An assembly zone antigen of the insect cuticle

The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.     

Steroidogenesis by the human oocyte-cumulus cell complex in vitro

The ability of the human oocyte-cumulus cell complex to synthesize progesterone, androgens and estrogens and to modify its endocrine environment in vitro was investigated. Germinal-vesicle stage oocytes with adhering layers of cumulus cells were recovered from human ovaries and maintained for 40–50 h in vitro in a culture medium with or without antral fluid.The results show that oocyte-cumulus (0-C) cell complexes were capable of synthesizing progesterone, androgens and estrogens. Oocytes with the capacity of resuming meiosis in vitro were part of an 0-C complex producing significantly more progesterone than those 0-C complexes containing oocytes incapable of resuming meiosis. Irrespective of the stage of oocyte maturation at the end of culture, testosterone and estrone were respectively the major androgen and estrogen produced.It is concluded that the oocyte-cumulus compartment of the antral follicle is a steroidogenically competent unit and that it has the capacity to modify the endocrine microenvironment of the follicle.     

Dual Actions of Sphingosine-1-Phosphate: Extracellular through the Gi-coupled Receptor Edg-1 and Intracellular to Regulate Proliferation and Survival

Sphingosine-1-phosphate (SPP), a bioactive lipid, acts both intracellularly and extracellularly to cause pleiotropic biological responses. Recently, we identified SPP as a ligand for the G protein–coupled receptor Edg-1 (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. 1998. Science. 279:1552–1555). Edg-1 binds SPP with remarkable specificity as only sphinganine-1-phosphate displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-1 resulted in inhibition of forskolin-stimulated cAMP accumulation, in a pertussis toxin–sensitive manner. In contrast, two well-characterized biological responses of SPP, mitogenesis and prevention of apoptosis, were clearly unrelated to binding to Edg-1 and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p125^FAK, independently of edg-1 expression. Moreover, DNA synthesis in Swiss 3T3 fibroblasts was significantly and specifically increased by microinjection of SPP. Finally, SPP suppresses apoptosis of HL-60 and pheochromocytoma PC12 cells, which do not have specific SPP binding or expression of Edg-1 mRNA. Conversely, sphinganine-1-phosphate, which binds to and signals via Edg-1, does not have any significant cytoprotective effect. Thus, SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers.     

Neurotensin metabolism in the rat: Contribution of the kidney

The kidney plays a key role in the metabolism of neurotensin (NT). We have examined the renal mechanisms of NT clearance by measuring plasma NT basally and after 45 min infusion of NT(1–13) in intact rats, anephric rats (no glomerular filtration, no peritubular metabolism) and ureteral ligated rats (reduced filtration). Plasma NT was measured by radioimmunoassay with both C (biologically active end) and N terminal directed antisera. In anephric and ureteral ligated rats, basal plasma NT like immunoreactivity measured with either antisera was increased 3-fold compared with unoperated rats. C terminal concentrations were higher than N indicating that a C terminal variant of NT was present in basal plasma. Infusion of NT(1–13) increased N terminal NT from 36±3 to 249±35 pmol/l (p<0.01) in unoperated rats with significantly larger increases in the renally compromised groups. This was reflected in the reduced metabolic clearance rates (measured with the N terminal directed antisera) in the anephric (16±1 ml/kg/min) and ureteral ligated (17±3 ml/kg/min) rats when compared with the control rats (26±4 ml/kg/min). The similar reductions in the anephric and ureteral ligated rats suggested that the decrease in N terminal NT metabolism was from the absence of filtration. Infusion of NT did not increase C terminal NT immunoreactivity in intact, anephric and ureteral ligated rats showing that the C terminal end was extremely labile. However when endogenous converting enzyme activity was blocked by captopril administration there was a significant increase in C terminal immunoreactivity suggesting a role for converting enzyme like proteases in the clearance of the biologically active end of NT. The results indicate that glomerular filtration is important for the clearance of N terminal NT while the C terminal part can be rapidly cleared by non-renal mechanisms. The higher concentration of C than N terminal immunoreactivity in basal plasma of intact rats and the further increase when renal function is reduced suggests that a NT variant sharing the C terminal end may be of physiological significance.     

Quantification by flow cytometry of chromosome-17 deletions in Smith-Magenis syndrome patients

We have used bivariate flow karyotyping to quantify the deletions involving chromosome 17 in sixteen patients with Smith-Magenis syndrome (SMS). The fluorescence intensities of mitotic chromosomes stained with Hoechst 33258 and chromomycin were quantified in a dual-beam flow cytometer. For each patient, the position of the peak representing the deleted chromosome 17 was compared to those of the normal homologs of an unaffected parent. The patients could be classified into four groups based on the size of their deletions. The deletions ranged from ∼9–10 Mb (∼10–11% of the chromosome) to below the detection limit of the technique (2 Mb). Different deletion sizes were detected among patients whose high-resolution banding results were similar. Some deletions detected by banding were not detected by flow analyses. Deletion estimates are largely consistent with the results of molecular analyses. Patients with larger deletions that extend into band 17p12 have abnormal electrophysiologic studies of peripheral nerves. Deletion size does not appear to correlate with the degree of mental retardation, presence of behavioral abnormalities, craniofacial anomalies or common skeletal findings in SMS. By identifying patients with varying deletion sizes, these data will aid the construction of a long-range deletion-based map of 17p11.2 and identification of the genes involved in this syndrome. Received: 19 March 1996 / Revised: 21 June 1996     

Genetic variations in human testosterone-estradiol binding globulin

The human testosterone-estradiol-binding globulin (hTeBG) is a plasma heterogeneous glycoprotein with high affinity for a number of circulating steroid hormones. The heterogeneity originates from differential glycosylation of a common protein precursor. Analysis of desialylated hTeBG by isoelectric focusing (IEF) has revealed that microheterogeneity could be partly attributed to variability in sialic acid content or rearrangement of amino acid composition. We have studied this possibility by the analysis of desialylated serum hTeBG by Western blotting of proteins previously separated on IEF-gels. Two distinct well-defined IEF patterns were identified. The most frequent consisted of two major IEF-bands of equal color intensity. The other pattern consisting of four IEF-bands was present in only 5.55% of the total serum samples analyzed. Family studies showed that these phenotypes were autosomally inherited with a simple Mendelian transmission and allele frequencies had an excellent agreement between the observed and expected phenotypes. Androgen affinity constants and serum concentrations of hTeBG variant were similar to those of normal hTeBG. Molecular analyses of each of the exons of hTeBG gene by denaturing gradient gel electrophoresis revealed the presence of a point mutation in exon 8. The studies presented herein confirm and extend previous reports on the existence of structural variants of hTeBG. In addition, the mutation reported in this study is probably the same as that recently identified within numerous ethnic groups throughout the world, thus further supporting the concept of a two allele gene worldwide concoding hTeBG.