Non-coding variation in disorders of sex development.

Genetic studies in Disorders of Sex Development (DSD), representing a wide spectrum of developmental or functional conditions of the gonad, have mainly been oriented towards the coding genome. Application of genomic technologies, such as whole-exome sequencing, result in a molecular genetic diagnosis in ∼50% of cases with DSD. Many of the genes mutated in DSD encode transcription factors such as SRY, SOX9, NR5A1, and FOXL2, characterized by a strictly regulated spatiotemporal expression. Hence, it can be hypothesized that at least part of the missing genetic variation in DSD can be explained by non-coding mutations in regulatory elements that alter gene expression, either by reduced, mis- or overexpression of their target genes. In addition, structural variations such as translocations, deletions, duplications or inversions can affect the normal chromatin conformation by different mechanisms. Here, we review non-coding defects in human DSD phenotypes and in animal models. The wide variety of non-coding defects found in DSD emphasizes that the regulatory landscape of known and to be discovered DSD genes has to be taken into consideration when investigating the molecular pathogenesis of DSD.

Calcium and bone homeostasis in heterozygous carriers of CYP24A1 mutations: A cross-sectional study.

BACKGROUND: Bi-allelic CYP24A1 mutations can cause idiopathic infantile hypercalcemia (IIH), adult-onset nephrocalcinosis, and possibly bone metabolism disturbances. It is currently unclear if heterozygous carriers experience clinical problems or biochemical abnormalities. Our objective is to gain insight in the biochemical profile and health problems in CYP24A1 heterozygotes. STUDY DESIGN: Cross-sectional evaluation of participants. Data of previously reported carriers are reviewed. SETTING AND PARTICIPANTS: Outpatient clinic of a tertiary care hospital. Participants were eight family members of an infant with a well-characterized homozygous CYP24A1 mutation c.1186C>T p.(Arg396Trp). OUTCOMES: Serum vitamin D metabolites. Symptoms or biochemical signs of hypercalcemia, hypercalciuria or nephrocalcinosis. Bone health in heterozygous as compared to wild type (WT) subjects. MEASUREMENTS: Genotyping by Sanger sequencing; vitamin D metabolites by liquid chromatography tandem mass spectrometry; renal, calcium and bone markers by biochemical analyses; presence of nephrocalcinosis by renal ultrasound; bone health by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. RESULTS: Six participants were heterozygous carriers of the mutation. None of the heterozygous subjects had experienced IIH. One had a documented history of nephrolithiasis, two others had complaints compatible with this diagnosis. No major differences between WT and heterozygous subjects were found regarding bone health, serum or urinary calcium or 25OHD/24,25(OH)2D ratio. Literature reports on three out of 33 heterozygous cases suffering from IIH. In all three, the 25OHD/24,25(OH)2D ratio was highly elevated. Nephrocalcinosis was frequently reported in family members of IIH cases. LIMITATIONS: Small sample size, lack of a large control group. CONCLUSIONS: Our and literature data suggest that most heterozygous CYP24A1 mutation carriers have a normal 25OHD/24,25(OH)2D ratio, are usually asymptomatic and have a normal skeletal status but may possibly be at increased risk of nephrocalcinosis. A review of the available literature suggests that an elevated 25OHD/24,25(OH)2D ratio may be associated with symptoms of IHH, irrespective of carrier status.

Expression and localization of VEGF-C and VEGFR-3 in glioblastomas and haemangioblastomas.

Primary human brain tumours account for approximately 2% of all cancers. High levels of expression of vascular endothelial growth factor-A (VEGF-A), a potent angiogenic factor, are linked to poor prognosis. In contrast, the potential role in human brain tumour biology of newer VEGF family members, VEGF-C and VEGF-D, both of which are lymphangiogenic factors, is poorly understood. In the present study, the expression of all VEGFs (VEGF-A, -B, -C, and -D) and their receptors (VEGFR-1, -2, and -3) has been assessed in 39 primary human brain tumours. The well-established findings were confirmed with VEGF-A. Surprisingly, however, VEGF-C and VEGF-D, as well as VEGFR-3, were expressed in some tumour types such as haemangioblastomas and glioblastomas, despite their lack of lymphatic vessels. VEGF-C and VEGFR-3 transcripts were localized to the tumour palisade around necrotic areas in glioblastomas and were evenly distributed throughout haemangioblastomas. VEGF-C protein was localized by immunohistochemistry to the palisade layer in glioblastomas. More than 50% of VEGF-C-positive cells also expressed the intermediate-stage inflammatory macrophage marker CD163; however, a significant proportion of VEGF-C-positive cells were CD163-negative. These data demonstrate the presence of molecules, primarily described as regulators of lymphangiogenesis, in normal human brain and brain tumours that are devoid of lymphatics. Their localization in macrophages points to a role in tumour-associated inflammation.

Gas chromatographic analysis of polyhydroxybutyrate in activated sludge: a round-robin test.

Polyhydroxyalkanoates (PHA) and poly-beta-hydroxybutyrate (PHB) in particular have become compounds which is routinely investigated in wastewater research. The PHB analysis method has only recently been applied to activated sludge samples where PHA contents might be relatively low. This urges the need to investigate the reproducibility of the gas chromatographic method for PHB analysis. This was evaluated in a round-robin test in 5 European laboratories with samples from lab-scale and full-scale enhanced biological phosphorus removal systems. It was shown that the standard deviation of measurements in each lab and the reproducibility between the labs was very good. Experimental results obtained by different laboratories using this analysis method can be compared. Sludge samples with PHB contents varying between 0.3 and 22.5 mg PHB/mg sludge were analysed. The gas chromatographic method allows for PHV, PH2MB and PH2MV analysis as well.

Coupled particle light scattering: a new technique for serodiagnosis of Epstein-Barr virus infection.

The Coupled Particle Light Scattering technique was evaluated for serological diagnosis of Epstein-Barr Virus (EBV) infection. Two hundred ninety-six patient sera selected from several clinical categories (acute infection, non-primary infection, interfering non-EBV infection, non-infected) were tested for IgM and IgG antibodies (anti-VCA, anti-EBNA and anti-EA). Determination of EBV IgG with Copalis multiplex was accurate when compared with Enzygnost Anti-EBV/IgG ELISA. Although the sensitivity of Copalis IgM for acute infections was 100% a positive IgM result did not always indicate an acute infection. Strong reactivity to IgG EA (ratio 3, 1) and IgG VCA (ratio 13, 3) correlated with persistent infection or reactivation. The CopalisI has many advantages over the existing methods, such as the possibility to measure three semi-quantitative IgG responses to three different EBV antigens simultaneously.

Delivery of FGF-2 but not VEGF by encapsulated genetically engineered myoblasts improves survival and vascularization in a model of acute skin flap ischemia.

Stimulating angiogenesis by gene transfer approaches offers the hope of treating tissue ischemia which is untreatable by currently practiced techniques of vessel grafting and bypass surgery. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) are potent angiogenic molecules, making them ideal candidates for novel gene transfer protocols designed to promote new blood vessel growth. In this study, an ex vivo gene therapy approach utilizing cell encapsulation was employed to deliver VEGF and FGF-2 in a continuous and localized manner. C(2)C(12) myoblasts were genetically engineered to secrete VEGF(121), VEGF(165) and FGF-2. These cell lines were encapsulated in hollow microporous polymer membranes for transplantation in vivo. Therapeutic efficacy was evaluated in a model of acute skin flap ischemia. Capsules were positioned under the distal, ischemic region of the flap. Control flaps showed 50% necrosis at 1 week. Capsules releasing either form of VEGF had no effect on flap survival, but induced a modest increase in distal vascular supply. Delivery of FGF-2 significantly improved flap survival, reducing necrosis to 34.2% (P < 0.001). Flap vascularization was significantly increased by FGF-2 (P < 0.01), with numerous vessels, many of which had a large lumen diameter, growing in the proximity of the implanted capsules. These results demonstrate that FGF-2, delivered from encapsulated cells, is more efficacious than either VEGF(121) or VEGF(165) in treating acute skin ischemia and improving skin flap survival. Furthermore, these data attest to the applicability of cell encapsulation for the delivery of angiogenic factors for the treatment and prevention of tissue ischemia.

Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.

Regulation of VEGF and VEGF receptor expression in the rodent mammary gland during pregnancy, lactation, and involution.

Vascular endothelial growth factors (VEGFs) are endothelial cell-specific mitogens with potent angiogenic and vascular permeability-inducing properties. VEGF, VEGF-C, and VEGFRs -1, -2, and -3 were found to be expressed in post-pubertal (virgin) rodent mammary glands. VEGF was increased during pregnancy (5-fold) and lactation (15-19-fold). VEGF-C was moderately increased during pregnancy and lactation (2- and 3-fold respectively). VEGF levels were reduced by approximately 75% in cleared mouse mammary glands devoid of epithelial components, demonstrating that although the epithelial component is the major source of VEGF, approximately 25% is derived from stroma. This was confirmed by the findings (a) that VEGF transcripts were expressed predominantly in ductal and alveolar epithelial cells, and (b) that VEGF protein was localized to ductal epithelial cells as well as to the stromal compartment including vascular structures. VEGF was detected in human milk. Finally, transcripts for VEGFRs -2 and -3 were increased 2-3-fold during pregnancy, VEGFRs -1, -2 and -3 were increased 2-4-fold during lactation, and VEGFRs -2 and -3 were decreased by 20-50% during involution. These results point to a causal role for the VEGF ligand-receptor pairs in pregnancy-associated angiogenesis in the mammary gland, and suggest that they may also regulate vascular permeability during lactation.

Lipotoxic heart disease in obese rats: implications for human obesity.

To determine the mechanism of the cardiac dilatation and reduced contractility of obese Zucker Diabetic Fatty rats, myocardial triacylglycerol (TG) was assayed chemically and morphologically. TG was high because of underexpression of fatty acid oxidative enzymes and their transcription factor, peroxisome proliferator-activated receptor-alpha. Levels of ceramide, a mediator of apoptosis, were 2-3 times those of controls and inducible nitric oxide synthase levels were 4 times greater than normal. Myocardial DNA laddering, an index of apoptosis, reached 20 times the normal level. Troglitazone therapy lowered myocardial TG and ceramide and completely prevented DNA laddering and loss of cardiac function. In this paper, we conclude that cardiac dysfunction in obesity is caused by lipoapoptosis and is prevented by reducing cardiac lipids.

Troglitazone prevents mitochondrial alterations, beta cell destruction, and diabetes in obese prediabetic rats.

To determine whether the antidiabetic action of troglitazone (TGZ), heretofore attributed to insulin sensitization, also involves protection of beta cells from lipoapoptosis, we treated prediabetic Zucker Diabetic Fatty rats with 200 mg/kg per day of TGZ. Their plasma-free fatty acids and triacylglycerol fell to 1.3 mM and 111 mg/dl, respectively, compared with 2.0 mM and 560 mg/dl in untreated controls. Their islet triacylglycerol content was 34% below controls. In islets of control rats, beta cells were reduced by 82% and the islet architecture was disrupted; beta-cell glucose transporter-2 was absent, 85% of their mitochondria were altered, and they were unresponsive to glucose. In treated rats, the loss of beta cells was prevented, as were the loss of beta cell glucose transporter-2, the mitochondrial alterations, and the impairment of glucose-stimulated insulin secretion. We conclude that the antidiabetic effect of TGZ in prediabetic Zucker Diabetic Fatty rats involves prevention of lipotoxicity and lipoapoptosis of beta cells, as well as improvement in insulin sensitivity.

A sudden awakening from a near coma after combined intake of gamma-hydroxybutyric acid (GHB) and ethanol.

OBJECTIVE: A case of a sudden awakening from a near coma after combined intake or gamma-hydroxybutyric acid (GHB) (125 micrograms/mL), ethanol (134 mg/dL), and cannabinoids is described. METHODS: GHB was determined by gas chromatography-mass spectrometry after acetonitrile precipitation and derivation with N-methyl-N-trimethylsilyltrifluoroacetamide, using valproic acid as the internal standard. CONCLUSION: The described case illustrates the consequences of GHB overdose. GHB overdose should be considered in every case of unexplained sudden coma, i.e., without any evidence of head injury, intake of coma-inducing drugs, or increasing intracranial pressure. GHB overdose will be missed by routine toxicological screening.

Identification and characterization of alpha 3 beta 1 integrin on primary and transformed rat islet cells.

Dispersed rat islet cells embedded in a matrix of collagen I are known to form aggregates in vitro reminiscent of native islets. Furthermore, it appears that islet function and survival are better maintained in vitro when cells are grown in the presence of extracellular matrix. These studies suggest an important role of cell--matrix interactions in the formation and maintenance of islet structure and function. The molecular basis of these interactions is mostly unknown. In the present study, we confirm the presence of beta 1 integrins on primary and transformed (RIN-2A line) rat islet cells. Perturbation studies in vitro show that beta 1 integrins play a role in islet cell attachment and spreading on bovine extracellular matrix and on the matrix produced by A-431 cells. The alpha 3 integrin subunit is coimmunoprecipitated with beta 1 from extracts of both primary and transformed islet cells, and immunodepletion studies suggest that alpha 3 beta 1 represents nearly half of the total beta 1 integrins expressed on primary islet cells. In situ, alpha 3 and beta 1 are expressed on the surface of all islet cell types, as shown by indirect immunocytochemistry on paraformaldehyde-fixed sections of rat pancreas. In conclusion, the study demonstrates the presence of alpha 3 beta 1 on primary and transformed rat islet cells, and an important role of beta 1 integrins in islet cell attachment and spreading in vitro.

Activity-dependent mobilization of the adhesion molecule polysialic NCAM to the cell surface of neurons and endocrine cells.

The alpha-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.

Expression of neural cell adhesion molecule (N-CAM) in rat islets and its role in islet cell type segregation.

Endocrine cell types are non-randomly distributed within pancreatic islets of Langerhans. In the rat, insulin-secreting B-cells occupy the core of the islets and are surrounded by A-, D- and PP-cells, secreting glucagon, somatostatin and pancreatic polypeptide, respectively. Furthermore, dissociated islet cells have the ability in vitro to form aggregates with the same cell-type organization as native islets (pseudoislets). These observations suggest that a differential expression of cell adhesion molecules (CAMs) might characterize B- and non-B-cells (A-, D- and PP-cells), and be in part responsible for the establishment and maintenance of islet architecture. Indirect immunofluorescence using antibodies against CAMs and islet hormones was performed on serial sections of the splenic and duodenal parts of the rat pancreas. Staining for the Ca(2+)-dependent CAM E-cadherin was detected on both exocrine and endocrine tissue and was uniform over the entire islet section, in both pancreatic regions. By contrast, staining for the Ca(2+)-independent neural CAM (N-CAM) was restricted to endocrine tissue and nerve endings. Furthermore, N-CAM staining of endocrine cells was stronger in the islet periphery, a region composed mostly of non-B-cells. Serial sections demonstrate that cells staining strongly for N-CAM in the splenic part correspond to glucagon cells and in the duodenal part to pancreatic polypeptide cells. Within pseudoislets in vitro a stronger staining for N-CAM was also observed on peripheral cells, corresponding to non-B-cells.

Enzymic and metabolic anomalies in islets of diabetic rats: relationship to B cell mass.

A preferential impairment of the pancreatic B cell secretory response to D-glucose occurs in adult rats injected with streptozotocin during the neonatal period. Three possible explanations for such a preferential defect were investigated in the present study. First, the time course for 3-O-methyl-D-glucose uptake by islets suggested that the anomaly in hexose transport was mainly attributable to a decrease in the space accessible to the D-glucose analog commensurate with the decrease in B cell mass, rather than to a delayed equilibration of hexose concentration across the B cell plasma membrane. Second, the activity of glucose-6-phosphatase was found to be equally low in islets from diabetic and control rats, ruling out the futile cycling between D-glucose and D-glucose 6-phosphate as a cause for the preferential alteration of the secretory response to the hexose. Third, the activity of flavine adenine dinucleotide-linked glycerophosphate dehydrogenase was found to be decreased to a greater relative extent than the B cell mass. This coincided with an impaired generation of 3HOH from L-[2-3H] glycerol in intact islets. It is proposed, therefore, that an altered circulation in the glycerol phosphate shuttle may play a major role in the impaired process of glucose-stimulated insulin release in this model of noninsulin-dependent diabetes.

Evidence that down-regulation of beta-cell glucose transporters in non-insulin-dependent diabetes may be the cause of diabetic hyperglycemia.

Non-insulin-dependent diabetes mellitus (NIDDM) is attributed to a failure of pancreatic beta cells to maintain insulin secretion at a level sufficient to compensate for underlying insulin resistance. In the ZDF rat, a model of NIDDM that closely resembles the human syndrome, we have previously reported profound underexpression of GLUT-2, the high-Km facilitative glucose transporter expressed by beta cells of normal animals. Here we report that islets of diabetic rats exhibit a marked decrease in the volume of GLUT-2-positive beta cells and a reduction at the electron-microscopic level in the number of GLUT-2-immunoreactive sites per unit of beta-cell plasma membrane. The deficiency of GLUT-2 cannot be induced in normal beta cells by in vivo or in vitro exposure to high levels of glucose nor can it be prevented in beta cells of prediabetic ZDF rats by elimination of hyperglycemia. We conclude that this dearth of immunodetectable GLUT-2 in NIDDM is not secondary to hyperglycemia and therefore that it may well play a causal role in the development of hyperglycemia.

Reduced beta-cell glucose transporter in new onset diabetic BB rats.

Previous studies from our laboratories have suggested a defect in glucose transport in islets isolated from BB rats on the first day of overt diabetes. To quantitate by immunostaining the glucose transporter of beta-cells (GLUT-2) before and at the onset of autoimmune diabetes we employed an antibody to its COOH-terminal octapeptide. On the first day of overt diabetes, defined as the day the daily blood glucose first reached 200 mg/dl, the volume density ratio of GLUT-2-positive to insulin-positive beta-cells was only 0.48 +/- 0.06, compared to 0.91 +/- 0.02 in age-matched nondiabetic diabetes-resistant controls (P less than 0.001). In age-matched nondiabetic diabetes-prone rats, most of which would have become diabetic, the ratio was 0.85 +/- 0.02, also less than the controls (P less than 0.05). Protein A-gold labeling of GLUT-2 in beta-cells of day 1 diabetic rats revealed 2.17 +/- 0.16 gold particles per micrometer length of microvillar plasma membranes compared to 3.91 +/- 0.14 in controls (P less than 0.001) and 2.87 +/- 0.24 in the nondiabetic diabetes-prone rats (P less than 0.02). Reduction in GLUT-2 correlates temporally with and may contribute to the loss of glucose-stimulated insulin secretion that precedes profound beta-cell depletion of autoimmune diabetes.

Compensatory capabilities of islets of BB/Wor rats exposed to sustained hyperglycemia.

To determine if discordance for autoimmune diabetes in genetically homogeneous animals might reflect differences in the compensatory capacity of their beta cells, the glycemic responses of diabetes-prone BB/Wor rats during a high rate infusion of 50% glucose were compared with normal and with 40% pancreatectomized Wistar rats similarly infused. In all three groups, the initially severe hyperglycemia declined after the first 48 hours to below the target level of 300 mg/dL despite an increasing rate of glucose infusion. The glycemic profile did not differ from controls and was lower than that of the partially depancreatized rats. Five of 20 hyperglycemic BB/Wor rats became diabetic during the 12-day infusion of 50% glucose; there was no difference between their glucose profiles and those of the 15 prediabetic BB/Wor rats that remained nondiabetic throughout the period of hyperglycemic infusion. The latter group of BB/Wor rats, many of which would ultimately have become diabetic, exhibited a 2.4-fold increase in the volume density of their beta cells, compared with a 2.1-fold increase in the Wistar controls. This clinical and morphologic evidence of beta-cell compensation in diabetes-prone rats, even in on the verge of overt diabetes, excludes the possibility that subnormal compensation by beta cells contributes to diabetes in the BB/Wor rat.

Morphologic and functional changes in sympathetic nerve relationships with pancreatic alpha-cells after destruction of beta-cells in rats.

Insulin-dependent diabetes mellitus (IDDM) in humans is accompanied by an attenuation of the response of glucagon to hypoglycemia. To identify an animal model of IDDM with alpha-cell unresponsiveness to glucopenia in which to pursue morphologic and in vitro functional investigation of the lesion, pancreases isolated from rats with IDDM induced by streptozocin (STZ) or occurring spontaneously in BB/W rats were perfused with buffer containing 150, 25, and 150 mg/dl of glucose. In both forms of IDDM the normal glucagon rise during glucopenia was markedly impaired, suggesting an abnormality comparable to that of human IDDM. Studies of the insular sympathetic apparatus were conducted in these rat models. Electron-microscopic examination of peri-insular nerve endings disclosed no discernible abnormality in either form of rat IDDM. However, morphometric analysis of contacts between [3H]norepinephrine-labeled sympathetic nerve terminals and alpha-cells in pancreases from STZ-induced diabetic (STZ-D) rats revealed a 65-70% reduction in direct contacts. An 80% reduction in the number of nerve endings (not labeled) in direct contact with alpha-cells was also noted in the BB/W diabetic rats. Norepinephrine reuptake, studied only in the STZ-D group, was not impaired. The availability of local endogenous norepinephrine to alpha-cells and their sensitivity to exogenous norepinephrine was determined by perfusing 2, 5, or 10 micrograms/ml of tyramine, a releaser of endogenous norepinephrine, and norepinephrine at a concentration that in pancreases from nondiabetic rats gave a quantitatively similar glucagon response.(ABSTRACT TRUNCATED AT 250 WORDS)