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.     

Age and hormone dependent activity of Delta5, 3 beta-hydroxysteroid dehydrogenase in isolated Leydig cells from mouse testes

Mouse males aged 2, 4 weeks, and 2 months were given PMS and HCG hormones, and testosterone in intramuscular injections. Leydig cells obtained from treated animals were histochemically investigated for the activity of delta 5, 3 beta-hydroxysteroid dehydrogenase). PMS caused no statistically significant changes in the activity of delta 5, 3 beta-OH-SDH in any of the investigated age groups of mice. However, in all tested groups treated with HCG the amount of cells having very strong enzyme activity increased. Testosterone injected into mice induced decrease of delta 5, 3 beta-OH-SDH activity. Both under the influence of HCG and testosterone differences between control group and experimental ones were statistically significant.     

Comparative analysis of chemotaxis in Dictyostelium using a radial bioassay method: protein tyrosine kinase activity is required for chemotaxis to folate but not to cAMP

The role of signal transduction during chemotaxis of Dictyostelium discoideum cells to cAMP and folic acid was investigated using a radial bioassay technique. The effects of signalling agonists were assessed by measuring the diameters of visible rings formed by the outward migration of amoebae up radial gradients of chemoattractant. This rapid and simple bioassay method yields chemotactic rates equivalent to more complex assay systems. In support of previous studies, chemotaxis toward both cAMP and folic acid was inhibited in a dose-dependent manner by LaCl3, EDTA, chlorotetracycline and A1F3, supporting the importance of calcium ions and G protein-mediated signalling in both chemotactic events. The work was extended by examining the effects of the protein tyrosine kinase inhibitor genistein. This agent inhibited chemotaxis to folate in a dose-dependent manner but had no observable effect on chemotaxis toward cAMP. The notion that phosphorylation of proteins on tyrosine residues is critical for chemotaxis to folic acid was supported by Western blotting experiments with monoclonal anti-phosphotyrosine antibodies which detected two candidate proteins of M(r) 52,000 and 38,000 in the membranes of folate-responsive amoebae. These two bands disappeared with starvation which leads to the loss of responsiveness of folic acid and the acquisition of responsiveness to cAMP. Time-lapse videomicrography also revealed some unique differences in chemotactic response. Starved cells responded to cAMP as individuals but feeding cells chemoattracted to folic acid on a populational basis. The ability to compare two different types of chemotaxis using a simple, rapid and accurate bioassay system should enhance future studies of chemotaxis in wild-type and mutant strains of D. discoideum.     

The cellular mechanisms of corticotropin-releasing hormone (CRH)-stimulated steroidogenesis in mouse Leydig cells are similar to those for LH

An orderly scheme of action is proposed to allow for the practical solution of clinical ethical problems. This scheme depends on understanding and discussion, between patient and doctor, of the ethical assumptions involved in any dilemma. Instead of the more usual ethical principles, arguments are presented for six basic ethical assumptions (and their associated corollaries) in favour of Life, Autonomy, Beneficence, Equity, Truth, and Law. Because these assumptions are dependent on the different personal viewpoints of the people involved and not immutable principles, such ethical assumptions are able to be set in different hierachical orders on different occasions permitting in most cases a particular solution specific for that dilemma.     

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity

Protein tyrosine phosphorylation accompanies the integrin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Readhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.     

Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation

Polycystic ovary syndrome is the most common cause of anovulatory infertility. Anovulation in polycystic ovary syndrome is characterized by the failure of selection of a dominant follicle with arrest of follicle development at the 5-10 mm stage. In an attempt to elucidate the mechanism of anovulation associated with this disorder we have investigated at what follicle size human granulosa cells from normal and polycystic ovaries respond to LH. Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA. In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5-3.9) and P, 1.0 (0.3-2.5) in ovulatory women and E2, 1.4 (0.7-25.4) and P, 1.3 (0.3-7.0) in anovulatory women. Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.     

Tctex3, related to Drosophila polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6.     

Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up-regulate their responsiveness to estrogens

We have reported that pretreatment with 1 alpha, 25(OH)2D3(1, 25) up-regulates responsiveness and sensitivity to 17 beta estradiol (E2) in osteoblast-like cells, as measured by parallel stimulation of [3H]thymidine incorporation into DNA and the specific activity of creatine kinase BB (CK). Increased responsiveness was correlated with increased E2 receptor concentration. In this study, we have extended these observations to new nonhypercalcemic analogs of 1,25. We compared the analogs hexafluoro vitamin D3 (FL), and the side chain modified derivatives: EB 1089 (EB), CB 1093 (CB) and MC 1288 (MC) with 1,25 and 25 (OH)D3(25 D3). Treatment with 30 nM E2 for 4 h stimulated CK activity in ROS 17/2-8 cells by 40%; there was no further increase after 3 daily additions of E2. Treatment by 3 daily additions, at 1 nM, of all analogs except 25 D3 caused a 2-3-fold increase in CK specific activity. This schedule of treatment also upregulated the response to 4 h exposure to 30 nM E2 by 30-70% above the response to vitamin D analogs alone, and by up to 2 fold compared to E2 without pretreatment. At 1 pM, the analogs doubled CK activity, and, except for 1,25, upregulated the response to E2 to levels characteristic of each analog. Pretreatment with vitamin D analogs also increased the sensitivity to E2 by lowering the dose for a comparable response to E2 by one or two orders of magnitude. Stimulation of specific activity of CK by the analogs was paralleled by increases in the steady state level of mRNA for CKB, but not in its half life. Whereas pretreatment by vehicle followed by E2 for 2 h was unable to increase CKB mRNA, pretreatment with the analogs made possible detection of mRNA responsiveness to E2. These results add to the evidence for the interaction of estrogens and antiestrogens with vitamin D metabolites in regulation of bone growth in vitro. They also strengthen the potential for treatment of bone loss, as occurs in postmenopausal osteoporosis, by a combination of nonhypercalcemic vitamin D analogs and estrogens.     

Truncation of the thyrotropin-releasing hormone receptor carboxyl tail causes constitutive activity and leads to impaired responsiveness in Xenopus oocytes and AtT20 cells

We studied the activity of a truncated thyrotropin-releasing hormone receptor (TRH-R), which lacks the last 59 amino acids of the carboxyl tail, where Cys-335 was mutated to a stop codon (C335Stop) (Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392). In Xenopus laevis oocytes expressing C335Stop TRH-Rs, TRH binding was higher, whereas chloride current, 45Ca2+ efflux, and [Ca2+]i responses evoked by TRH were 23, 39, and 21%, respectively, of those in oocytes expressing wild type mouse pituitary TRH-Rs (WT TRH-Rs). In oocytes expressing C335Stop TRH-Rs, basal 45Ca2+ efflux and [Ca2+]i were twice those in oocytes expressing WT TRH-Rs; chelation of Ca2+ caused a rapid increase in holding current, which is consistent with basal activation; and coexpression with other receptors caused inhibition of the responses to the other cognate agonists. In AtT20 pituitary cells stably expressing C335Stop TRH-Rs, thyrotropin-releasing hormone (TRH)-independent inositol phosphate formation was 1.32 +/- 0.11-fold higher, basal [Ca2+]i was 1.8 +/- 0.2-fold higher, and the [Ca2+]i response to TRH was much lower than in cells expressing WT TRH-Rs. We conclude that a TRH-R mutant truncated at Cys-335 exhibits constitutive activity that results in desensitization of the response to TRH.     

Paclitaxel is preferentially cytotoxic to human cervical tumor cells with low Raf-1 kinase activity: implications for paclitaxel-based chemoradiation regimens

Little is known about the association of echocardiographic estimates of right ventricular (RV) function with survival, in relation to hemodynamic and exercise-derived predictors of outcome in congestive heart failure. We prospectively studied 40 patients (age 55+/-10 years, in New York Heart Association functional class III [70%] and IV [30%]), with left ventricular (LV) ejection fraction <30%. At enrollment, all patients underwent echocardiographic evaluation of LV dimensions and function. RV shortening was measured as the difference of the end-diastolic distance - the end-systolic distance between the tricuspid annulus and the RV apex. Thirty-five patients (88%) were able to perform a maximal symptom-limited exercise test. Peak oxygen consumption (peak VO2) and percent peak age- and gender-adjusted predicted oxygen consumption (%peak VO2) were calculated. Of 40 patients, 10 died during a mean follow-up period of 14+/-10 months. On univariate analysis, nonsurvivors had lower RV shortening (p=0.0001), higher pulmonary artery wedge pressure (p=0.009), higher pulmonary vascular resistance (p=0.02), and lower mean aortic pressure (p=0.05). Cox proportional-hazards model revealed that the only independent associate of survival was RV shortening (p=0.0005), with a trend toward significance for mean aortic pressure (p=0.08). The best cutoff point of RV shortening identified by the receiver-operating curve was 1.25 cm. This value had a sensitivity of 90%, specificity of 80%, and overall predictive accuracy of 83% to distinguish survivors from nonsurvivors. In patients with advanced heart failure, preserved RV function as indicated by an echocardiographically derived RV shortening > 1.25 cm is a strong predictor of survival.     

Pseudohypoparathyroidism with osteitis fibrosa cystica: direct demonstration of skeletal responsiveness to parathyroid hormone in cells cultured from bone

The role of aberrant neurodevelopment in the etiology of schizophrenia is reviewed in light of recent neuropathologic, neurochemical, and neuroimaging evidence of cerebral abnormalities in schizophrenic patients. There may exist some genetic defect in the control of brain development. Clinical epidemiologic surveys highlight the importance of obstetric complications, and prenatal exposure to influenza epidemics in contributing to these abnormalities. It is suggested that such environmental hazards and aberrations in the control of early brain development produce the neuronal phenotype that manifests as schizophrenia with early age of onset of symptoms associated with soft neurologic signs and is more common in young males.     

Possible involvement of differential splicing in regulation of the activity of Arabidopsis ANP1 that is related to mitogen-activated protein kinase kinase kinases (MAPKKKs)

Retrospective study about 10 verrucous carcinomas of the larynx surgically treated in a 20-year-term. This variety accounted por the 1.9 percent of the cancers seen in that period of time. Tabagism and alcoholism predominated in 8 and 6 patients respectively. Glottis was the localization and dysphony the paramount symptom. In the paper are emphasized the most important histologic features. Koilocytosis was present in 6 cases. Four patients developed a second tumor of epidermoid carcinoma type. No one exitus due to the verrucous growth. Only in 3 the death was attributed to the second malignancy.     

The activated form of the Lck tyrosine protein kinase in cells exposed to hydrogen peroxide is phosphorylated at both Tyr-394 and Tyr-505

Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H2O2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H2O2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members.     

Bruton's tyrosine kinase activity and inositol 1,4,5-trisphosphate production are not altered in DT40 lymphoma B cells exposed to power line frequency magnetic fields

Exposure of wild-type DT40 lymphoma B cells or Bruton's tyrosine kinase (BTK)-deficient DT40 cells reconstituted with the human btk gene to a 1-gauss 60-Hz electromagnetic field (EMF) has been reported to rapidly increase inositol 1,4,5-trisphosphate (Ins 1,4, 5-P3) production (1,2). Here we have used BTK-deficient DT40 B cells reconstituted with the human btk gene to evaluate the reproducibility of these findings. An experimental design with blinded exposures and anti-IgM treatment to induce Ins 1,4,5-P3 production as a positive control, showed no significant effect of a 1-gauss 60-Hz EMF on Ins 1,4,5-P3 production. Because recent work has shown that the activation of BTK was required for EMF-responsiveness (2), we also evaluated the reproducibility of this finding in wild-type DT40 cells. BTK was activated in a dose- and time-dependent manner by treatment with the tyrosine phosphatase inhibitor pervanadate. However, the ability to detect BTK activation, as measured by increased autophosphorylation by immune complex kinase assay, was dependent on the kinase buffer. Using cells from the original investigators, no evidence was obtained to support the hypothesis that exposure to a 1-gauss 60-Hz EMF had a causal effect on protein-tyrosine kinase activities affecting Ins 1,4,5-P3 production.     

Flow cytometric analysis of basophil numbers in chronic urticaria: basopenia is related to serum histamine releasing activity

BACKGROUND: Peripheral blood basophils are reduced in some chronic urticaria patients when counted with granule stains. Approximately 30% of patients with severe chronic urticaria have functional autoantibodies which release histamine from healthy donor basophils in vitro but the relationship between basophil numbers in vivo and serum histamine releasing activity has not been studied. OBJECTIVE: To determine the relationship between basophil numbers and serum basophil histamine releasing activity and to assess whether basophils are present, but undetectable, in peripheral blood with granule stains by using a new flow cytometric method based on surface immunophenotype. METHODS: Basophils were counted manually by a chamber method using a granule stain and by flow cytometry using dual staining with anti-IgE and anti-Fc epsilonRI in 25 chronic idiopathic urticaria patients and 25 healthy controls. Serum histamine releasing activity was assessed on healthy donor basophils in vitro and by the weal response to autologous serum skin testing in vivo (patients only). RESULTS: Basophils were significantly reduced in chronic urticaria by manual counting and flow cytometry. A subgroup of seven patients with in vitro histamine releasing activity showed a marked reduction or absence of basophils by both methods. There were no obvious distinguishing clinical characteristics between these patients and the others; six of them showed positive autologous serum skin-test responses. On comparing the two methods, the manual basophil counts were generally lower than flow cytometric counts. Agreement over the full range of values was not strong and therefore counts obtained by the two methods are not directly interchangeable. CONCLUSION: Basopenia in chronic idiopathic urticaria is associated with serum basophil histamine releasing activity in a subgroup of patients. The lack of granule and surface immunophenotype staining suggests a reduction in numbers rather than an inability to detect circulating degranulated cells by conventional counting methods.