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1.
Eur J Neurosci ; 10(10): 3237-45, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9786217

ABSTRACT

While the role of heat shock proteins under experimental stress conditions is clearly characterized, their expression in unstressed cells and tissues and their functions in normal cell physiology, besides their chaperone action, remain largely undetermined. We report here the identification in chicken of the antigen recognized by the monoclonal antibody PM1 [Hernández-Sánchez et al. (1994) Eur. J. Neurosci., 6,1801-1810] as the noninducible chaperone heat-shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno-crossreactivity and two-dimensional gel-electrophoresis. In addition, we examined its expression during chick embryo retinal neurogenesis. The early widespread Hsc70 immunostaining corresponding to most, if not all, of the neuroepithelial cells becomes restricted to a subpopulation of these cells in the peripheral retina as development proceeds. On the other hand, retinal ganglion cells, differentiating in the opposite central-to-peripheral gradient, retained Hsc70 immunostaining. Other molecular chaperones, the heat-shock proteins Hsp40, Hsp60 and Hsp90, did not seem to compensate the loss of Hsc70. They also showed decreasing immunostaining patterns as neurogenesis proceeds, although distinctive from that of Hsc70, whereas Hsp70 was not detected in the embryonic retina. This precise cellular and developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools and a further insight on neural precursor heterogeneity, and suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.


Subject(s)
Carrier Proteins , HSP70 Heat-Shock Proteins , Molecular Chaperones/biosynthesis , Neurons/cytology , Retina/growth & development , Adenosine Triphosphatases , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Carrier Proteins/biosynthesis , Carrier Proteins/chemistry , Carrier Proteins/genetics , Chick Embryo , Clathrin , Coated Pits, Cell-Membrane , Genes, Tumor Suppressor , HSC70 Heat-Shock Proteins , Molecular Chaperones/analysis , Molecular Sequence Data , Retina/cytology , Retina/embryology , Retinal Ganglion Cells/chemistry , Retinal Ganglion Cells/cytology
2.
Proc Natl Acad Sci U S A ; 95(17): 9950-5, 1998 Aug 18.
Article in English | MEDLINE | ID: mdl-9707581

ABSTRACT

Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis.


Subject(s)
Apoptosis/physiology , Carrier Proteins/physiology , Heat-Shock Proteins , Molecular Chaperones/physiology , Proinsulin/physiology , Amino Acid Sequence , Animals , Apoptosis/genetics , Base Sequence , Carrier Proteins/genetics , Chick Embryo , Cloning, Molecular , Cysteine Endopeptidases/physiology , DNA Primers/genetics , DNA, Complementary/genetics , Endoplasmic Reticulum Chaperone BiP , Gene Expression Regulation, Developmental , HSC70 Heat-Shock Proteins , HSP70 Heat-Shock Proteins/genetics , Humans , Molecular Chaperones/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino Acid
3.
J Vasc Interv Radiol ; 9(3): 401-6, 1998.
Article in English | MEDLINE | ID: mdl-9618097

ABSTRACT

PURPOSE: To investigate current antibiotic prophylactic usage for arteriography, angioplasty, vascular stent placement, transjugular intrahepatic portosystemic shunt placement (TIPS), tunneled-port placement, inferior vena cava (IVC) filter placement, biliary drainage, genitourinary drainage, abdominal drainage, and enteral tube placement with an aim to better clarify indications and regimens for prophylaxis. METHODS: A questionnaire regarding antibiotic prophylactic usage was sent to 2,039 members of the Society of Cardiovascular and Interventional Radiology (SCVIR). There were 401 respondents. Replies were evaluated for frequency and indications of prophylaxis, specific prophylaxis used, and clarity of indications for prophylaxis. RESULTS: A majority of responders never used prophylaxis for arteriography, angioplasty, vascular stent placement, IVC filter placement, abdominal drainage, and enteral tube placement. Infective complication rates from nonusage ranged between 1% and 15%. Approximately 45% always used prophylaxis for tunneled-port placement and TIPS with a 13%-16% infective complication rate among nonusers. In contrast, a majority of responders always used prophylaxis for biliary and genitourinary drainage, with a 40%-58% infective complication rate in nonusers. More than 70% of responders believed that the indications for prophylaxis were not clear for arteriography, angioplasty, vascular stent placement, tunneled-port placement, TIPS, IVC filter placement, and enteral tube placement, and in contrast, that the indications for prophylaxis for biliary and genitourinary drainage were clear. Fifty-one percent of responders believed that indications for prophylaxis for abdominal drainage were clear. CONCLUSIONS: Indications for antibiotic prophylaxis are not clear to interventionalists for a large number of vascular and nonvascular interventional procedures. Prophylaxis appears unnecessary for routine arteriography, angioplasty, IVC filter placement, vascular stent placement, or enterostomy tube placement. Antibiotic prophylaxis is warranted for TIPS and tunneled-port placement. Conversely, indications for antibiotic prophylaxis are clear to interventionalists for biliary and genitourinary drainage procedures. Routine prophylaxis remains warranted for both.


Subject(s)
Antibiotic Prophylaxis , Cardiovascular Diseases/therapy , Practice Patterns, Physicians'/statistics & numerical data , Radiography, Interventional , Antibiotic Prophylaxis/statistics & numerical data , Data Collection , Humans , Radiography, Interventional/methods , Radiography, Interventional/statistics & numerical data
4.
Article in English | MEDLINE | ID: mdl-9972280

ABSTRACT

Evidence that the insulin-like growth factors play a role in embryonic as well as postnatal growth and central nervous system development has accumulated recently from studies using knock-out mice models. However, no effects of IGF-I and II have been demonstrated prior to organogenesis in these studies. We summarize here results supporting the role of insulin (or its precursor proinsulin) in vertebrate development prior to the expression of IGFs. (Pro)insulin mRNA is expressed in the chick embryo during neurulation and early organogenesis and its inhibition by antisense oligodeoxynucleotides increase apoptosis. In another system, proliferative neuroretina, (pro)insulin expression predominates over IGF-I expression. Modulation of apoptosis by (pro)insulin in retina may be largely responsible for the observed stimulation of DNA synthesis and neuronal differentiation. These effects are elicited as well by IGF-I, expressed later in neuroretina. Thus, these polypeptides have complementary expression in early embryos which suggests coordinated actions during development.


Subject(s)
Insulin-Like Growth Factor I/genetics , Insulin/genetics , Proinsulin/genetics , Animals , Apoptosis/drug effects , Chick Embryo , Gene Expression Regulation, Developmental , Mice , Mice, Knockout , Nervous System/embryology , Nervous System/metabolism , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism
6.
Endocrinology ; 138(9): 3967-75, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9275088

ABSTRACT

The characterization of (pro)insulin as an early embryonic growth factor requires demonstration of its expression and cellular effects in vivo. By in situ hybridization, we found widespread preproinsulin transcripts in the chick embryo throughout gastrulation and neurulation, before the beginning of preproinsulin-like growth factor I expression and pancreatic organogenesis. To analyze the prepancreatic (pro)insulin effect on apoptotic cell death, we treated embryos with antisense oligodeoxynucleotides in ovo and in vitro. The specific effect of two preproinsulin messenger RNA (mRNA) antisense oligodeoxynucleotides was confirmed by the decrease in a biosynthetically labeled protein immunoprecipitated with antiinsulin Igs. Insulin receptor mRNA antisense oligodeoxynucleotide applied in ovo increased by 2.7-fold the level of apoptosis in the 1.5-day embryo (neurulation) compared with that in its random sequence control. In a whole embryo culture, apoptosis increased by 25-35% with the addition of preproinsulin or insulin receptor mRNAs antisense oligodeoxynucleotides, respectively, whereas it decreased by 64% after 10 h in the presence of 10(-8) M chicken insulin. Exogenous insulin also rescued the death induced by preproinsulin antisense oligonucleotides. These findings provide evidence for an autocrine/paracrine role ofpreproinsulin gene products acting through the insulin receptor in the control of cell survival/death during early embryonic development.


Subject(s)
Apoptosis , Chick Embryo/cytology , Embryo, Mammalian/cytology , Embryo, Nonmammalian , Proinsulin/physiology , Receptor, Insulin/physiology , Animals , Apoptosis/drug effects , Chick Embryo/growth & development , Culture Techniques , Embryonic and Fetal Development , In Situ Hybridization , Insulin/pharmacology , Insulin-Like Growth Factor I/genetics , Oligonucleotides, Antisense/pharmacology , Pancreas/embryology , Proinsulin/genetics , Protein Precursors/genetics , RNA, Messenger/analysis , Receptor, Insulin/genetics , Signal Transduction , Time Factors
7.
Dev Dyn ; 206(4): 343-53, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8853984

ABSTRACT

cCdx-B (formerly cHox-cad 2) is a chick homeobox-containing gene related to the Drosophila caudal. Compared with other caudal homologues, its similarity is highest with the murine Cdx-4. In the present study, we characterize the localization of cCdx-B transcripts to the caudal region of the embryo by using reverse transcription-polymerase chain reaction (RT-PCR) and, in detail, by using in situ hybridization. Chick embryos from gastrulation to early organogenesis were hybridized with digoxigenin-labeled riboprobes, and the pattern of expression of cCdx-B mRNA was analyzed in wholemount embryos and in tissue sections. In the early gastrula, transcripts were localized in a gradient through the caudal half of the embryo, in the epiblast and the mesoderm cells, but not including Hensen's node. During neurulation, cCdx-B transcripts were found more rostrally, with high levels localized in Hensen's node and the posterior neural plate. Expression was also high in paraxial mesoderm, with a rostral limit in the most recently formed somite. There was no expression in definitive endoderm. During late neurulation and tail bud formation, cCdx-B mRNA expression regressed posteriorly and was finally confined to the tail bud region. This pattern of expression of cCdx-B, regulated in time and space, is different from that of the other known chick caudal homologue, cCdx-A. Both genes may play a coordinated role in the posterior axial patterning of the chick embryo, whereas cCdx-B may specify further the identity of the tail region.


Subject(s)
Avian Proteins , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Genes, Homeobox , Homeodomain Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Chick Embryo , Molecular Sequence Data
9.
Endocrinology ; 135(6): 2342-50, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7988416

ABSTRACT

Despite the absence of a pancreas, which develops between embryonic day 3 (E3) to E4, previous studies showed that insulin receptors are widely expressed in chicken embryos from the blastoderm stage (unincubated embryo, E0) through gastrulation (E0.5-E1), neurulation (E1.5-E2), and organogenesis. We now characterize prepancreatic preproinsulin gene expression and its regulation, using a highly sensitive modification of the polymerase chain reaction. We found preproinsulin messenger RNA (mRNA) expression at all stages, from the unincubated chicken blastoderm through early organogenesis, with the highest expression in embryos undergoing gastrulation. In situ hybridization analysis of E1-E1.5 embryos in toto showed widespread distribution of preproinsulin mRNA in a pattern similar to that of insulin receptor mRNA. In contrast, insulin-like growth factor-I mRNA expression appeared later than preproinsulin mRNA in the embryo; it was first demonstrable in the head portion of E3 and was found in head, trunk, and caudal regions by E4. With a novel culture system for chicken embryos during neurulation, we examined whether glucose regulated prepancreatic preproinsulin mRNA expression. Embryos cultured in glucose-free medium had increased preproinsulin mRNA with respect to the value in ovo, but the addition of 17 mM glucose had no stimulatory effect. In marked contrast, in organ cultures of E13 pancreas, insulin mRNA expression decreased in glucose-free medium by 50% relative to that in ovo. The addition of glucose restored the levels to a concentration similar to that found in ovo. Exogenous insulin added to cultured E1.5 embryos increased protein and DNA synthesis. We conclude that the preproinsulin gene is widely expressed in chicken embryo structures throughout gastrulation and neurulation. This prepancreatic preproinsulin mRNA is differentially regulated compared to the pancreatic mRNA. Preproinsulin gene products may have a role in cell proliferation, differentiation, or survival in very early avian embryos at a time when insulin-like growth factor-I expression is absent or undetectable.


Subject(s)
Embryonic and Fetal Development , Gastrula/physiology , Gene Expression Regulation, Developmental , Nervous System/embryology , Proinsulin/genetics , Protein Precursors/genetics , Animals , Base Sequence , Chick Embryo/metabolism , Chromatography, Thin Layer , In Situ Hybridization , Insulin , Insulin-Like Growth Factor I/genetics , Molecular Sequence Data , Oligonucleotide Probes/genetics , Pancreas/embryology , Polymerase Chain Reaction , RNA, Messenger/metabolism , Transcription, Genetic
10.
Arch Environ Health ; 33(1): 12-5, 1978.
Article in English | MEDLINE | ID: mdl-629591

ABSTRACT

In May 1976 an investigation of a factory in Puerto Rico which formulates oral contraceptives revealed that during the previous 12 months five of the company's twenty-five employees (20%), and twelve of the company's thirty female employees (40%) had experienced symptoms associated with hyperestrogenism. The affected males had gynecomastia and three of them also reported a history of decreased libido or impotence. The affected females each had had at least one episode of intermenstrual bleeding during the preceding 12 months. There was an estimated relative risk of 4.3 for intermenstrual bleeding in nonclerical female employees compared with matched controls who did not work at the plant. Elevated levels of plasma ethinyl estradiol were twice as frequent in the two highest-risk job categories compared with the rest of the factory population, but the difference in prevalence of elevated levels was not statistically significant (P = 0.08). Wide variations in mestranol concentration were noted in the environmental dust samples. Prompt consideration should be given to establishing health standards for persons occupationally exposed to estrogens in view of the possible long-term sequelae of such exposure.


Subject(s)
Contraceptives, Oral/toxicity , Occupational Diseases/chemically induced , Drug Industry , Estrogens/toxicity , Ethinyl Estradiol/blood , Female , Gynecomastia/chemically induced , Humans , Male , Menstruation Disturbances/chemically induced
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