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We have reported that the rate of de novo triglyceride (TG) synthesis by omental, but not subcutaneous, adipose tissue was higher in African-American women (AAW) than in Caucasian women (CAW). The purpose of this study was to explore the potential mechanisms underlying this increase. Toward that end, we determined the activities of key enzymes in the pathway of TG synthesis, the rates of uptake of fatty acids by adipocytes, mRNA and protein levels of the fatty acid-transporting proteins FAT/CD36 and FATP, and mRNA and protein levels of PPARgamma in omental fat of AAW and CAW. The results showed 1) no difference in the activity of phosphofructokinase, glycerol-3-phosphate dehydrogenase, or diacylglycerol acyltransferase; 2) a higher rate of fatty acid uptake by adipocytes of the AAW; 3) an increase in the mRNA and protein levels of CD36 and FATP4 in the fat of the AAW; and 4) an increase in the mRNA and protein levels of PPARgamma, which can stimulate the expression of CD36 and FATP. These results suggest that the increase in the transport of fatty acid, which is mediated by the overexpression of the transport proteins in the omental adipose tissue of the AAW, might contribute to the higher prevalence of obesity in AAW.

The total syntheses of five new porphyrin-cobaltacarborane conjugates (1-5) have been achieved in 88-98% yields in a single-step reaction between a nucleophilic meso-pyridyl-containing porphyrin and zwitterionic cobaltacarborane [3,3'-Co(8-C(4)H(8)O(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))]. These unique zwitterionic compounds have one to four cobaltabisdicarbollide anions conjugated to the porphyrin macrocycle via (CH(2)CH(2)O)(2) chains. The X-ray structure of one of these conjugates (1) is presented and discussed. The cellular uptake, cytotoxicity, and subcellular localization of cobaltacarboraneporphyrins 1-5 were investigated in human HEp2 cells. The number and distribution of cobaltacarborane residues linked to the porphyrin macrocycle has a significant effect on the cellular uptake of the conjugates.

The basic helix-loop-helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in beta-cells and either absent or expressed below the limit of lacZ detection in mature alpha-, delta-, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM-6/PDX-1, a human pancreatic delta-cell line, resulted in potent repression of somatostatin concomitant with induction of the beta-cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in beta- but not delta-cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously.

Two porphyrin-cobaltacarborane conjugates and were prepared in high yields via a nucleophilic ring-opening reaction of . These novel boron-rich and fluorescent compounds have potential application as boron delivery agents for the boron neutron capture therapy of tumors.

In this paper we overview our recent studies of anisotropic noble metal (e.g. gold and silver) nanoparticles, in which a combination of theory and experiment has been used to elucidate the extinction spectra of the particles, as well as information related to their surface enhanced Raman spectroscopy. We used wet-chemical methods to generate several structurally well-defined nanostructures other than solid spheres, including silver nanodisks and triangular nanoprisms, and gold nanoshells and multipods. When solid spheres are transformed into one of these shapes, the surface plasmon resonances in these particles are strongly affected, typically red-shifting and even splitting into distinctive dipole and quadrupole plasmon modes. In parallel, we have developed computational electrodynamics methods based on the discrete dipole approximation (DDA) method to determine the origins of these intriguing optical features. This has resulted in considerable insight concerning the variation of plasmon wavelength with nanoparticle size, shape and dielectric environment, as well as the use of these particles for optical sensing applications.

Iron oxide nanoparticles have been shown to magnetically label cells in order to visualize them in vivo via MR imaging. This technology has yet to be implemented in insulin secreting cells, thus it is not known whether the presence of these nanoparticles in the cytoplasm of the cells affects insulin secretion. This study investigates the effectiveness and consequence of labeling mouse insulinoma betaTC3 and betaTC-tet cells with monocrystalline iron oxide nanoparticles (MION). Our data show that MION can be internalized in both betaTC3 and betaTC-tet cells following a 24h exposure to 0.02mg/ml MION solution. The metabolic and secretory activities of both MION-labeled cell lines were statistically indistinguishable from sham treatment. Furthermore, cell viability and apoptosis remained constant throughout the cell's exposure to MION. Finally, MR images demonstrated significant contrast between labeled and sham-treated cells. Thus, labeling murine insulinoma cell lines with magnetic iron oxide nanoparticles does not hinder their insulin secretion, while it provides MR imaging contrast.

Imaging of adoptively transferred cells in vivo by magnetic resonance imaging (MRI) could provide important information on disease-related patterns of lymphocyte homing in nonhuman primate models of AIDS. As a preliminary study to assess the feasibility of visualizing activated rhesus T cells by MRI, anti-CD3/CD28-expanded CD4+ T lymphocytes were labeled in vitro with monocrystalline iron oxide nanoparticles (MION). Intracellular incorporation of MION was determined by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrography (ICP-MS). Pretreatment with colchicine did not affect MION labeling, suggesting that cellular uptake of MION occurred by adsorptive pinocytosis or receptor-mediated endocytosis. TEM analysis revealed that MION were intracellularly compartmentalized exclusively in the cytoplasm and did not cause any measurable physiologic effects on T-cell function, including viability, proliferation, synthesis of select cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma), activation antigens (CD25 and CD69), adhesion molecules (alpha4beta7 and CD49d), and susceptibility to in vitro infection with simian immunodeficiency virus mac239. A sensitivity of 0.05% (1 MION-labeled T cell in 2000 unlabeled cells) could be achieved using T2-weighted gradient echo imaging. Furthermore, under these experimental conditions, the MRI signal did not decrease in proliferating MION-labeled CD4+ T cells over a period of 120 hours. These results indicate that intracellular labeling with MION can be a useful technique for noninvasively monitoring trafficking patterns of adoptively transferred leukocyte subsets in real-time by MRI in nonhuman primate models of AIDS.

We use the discrete dipole approximation to investigate the electromagnetic fields induced by optical excitation of localized surface plasmon resonances of silver nanoparticles, including monomers and dimers, with emphasis on what size, shape, and arrangement leads to the largest local electric field (E-field) enhancement near the particle surfaces. The results are used to determine what conditions are most favorable for producing enhancements large enough to observe single molecule surface enhanced Raman spectroscopy. Most of the calculations refer to triangular prisms, which exhibit distinct dipole and quadrupole resonances that can easily be controlled by varying particle size. In addition, for the dimer calculations we study the influence of dimer separation and orientation, especially for dimers that are separated by a few nanometers. We find that the largest /E/2 values for dimers are about a factor of 10 larger than those for all the monomers examined. For all particles and particle orientations, the plasmon resonances which lead to the largest E-fields are those with the longest wavelength dipolar excitation. The spacing of the particles in the dimer plays a crucial role, and we find that the spacing needed to achieve a given /E/2 is proportional to nanoparticle size for particles below 100 nm in size. Particle shape and curvature are of lesser importance, with a head to tail configuration of two triangles giving enhanced fields comparable to head to head, or rounded head to tail. The largest /E/2 values we have calculated for spacings of 2 nm or more is approximately 10(5)., ((c) 2004 American Institute of Physics)

Achieving normoglycemia is the goal of diabetes therapy. Potentially, there are many ways to achieve this goal, including transplantation of cells exhibiting glucose-responsive insulin secretion. However, to be applicable to the large number of people who might benefit from beta cell replacement, an unlimited supply of beta cells must be found. To address this problem, we have been developing cell lines from the human endocrine pancreas. In one case, a cell line, betalox5, has been developed from human islets that can be induced under some circumstances to differentiate into functional beta cells exhibiting appropriate glucose-responsive insulin secretion. Inducing differentiation is complex, requiring the activation of multiple signaling pathways, including those downstream of those involved in cell-cell contact and the glucagon-like peptide-1 receptor. In addition, transfer of the PDX-1 gene is also necessary to render the cells competent for differentiation. However, it is clear that many other genes are involved in maintaining the commitment of betalox5 cells towards the beta cell lineage. Understanding the complement of genes required to establish and maintain a beta cell lineage commitment would be enormously helpful in efforts to develop a cell line that can be used for beta cell replacement therapies. Here, we provide further information on the characteristics of cell lines that we have developed from the human pancreas that are relevant to the development of a beta cell replacement therapy for diabetes.

Inorganic nanoparticles exhibit size-dependent properties that are of interest for applications ranging from biosensing and catalysis to optics and data storage. They are readily available in a wide variety of discrete compositions and sizes. Shape-selective synthesis strategies now also yield shapes other than nanospheres, such as anisotropic metal nanostructures with interesting optical properties. Here we demonstrate that the previously described photoinduced method for converting silver nanospheres into triangular silver nanocrystals--so-called nanoprisms--can be extended to synthesize relatively monodisperse nanoprisms with desired edge lengths in the 30-120 nm range. The particle growth process is controlled using dual-beam illumination of the nanoparticles, and appears to be driven by surface plasmon excitations. We find that, depending on the illumination wavelengths chosen, the plasmon excitations lead either to fusion of nanoprisms in an edge-selective manner or to the growth of the nanoprisms until they reach their light-controlled final size.

We report the synthesis of Ag nanodisk using polystyrene mesospheres as templates. TEM measurements show that the average disk thickness is 9.0 +/- 1.0 nm and the diameter is 36 +/- 8 nm. The particles exhibit an intense electronic spectrum that differs markedly from that for spheres (purple vs yellow solutions). We present electromagnetic theory calculations which exhibit a qualitative match with the observed spectrum, enabling us to assign several of the plasmon resonance features.

Background: The recent results of clinical islet transplantation have improved substantially with the introduction of a more potent but less diabetogenic immunosuppressant protocol. The successful development of this protocol was based in part on the outcomes of studies reported herein, addressing the diabetogenic potential of a series of immunosuppressant agents used alone or in combination in a canine islet autograft model. Although it is recognized that failure to achieve long-term insulin independence in human islet allotransplantation has been multifactorial, with low engraftment mass, acute or chronic rejection, autoimmune recurrence, loss of islet-acinar integrity, heterotopic site, denervation, and insulin resistance all being implicated to varying degrees, avoidance of diabetogenic immunosuppression has been pivotal to the enhanced outcomes of clinical islet transplantation. We here explore the effects of clinically relevant doses of cyclosporine or tacrolimus when given alone or in combination with glucocorticoids on long-term canine islet autograft function., Method: Dogs (n=8) underwent total pancreatectomy, islet isolation, and intrasplenic autotransplantation and were normoglycemic with stable long-term graft function 3 months to 8 years posttransplant. The frequently sampled intravenous glucose tolerance test (FSIGT) was performed predrug (baseline), at 1 month of therapy (on drug), and again 1 month after withdrawal of therapy (postdrug)., Results: Monotherapy treatments with low- or high-dose prednisone, Neoral, or tacrolimus had minimal impact on islet autograft function. The combination of Neoral and prednisone led to a marked impairment in glucose decay (25% decline from 1.77+/-0.2 to 1.24+/-0.2, P<0.05), without significant change in insulin responsiveness or glucose effectiveness. However, insulin sensitivity was markedly impaired while on therapy (7.10+/-1.2 to 3.10+/-0.5, P<0.01). Importantly, glucose decay and insulin sensitivity failed to return to baseline after withdrawal of therapy. The combination of tacrolimus and glucocorticoids led to permanent and irreversible diabetes in all recipients (n=6, P<0.001). Similar treatment of healthy control dogs led to a 44% decrease in glucose decay (P<0.01)., Conclusions: Immunosuppression must be specifically tailored for islet transplantation and be glucocorticoid free if insulin independence is to be sustained clinically.

Human islet expansion in monolayer culture leads to loss of function and senescence. By maintaining the 3-D configuration of islets in fibrin gels, it is feasible to expand beta-cells in response to hepatocyte growth factor (HGF) while preserving physiologic glucose responsiveness both in vitro and in vivo after transplantation into nude mice. Islets were cultured free floating with or without growth factors and nicotinamide and in fibrin gels with the same conditions. Proliferation was observed only in islets cultured in fibrin gels and the cocktail; total insulin increased by threefold, with a concomitant increase in beta-cell mass by morphometry. Insulin release after glucose challenge was also preserved. Islets in fibrin gels gave rise in vivo to large grafts rich in insulin and glucagon, and grafts from free-floating islets were smaller with fewer endocrine cells. Circulating human C-peptide levels were higher than in the mice receiving free-floating islets. In summary, fibrin allows for HGF-mediated cell proliferation while preserving glucose responsiveness in an environment that preserves cell-cell contacts. Limited islet ex vivo expansion under these conditions may improve recipient-donor tissue ratios to equal the functional results of whole-organ transplants.

The purpose of this study was to determine the potential causes of the lower lipolytic rates in obese Black American women compared to obese Caucasian women. Subcutaneous and omental adipose tissue were obtained from subjects during abdominal surgery, and hormone-sensitive lipase (HSL) mass, mRNA, and activity were determined. HSL mRNA levels did not differ between the Black American and Caucasian women in either subcutaneous or omental adipose tissue. However, HSL mass was approximately 35% lower (P <.05) in both subcutaneous and omental adipose tissue of the Black Americans. Because of these differences, we measured HSL activity in frozen subcutaneous and omental adipose tissue, and also measured basal and isoproterenol-stimulated lipolytic rates in tissue fragments. No racial differences were found in the activity of HSL in either subcutaneous or omental adipose tissue. However, basal lipolytic rates in the Black Americans were 53% and 44% lower (P <.05) in the subcutaneous and omental fat, respectively, compared to the Caucasian women, despite a lack of difference in cell size between the 2 groups. Interestingly, the degree of stimulation by isoproterenol was higher in both the subcutaneous and omental adipose tissue of the Black American than those of the Caucasian women, resulting in equal stimulation by isoproterenol in the 2 groups. These results indicate that despite the lower mass and lower basal HSL activity in the obese Black American women, stimulation of HSL results in equal activity of the enzyme in the 2 races. This suggests that the signaling pathway of HSL stimulation is more efficient in the Black American women., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

Background: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation., Methods: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred., Results: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%., Conclusions: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

ZnSe and Cu-doped ZnSe nanoparticle aqueous suspensions were prepared in the presence of mercaptopropionic acid (MPA). Cu-doped ZnSe nanoparticles exhibited a strong blue emission that was strongly dependent upon the Cu dopant level and the chemical surface passivation produced by zinc-mercaptopropionic acid complexes. These Cu-doped ZnSe nanoparticles were further assembled into ultrathin polymer-supported films using electrostatic interactions and the layer-by-layer assembly method. UV-visible spectroscopy and X-ray photoelectron spectroscopy (XPS) provided evidence for the presence and optical activity of Cu-doped ZnSe nanoparticles within the polymer ultrathin films. Moreover, XPS data supported the presence of zinc mercaptopropionic acid complexes on nanoparticle surfaces and the presence of Cu(+) ions with high luminescent activity in the doped nanoparticles. Copyright 2001 Academic Press.

Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human beta-cells, inducibly transformed pancreatic beta-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of beta-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional beta-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human beta-cells is a major step toward developing a cell transplantation therapy for diabetes.

Because cholesteryl ester transfer protein (CETP) is considered a potential target in the treatment of atherosclerosis, several reports have focused on the regulation of this enzyme, and there is evidence that insulin may be a regulatory factor. The present study examines the differential expression of the human CETP gene between physiologic conditions that are accompanied by low (fasted) and high (fed) insulin levels. CETP expression was examined in plasma and tissues of transgenic mice expressing the human CETP minigene after 12 hours of fasting (n = 20) or ad libitum feeding (n = 20) with normal mouse chow. Plasma cholesteryl ester transfer activity (CETA) was 20% higher in fed than in fasted mice, reflecting higher levels of CETP (P < 0.05). This observation was accompanied by higher liver mRNA in fed mice (100%, P < 0.05), as determined by ribonuclease protection assays, as well as by higher CETA (23%, P < 0.05) and CETP mass (29%, P < 0.05) in the particulate fraction of liver homogenates. These parameters of liver CETP expression correlated well with each other, as well as with plasma CETA. CETP in the liver particulate fraction was found as a doublet (approximately 70 and 65 kDa), which resolved to a single band (approximately 60 kDa) upon deglycosylation. No differences in CETP expression were observed in pooled adipose tissue samples from fed and fasted mice. Insulin and glucose were not related to any plasma or tissue parameter of CETP expression. In summary, the concerted, differential expression of CETP in the liver of fed and fasted transgenic mice appears to contribute to higher plasma CETP levels in fed mice, but the precise role of insulin and glucose in regulating CETP expression under fasted and fed conditions needs to be defined.

The synthesis and photophysical characterization of ZnS:Ag colloid are reported. The presence of mercaptoacetic acid has an important effect not only on the formation of doped and undoped ZnS but also on the photophysical properties. ZnS colloid doped with silver shows a strong green emission upon ultraviolet excitation, the intensity of which was enhanced significantly compared with that of the undoped colloid. The green emission was ascribed to a transition from a donor level such as anion vacancy to the levels of the Ag impurities. Copyright 1998 Academic Press.

Objective: To measure cystic fibrosis transmembrane regulator (CFTR) gene expression in cervical secretions during the menstrual cycle., Design: Prospective, descriptive clinical study., Patients: Thirteen healthy women with ovulatory menstrual cycles., Interventions: Endocervical cells and secretions were obtained by cytobrushings during the midfollicular, midcycle, and luteal phases. The cells were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for CFTR and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression., Main Outcome Measures: Detection of CFTR gene expression and a quantitative comparison of the levels of CFTR to GAPDH gene expression., Results: All endocervical samples exhibited some degree of CFTR gene expression throughout the menstrual cycle; however, the levels of expression were variable. Cystic fibrosis transmembrane regulator gene expression did not correlate with E2 or P levels., Conclusion: The production of copious cervical secretions at the time of ovulation in part may be because of the transport of sodium and water across endocervical cell membranes as a result of E2-stimulated CFTR mRNA and protein. Although cervical mucus becomes thick and scant during the luteal phase, CFTR gene expression is present in these secretions.

Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observe 'blinded' to the method of isolation used, and only intact islets of diameter > or = 100 microns were included. Stationary gave 719 +/- 114 islets per pancreas (mean +/- SD, n = 21 isolations) vs. 487.5 +/- 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (< or = 8 mmol/L) by 3.3 +/- 4.8 days vs. 1.6 +/- 1.5 days for Chopped recipients (p = 0.4 ns, mean +/- SEM). IVGTT performed at 1 mo posttransplant gave K-values for Stationary of 2.64 +/- 0.8 vs. 2.62 +/- 0.8 for Chopped (mean +/- SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 +/- 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.

We have surveyed a population size of 6633315 from Diseases Surveillance Points (DSP) system in Gansu province for the last eleven years. The annual birth rate was 18.20% with an annual standard mortality rate 545.80/10(5). The annual standard mortality for male and female were 607.53/10(5) and 483.29/10(5) respectively. The major causes of death were Respiratory system diseases, Cardiovascular diseases, Neoplasms, Injuries, Digestive system diseases, Pediatric diseases, Infectious diseases in sequence. In eleven years, there seemed to be a rising trend in the mortalities of following diseases as: Cerebrovascular diseases, Ischemic heart diseases, Rheumatic fever and heart disease, Lung Cancer, Liver Cancer, Cancer of the Esophagus, Intestinal cancer, Cervical cancer, Injury, Congenital abnomalities, to different degrees. However, an obvious descending trend on the morbidity and mortality of infectious diseases was moticed. The average life expectancy was 71.05 years in DSP, with male 69.57 years, and female 72.72 years. Diseases with higher PYLL were Injuries, Neoplasms, Respiratory system diseases and the like. Data suggested not only the prevention andcontrol of infectious diseases, but also the surveillance of injuries and the prevention and control of chronic diseases should be strengthened.

Syndromes of resistance to thyroid hormones are caused by mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. The S receptor (deletion of THR332) is a potent dominant negative protein cloned from a kindred with generalized resistance to thyroid hormones. The G-H receptor (ARG311HIS) has compromised dominant negative function and was found in both normal individuals and in a patient with severe pituitary resistance to thyroid hormones. We have investigated the mechanism responsible for the difference in receptor phenotypes by analyzing the binding of S and G-H receptors to thyroid hormone response elements with electrophoretic mobility shift analysis. Wild-type human c-erbA beta 1 (WT), S, and G-H receptors were synthesized in reticulocyte lysate, reacted with a thyroid hormone response element consisting of a direct repeat with 4 base pairs (DR+4; AGGTCA CAGG AGGTCA), and the products analyzed by gel shift. G-H receptor homodimerization was greatly impaired; G-H formed predominantly monomeric complex compared with monomeric and homodimeric WT complexes. The G-H receptor was able to form heterodimeric complexes with cellular thyroid hormone receptor auxiliary protein (TRAP) factors including the human retinoid X receptor-alpha. When TRAP was limiting, the levels of G-H heterodimeric complex were 2- to 3-fold reduced compared with WT receptor. In contrast to the WT and G-H receptors, the S receptor formed almost exclusively homodimeric complex with DR+4; the approximate ratio of S:WT:G-H homodimeric complexes at equivalent concentrations of receptors was 60:20:1. A measurable increase (1.2- to 2.6-fold) in heterodimeric complex formation was observed with the S receptor relative to WT when TRAP was at limiting concentration. As reported previously by others, thyroid hormone significantly reduced the WT homodimeric complex with DR+4. There was no effect on the S homodimeric complex. Finally, the WT, S, and G-H receptors formed different complexes with the element consisting of an inverted repeat with 5 base pairs (IR+5; AGGTCA ACAGT TGACCT) and the IR element (AGGTCA TGACCT), which were differently regulated by thyroid hormone. The S receptor bound as a homodimer with IR+5, whereas the WT receptor bound as a homodimer only with thyroid hormone. No homodimeric complex formed with IR+5 and the G-H receptor. Qualitatively similar results were observed with the IR element. We conclude that the ARG311HIS mutation severely perturbs the homodimerization and, to a much less degree, heterodimerization functions of the c-erbA beta 1 receptor. Furthermore, the THR332 deletion mutation augments homodimerization of the c-erbA beta 1 receptor. These results indicate that different mutations in the c-erbA beta 1 thyroid hormone receptor have divergently affected dimerization activities which seem to influence the level of dominant negative activity in man.

Immediate and sustained signal transduction is involved in mediating phorbol ester-induced changes in growth and differentiation. Activation of protein kinase C (PKC) is the initial step in phorbol ester-induced signal transduction. By virtue of preferential down-regulation of individual isoforms and generation of proteolytically derived kinase activities, the signal transduced by sustained activation of this pathway may differ substantially from that generated initially upon application of the phorbol ester. To examine the effect of chronic phorbol ester-induced activation of this pathway, the relationship between PKC activity/content and AP-1 binding activity and gene expression was studied in the U937 cell. Phorbol ester-induced differentiation of the U937 cell into a monocyte/macrophage-like cell requires sustained activation of the PKC pathway. AP-1 binding activity was enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and in a temporally dependent manner, with conversion of a high to low mobility band shift occurring after a 12-h exposure to TPA. After a 72-h exposure, AP-1 binding activity was maximally increased by 1 nM TPA and remained elevated to a similar degree even after treatment with 600 nM TPA. Enhanced AP-1 binding activity was dependent upon continuous exposure to TPA and was not secondary to differentiation. A 72-h treatment with one nM TPA maximally increased expression of c-jun, krox-24, and jun-B mRNA transcripts. Exposure to higher TPA concentrations decreased the content of these transcripts. Maximal expression of collagenase and plasminogen activator receptor transcripts required exposure to much higher TPA concentrations (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

C-erbA receptors and v-erbA have been shown to functionally interact with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-inducible gene expression. These proteins enhance trans-activation by c-jun, and the c-erbA receptors in the presence of thyroid hormone repress TPA and c-jun induction of transcription. Also, v-erbA can abrogate T3-mediated repression. We have examined how dominant negative (S and CL) and nondominant negative (G-H) receptors cloned from various patients with thyroid hormone resistance syndromes affect expression of the collagenase promoter induced with TPA. The CL receptor (ARG315HIS mutation) has a 2-fold reduction in T3-binding affinity compared with human c-erbA beta 1 wild-type (WT) receptor, whereas the G-H receptor (ARG311HIS) and S receptor (deletion, THR codon 332) have T3-binding affinities reduced by 100-fold and greater than 100-fold, respectively. These mutant receptors were cotransfected with a collagenase promoter (-1200 to +63 base pairs) chloramphenicol acetyltransferase reporter gene (Col-CAT) into COS-7 cells. Levels of CAT reporter gene expression after transient transfection were determined in the presence or absence of 3-10 nM T3 and the presence or absence of 100 nM TPA. Unoccupied CL receptor and G-H and S receptors stimulated TPA-induced Col-CAT expression 1.5- to 9-fold. The CL receptor with thyroid hormone totally repressed TPA induction of the collagenase receptor. In the presence of thyroid hormone, the enhancing effects by S and G-H receptors on TPA-induced Col-CAT expression were unaffected and minimally diminished, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the c-erbA beta thyroid hormone receptor gene in a kindred, G.H., with a member, patient G.H., who had a severe form of selective pituitary resistance to thyroid hormones (PRTH). This patient manifested inappropriately normal thyrotropin-stimulating hormone, markedly elevated serum free thyroxine (T4) and total triiodothyronine (T3), and clinical hyperthyroidism. The complete c-erbA beta 1 coding sequence was examined by a combination of genomic and cDNA cloning for patient G.H. and her unaffected father. A single mutation, a guanine to adenine transition at nucleotide 1,232, was found in one allele of both these members, altering codon 311 from arginine to histidine. In addition, a half-sister of patient G.H. also harbored this mutant allele and, like the father, was clinically normal. The G.H. receptor, synthesized with reticulocyte lysate, had significantly defective T3-binding activity with a Ka of approximately 5 x 10(8) M-1. RNA phenotyping using leukocytes and fibroblasts demonstrated an equal level of expression of wild-type and mutant alleles in patient G.H. and her unaffected father. Finally, the G.H. receptor had no detectable dominant negative activity in a transfection assay. Thus, in contrast to the many other beta-receptor mutants responsible for the generalized form of thyroid hormone resistance, the G.H. receptor appeared unable to antagonize normal receptor function. These results suggest that the arginine at codon 311 in c-erbA beta is crucial for the structural integrity required for dominant negative function. The ARG-311-HIS mutation may contribute to PRTH in patient G.H. by inactivating a beta-receptor allele, but it cannot be the sole cause of the disease.

The present report studies a large kindred (WR) with generalized thyroid hormone resistance that has varying degrees of neuropsychological dysfunction, hyperactivity, poor attention span, decreased IQ and/or abnormalities in spatial perception. In this kindred, there has been found tight linkage of the syndrome with the c-erb A beta gene. The present study was performed to identify the presence of a possible gene mutation as a cause for this syndrome. DNA from peripheral leukocytes was isolated from 15 unaffected and 8 affected individuals from the kindred. Primers encompassing exons 9 (nucleotides 1171-1429) and 10 (nucleotides 1430-1698) were synthesized and used in PCR reactions to amplify these exons. Direct sequencing revealed a consistent substitution in each affected subject, but in none of the unaffected individuals, of a C to T change in one allele from nucleotide 1243, resulting in an arg to cys change in codon 315. The mutant and wild-type human beta 1 receptors were prepared and their translated proteins were analyzed for T3 binding. The WR T3 receptor from affected patients had reduced T3 binding affinity, with values approximately 2.5 x 10(10) M-1 compared to about 5 x 10(10) M-1 in normals. In summary, we have: i) identified a consistent and reproducible mutation of a C to T change in nucleotide 1243 in each of the affected but in none of the unaffected individuals of a large well characterized kindred with generalized thyroid hormone resistance; and ii) noted that the WR allele causes an approximate 50% decrease in the T3 binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Generalized resistance to thyroid hormones results from diverse mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor, and different kindreds have variable phenotypes. However, the T3-binding affinities of these mutant receptors studied in vitro have all been severely reduced compared to wild type. We report here a new kindred, CL, with a mutation further upstream than previously reported, a guanine to adenine base substitution at nucleotide 1244 in codon 315 changing an arginine to histidine. This base substitution was the only one found in codons 90-456 of genomic sequence and was formally shown to be a mutation by screening 51 random individuals. The kindred CL receptor complementary DNA was recreated, and the mutant receptor synthesized with rabbit reticulocyte lysate had a T3-binding affinity of 2.4 +/- 0.9 x 10(10) M-1 compared to the wild-type human placental receptor affinity of 5.2 +/- 1.6 x 10(10) M-1. Affected members of this kindred appeared clinically to have a relatively mild degree of resistance with mean total thyroxine of only 192 +/- 24 nmol/L and inappropriately normal TSH levels. Kindred CL is an example of mild generalized resistance to thyroid hormones correlated with a mutation in the beta-receptor that resulted in only a modest deficiency in T3-binding activity.

Mutations in the gene encoding the human beta 1 T3 receptor (hTR beta 1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR beta 1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T3 affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T3 affinity required 5- and 15-fold higher T3 concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTR beta 1s were able to inhibit the function of transfected normal hTR beta 1 and endogenous retinoic acid receptor in activating a palindromic positive T3 response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T3 concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTR beta 1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTR beta 1 was assessed by immunocytochemistry. The hTR beta 1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells. These data suggest that different mutations in the ligand-binding domain of the human hTR beta 1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T3 receptor dimers.

GH-releasing peptide (GHRP; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2), a hexapeptide derived from enkephalin, has been shown to have GH-releasing activity in man and several animal species. To characterize the GHRP dose-response curve and compare it with that of GH-releasing hormone [GHRH-(1-44)NH2], six unanesthetized young adult cynomolgus macaques were tested with a range of iv doses of GHRP or GHRH in random order. Animals were fitted with vests and tethers. Blood samples were obtained before and at 15-min intervals after the administration of drugs. Doses ranged from 0.03-3 mg/kg for GHRP and from 1-30 micrograms/kg for GHRH. The dose-response curves for the two peptides were not parallel. GHRP had lower potency, but evoked a much higher peak GH response than GHRH (greater than 55 vs. 12 micrograms/L). Because one of the proposed mechanisms of action of GHRP is the inhibition of somatostatin (SS), we tested the effects of propranolol, which inhibits SS, on the GH responses to GHRH and GHRP. Propranolol was given at a dose of 14 micrograms/kg, iv, 10 min before the injection of saline, GHRH (10 micrograms/kg), or GHRP (1 mg/kg). GH responses to propranolol alone did not differ from those to placebo (peak, 6 +/- 2 vs. 8 +/- 2 micrograms/L). However, propranolol pretreatment doubled the GH responses to both GHRH and GHRP compared with those to GHRH or GHRP alone 28 +/- 5 micrograms/L vs. 14 +/- 5 (P less than 0.05) and 54 +/- 2 vs. 25 +/- 6 micrograms/L (P less than 0.001), respectively]. These results show that GHRP causes a potent dose-dependent release of GH in this primate species. Since GHRP can produce a greater maximal GH response than GHRH, mechanisms other than release of endogenous GHRH must be involved.

To define the role of noradrenergic regulation of growth hormone (GH) secretion in a primate species, spontaneous and GH-releasing hormone (GHRH) stimulated GH secretion was studied in 6 chronically catheterized adult male cynomolgus monkeys before and after inhibition of norepinephrine synthesis. Blood samples were obtained at 15-min intervals during an 8-hour period to characterize the pattern of GH secretion, and the GH response to GHRH, 10 micrograms/kg i.v., was determined. These measurements were repeated 2 weeks later, 2 h after the intravenous administration of 12.5 mg/kg of the dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC), which has been shown to be an effective norepinephrine synthesis inhibitor in the rat. Spontaneous and stimulated GH secretory patterns before and after DDTC administration were compared. Both the frequency and the amplitude of spontaneous GH pulses were markedly reduced by DDTC [3.8 +/- 0.4 before vs. 1.8 +/- 0.4 peaks/8 h after DDTC (p less than 0.005) and 5.5 +/- 0.7 vs. 2.0 +/- 0.6 ng/ml (p less than 0.01)]. Areas under the curve were also reduced by DDTC treatment [10.8 +/- 1.0 vs. 5.7 +/- 1.1 ng.h/ml (p less than 0.01)], and DDTC administration diminished the peak GH responses to GHRH [12 +/- 6 vs. 4 +/- 2 ng/ml (p less than 0.05)]. These results are consistent with the belief that DDTC is a potent inhibitor of spontaneous and GHRH-induced GH secretion. The action of DDTC could be mediated by a reduction in GHRH due to reduced norepinephrine synthesis, by an increase in somatostatin release through a dopaminergic stimulus, or by a direct dopaminergic effect on somatotrophs.