Simon C.M. Kwok

Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta

The purpose of this investigation was to identify the cellular origin, and the temporal and regional characteristics of placental lactogen-I (PL-I) and placental lactogen-II (PL-II) expression during placental development in the rat. PL-I and PL-II mRNA expression were assessed by Northern blot analysis and in situ hybridization. PL-I and PL-II protein expression were determined by Western blot and immunocytochemical analyses. PL-I mRNA was first detected by in situ hybridization at Day 6 of gestation in mural trophoblast giant cells and a day later, PL-I protein was first detected by immunocytochemistry. PL-I immunostaining extended to the polar trophoblast giant cells as gestation advanced. Polar trophoblast giant cell staining for PL-I was not as intense as the mural trophoblast giant cell staining. Northern and Western blot analyses confirmed the asymmetric distribution of PL-I expression. PL-I mRNA migrated as a 1-kb species and PL-I protein migrated as 30- and 36-40-kDa forms. PL-I expression abruptly declined at Day 12, and by Day 13, PL-I was not detectable. PL-II protein was first detectable at Day 11 of gestation and was localized to trophoblast giant cells. PL-II mRNA could be detected at Day 10 of gestation. Northern and Western blot analyses indicated that PL-II expression significantly increased as gestation advanced and that PL-II expression was asymmetrically distributed similar to PL-I. PL-II mRNA migrated as a 1-kb species and PL-II protein migrated as a 25-kDa species. Blastocysts recovered on Day 4 of gestation initially showed no detectable expression of PL-I or PL-II; however, after 2 days of culture PL-I protein expression was detectable. Biochemical characteristics of PL-I synthesized and secreted by blastocyst outgrowths were similar to PL-I synthesized and secreted by Day 10 placental explants. In summary, (1) PL-I and PL-II are produced by trophoblast giant cells of the developing placenta, (2) PL-I and PL-II exhibit distinct temporal and regional patterns of expression during placental morphogenesis, and (3) PL-I expression by blastocyst outgrowths can be induced in vitro, whereas a more complex array of signals appears necessary for induction of PL-II expression.

Placental lactogen-I variant utilizes the prolactin receptor signaling pathway

Placenta lactogen-I variant (PL-Iv) is a member of a family of proteins expressed by the rat placenta with characteristics similar to prolactin (PRL). In this report, we present the molecular cloning, chromosomal localization, and heterologous expression of PL-Iv. Nucleotide sequence analysis of the PL-Iv cDNA clone predicted a precursor protein of 223 amino acids, including a 28-amino acid signal sequence. The PL-Iv gene was localized to chromosome 17 of the rat genome, which also carries other members of the PRL gene family. PL-Iv heterologously expressed in Chinese Hamster ovary (CHO) cells exhibited similar immunoreactive and electrophoretic characteristics with PL-Iv produced by the rat placenta. N-terminal sequencing verified the identity and purity of the recombinant PL-Iv species and the site of cleavage of the signal peptide from the mature secreted PL-Iv species. Recombinant PL-Iv was shown to bind to ovarian and liver PRL receptors, stimulate the proliferation of Nb2 lymphoma cells, and activate Jak2. Each of these actions is consistent with PL-Iv utilizing the PRL receptor signal transduction pathway.

Identification of a Point Mutation in the Human Insulin Gene Giving Rise to a Structurally Abnormal Insulin (Insulin Chicago)

Both insulin gene alleles of a diabetic patient with a mutant insulin were cloned in a lambda vector and their nucleotide sequences were determined. Nucleo-tide sequence analysis revealed, in one allele, a C (cytidylate) to G (guanylate) transversion in the codon for phenylalanine at position 25 of the insulin B-chain. This point mutation leads to the substitution of a leucine for phenylalanine accompanied by the loss of a restriction endonuclease Mboll recognition site and the creation of a new Rsal cleavage site at this position.

Cloning and nucleotide sequence analysis of the human β-microseminoprotein gene

Abstract β-microseminoprotein (MSP) is a small protein (94 amino acids) synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. Restriction endonuclease mapping of human genomic DNA with a human MSP cDNA probe identified a 19 kilobase (Kb) hybridizing band in both EcoRI and BamHI digestions. Subsequently, the 19 Kb EcoRI fragment of human genomic DNA containing the MSP gene was isolated and cloned into an EMBL4 phage vector. Screening of the recombinant phages resulted in the isolation of one clone containing the MSP gene. Restriction endonuclease mapping and sequence analysis of this clone revealed the human MSP gene of approximately 15 Kb in length. The gene contains four exons and three large introns of approximately 6, 1, and 7 Kb.

Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active

Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.

The Biosynthesis of Insulin: Some Genetic and Evolutionary Aspects

In this article we review the present state of our knowledge regarding insulin formation in the beta-cells of the islets of Langerhans, and of disorders that affect these processes. We also briefly consider some evolutionary aspects of preproinsulin and its associated family of anabolic hormones and/or growth factors. Insight into the biosynthetic mechanism for insulin has provided the key to understanding how many other small peptide hormones and neurosecretory peptides are formed in the body, and thus insulin has now become a useful model protein in the widening search for efficient ways of producing eukaryotic proteins in bacteria. Insulin is a remarkably complex small globular protein, as revealed by the pioneering protein sequencing studies of Fred Sanger combined with the elegant x-ray crystallographic analyses of Dorothy Hodgkin and her co-workers at Oxford.* Figure 1 illustrates the three-dimensional structure of insulin in its dissociated monomeric form. In crystals, however, the hormone is usually present in the form of globular zinc-stabilized hexamers, which are made up of three slightly asymmetric dimers arranged around a threefold axis. The dimers are held together essentially by hydrophobic interactions and hydrogen bonds between conserved regions of the B-chains of each opposing monomer.

Expression of alkaline phosphatase in differentiated rat labyrinthine trophoblast tissue

In this report, we describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristics of alkaline phosphatase expression during maturation of the rat chorioallantoic placenta. An antipeptide antiserum was generated to the amino terminal 15 amino acids of rat alkaline phosphatase. The antiserum specifically recognized alkaline phosphatase. Alkaline phosphatase expression was monitored in the junctional and labyrinth zones of the chorioallantoic placenta by Western and Northern blot analyses. Alkaline phosphatase protein and mRNA were present in both the junctional and labyrinth zones on day 13 of gestation. As gestation advanced, alkaline phosphatase mRNA and protein expression decreased below the limits of detection in the junctional zone, while alkaline phosphatase expression increased in the labyrinth zone. Labyrinthine alkaline phosphatase migrated predominantly as a 95-kDa species, whereas rat kidney expressed exclusively the 75-kDa species. Enzymatic deglycosylation of the 75- and 95-kDa alkaline phosphatase species resulted in the generation of a 55-kDa species. In summary, alkaline phosphatase expression is a useful indicator of trophoblast differentiation.

Clinical report of microphthalmia and optic nerve coloboma associated with a de novo microdeletion of chromosome 16p11.2.

Anophthalmia and microphthalmia are etiologically and clinically heterogeneous. We present a 13‐year‐old boy with microphthalmia and multiple anomalies who was evaluated as part of our research into the etiology of microphthalmia. His clinical features included left microphthalmia, persistent hyperplastic primary vitreous and posterior coloboma, right posterior pole coloboma, pectus excavatum, mild hypotonia, mild delays in speech and motor development, and an anxiety disorder with social difficulties. Investigations with a chromosome microarray revealed a de novo deletion of chromosome 16p11.2 of approximately 882 kb in size. Deletions of this region of chromosome 16p11.2 are a newly delineated microdeletion syndrome, but this is the first report of microphthalmia and coloboma associated with monosomy for 16p11.2, and emphasizes the clinical variability that can be present with this deletion. This report contributes to the growing knowledge regarding this microdeletion and suggests that rare copy number changes may be a cause of microphthalmia and other eye anomalies.

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.

Atorvastatin activates heme oxygenase‐1 at the stress response elements

Statins are known to inhibit growth of a number of cancer cells, but their mechanism of action is not well established. In this study, human prostate adenocarcinoma PC‐3 and breast adenocarcinoma MCF‐7 cell lines were used as models to investigate the mechanism of action of atorvastatin, one of the statins. Atorvastatin was found to induce apoptosis in PC‐3 cells at a concentration of 1 μM, and in MCF‐7 cells at 50 μM. Initial survey of possible pathway using various pathway‐specific luciferase reporter assays showed that atorvastatin‐activated antioxidant response element (ARE), suggesting oxidative stress pathway may play a role in atorvastatin‐induced apoptosis in both cell lines. Among the antioxidant response genes, heme oxygenase‐1 (HO‐1) was significantly up‐regulated by atorvastatin. Pre‐incubation of the cells with geranylgeranyl pyrophosphate blocked atorvastatin‐induced apoptosis, but not up‐regulation of HO‐1, suggesting that atorvastatin‐induced apoptosis is dependent on GTPase activity and up‐regulation of HO‐1 gene is not. Six ARE‐like elements (designated StRE1 [stress response element] through StRE6) are present in the HO‐1 promoter. Atorvastatin was able to activate all of the elements. Because these StRE sites are present in clusters in HO‐1 promoter, up‐regulation of HO‐1 by atorvastatin may involve multiple StRE sites. The role of HO‐1 in atorvastatin‐induced apoptosis in PC‐3 and MCF‐7 remains to be studied.