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Hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF1beta (or vHNF1) are closely related transcription factors expressed in liver, kidney, gut, and pancreatic beta-cells. Many HNF1 target genes are involved in carbohydrate metabolism. Human mutations in HNF1alpha or HNF1beta lead to maturity-onset diabetes of the young (MODY3 and MODY5, respectively), and patients present with impaired glucose-stimulated insulin secretion. The underlying defect in MODY5 is not known. Analysis of HNF1beta deficiency in mice has not been possible because HNF1beta null mice die in utero. To examine the role of HNF1beta in glucose homeostasis, viable mice deleted for HNF1beta selectively in beta-cells (beta/H1beta-KO mice) were generated using a Cre-LoxP strategy. beta/H1beta-KO mice had normal growth, fertility, fed or fasted plasma glucose and insulin levels, pancreatic insulin content, and insulin sensitivity. However, beta/H1beta-KO mice exhibited impaired glucose tolerance with reduced insulin secretion compared with wild-type mice but preserved a normal insulin secretory response to arginine. Moreover, beta/H1beta-KO islets had increased HNF1alpha and Pdx-1, decreased HNF4 mRNA levels, and reduced glucose-stimulated insulin release. These results indicate that HNF1beta is involved in regulating the beta-cell transcription factor network and is necessary for glucose sensing or glycolytic signaling.

The homeodomain transcription factor, pancreas duodenum homeobox (PDX)-1, is essential for pancreas development, insulin production, and glucose homeostasis. Mutations in pdx-1(ipf-1) are associated both with maturity-onset diabetes of the young and type 2 diabetes. PDX-1 interacts with multiple transcription factors and coregulators, including the coactivator p300, to activate the transcription of the insulin gene and other target genes within pancreatic beta-cells. In characterizing the protein-protein interactions of PDX-1 and p300, we identified mutations in PDX-1 that disrupt its function and are associated with increased or decreased interactions with p300. Several mutant PDX-1 proteins that are associated with heritable forms of diabetes in humans, in particular the mutant P63fsdelC, exhibited increased binding to a carboxy-terminal segment of p300 in the setting of decreased DNA-binding activities, suggesting that sequestration of p300 by mutant PDX-1 proteins may be an additional mechanism by which insulin gene expression is reduced in heterozygous carriers of pdx-1(ipf-1) mutations. The introduction of the point mutations S66A/Y68A in the highly conserved amino-terminal PDX-1 transactivation domain reduced the ability of PDX-1 to interact with p300, substantially diminished the transcriptional activation of PDX-1, and reduced the synergistic activation of glucose-responsive insulin promoter enhancer sequences by PDX-1, E12, and E47. We propose that interactions of PDX-1 with p300 are required for the transcriptional activation of PDX-1 target genes. Impairment of interactions between PDX-1 and p300 in pancreatic beta-cells may limit insulin production and lead to the development of diabetes.

In a transgenic mouse model of the neurodegenerative disorder Huntington's disease (HD), age-dependent neurologic defects are accompanied by progressive alterations in glucose tolerance that culminate in the development of diabetes mellitus and insulin deficiency. Pancreatic islets from HD transgenic mice express reduced levels of the pancreatic islet hormones insulin, somatostatin, and glucagon and exhibit intrinsic defects in insulin production. Intranuclear inclusions accumulate with aging in transgenic pancreatic islets, concomitant with the decline in glucose tolerance. HD transgenic mice develop an age-dependent reduction of insulin mRNA expression and diminished expression of key regulators of insulin gene transcription, including the pancreatic homeoprotein PDX-1, E2A proteins, and the coactivators CBP and p300. Disrupted expression of a subset of transcription factors in pancreatic beta cells by a polyglutamine expansion tract in the huntingtin protein selectively impairs insulin gene expression to result in insulin deficiency and diabetes. Selective dysregulation of gene expression in triplet repeat disorders provides a mechanism for pleiotropic cellular dysfunction that restricts the toxicity of ubiquitously expressed proteins to highly specialized subpopulations of cells.

The objective of this literature review is to explore and synthesize research related to Amish women's health issues. Literature searches were conducted on an extensive list of Internet-based databases, with a total of 767 articles identified. Inclusion criteria consisted of published, gender-based, and evidence-based research. The research reviewed covered a broad spectrum of health-related topics, and nearly all authors cited cultural factors as an explanation for the differences in Amish and non-Amish populations. However, the lack of evidence-based research and diversity of the topics prevented clear patterns of health-related issues to be discussed. Many researchers made broad-sweeping generalizations about the Amish, which can perpetuate the stereotypes and myths associated with their way of life. It is imperative that more evidence-based research be conducted to determine the cultural aspects and other factors that may influence health in this population.

Monogenic forms of diabetes can result from mutations in genes encoding transcription factors. Mutations in the homeodomain transcription factor IDX-1, a critical regulator of pancreas development and insulin gene transcription, confer a strong predisposition to the development of diabetes mellitus in humans. To investigate the role of IDX-1 expression in the pathogenesis of diabetes, we developed a model for the inducible impairment of IDX-1 expression in pancreatic beta cells in vivo by engineering an antisense ribozyme specific for mouse IDX-1 mRNA under control of the reverse tetracycline transactivator (rtTA). Doxycycline-induced impairment of IDX-1 expression reduced activation of the Insulin promoter but activated the Idx-1 promoter, suggesting that pancreatic beta cells regulate IDX-1 transcription to maintain IDX-1 levels within a narrow range. In transgenic mice that express both rtTA and the antisense ribozyme construct, impaired IDX-1 expression elevated glycated hemoglobin levels, diminished glucose tolerance, and decreased insulin/glucose ratios. Metabolic phenotypes induced by IDX-1 deficiency were observed predominantly in male mice over 18 months of age, suggesting that cellular mechanisms to protect IDX-1 levels in pancreatic beta cells decline with aging. We propose that even in the absence of Idx-1 gene mutations, pathophysiological processes that decrease IDX-1 levels are likely to impair glucose tolerance. Therapeutic strategies to attain normal glucose homeostasis by restoring normal IDX-1 levels may be of particular importance for older individuals with diabetes mellitus.

Insulin gene expression in pancreatic beta-cells is regulated by signals from developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5'-deletion insulin promoter-reporter constructs in transient transfections of clonal INS-1 beta-cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response elements Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by ectopic Hh expression increased (and inhibition of Hh signaling with the Hh-specific inhibitor cyclopamine decreased) transcriptional activation of a multimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nuclear extracts from INS-1 cells activated by ectopic Hh expression increased (and extracts from INS-1 cells treated with cyclopamine decreased) protein binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum directed against the transcription factor islet duodenum homeobox-1 (IDX-1), a regulator of pancreas development and activator of the insulin gene promoter, attenuated the binding activity of Hh-responsive protein complexes. Nuclear IDX-1 protein levels on Western blots were increased by ectopic Hh expression, thereby providing a mechanism for Hh-mediated regulation of the insulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 messenger RNA expression. In transient transfections of a -4.5-kb mouse IDX-1 promoter-reporter construct, ectopic Hh expression increased (and cyclopamine administration decreased) transcriptional activation of the IDX-1 promoter in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory target of Hh signaling in insulin-producing pancreatic beta-cells. We propose that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is essential for insulin gene expression, pancreatic beta-cell development, and normal glucose homeostasis, our findings that Hh signaling regulates IDX-1 expression in the endocrine pancreas suggest possible novel therapeutic approaches for diabetes mellitus.

The endocrine cells of the rat pancreatic islets of Langerhans, including insulin-producing beta-cells, turn over every 40-50 days by processes of apoptosis and the proliferation and differentiation of new islet cells (neogenesis) from progenitor epithelial cells located in the pancreatic ducts. However, the administration to rats of islet trophic factors such as glucose or glucagon-like peptide 1 for 48 h results in a doubling of islet cell mass, suggesting that islet progenitor cells may reside within the islets themselves. Here we show that rat and human pancreatic islets contain a heretofore unrecognized distinct population of cells that express the neural stem cell-specific marker nestin. Nestin-positive cells within pancreatic islets express neither the hormones insulin, glucagon, somatostatin, or pancreatic polypeptide nor the markers of vascular endothelium or neurons, such as collagen IV and galanin. Focal regions of nestin-positive cells are also identified in large, small, and centrolobular ducts of the rat pancreas. Nestin-positive cells in the islets and in pancreatic ducts are distinct from ductal epithelium because they do not express the ductal marker cytokeratin 19 (CK19). After their isolation, these nestin-positive cells have an unusually extended proliferative capacity when cultured in vitro (approximately 8 months), can be cloned repeatedly, and appear to be multipotential. Upon confluence, they are able to differentiate into cells that express liver and exocrine pancreas markers, such as alpha-fetoprotein and pancreatic amylase, and display a ductal/endocrine phenotype with expression of CK19, neural-specific cell adhesion molecule, insulin, glucagon, and the pancreas/duodenum specific homeodomain transcription factor, IDX-1. We propose that these nestin-positive islet-derived progenitor (NIP) cells are a distinct population of cells that reside within pancreatic islets and may participate in the neogenesis of islet endocrine cells. The NIP cells that also reside in the pancreatic ducts may be contributors to the established location of islet progenitor cells. The identification of NIP cells within the pancreatic islets themselves suggest possibilities for treatment of diabetes, whereby NIP cells isolated from pancreas biopsies could be expanded ex vivo and transplanted into the donor/recipient.

Hedgehogs (Hhs) are intercellular signaling molecules that regulate tissue patterning in mammalian development. Mammalian Hhs include Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). The absence of Shh expression is required for the early development of the endocrine and exocrine pancreas, but whether Hh signaling functions in the fully developed adult endocrine pancreas is unknown. Here we report that Hhs Ihh and Dhh and their receptors patched (Ptc) and smoothened are expressed in the endocrine islets of Langerhans of the fully developed rat pancreas and in the clonal gamma-cell line INS-1. We demonstrate the coexpression of Ptc with insulin in beta-cells of mouse pancreatic islets, indicating that beta-cells are targets of active Hh signaling. The administration of cyclopamine, a Hh signaling inhibitor, decreases both insulin secretion from and insulin content of INS-1 cells. The effects of Hh signaling on insulin production occur at the transcriptional level because activation of Hh signal transduction by ectopic expression of Shh increases rat insulin I promoter activation in a dose-dependent manner in transient transfections of INS-1 and MIN6 beta-cell lines. In contrast, inhibition of Hh signaling with increasing concentrations of cyclopamine progressively reduces insulin promoter activity. Furthermore, the treatment of INS-1 cells with cyclopamine diminishes endogenous insulin mRNA expression. We propose that Hh signaling is not restricted to patterning in early pancreas development but also continues to signal in differentiated beta-cells of the endocrine pancreas in regulating insulin production. Thus, defective Hh signaling in the pancreas should be considered as a potential factor in the pathogenesis of type 2 diabetes.

The disorders of vitamin D metabolism are inherited metabolic abnormalities involving mutations of the vitamin D receptor or enzymes involved in the metabolism of vitamin D to its biologically active form 1,25-dihydroxyvitamin D. Although these mutations are rare, studies in affected patients and animal models have helped to identify critical actions of vitamin D and the mechanism by which it exerts its effects. Vitamin D deficiency, however, is an increasingly recognized problem among the elderly and in the general population. Screening for vitamin D deficiency only in those patients with known risk factors will result in a large proportion of unrecognized affected patients.

Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neurogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancreatic beta cell, E2A proteins, in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for beta-cell function. By yeast two-hybrid screening of a cDNA library prepared from rat insulinoma (INS-1) cells, we identified a novel protein, Bridge-1, that interacts with E2A proteins and functions as a coactivator of gene transcription mediated by E12 and E47. Bridge-1 contains a PDZ-like domain, a domain known to be involved in protein-protein interactions. Bridge-1 is highly expressed in pancreatic islets and islet cell lines and the expression pattern is primarily nuclear. The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro-translated Bridge-1 with E12 or E47 and by mammalian two-hybrid studies. The PDZ-like domain of Bridge-1 is required for interaction with the carboxy terminus of E12. In both yeast and mammalian two-hybrid interaction studies, Bridge-1 mutants lacking an intact PDZ-like domain interact poorly with E12. An E12 mutant (E12DeltaC) lacking the carboxy-terminal nine amino acids shows impaired interaction with Bridge-1. Bridge-1 has direct transactivational activity, since a Gal4 DNA-binding domain-Bridge-1 fusion protein transactivates a Gal4CAT reporter. Bridge-1 also functions as a coactivator by enhancing E12- or E47-mediated activation of a rat insulin I gene minienhancer promoter-reporter construct in transient-transfection experiments. Substitution of the mutant E12DeltaC for E12 reduces the coactivation of the rat insulin I minienhancer by Bridge-1. Inactivation of endogenous Bridge-1 in insulinoma (INS-1) cells by expression of a Bridge-1 antisense RNA diminishes rat insulin I promoter activity. Bridge-1, by utilizing its PDZ-like domain to interact with E12, may provide a new mechanism for the coactivation and regulation of transcription of the insulin gene.

Three methods have been used to assess the conformational effects associated with ligand binding to two unrelated cyclic nucleotide receptor proteins: the cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) and the cGMP-dependent protein kinase (PKG). The methods should be applicable to other proteins and to other types of modification such as phosphorylation. The procedures use either ion-exchange chromatography, size-exclusion chromatography, or native gel electrophoresis of these proteins in the absence and presence of regulatory ligands. Measurements from these respective approaches allow documentation of changes in the quaternary structure, surface electronegativity, and relative compactness (Stokes radius) of the protein molecule. The combined data allow the changes in protein conformation to be quantitated in terms of alterations in the axial ratio or length/width dimension of the molecule. The methods can be applied to partially purified proteins and to proteins that are available in limited quantities. Conformational changes due to stable modifications of proteins can be potentially examined in crude extracts of intact cells. Each of the methods can be tailored to optimize resolution of a particular protein under a variety of conditions. Activity measurements, Coomassie brilliant blue or silver staining of gels, radioautography, or Western blot analysis can be used for detection of the protein., (Copyright 1998 Academic Press.)

The homeodomain protein IDX-1 appears to be a "master regulator" of pancreas development and beta-cell differentiation and function. In murine gene inactivation models and in a human subject with a homozygous mutation of the IDX-1 gene, the pancreas fails to develop. In the adult endocrine pancreas, IDX-1 is primarily expressed in beta cells, where it is a key factor in the upregulation of insulin gene transcription and appears to have a role in the regulation of the somatostatin, glucokinase, glucose transporter-2, and islet amyloid polypeptide genes. Recent studies also suggest a role for IDX-1 in the neogenesis and proliferation of beta cells. The observed functions of IDX-1 and its downregulation in parallel with insulin in glucose-toxicity models implicate IDX-1 as a potential factor contributing to the pathogenesis of diabetes mellitus. Future directions include the use of conditional gene inactivation to determine more precisely the role of IDX-1 throughout endocrine pancreas differentiation and the exploration of IDX-1 as a potential target for gene therapy of diabetes mellitus.

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.

The prothoracicotropic hormones (PTTH) are cerebral peptides that control insect postembryonic development by stimulating the prothoracic glands to synthesize ecdysteroids. Using immunoaffinity chromatography and SDS-PAGE, a 25.5 kDa big PTTH has been purified from Manduca sexta. Based upon SDS-PAGE and Western blot analysis, the native form of big PTTH appears to be a dimer with monomers of 16.5 kDa. Four HPLC-separated fragments of this acidic peptide were sequenced and exhibited no sequence similarity with Bombyx mori PTTH. In agreement with this finding, the basic Bombyx PTTH had no PTTH bioactivity in Manduca. One sequenced fragment of the Manduca PTTH is approximately 70% similar to the vertebrate cellular retinoid binding proteins, suggesting these binding proteins may be present in insects.

The clinical course of 87 infants with bronchopulmonary dysplasia (BPD) on home oxygen therapy was reviewed to determine the occurrence of systemic hypertension (HTN) and to evaluate associated clinical features. Eleven of 87 (13%) infants developed systemic HTN either in the neonatal intensive care unit or following discharge. Clinical features that distinguished the hypertensive from the normotensive group were as follows: greater use of bronchodilators, 91% vs 37% (p < 0.001), and diuretics, 91% vs 55% (p < 0.05), longer duration of home oxygen therapy 21.6 +/- 9.9 vs 9.2 +/- 5.8 months (p < 0.05), and greater mortality, 36% vs 1% (p < 0.001). The course of systemic HTN in the surviving patients (7 of 11) was benign and resolved in all patients prior to weaning from home oxygen therapy. Systemic HTN is frequently present in infants with severe BPD and appears to be related to the clinical severity of lung disease.

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.

To determine whether long-term renal sequelae follow the use of furosemide in preterm infants, we evaluated renal function in 27 former very low birth weight infants (less than 1500 gm) at 1 to 2 years of age. Patients were classified into three groups on the basis of status at the time of discharge from the hospital: group 1 (n = 7) had no furosemide treatment or renal calcifications, group 2 (n = 10) had furosemide therapy but no calcifications, and group 3 (n = 10) had furosemide therapy with renal calcifications. Renal ultrasonography at the time of the study demonstrated resolution of the calcifications in six patients in group 3. No differences in renal function were observed between groups 1 and 2. Creatinine clearance (mean +/- SEM) in group 3 (83.6 +/- 7.8 ml/min per 1.73 m2) was significantly lower than clearance in groups 1 and 2 (103.2 +/- 6.5 and 109.1 +/- 5.1, respectively; p less than 0.05). Children in group 3 had significantly higher urinary calcium/creatinine ratios and fractional excretion of sodium and lower tubular reabsorption of phosphate than children in the two other groups had. Urine-blood difference in carbon dioxide tension after oral acetazolamide load, which indicates the ability of the distal tubule to secrete hydrogen ions, was 8.4 +/- 3.4 mm Hg in group 3, significantly lower than values in groups 1 and 2 (22.6 +/- 3.1 and 28.0 +/- 4.3 mm Hg, respectively, p less than 0.05). Within group 3 the four children with persistent renal calcifications had significantly lower urine-blood carbon dioxide tension differences than did those with resolution of calcifications (p = 0.02). We conclude that furosemide-related renal calcifications in very low birth weight infants may lead to glomerular and tubular dysfunction; further long-term follow-up of this population is recommended.

This survey may provide a starting point for the improvement of the acceptability of headgears for children and teenagers during orthodontic treatment by taking aesthetic and material factors into consideration when selecting these appliances. The extent to which these findings are allowed to influence the planning of future treatment must be decided on an individual base; it is to be expected that such considerations will contribute both to the success of therapeutic aims and a reduction of costs in orthodontics.

A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.

A purified bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) was rapidly phosphorylated by purified bovine lung cGMP-dependent protein kinase (cGK). Within a physiological concentration range, cGK catalyzed phosphorylation of cG-BPDE at a rate approximately 10 times greater than did equimolar concentrations of purified catalytic subunit of cAMP-dependent protein kinase (cAK). cG-BPDE was a poor substrate for either purified protein kinase C or Ca2+/calmodulin-dependent protein kinase II. Binding of cGMP to the cG-BPDE binding site was required for phosphorylation since (a) phosphorylation of cG-BPDE by the catalytic subunit of cAK was cGMP-dependent, (b) phosphorylation of cG-BPDE in the presence of a cGMP analog specific for activation of cGK was cGMP-dependent, and (c) occupation of the cG-BPDE hydrolytic site with competitive inhibitors did not produce the cGMP-dependent effect. cGMP-dependent phosphorylation of cG-BPDE by both cGK and cAK occurred at serine. Proteolytic digestion of cG-BPDE phosphorylated by either cGK or cAK revealed the same phosphopeptide pattern, suggesting that phosphorylation by the two kinases occurred at the same or adjacent site(s). Tryptic digestion of cG-BPDE phosphorylated by cGK and [gamma-32P]ATP produced a single major phosphopeptide of approximately 2 kDa with the following amino-terminal sequence: Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg- Radioactivity was released during the third cycle of Edman degradation. cG-BPDE is one of few specific in vitro cGK substrates of known function to be identified. Elevation of intracellular cGMP may cause phosphorylation of cG-BPDE by modulating the substrate site availability as well as by activating cGK. Such regulation would greatly increase the selectivity of the phosphorylation of cG-BPDE and would represent a unique mechanism of action of a cyclic nucleotide or other second messenger.

2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.

In an insect, the tobacco hornworm Manduca sexta, the cerebral neuropeptide prothoracicotropic hormone (PTTH), the primary effector of postembryonic development, exists as two molecular forms. These two PTTH's elicit characteristic in vitro dose responses of activation of prothoracic glands from different developmental stages, an indication that during development the glands change in their sensitivity to the neurohormones. Both PTTH's are active in a specific in situ bioassay. Since they may be released in situ at stage-specific times to evoke distinctly different developmental responses, the PTTH neuroendocrine axis appears to be an effective system for determining the functions of molecular forms of a neurohormone in the regulation of growth and development.

Ecdysteroidogenesis in Manduca sexta prothoracic glands is regulated by a set of bioregulatory molecules, including prothoracicotropic hormone (PTTH) and a protein factor present in larval hemolymph, and by the competence of the glands to synthesize ecdysteroids in response to those molecules. A larval molting bioassay was used to assess the in vivo activity of Manduca PTTHs. Crude PTTH, big PTTH, and small PTTH each elicited a larval molt in head-ligated larvae. However, big PTTH was approximately 10-fold more potent than crude PTTH, which was, in turn, several orders of magnitude more potent than small PTTH. When big and small PTTH were combined, the molting response was similar to that elicited with crude PTTH. The chemical nature of the hemolymph protein factor was also investigated. Injection of [3H]cholesterol into last-instar larvae and fractionation of the radiolabeled hemolymph by gel filtration chromatography revealed three peaks of radioactivity. One peak eluted in fractions containing the hemolymph protein factor, a result consistent with the notion that the factor transports a sterol substrate. The possibility that the factor is a 3(2)-ketoreductase was investigated by assessing the effect of the factor on the accumulation of RIA-detectable ecdysteroids in prothoracic-gland-conditioned medium. Three of five preparations of the factor significantly enhanced the amount of RIA-detectable ecdysteroids in conditioned medium, indicating that at least some preparations of the factor may contain ketoreductase activity. The above findings are discussed in the context of current hypotheses of how bioregulatory molecules interact with the prothoracic glands to regulate ecdysteroidogenesis in Manduca.