Expression of the leptin receptor in human leukaemic blast cells

The highly conserved residue F208 in protein R2 of E. coli ribonucleotide reductase is close to the binuclear iron center, and found to be involved in stabilizing the tyrosyl radical Y122. in wild type R2. Upon the reconstitution reaction of the mutant R2 F208Y with ferrous iron and molecular oxygen, we observed a new EPR singlet signal (g = 2.003) formed concomitantly with decay of the transient tyrosyl radical Y122. (g = 2.005). This new paramagnetic species (denoted Z) was stable for weeks at 4 degrees C and visible by EPR only below 50 K. The EPR singlet could not be saturated by available microwave power, suggesting that Z may be a mainly metal centered species. The maximum amount of the compound Z in the protein purified from cells grown in rich medium was about 0.18 unpaired spin/R2. An identical EPR signal of Z was found also in the double mutant R2 F208Y/Y122F. In the presence of high concentration of sodium ascorbate, the amounts of both the transient Y122. and the new species Z increased considerably in the reconstitution reaction. The results suggest that Z is most likely an oxo-ferryl species possibly in equilibrium with a Y208 ligand radical.     

Expression of functional P2-purinergic receptors in primary cultures of human colorectal carcinoma cells

Primary cell cultures of human colorectal carcinomas were established and characterized immunocytochemically. In the isolated cancer cells intracellular Ca2+ concentrations ([Ca2+]i) were measured by the fura-2 method. Stimulation with either extracellular ATP or UTP caused a biphasic rise of [Ca2+]i in a dose-dependent manner and cross-desensitization between both nucleotides was observed. The rank order of potency was ATP >== UTP > ATP-gamma-S > ADP > adenosine which is characteristic for a P2U-receptor subtype. Selective agonists of P1-, or P2X- purinoceptors had no effect on [Ca2+]i. The initial rise in [Ca2+]i was independent of extracellular calcium [Ca2+]e, whereas the second phase was not observed under [Ca2+]e-free conditions suggesting a capacitative Ca2+-entry-mechanism. Intracellular Ca2+ mobilization was proven by use of the Ca2+-ATPase inhibitor thapsigargin. P2U-specific mRNA could be detected by RT-PCR in both colorectal tumor tissues and in the human colorectal cancer cell line HT 29. In HT 29 cells, the hydrolysis-resistant ATP analog ATP-gamma-S inhibited cell proliferation and, also, induced apoptosis in a dose-dependent manner. Thus, human colorectal cancer cells express functional P2U-receptors which may play a role in the regulation of cell proliferation and apoptosis.     

Expression of human muscarinic cholinergic receptors in tobacco

The T lymphocyte beta 2-adrenergic receptor (beta 2AR) density and function were compared in 15 patients suffering acute myocardial infarction and 10 patients with stable coronary artery disease (CAD). Density was determined using radioligand binding with 125IPIN, and function by in vitro cyclic adenosine 3',5'-monophosphate (cAMP) production. In patients suffering acute myocardial infarction, T lymphocyte beta 2AR density (823.8 +/- 480 sites/cell) was slightly but not significantly different from that in patients with stable CAD (629 +/- 301 sites/cell). There was no difference in T lymphocyte cAMP production at baseline (1.11 +/- 0.70 vs 1.04 +/- 0.49 pM/10(6) cells) or after isoproterenol stimulation (2.53 +/- 1.63 vs 2.62 +/- 2.05 pM/10(6) cells), respectively. Further study is necessary to determine if beta 2AR numbers on T lymphocytes are significantly increased after acute myocardial infarction.     

Expression of retinoid X receptors in human dermal fibroblasts

Retinoic acid (RA) is known to exert profound effects on growth and differentiation in human dermal fibroblasts. In the observations presented here, we examined the regulation of expression of members of the RXR multigene family in human dermal fibroblasts. We showed that the messenger RNAs for both RXR alpha and RXR beta are expressed in human fibroblasts, but that the messenger RNA for RXR gamma is not detectable in these cells. Electrophoretic mobility shift studies of binding to the beta 2RARE in human dermal fibroblasts demonstrated that a single complex binds to beta 2RARE in the absence of RA. Stimulating cells with all-trans RA induced a second complex. An antibody to the RXR beta protein supershifted both complexes, while an antibody to the RXR alpha S/B protein had no effect on the binding. These data demonstrate that RXR beta plays an important role in retinoid-regulated signal transduction pathways in human dermal fibroblasts and the regulation of expression of the RXR gene family is different from that of the RAR gene family.     

Expression of somatostatin receptors in peritumoral veins of human tumors

We prospectively evaluated the relation of upper airway, lower airway, and gastric colonization patterns with the development of pneumonia and its etiology in 48 patients with surgical (n = 25) and medical (n = 23) head injury. Initial colonization was assessed by cultures of nasal and pharyngeal swabs, tracheobronchial aspirates, gastric juice, and bronchoscopically retrieved protected specimen brush. Follow-up colonization was determined until the end points extubation, suspected ventilator-associated pneumonia (VAP), or death. The initial colonization rate at any site at ICU admission was 39/47 (83%). It mainly accounted for Group I pathogens (Streptococcus pneumoniae, Staphylococcus aureus, Hemophilus influenzae) of the upper and lower airways. At follow-up, colonization rates with Group II pathogens (Gram-negative enteric bacilli and Pseudomonas spp.) increased significantly. The high initial bacterial load with Group I pathogens of the upper airways and trachea decreased during Days 2 to 4, whereas that of Group II pathogens increased. Upper airway colonization was an independent predictor of follow-up tracheobronchial colonization (odds ratio [OR], 9.9; 95% confidence interval [CI], 1.8 to 56.3 for initial colonization with Group I pathogens; OR, 23.9; 95% CI, 3.8 to 153.3 for follow-up colonization with Group II pathogens). Previous (short-term) antibiotics had a protective effect against colonization with Group I pathogens of the lower respiratory tract (OR, 0.2; 95% CI, 0.05 to 0.86), but they were a risk factor for colonization with Group II pathogens (OR, 6.1; 95% CI, 1.3 to 29). Initial tracheobronchial colonization with Group I pathogens was associated with a higher probability of early onset pneumonia (OR, 4. 1; 95% CI, 0.7 to 23.3), whereas prolonged antibiotic treatment (> 24 h) independently predicted late-onset pneumonia (OR, 9.2; 95% CI, 1.7 to 51.3). We conclude that patients with head injury are colonized in the airways mainly by Group I pathogens early in the evolution of illness. The upper airways represent the main reservoir for subsequent lower airway colonization with Group I pathogens. Previous (short-term) antibiotic treatment is protective against initial tracheobronchial colonization with Group I pathogens, but it represents a risk factor for subsequent lower airway colonization by Group II pathogens.     

Expression of granulocyte-macrophage colony-stimulating factor receptors in human prostate cancer

This investigation examined the effects of NaHCO3 loading on lactate concentration ([La]), acid-base balance, and performance for a 603. 5-m sprint task. Ten greyhounds completed a NaHCO3 (300 mg/kg body weight) and control trial in a crossover design. Results are expressed as means +/- SE. Presprint differences (P < 0.05) were found for NaHCO3 vs. control, respectively, for blood pH (7.47 +/- 0.01 vs. 7.42 +/- 0.01), HCO-3 (28.4 +/- 0.4 vs. 23.5 +/- 0.3 meq/l), and base excess (5.0 +/- 0.3 vs. 0.2 +/- 0.3 meq/l). Peak blood [La] increased (P < 0.05) in NaHCO3 vs. control (20.4 +/- 1.6 vs. 16.9 +/- 1.3 mM, respectively). Relative to control, NaHCO3 produced a greater (P < 0.05) reduction in blood base excess (-18.5 +/- 1.4 vs. -14.1 +/- 0.8 meq/l) and HCO-3 (-17.4 +/- 1.2 vs. -12.8 +/- 0.7 meq/l) from presprint to postexercise. Postexercise peak muscle H+ concentration ([H+]) was higher (P < 0.05) in NaHCO3 vs. control (158.8 +/- 8.8 vs. 137.0 +/- 5.3 nM, respectively). Muscle [H+] recovery half-time (7.2 +/- 1.6 vs. 11.3 +/- 1.6 min) and time to predose values (22.2 +/- 2.4 vs. 32.9 +/- 4.0 min) were reduced (P < 0.05) in NaHCO3 vs. control, respectively. No differences were found in blood [H+] or blood [La] recovery curves or performance times. NaHCO3 increased postexercise blood [La] but did not reduce the muscle or blood acid-base disturbance associated with a 603.5-m sprint or significantly affect performance.     

Immunolocalization of retinoic acid receptors in rat, mouse and human ovary and uterus

We raised an antibody against a synthetic peptide corresponding to amino acids 155-174 of human retinoic acid receptor alpha (RAR-alpha). The sequence is highly homologous in all RARs and their isoforms. When mouse and human RARs (alpha, beta and gamma) expressed in Cos cell were analysed with immunoblot, all receptors gave a specific 51 K signal. Mouse RAR-gamma gave an additional signal corresponding to 58 K. In human teratocarcinoma cells (F9) both 51 and 58K molecule sizes were detected. The RAR expression in F9 cells was slightly down-regulated in charcoal-stripped culture medium and returned to normal level after retinoic acid treatment. The 51 K protein was found in all ovarian and uterine samples, but the quantity of the 58 K protein varied in different species and organs, being highest in the mouse uterus and the rat and human ovary. Using immunohistochemistry the RARs were found in the nuclear compartment. In the rat uterus, positive immunoreaction was found mainly in the nuclei of epithelial, uterine glandular and stromal cells. In the rat ovary, positive reaction was found in the nuclei of germinal epithelial, follicular and stromal cells.     

Expression and recovery of functional G-protein-coupled receptors using baculovirus expression systems

BACKGROUND: Enterococcus faecium has received increased attention, primarily due to the emergence of vancomycin resistance. The purpose of this investigation was to study the epidemiological characteristics of vancomycin-resistant E faecium (VRE) bacteremia and to determine the clinical impact of vancomycin resistance on the outcome of patients with this infection. METHODS: We retrospectively analyzed the clinical features and outcome of 53 patients with E faecium bacteremia. RESULTS: From January 1992 until December 1995, there were 32 episodes of bacteremia caused by vancomycin-susceptible E faecium (VSE) and 21 caused by VRE. An intra-abdominal site was the most common source of bacteremia in both groups. All of the VRE and 78% of VSE bacteremia cases were nosocomially acquired. Previous administration of vancomycin was associated with VRE bacteremia (P<.001), as were indwelling bladder catheters (P=.01). Fifty-nine percent of the patients with VSE bacteremia survived vs 24% with VRE (P=.009), despite similar severity-of-illness scores. In 62% of the patients with VRE sepsis, death was related to the bacteremia (P=.01). Patients infected with VRE had longer hospitalizations than those with VSE (34.8 vs 16.7 days, respectively) (P=.004), were more likely to be on the medical service (P=.03), and on the average, had hospitalization costs of more than $27,000 per episode than did patients with VSE bloodstream infection ($83,897 vs $56,707, respectively) (P=.04). CONCLUSIONS: Vancomycin-resistant E faecium bacteremia is a complication of prolonged hospitalization in debilitated patients. Vancomycin resistance has a negative impact on survival in patients with E faecium bacteremia and leads to higher health care costs.     

Serum immunoreactive leptin concentrations in women with polycystic ovary syndrome

Recent data in the mouse demonstrate that leptin, a protein hormone produced by fat cells, is required for fertility. In the absence of leptin the mice become obese, diabetic and infertile. Polycystic ovary syndrome (PCOS), a common cause of infertility in women, is associated with obesity and insulin resistance. Because of the increased frequency of PCOS in obese women we tested the hypothesis that alterations in serum leptin concentrations might be associated with PCOS. Immunoreactive leptin concentrations were measured in 58 women with PCOS and 70 regularly menstruating (control) women. As has previously been shown there was a positive correlation between leptin levels and body mass index (BMI). Although the leptin levels in the majority of women with PCOS fell within the control range, 29% of PCOS women had leptin levels above the 99% prediction interval for their BMI and none had low leptin levels. There were also positive correlations of leptin levels with free testosterone and insulin sensitivity in control women. In women with PCOS, 13% and 9.5% exhibited higher than expected leptin concentrations with respect to free testosterone and insulin sensitivity, respectively. Insulin resistant PCOS women had higher leptin levels than controls. The data demonstrate that a substantial proportion of women with PCOS have leptin levels that are higher than expected for their BMI, free testosterone and insulin sensitivity. These results suggest that abnormalities in leptin signaling to the reproductive system may be involved in certain cases of PCOS.     

Progesterone receptors: expression and regulation in the mammalian ovary

The authors have briefly discussed the molecular structure, regulation, and function of progesterone receptors in the mammalian ovary. Particularly important is the contrast in the regulatory mechanisms of PR induction in the ovary (gonadotropins/membrane receptor mediated) and other well-known progesterone target tissues, such as the uterus and mammary gland (estrogen/nuclear receptor mediated). Future research will focus on how the PR gene responds to these hormonal regulatory signals in this cell-specific manner. Equally important in this discussion has been the mounting evidence indicating that PRs are an essential component of the ovulatory process. The observation that PR-/- knockout mice are incapable of undergoing ovulation, even in response to gonadotropin challenge, further supports the previous physiological evidence indicating that PRs in preovulatory follicles are induced before, and are necessary for, ovulation. Further studies are required to determine the identity of PR-regulated target genes during the periovulatory period. Although our knowledge of PR structure, regulation, and function has increased dramatically during the past decade, many exciting questions remain related to the regulation and function(s) of PRs in the ovary and other tissues.     

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or coincubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentration-dependently inhibited twitch responses (IC50 73 nM), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction. These results provide functional evidence of the existence of prejunctional cannabinoid CB1-receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.     

A role for neurotransmitters in early follicular development: induction of functional follicle-stimulating hormone receptors in newly formed follicles of the rat ovary

The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a beta-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an alpha-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.     

Expression of a functional endothelin (ETA) receptor in human meningiomas

Endothelin (ET) receptor subtypes (ETA and ETB) in human meningiomas were characterized using quantitative receptor autoradiography. A single class of high-affinity 125I-ET-1 binding sites was localized in all meningioma tissue studied (dissociation constant: 2.4 +/- 0.3 nM, maximum binding capacity: 319 +/- 66 fmol/mg (mean +/- standard error of the mean for 13 tumors)). Unlabeled ET-1 showed a strong affinity for 125I-ET-1 binding to tissue sections of the tumors with a 50% inhibiting concentration (IC50) of 2.9 +/- 0.7 x 10(-9) M, whereas ET-3 showed a much lower affinity (IC50: 8.4 +/- 2.5 x 10(-6) M). Sarafotoxin S6c, a selective agonist for the ETB receptor, could not compete for 125I-ET-1 binding to meningiomas. Endothelin-1 significantly stimulated deoxyribonucleic acid (DNA) synthesis in a dose-dependent manner in cultured human meningioma cells. In contrast, no significant stimulation of DNA synthesis occurred with an S6c concentration up to 10(-7) M. Pretreatment of the meningioma cells with pertussis toxin, a bacterial toxin that adds adenosine 5'-diphosphate-ribose to the alpha subunit of guanine nucleotide binding (G) proteins such as Gi or G(o), induced a concentration-dependent reduction in ET-stimulated DNA synthesis in meningioma cells, but did not affect the epidermal growth factor-induced DNA synthesis. These observations suggest that the ETA receptor is predominantly expressed in human meningioma tissue and that ET may act as a growth factor on the meningioma cells by interacting with the ETA receptor and by pertussis toxin-sensitive mechanisms.     

Expression of leptin receptor isoforms in rat brain microvessels

Current evidence suggests that host defense in respiratory mycoplasmosis is dependent on both innate and humoral immunity. To further delineate the roles of innate and adaptive immunity in antimycoplasmal defenses, we intranasally infected C3H/HeSnJ-scid/scid (C3H-SCID), C3H/HeSnJ (C3H), C57BL/6J-scid/scid (C57-SCID), and C57BL/6N (C57BL) mice with Mycoplasma pulmonis and at 14 and 21 days postinfection performed quantitative cultures of lungs and spleens, quantification of lung lesions, and histopathologic assessments of all other major organs. We found that numbers of mycoplasmas in lungs were associated with genetic background (C3H susceptible, C57BL resistant) rather than functional state of adaptive immunity, indicating that innate immunity is the main contributor to antimycoplasmal defense of the lungs. Extrapulmonary dissemination of mycoplasmas with colonization of spleens and histologic lesions in multiple organs was a common occurrence in all mice. The absence of adaptive immune responses in severe combined immunodeficient (SCID) mice resulted in increased mycoplasmal colonization of spleens and lesions in extrapulmonary sites, particularly spleens, hearts, and joints, and also reduced lung lesion severity. The transfer of anti-M. pulmonis serum to infected C3H-SCID mice prevented extrapulmonary infection and disease, while the severity of lung lesions was restored by transfer of naive spleen cells to infected C3H-SCID mice. Collectively, our results strongly support the conclusions that innate immunity provides antimycoplasmal defense of the lungs and humoral immunity has the major role in defense against systemic dissemination of mycoplasmal infection, but cellular immune responses may be important in exacerbation of mycoplasmal lung disease.