Circadian Changes in the Surface Area of Renal Glomeruli from Normal Rats

In an attempt to further investigate circadian changes in kidney function, the planar surface area (PSA) (µm 2) of renal isolated glomeruli from normal rats was monitored using a computerized image analyzer method. Eight male Sprague-Dawley (SPRD) rats, aged 12û14 weeks, were entrained to a 12-h light/12-h dark cycle in controlled environmental conditions of temperature (22±2°C) two weeks before the experiments started. Isolated glomerular preparations were obtained, using a mechanical sieving technique, at four different circadian times: 07:00, 13:00, 19:00 and 01:00. It was the finding of the present study that the PSA of the glomeruli varied significantly over the 24-h period, but showed a weak amplitude. The size of the glomeruli reached the highest values at night (21358.59±456.72 µm 2 at 21:17) with an acrophase at 21:39, as do all the other renal parameters, but also blood pressure and many vasoactive compounds involved in the regulation of the mesangial cell physiology, contractile element of the glomerulus. Such chronobiological data not only provided a clear example of complementarity between in vivo and ex vivo experiments, but evidenced that temporal changes do exist in kidney structure and could be correlated with rhythms in renal physiology.     

Dystrophin (Xp21), a New Phenotype Marker of Cultured Rat Aortic Myocytes

Dystropbin is a low-abundance cytoskeletal protein involved in the maintenance of membrane integrity in striated muscle. Very little is known about its role in smooth muscle. Utrophin (a dystropbin-related protein) is an ubiquitous protein whose role is still unclear. Changes in the expression of both proteins (if any) during phenotypic modulation of smooth muscle have not yet been reported. In contrast, modulated expression of heavy-molecular-weight caldesmon (h-CaD), a well-known specific regulatory protein of the contractile apparatus in smooth muscle, is well documented, along with its nonmuscle isoform, low-molecular-weight caldesmon (l-CaD), and other cytoskeletal proteins. We investigated three properties of cultured rat aortic smooth muscle cells: morphology, contractile ability, and expression of dystrophin, utrophin, h-CaD, and l-CaD. Cells were grown either in serum substitute supplemented medium (U-medium), where they reexpressed contractility, or in fetal calf serum-supplemented medium (F-medium), where they did not. It was found that only cultures grown in U-medium continued expressing dystrophin, even during the proliferation phase, contrary to cells grown in F-medium. However, when F-medium was changed for U-medium the cells recovered their contractility and reexpressed dystrophin. Expression of utrophin, h-CaD, and l-CaD was similar in both culture types. Dystrophin was demonstrated to be a true phenotype marker of cultured rat aortic smooth muscle cells, particularly with respect to their actual contractility.     

Structural Differences Between Dopamine D2 Receptors Present in a Rat Pituitary Adenoma and in Transplantable Rat Pituitary Tumors 7315a and MtTW15

We have investigated the structure of dopamine (DA) D2 receptors present in an estrone-induced, prolactin (PRL)-secreting, DA-sensitive adenoma and in two PRL-secreting and DA-insensitive transplantable tumors 7315a and MtTW15, in order to identify better the anomalies present in DA-resistant lactotrophs. D2 receptors were found in both a high- and a low-affinity state in adenomatous lactotrophs as shown by displacement studies with the agonist N-propylnorapomorphine (NPA), but only in the low-affinity state in the two DA-resistant tumors. Treatment with the alkylating agent N-ethylmaleimide induced a disappearance of the high-affinity state of the D2 receptor in the adenoma and a reduction in receptor concentration, but did not have any effect on the affinity of receptors present in DA-resistant tumors. Moreover, target size analysis and radiation inactivation studies of D2 receptors, using membranes preincubated with NPA and [3H]spiperone as ligand or using [3H]NPA as ligand on membranes preparations, have shown the presence of distinct structural differences between adenomatous and tumoral D2 receptors and between the two tumoral receptors themselves; these results suggest that the normal functional unit of the D2 receptor is a dimer associated with a guanine nucleotide-binding protein (G protein) subunit and that tumoral D2 receptors may exist in various polymeric forms unassociated with G proteins. The anomalies found to be present in tumoral D2 receptor complexes may be responsible for the insensitivity of these tumors to dopaminergic agonists' inhibitory activity on PRL release and tumor growth.     

Molecular Weight of Human Brain Neutral Sphingomyelinase Determined In Situ by the Radiation Inactivation Method

The radiation inactivation method was used to determine the molecular weight of membrane-bound neutral sphingomyelinase from normal human brain. Inactivation curves showed a molecular mass of 167,000 +/- 32,000. Molecular weights of two control enzymes, beta-N-acetylglucosaminidase and nonspecific beta-glucosidase, determined by the same procedure, were consistent with previous reports.     

The radiation inactivation method provides evidence that membrane-bound mitochondrial creatine kinase is an oligomer

Lyophilized suspensions of rabbit heart mitochondria have been irradiated with varying doses of gamma rays. Mitochondrial creatine kinase activity was inactivated exponentially with a radiation inactivation size of 352 or 377 kDa depending upon the initial medium. These values are in good agreement with the molecular mass previously deduced from by permeation experiments: 357 kDa. This is the first direct evidence showing that the native form of mitochondrial creatine kinase is associated to the inner membrane as an oligomer, very likely an octamer.     

Estrone sulfate-sulfatase and 17β-hydroxysteroid dehydrogenase activities: a hypothesis for their role in the evolution of human breast cancer from hormone-dependence to hormone-independence

The evaluation of estrogens (estrone, estradiol, and their sulfates) in the breast tissue of post-menopausal patients with breast cancer indicates high levels, particularly of estrone sulfate (E₁ S) which is 15–25 times higher than in the plasma. Breast cancer tissue contains the enzymes necessary for local synthesis of estradiol and it was demonstrated that, despite the presence of the sulfatase and its messenger in hormone-dependent and hormone-independent breast cancer cells, this enzyme operates particularly in hormone-dependent cells. Different progestins: Nomegestrol acetate, Promegestone, progesterone, as well as Danazol, can block the conversion of E₁ S to E₂ very strongly in hormone-dependent breast cancer cells. The last step in the formation of estradiol is the conversion of E₁ to this estrogen by the action of 17β-hydroxysteroid dehydrogenase. This activity is preferentially in the reductive direction (formation of E₂) in hormone-dependent cells, but oxidative (E₂ → E₁) in hormone-independent cells. Using intact hormone-dependent cells it was observed that Nomegestrol acetate can block the conversion of E₁ to E₂. It is concluded, firstly, that in addition to ER mutants other factors are involved in the transformation of hormone-dependent breast cancer to hormone-independent, this concerns the enzymatic activity in the formation of E₂; it is suggested that stimulatory or repressive factor(s) involved in the enzyme activity are implicated as the cancer evolves to hormone-independence; secondly, different drugs can block the conversion of E₁ S to E₂. Clinical trials of these “anti-enzyme” substances in breast cancer patients could be the next step to investigate new therapeutic possibilities for this disease.     

Structure of the lysosomal neuraminidase-beta-galactosidase-carboxypeptidase multienzymic complex.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed     

The target sizes of the in situ and solubilized forms of human placental steroid sulfatase as measured by radiation inactivation

Whole microsomal membrane preparations and Triton X-100-solubilized human placental steroid sulfatase were subjected to radiation inactivation analysis using gamma rays from a 60Co irradiator in order to assess the size of the physiological form of the enzyme. The data indicate that the enzyme exists as a monomer of molecular weight 78600 in Triton-containing buffers and as a polymer of molecular weight 533000 within the microsomal membrane.     

Organization of the yeast URA2 gene: identification of a defective dihydroorotase-like domain in the multifunctional carbamoylphosphate synthetase-aspartate transcarbamylase complex

The 6636 bp of the yeast URA2 gene encoding the carbamoylphosphate synthetase-aspartate transcarbamylase complex have been sequenced. The protein is organized into four regions, three of which are functional domains as indicated previously by genetic analysis. The fourth domain corresponds to a defective dihydroorotase called DHOase-like. The URA2 gene complex with the same organization as the equivalent genes in higher eukaryotes suggests an evolution from a common ancestral gene.