Randall G. Richards

Studies of the molecular pathogenesis of hexane neuropathy: II. Evidence that pyrrole derivatization of lysyl residues leads to protein crosslinking☆☆☆

Abstract In the reaction between ethanolamine and 2,5-hexanedione, 1-(2-hydroxyethyl)-2,5-dimethylpyrrole was formed, and the pyrrole was found to autoxidize to form an orange chromophore. Similar orange chromophores were observed in the reaction of 2,5-hexanedione, 2,5-heptanedione, and 3,6-octanedione with a variety of primary amines and with proteins. The development of the orange chromophore in the reaction of 2,5-hexanedione with proteins was attended by a proportional derivatization of lysyl residues and by extensive intramolecular and intermolecular crosslinking. These observations suggest that the sequence of events in the crosslinking of neurofilaments during chronic n -hexane intoxication may be metabolism to 2,5-hexanedione, formation of an imine with lysyl residues, cyclization to form a pyrrole, autoxidation of the pyrrole, and finally covalent crosslinking involving pyrrole rings.

The physiology of decidual prolactin and other decidual protein hormones

Human decidual tissue, the specialized endometrium of the luteal phase of the menstrual cycle and pregnancy, synthesizes and releases prolactin, relaxin, renin, and at least three insulinlike growth factor (IGF)-binding proteins. Although the physiologic roles of these decidual protein hormones during pregnancy are unclear, numerous studies suggest that the hormones may act locally to affect the function of the placenta, decidua, and fetal membranes. In addition, the synthesis and release of these hormones appear to be regulated locally by factors produced by the placenta, decidua, and fetal membranes.

Purification of decidual prolactin-releasing factor, a placental protein that stimulates prolactin release from human decidual tissue.

Decidual prolactin-releasing factor (PRL-RF), a placental protein that stimulates the release of prolactin from human decidual tissue, has been purified from conditioned medium of human placental explants. The purification scheme consisted of ethanol extraction, anion exchange chromatography on DEAE-cellulose, size exclusion chromatography on Spherogel TSK-3000, and either a) immunoaffinity chromatography using an antiserum to a partially purified PRL-RF preparation or b) acetic acid-urea/SDS 2-dimensional PAGE. The apparent molecular weight of the purified releasing factor, estimated by SDS-PAGE, was 23,500 Da; and the half-maximal dose for the acute stimulation of prolactin release from human decidual cells was 0.05-0.1 ug/ml (2.2-4.4 nM).

Autocrine/paracrine regulation of prolactin release from human decidual cells.

Studies from this laboratory indicate that the synthesis and release of prolactin from human decidual cells are regulated by factors released by the placenta, decidua and fetal membranes. A 23.5 kD protein (decidual prolactin-releasing factor, PRL-RF) has been purified to homogeneity from human placental tissue and placental conditioned medium. The releasing factor stimulates an acute release of prolactin that occurs within the first few minutes of exposure and a prolonged release, secondary to new hormone synthesis, that begins about 6-8 hours later. In addition, the synthesis and release of decidual prolactin are stimulated by IGF-I, insulin and relaxin, each acting through distinct plasma membrane receptors. In contrast, the synthesis and release of decidual prolactin are inhibited by arachidonic acid, lipocortin I and a 35-45 kD decidual protein (prolactin-releasing inhibitory factor) that has been partially purified from decidual conditioned medium. Studies of the second messengers involved in the regulation of decidual prolactin release strongly suggest that decidual prolactin release may be mediated, at least in part, by activation of phosphoinositide metabolism and stimulation of adenylate cyclase. The demonstration that the synthesis and release of decidual prolactin are regulated by PRL-RF, IGF-I, insulin, relaxin, arachidonic acid, PRL-IF and lipocortin I strongly suggests that there is novel autocrine/paracrine feedback regulation between the placenta, fetal membranes, and decidua in the regulation of decidual prolactin.

In situ protamine release: A versatile sample preparation method for the electrophoretic analysis of nuclear proteins on acid/urea-based gels

Abstract Protamine sulfate is used to release histones and other basic proteins from the DNA of chromatin. This phenomenon becomes the basis of a versatile method for analysis of the nucleic acid and protein composition of nucleoprotein samples, which is termed here in situ protamine release. When protamine is added to a nucleoprotein sample in 5% acetic acid and 8 m urea, at a concentration of 1.0%, ≥94% of the histones are released from the DNA of chromatin, comparable to the release of histones using sodium dodecyl sulfate. This makes in situ protamine release the method of choice for the analysis of acid-soluble proteins on acid/urea-based gels, where the DNA must be removed from the protein prior to electrophoresis. Compared to DNase I release or acid extraction, protamine release is found to be the simplest, most reliable, and most effective method for removing the acid-soluble proteins from DNA. Protamine is either added to the sample (very much like the detergent, sodium dodecyl sulfate), or electrophoresed through a gel containing nucleoprotein, thus displacing proteins in its path. A serendipitous advantage of protamine is that it can also serve as a carrier for the precipitation of dilute nucleoprotein samples with ethanol, and 3 m m Mg 2+ , to concentrate the nucleoprotein in preparation for analysis. A unique feature of the in situ protamine-release method is that the DNA is not lost or destroyed and can therefore be used for subsequent analysis.

Autocrine/Paracrine Regulation of Decidual Prolactin Expression

For many years theprolactin (PRL) gene was thought to be expressed only in the pituitary. However, more recent studies indicate that the PRL gene is also expressed in human decidual cells that are present in the uterus during the luteal phase of the menstrual cycle and during pregnancy (1–3). The mature secreted form of decidual PRL is identical to that of pituitary PRL (4, 5), and both PRLs are synthesized and released in glycosylated and nonglycosylated forms (6). However, the physiological roles of decidual PRL appear to be different than those demonstrated for pituitary PRL, and the factors involved in the regulation of decidual PRL expression are, in large part, different from those involved in the regulation of pituitary PRL expression. The PRL gene has also been shown to be expressed in lymphocytes (7) and ovarian, (8) lacrimal (9), and brain tissues (10), but the physiological roles of PRL and the factors that regulate PRL expression in these cells and tissues are unclear.

Preparation and Analysis of Core Particles and Nucleosomes: A Convenient Method for Studying the Protein Composition of Nucleosomes using Protamine-Release into Triton-Acid-Urea Gels

The nucleosomal repeat structure of chromatin consists of a core of 140 base pairs of DNA wrapped around an octamer of eight histones, and a spacer region which is variable in length and more susceptible to nuclease attack than the core1–10. Histone H1, which exhibits tissue specific variation, has been postulated to be associated with DNA in this spacer region3,4,10–13. The octameric histone complex in the core particle contains two copies each of histones H2A, H2B, H3 and H4. It is known that these core histones undergo postsynthetic covalent modifications such as acetylation and phosphorylation at different times during the cell cycle34. Recently, Cohen et al. 15 have shown in the developing sea urchin and Franklin and Zweidler 16 have shown in the differentiated tissues of mammals that there are also variations within the primary sequence of histones H2A, H2B, and H3. The molecular differences between variant histones arise from the conservative substitution of one or two amino acid residues in the polypeptide chain which can be detected on Triton/acid/urea gels. In the sea urchin system, different H2A and H2B variants are synthesized at different stages of embryonic development15. The existence of multiple forms of histones implies that there must be multiple forms of nucleosomes.

Physiology and Molecular Biology of Placental Lactogen in Human Pregnancy

Human placental lactogen (hPL) is a protein hormone that has striking homologies in its chemical and biological properties to human growth hormone (hGH) and human prolactin (hPRL) (1,2). In this chapter, we will briefly discuss the role of hPL in the regulation of fetal growth and the regulation of hPL secretion in normal and pathologic pregnancies. We will then focus on recent advances in the regulation of hPL gene expression, giving particular attention to roles of nuclear hormone receptors and cytokines in transactivation of the hPL promoter.

Paracrine and Autocrine Factors Involved in the Regulation of the Release of Human Decidual Prolactin and Human Placental Lactogen

During pregnancy, the decidua and placenta synthesise and secrete several protein hormones that have identical or nearly identical chemical and biological properties to protein hormones synthesised and secreted by the pituitary gland and other tissues. For example, human decidual tissue synthesises and releases protein hormones that are identical to pituitary prolactin1 and ovarian relaxin.2 The placenta synthesises and releases hCG and hPL which have striking chemical and biological similarities to LH and to growth hormone and prolactin, respectively.3 Nevertheless, despite the striking similarities between these hormones, the regulation of the synthesis and secretion of the protein hormones from the decidua and placenta is different from that of the pituitary protein hormones. Pituitary prolactin, growth hormone and most protein hormones are stored in large secretory granules, but ultrastructural and biochemical studies indicate that decidual prolactin4 and hPL5 are localised in the post-microsomal supernatants of decidual and placental tissue homogenates.