant C. bonariensis. Putrescine may play an important role in contributing to the base level of oxidant resistance found at the nonpeak period.

Journal Article↗