Connexins and cyclooxygenase-2 crosstalk in the expression of radiation-induced bystander effects.

BACKGROUND: Signalling events mediated by connexins and cyclooxygenase-2 (COX-2) have important roles in bystander effects induced by ionising radiation. However, whether these proteins mediate bystander effects independently or cooperatively has not been investigated. METHODS: Bystander normal human fibroblasts were cocultured with irradiated adenocarcinoma HeLa cells in which specific connexins (Cx) are expressed in the absence of endogenous Cx, before and after COX-2 knockdown, to investigate DNA damage in bystander cells and their progeny. RESULTS: Inducible expression of gap junctions composed of connexin26 (Cx26) in irradiated HeLa cells enhanced the induction of micronuclei in bystander cells (P<0.01) and reduced the coculture time necessary for manifestation of the effect. In contrast, expression of connexin32 (Cx32) conferred protective effects. COX-2 knockdown in irradiated HeLa Cx26 cells attenuated the bystander response due to connexin expression. However, COX-2 knockdown resulted in enhanced micronucleus formation in the progeny of the bystander cells (P<0.001). COX-2 knockdown delayed junctional communication in HeLa Cx26 cells, and reduced, in the plasma membrane, the physical interaction of Cx26 with MAPKKK, a controller of the MAPK pathway that regulates COX-2 and connexin. CONCLUSIONS: Junctional communication and COX-2 cooperatively mediate the propagation of radiation-induced non-targeted effects. Characterising the mediating events affected by both mechanisms may lead to new approaches that mitigate secondary debilitating effects of cancer radiotherapy.

Male pseudohermaphroditism due to a homozygous missense mutation of the luteinizing hormone receptor gene.

Leydig cell hypoplasia is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals. We have studied two Leydig cell hypoplasia patients (siblings born to consanguineous parents), and found them to be homozygous for a missense mutation (Ala593Pro) in the sixth transmembrane domain of the luteinizing hormone (LH) receptor gene. In vitro expression studies showed that this mutated receptor binds human choriogonadotropin with a normal KD, but the ligand binding does not result in increased production of cAMP. We conclude that a homozygous LH receptor gene mutation underlies the syndrome of autosomal recessive congenital Leydig cell hypoplasia in this family. These results have implications for the understanding of the development of the male genitalia.

HLA molecular study of patients in a public kidney transplant program in Guatemala.

Guatemala is a country located in Central America, and while it is one of the most populated countries in the region, the genetic diversity of the population has been poorly analyzed. Currently, there are no analyses of the distribution of human leukocyte antigen (HLA) system alleles in mixed ancestry (i.e., ladino) populations in Guatemala. The HLA system exhibits the most extensive polymorphism in the human genome and has been extensively analyzed in a large number of studies related to disease association, transplantation, and population genetics (with particular importance in the understanding of diversity in the human population). Here, we present HLA typing data from 127 samples of unrelated individuals from the kidney transplant program of the San Juan de Dios General Hospital (Guatemala City) using a PCR-SSOP-based (PCR-sequence specific oligonucleotide probes) typing method. We found 16 haplotypes that accounted for 39.76 % of the total haplotype diversity, of which thirteen have been reported previously in Native American populations and three have been reported in European populations. The analyses showed no deviations from Hardy-Weinberg equilibrium, and admixture estimates calculated with k = 3 ancestral components showed that Native American was the most represented component, followed by the European component. The African component was less prominent in the Guatemala mixed ancestry sample in comparison to samples from other countries in Central America. The HLA-based admixture results for Central America showed a continuum in the distribution of Native American, European and African ancestries throughout the region, which is consistent with the complex demographic history of the region.

Molecular profiling of osteosarcoma in children and adolescents from different age groups using a next-generation sequencing panel.

Osteosarcoma (OS) is a malignant bone tumor, with a peak of incidence in the second decade of life and possibly associated with the presence of germline mutations. Besides, clinicians have pointed to a second, rarer group of patients that develops OS before 10 years old. Here we access, through next-generation sequencing (NGS) strategy, the genetic alterations present in OS and blood samples from patients diagnosed before and during the second decade of life. A custom NGS panel, designed for the main alterations described in childhood and adolescence neoplasms, named Oncomine Childhood Cancer Research Assay (OCCRA©), was used. Of all 84 OS samples investigated, 42 (50%) presented some somatic variant, with TP53, MYC, CDK4, RB1 and PDGFRA genes harboring the most observed genetic variants. MYC CNVs were more frequent in tumors from patients diagnosed before 10 years old (X21= 5.18, p = 0.023). Additionally, patients diagnosed during the second decade of life presented a higher percentage of somatic and germline variants. Germline variants in TP53 and RB1 were found in 5 of the 11 (45.5%) patients analyzed. Clinical variables and tumor histopathological characteristics were also collected and correlated with our molecular findings.

The influence of thyroid hormones on periodontitis-related bone loss and tooth-supporting alveolar bone: a histological study in rats.

BACKGROUND AND OBJECTIVE: Recent studies have pointed to potentially periodontal risk indicators, however no information is available on the impact of changes in thyroid hormone levels on the progression of periodontitis and on the quality of alveolar bone. Thus, the aim of the present study was to evaluate histologically, in rats, the influence of thyroid hormones on the rate of periodontal bone loss resulting from ligature placement and on the quality of tooth-supporting alveolar bone. MATERIAL AND METHODS: Thirty-six male Wistar rats were randomly assigned to the following groups: healthy (control, n = 12), hypothyroidism (n = 12) and hyperthyroidism (n = 12). Once alterations were confirmed by total serum levels of triiodothyronine and thyroxine, ligatures were randomly placed around one of the first mandibular molars. Thirty days later, the animals were killed and specimens routinely processed for serial decalcified sections. The parameters assessed were periodontitis-related bone loss, quality of tooth-supporting alveolar bone and the number of cells positive for tartrate-resistant acid phosphatase (TRAP), a marker of bone resorption. RESULTS: At the ligated sites, intergroup analysis revealed that hypothyroidism significantly increased the bone loss resulting from ligature-induced periodontitis (p = 0.02) and the number of TRAP-positive cells on the linear surface of bone crest (p = 0.01). In addition, no significant differences were detected regarding the quality of the bone (p = 0.24) or the number of TRAP-positive cells in the area of the interradicular bone for ligated teeth among the groups (p = 0.17). CONCLUSION: It may be concluded that decreased serum levels of thyroid hormones may enhance periodontitis-related bone loss, as a function of an increased number of resorbing cells, whereas the tooth-supporting alveolar bone seems to be less sensitive to alterations in hormone levels.

Association of EGFR c.2073A>T polymorphism with decreased risk of diffusely infiltrating astrocytoma in a Brazilian case-control study.

Epidermal growth factor receptor (EGFR) gene overexpression has been implicated in the development of many types of tumors, including glioblastomas, the most frequent diffusely infiltrating astrocytomas. However, little is known about the influence of the polymorphisms of EGFR on EGFR production and/or activity, possibly modulating the susceptibility to astrocytomas. This study aimed to examine the association of two EGFR promoter polymorphisms (c.-191C>A and c.-216G>T) and the c.2073A>T polymorphism located in exon 16 with susceptibility to astrocytomas, EGFR gene expression and survival in a case-control study of 193 astrocytoma patients and 200 cancer-free controls. We found that the variant TT genotype of the EGFR c.2073A>T polymorphism was associated with a significantly decreased risk of astrocytoma when compared with the AA genotype [sex- and age-adjusted odds ratio 0.51, 95% confidence interval 0.26-0.98]. No association of the two promoter EGFR polymorphisms (or combinations of these polymorphisms) and risk of astrocytomas, EGFR expression or survival was found. Our findings suggest that modulation of the EGFR c.2073A>T polymorphism could play a role in future therapeutic approaches to astrocytoma.

High glucose induces DNA damage in cultured human endothelial cells.

Morphologic and functional abnormalities of vascular endothelium are well recognized in diabetes. In view of our previous finding that high glucose concentrations accelerate death and hamper replication of cultured human endothelial cells, we have investigated in the same model the possibility that exposure to high glucose may result in DNA damage. DNA from human endothelial cells--but not from fibroblasts--exposed to 30 mM glucose for 9-14 d manifested an accelerated rate of unwinding in alkali indicative of an increased number of single strand breaks (P less than 0.001 vs. control). Endothelial cells exposed to high glucose also manifested an increased amount of hydroxy-urea-resistant thymidine incorporation (333 +/- 153 cpm/10(5) cells vs. 88 +/- 42 in control cells, mean +/- SD, P = 0.04), which is indicative of increased DNA repair synthesis. Neither DNA damage nor repair synthesis were increased by medium hypertonicity achieved with 30 mM mannitol. These findings suggest the possibility that, under conditions of high ambient glucose, excess glucose entry in cells that are insulin independent for glucose transport may, directly or indirectly, perturb DNA function. Further, they suggest the possibility that different individual capabilities to repair DNA damage--a process that is under genetic control--may represent a mechanism for different individual susceptibilities to development of diabetic vascular complication.

Increased single strand breaks in DNA of lymphocytes from diabetic subjects.

Certain aspects of the chronic complications of diabetes suggest that, with time, the abnormal metabolic milieu leads to irreversible changes in some cell populations. Since we have previously observed that high glucose concentrations induce an increase in single strand breaks in the DNA of cultured human endothelial cells, we have investigated whether the same abnormality occurs in cells derived from the in vivo diabetic environment. Peripheral blood lymphocytes obtained from 21 type I diabetic patients and age- and sex-matched controls were tested for rate of unwinding in alkali (a measure of DNA single strand breaks). The patients were subdivided into two groups on the basis of glycohemoglobin values above or below 9%. The group with glycohemoglobin values of 12.9 +/- 2.4% (mean +/- SD), but not the group with glycohemoglobin values of 7.4 +/- 1.5%, showed accelerated unwinding of lymphocyte DNA when compared to controls (P less than 0.01). These studies suggest that poorly controlled diabetes may result in DNA lesions, whose impact on long-term complications deserves to be investigated.

Mutation analysis of glial cell line-derived neurotrophic factor (GDNF), a ligand for the RET/GDNF receptor alpha complex, in sporadic phaeochromocytomas.

Phaeochromocytomas usually occur sporadically but may also be a feature of three autosomal dominantly inherited cancer syndromes, multiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and, very rarely, type 1 neurofibromatosis. Germ-line missense mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2. In VHL, germ-line mutations in one of the three exons of the VHL tumor suppressor gene have been found in the majority of families. Whereas somatic mutations in the VHL gene have been common in sporadic renal cell carcinoma, a component cancer of VHL, somatic mutations in the RET and VHL genes together have been found in approximately 10% of sporadic phaeochromocytomas. Hence, other genes must also contribute to the pathogenesis of sporadic phaeochromocytomas. Recent data have suggested that glial cell line-derived neurotrophic factor (GDNF) is a ligand for RET and acts via a heterotetrameric receptor complex that includes GDNF receptor alpha, which provides ligand binding capabilities, and RET, which provides the signaling component. Thus, both GDNF and GDNFR-alpha are plausible candidate genes for involvement in the pathogenesis of phaeochromocytomas. To investigate the role of GDNF in sporadic phaeochromocytomas, we scanned a panel of 22 tumors. Among these samples, only a conservative sequence variant was detected in exon 2 of GDNF. No disease-associated somatic GDNF mutations or gross gene amplification were detected in these tumors, suggesting only a minor role for GDNF in the genesis of phaeochromocytomas.

High and low fluences of alpha-particles induce a G1 checkpoint in human diploid fibroblasts.

The effects of exposure to high and very low fluence alpha-particles on the G1 checkpoint were investigated in human diploid fibroblasts irradiated and released from density-inhibited confluent cultures by the use of the cumulative labeling index method. Transient and permanent arrests in G1 occurred in fibroblast populations exposed to mean doses as low as 1 cGy, suggesting that nontraversed bystander cells may contribute to the low dose response. In cells exposed to high fluences, the G1 checkpoint is at least as extensive as in gamma-irradiated cells. In contrast to gamma-irradiated cells, neither repair of potentially lethal damage nor a reduction in the fraction of cells transiently or permanently arrested in G1 were observed in cells held in confluence for 6 h after alpha-particle irradiation. Studies with isogenic wild-type, p53-/-, and p21Waf1-/- mouse embryo fibroblasts exposed to either gamma or alpha-particle radiation revealed a total lack of G1 arrest in either p53-/- or p21waf1-/- cells, indicating that the G1 checkpoint in wild-type cells is p53-dependent and that p21Wf1 fully mediates the role of p53 in its induction. In contrast to human cells, mouse embryo fibroblasts do not undergo a permanent G1 arrest. Except under conditions favoring potentially lethal damage repair, a comparable expression pattern of p53, p21Waf1, and other cell cycle-regulated proteins (pRb, p34cdc2, and cyclin B1) was observed in alpha-particle or gamma-irradiated human fibroblasts.

Curva de aprendizaje para el diagnóstico ecográfico específico de masas anexiales / Learning curve for the specific ultrasound diagnosis of adnexal masses

OBJETIVO: Determinar el tiempo que requiere una curva de aprendizaje para diagnóstico ecográfico específico histopatológico en masas anexiales basándonos en cálculos estadísticos no influidos por la prevalencia según diferentes grados de experiencia. MÉTODOS: Estudio observacional, descriptivo, transversal. Se estudiaron imágenes de 108 masas anexiales. La prueba estándar de oro fue el reporte histopatológico definitivo. Se comparó el rendimiento diagnóstico de 4 examinadores con la siguiente experiencia en diagnóstico ecográfico de patología anexial: A > 20 años, B ≤ 20 hasta > 10 años, C ≤ 10 hasta > 5 años y D ≤ 5 años, analizando solo imágenes y sin datos clínicos de las pacientes, para emitir un diagnóstico específico a libre escritura. RESULTADOS: Prevalencia de masas malignas 17,2 % (15/87). Nivel de confianza en los examinadores se consideró según falta de respuesta diagnóstica: alto (<6 %) con experiencia de más de 10 años y moderado a bajo ≤ 10 años. Examinadores con más de 5 años siempre mostraron likelihood ratio positivo mayor a 10, exactitud diagnóstica mayor a 90 % y Odds ratio diagnóstica mayor a 46, no así para examinador con menor tiempo de experiencia, quién presentó resultados con mala utilidad clínica. El cambio de probabilidad de acierto específico pre-test a post-test mejoró consistentemente con los años de experiencia. CONCLUSIÓN: Se necesitarían más de 10 años de experiencia con especial dedicación a ecografía ginecológica avanzada para un rendimiento diagnóstico específico deseado junto con alta confianza en ecografía de masas anexiales.

OBJECTIVE: To determine the time required for a learning curve of histopathological specific ultrasound diagnosis in adnexal masses based on statistical calculations not influenced by prevalence according to different degrees of experience. METHODS: Observational, descriptive, cross-sectional study. Images of 108 adnexal masses were studied. The gold standard test was the definitive histopathological report. The diagnostic performance of 4 examiners with the following experience in ultrasound diagnosis of adnexal pathology: A > 20 years, B ≤ 20 to > 10 years, C ≤ 10 to > 5 years and D ≤ 5 years was compared, analyzing only images and blinded of clinical data of the patients, to issue a specific diagnosis with free writing. RESULTS: Prevalence of malignant masses 17.2% (15/87). The level of confidence in the examiners was considered according to the lack of diagnostic response: high (<6%) with experience of more than 10 years and moderate to low ≤ 10 years. The examiners with more than 5 years always showed likelihood ratio positive greater than 10, diagnostic accuracy greater than 90% and diagnostic Odds ratio greater than 46, not so for the examiner with less experience time who presented results with little clinical utility. The change in specific probability from pre-test to post-test improved consistently with years of experience. CONCLUSION: More than 10 years of experience with special dedication to advanced gynecological ultrasound are probably needed for a desired specific diagnostic performance coupled with high confidence in adnexal mass ultrasound.

ATM complexes with HDM2 and promotes its rapid phosphorylation in a p53-independent manner in normal and tumor human cells exposed to ionizing radiation.

To further understand the mechanism(s) by which DNA damage activates p53, we analysed the expression levels of p53 and HDM2 (the human homolog of murine MDM2) in various human diploid fibroblast and tumor cell strains during the period that precedes activation of known downstream effectors of p53. In X-irradiated human cells, HDM2 protein was rapidly phosphorylated in serine/threonine residues in a p53, p14ARF and p73-independent manner. In p53 wild-type cells, HDM2 phosphorylation precedes a detectable increase in the levels of p53 and is not observed in ataxia telangiectasia (AT) fibroblasts. The transfection of AT cells with a vector expressing ATM restored the ability to rapidly phosphorylate HDM2 following X-irradiation, confirming a role for ATM in its phosphorylation. We also show that ATM complexes with HDM2. The DNA lesions signaling the early rapid phosphorylation of HDM2 are a result of X-ray and not UV-type damage. The ATM-promoted early covalent modification of HDM2 in X-irradiated human cells may provide a mechanism to activate p53.

Glucose toxicity for human endothelial cells in culture. Delayed replication, disturbed cell cycle, and accelerated death.

Functional and anatomical abnormalities of endothelium may represent a pathway to the increased vascular permeability and accelerated atherosclerosis characteristic of diabetes. To identify whether and how hyperglycemia may compromise the endothelial barrier, we have employed an in vitro system of human endothelial cells obtained from umbilical veins and cultured in elevated glucose concentrations (20 mM). Under these conditions, the achievement of saturation density was substantially delayed, with cell counts throughout most of the growth curve being 70-80% of control (P less than 0.002). More profound suppression of cell number was present in cultures exposed to 40 mM glucose. Similar, albeit slightly lesser, effects were observed in cultures exposed to 20 mM mannitol, mimicking the hypertonicity of the high glucose media. The effect of elevated glucose and mannitol was primarily mediated by a decrease in overall rate of replication of the endothelium as documented by the lower mitotic index (P less than 0.025). Analysis of the distribution of cells along phases of the cell cycle uncovered in the high glucose cultures a decreased proportion of cells in G0-G1 (70.5 +/- 5% versus 73.2 +/- 4% in controls, P less than 0.05) and an increased proportion of cells in S phase (16.5 +/- 2.7% versus 13.5 +/- 2.2% in controls, P less than 0.01), suggesting that the replicative delay is likely to occur between the phase of DNA synthesis and mitosis. Increased cellular death was specifically observed in the cultures exposed to elevated glucose concentrations (P less than 0.05), but it could account for only a minor portion of the deficit in cell number.(ABSTRACT TRUNCATED AT 250 WORDS)

High glucose prolongs cell-cycle traversal of cultured human endothelial cells.

There is evidence suggesting that the diabetic state adversely affects replication of certain cell populations. We document that exposure to high ambient glucose (20 mM) induces delay in various phases of the cell cycle of human endothelial cells in primary culture. Cells in S phase were labeled with bromodeoxyuridine (an analogue of thymidine), and the cell-cycle position of the labeled cohort was analyzed by flow cytometry at successive time points. The movement of cells exposed to high glucose for 7-8 days was retarded both in S and G2 phases so that the increase in bromodeoxyuridine-positive cells over 24 h was 1.6-fold, versus 2.0-fold in control cultures. In experiments in which mitotic arrest with vinblastine was used to investigate the movement of cells out of G1 phase without interference from reentering cells, depletion of the G1 compartment was significantly inhibited in cultures grown in high glucose. The effects of chronic high glucose on cell cycle occurred while total protein synthesis was not diminished. Acute exposure to high glucose had no effect on cell-cycle traversal or cell generation time. Cell-cycle abnormalities observed in this study may relate to the DNA damage we have previously observed in endothelial cells exposed to high glucose and, if occurring in vivo, could be of pathogenetic importance for the vascular lesions and teratogenicity of diabetes.

A homozygous mutation in the luteinizing hormone receptor causes partial Leydig cell hypoplasia: correlation between receptor activity and phenotype.

Leydig cell hypoplasia (LCH) is characterized by a decreased response of the Leydig cells to LH. As a result, patients with this syndrome display aberrant male development ranging from complete pseudohermaphroditism to males with micropenis but with otherwise normal sex characteristics. We have evaluated three brothers with a mild form of LCH. Analysis of their LH receptor (LHR) gene revealed a homozygous missense mutation resulting in a substitution of a lysine residue for a isoleucine residue at position 625 of the receptor. In vitro analysis of this mutant LHR, LHR(I625K), in HEK293 cells indicated that the signaling efficiency was significantly impaired, which explains the partial phenotype. We have compared this mutant LHR to two other mutant LHRs, LHR(A593P) and LHR(S616Y), identified in a complete and partial LCH patient, respectively. Although the ligand-binding affinity for all three mutant receptors was normal, the hormonal response of LHR(A593P) was completely absent and that of LHR(S616Y) and LHR(I625K) was severely impaired. Low cell surface expression explained the reduced response of LHR(S616Y), while for LHR(I625K) this diminished response was due to a combination of low cell surface expression and decreased coupling efficiency. For LHR(A593P), the absence of a reduced response resulted from both poor cell surface expression and a complete deficiency in coupling. Our experiments further show a clear correlation between the severity of the clinical phenotype of patients and overall receptor signal capacity, which is a combination of cell surface expression and coupling efficiency.

Stress signaling from irradiated to non-irradiated cells.

Evidence accumulated over the past two decades has indicated that exposure of cell populations to ionizing radiation results in significant biological effects occurring in both the irradiated and non-irradiated cells in the population. This phenomenon, termed the "bystander response", has been shown to occur both in vitro and in vivo. Experiments have indicated that genetic alterations, changes in gene expression and lethality occur in bystander cells that neighbor directly irradiated cells. Furthermore, cells recipient of growth medium harvested from irradiated cultures exhibit responses similar to those of the irradiated cells. Several mechanisms involving secreted soluble factors, gap-junction intercellular communication and oxidative metabolism have been proposed to regulate the radiation-induced bystander effect. In this review, our current knowledge of this phenomenon and its potential impact both on the estimation of risks of exposure to low doses/low fluences of ionizing radiation and on radiotherapy is discussed.

Insulin receptors mediate growth effects in cultured fetal neurons. I. Rapid stimulation of protein synthesis.

In this study we have examined the effects of insulin on protein synthesis in cultured fetal chick neurons. Protein synthesis was monitored by measuring the incorporation of [3H]leucine (3H-leu) into trichloroacetic acid (TCA)-precipitable protein. Upon addition of 3H-leu, there was a 5-min lag before radioactivity occurred in protein. During this period cell-associated radioactivity reached equilibrium and was totally recovered in the TCA-soluble fraction. After 5 min, the incorporation of 3H-leu into protein was linear for 2 h and was inhibited (98%) by the inclusion of 10 micrograms/ml cycloheximide. After 24 h of serum deprivation, insulin increased 3H-leu incorporation into protein by approximately 2-fold. The stimulation of protein synthesis by insulin was dose dependent (ED50 = 70 pM) and seen within 30 min. Proinsulin was approximately 10-fold less potent than insulin on a molar basis in stimulating neuronal protein synthesis. Insulin had no effect on the TCA-soluble fraction of 3H-leu at any time and did not influence the uptake of [3H]aminoisobutyric acid into neurons. The isotope ratio of 3H-leu/14C-leu in the leucyl tRNA pool was the same in control and insulin-treated neurons. Analysis of newly synthesized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that insulin uniformly increased the incorporation of 14C-leu into all of the resolved neuronal proteins. We conclude from these data that 1) insulin rapidly stimulates overall protein synthesis in fetal neurons independent of amino acid uptake and aminoacyl tRNA precursor pools; 2) stimulation of protein synthesis is mediated by the brain subtype of insulin receptor; and 3) insulin is potentially an important in vivo growth factor for fetal central nervous system neurons.

Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6.

As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and [gamma-32P]ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons.

Study of the multiple endocrine neoplasia type 1, growth hormone-releasing hormone receptor, Gs alpha, and Gi2 alpha genes in isolated familial acromegaly.

Familial acromegaly may occur as an isolated pituitary disorder or as a feature of hereditary syndromes, such as multiple endocrine neoplasia type 1 (MEN1) or the Carney complex. Herein, we characterized a newly identified kindred with isolated acromegaly and searched for germline mutation in genes that have been associated with endocrine tumors [i.e. MEN1, Gs alpha (GNAS1), and Gi2 alpha (GNAI2)], as well as the GHRH receptor (GHRH-R) gene. Genomic DNA was used to amplify exons 2-10 of MEN1, followed by dideoxy fingerprinting mutation analysis and direct sequencing. The GHRH-R gene was analyzed via direct sequencing of PCR-amplified fragments representing the coding exons and intron-exon junctions. To exclude mutation at hot spot areas of GNAS1 and GNAI2, exons 8 and 9 of GNAS1 and exons 5 and 6 of GNAI2 were amplified and screened for mutation via denaturing gradient gel electrophoresis. No mutations were detected in any of the four genes. The present data extend prior reports of the absence of mutation in MEN1, GHRH-R, and GNAS1 and describe the first family with isolated acromegaly in which germline mutation in GNAI2 has been searched.

An inactivating mutation of the luteinizing hormone receptor causes amenorrhea in a 46,XX female.

Hypergonadotropic hypogonadism is characterized by decreased gonadal function due to the inability of the gonads to respond to pituitary gonadotropins. Hypergonadotropic hypogonadism in females has many causes, among which are ovarian dysgenesis and abnormalities of the ovarian receptors for the pituitary gonadotropins. We evaluated a woman who presented with amenorrhea due to hypergonadotropic hypogonadism, but who had structurally normal ovaries. She is a sister of two previously identified 46,XY male pseudohermaphrodites with Leydig cell hypoplasia. Injection of hCG did not cause any change in plasma levels of estradiol or progesterone, suggesting complete ovarian resistance to LH. Analysis of the DNA sequence of the LH receptor gene revealed that the patient is homozygous for the same single base change as her two brothers. This mutation causes substitution of an alanine residue by a proline at position 593. In vitro analysis of the mutant LH receptor in cultured human embryonic kidney 293 cells documented that the receptor is unable to stimulate adenylyl cyclase in response to hCG. Plasma levels of estradiol and progesterone were low, whereas LH and FSH levels were increased. On histological analysis of the ovary, follicles were seen at all developmental stages. Nonetheless, primary amenorrhea had been present for 5 yr, and repeated measurements of plasma estradiol and progesterone indicate that ovulation does not occur. These results document the existence of inherited LH resistance as a cause of primary amenorrhea in women. The combined clinical and molecular observations are consistent with previous experimental data suggesting that in humans, LH is necessary for ovulation but follicular maturation can occur in the presence of FSH alone.