1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice.

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice.

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

Factors predictive of the healing of pancreatic pseudocysts treated by percutaneous evacuation.

BACKGROUND/AIMS: Pseudocyst formation is a well-known complication of pancreatitis which develops over 1 to 4 weeks in approximately 15% of patients. Nearly one-third of pancreatic pseudocysts resolve spontaneously; however, if there is no resolution within six weeks, evacuation must be performed. The aim of this study was to prospectively assess the reliability of the following: etiology; location; amount of pseudocyst liquid; and concentrations of certain biochemical parameters (LDH, glucose, proteins, sodium, potassium, bilirubin, lipase and amylase) in the pseudocyst content and patients' serum, in terms of the efficacy of ultrasound-guided percutaneous evacuation as a therapeutic approach. METHODOLOGY: Pseudocyst fluid was obtained by ultrasound-guided percutaneous evacuation in 67 patients, with a history of pancreatitis and pancreatic pseudocysts larger than five centimeters in diameter, with a matured membraneous wall that persisted for more than six weeks. RESULTS: There is a prognostic value associated with the location of the pseudocyst, the amount of pseudocyst liquid and the concentration of proteins, potassium, lipase and amylase in the evacuated material. CONCLUSION: Analysis of the aforementioned parameters provides an early forecast of the outcome of percutaneous evacuation.

The dynamics of endogenous zinc in different animal models of gastroduodenal ulceration.

The body possesses many various endogenous substances that enable it to protect itself from various noxious events. One of the nonspecific reactions is the increase of endogenous zinc concentrations in various tissues. The aim of this study was to focus on the changes of zinc serum and tissue concentrations in well-established models of gastric (24 h restraint stress and 5 min ethanol) and duodenal ulceration (24 h cysteamine). Ten female Wistar rats, weighing 180-250 g were used in each of the experimental groups. Upon sacrificing, the ulcer index was recorded. Zinc serum and tissue concentrations were determined using atomic absorption spectrophotometry. Ten unstressed animals served as a healthy control group. The results of our study clearly indicated certain changes in serum zinc in all these ulcer models. Changes in tissue concentrations, varying from model to model, were also observed. In conclusion, all this data confirm the fact that endogenous zinc represents a natural line of the body's defense system when exposed to stress.

Hepatitis C and human immunodeficiency virus coinfections.

Hepatitis C virus (HCV) has become a major contributor to morbidity and mortality in patients with human immunodeficiency virus (HIV). It is estimated that 30% to 50% of patients with HIV are coinfected with HCV. Advances in antiretroviral therapy and improved life expectancy of HIV patients have resulted in an emergence of HCV-induced liver disease as a leading cause of significant morbidity and death in this population. Clinically, hepatitis C is a more severe disease in HIV-infected individuals, characterized by rapid progression toward end-stage liver disease. Highly active antiretroviral therapy is the mainstay of current acquired immunodeficiency syndrome management. One of the limiting side effects of combination therapy for HIV is hepatotoxicity, which is more common and often more serious in patients with underlying liver disease. Management of coinfected patients has no strict guidelines, but it is generally accepted that HIV infection needs to be treated before HCV. Hepatitis C in coinfected individuals is probably best treated using combination therapy (interferon alpha and ribavirin). It appears that combination therapy can safely be administered to this population and that previous concerns about ribavirin/zidovudine antagonism are unsubstantiated in clinical practice. Although initial results using only interferon alpha showed poor results in HIV coinfected patients, combination therapy seems to be as effective as in the general population. All HIV-HCV coinfected patients should be vaccinated against hepatitis B and hepatitis A; vaccines are safe and effective.

Endogenous zinc concentrations in cysteamine-induced duodenal ulcers in the rat.

Exogenously administered zinc compounds have been shown to possess anti-ulcer activity against a wide variety of ulcerogenic agents, both in laboratory animal models and in human peptic ulcer disease. However, a strong possibility exists that endogenous zinc may also play an important role during noxious events by various mechanisms. Therefore, the aim of this study was to focus on the changes of endogenous zinc serum and tissue concentrations in cysteamine-induced duodenal lesions. We used atomic absorption spectrophotometry to determine the tissue and serum concentrations of zinc in normal (control) rats and those with cysteamine-induced duodenal ulcers. The results obtained in this study indicated that the onset, development and spontaneous healing of ulcer lesions were associated with certain shifts in zinc serum and tissue concentrations. Prior to ulcer formation, a significant increase was noted in serum zinc values. With the onset of duodenal lesions, zinc serum concentrations significantly decreased, while there was a significant increase in duodenal tissue concentrations when compared to healthy control animals. Zinc tissue concentrations decreased and returned to starting values by the end of the first week of spontaneous healing. This decrease in zinc tissue concentration corresponded to the healing rate of the duodenal ulcers. Serum zinc concentrations also returned to starting values within the first week period. These observations indicate and confirm that zinc could play an important role in duodenal ulcer disease and represent a natural defense system in the body.

The protective effect of zinc sulphate pretreatment against duodenal ulcers in the rat.

Exogenously administered zinc compounds have been shown to possess antiulcer activity in the development of gastric lesions. The aim of this study was to investigate the effects of zinc sulphate pretreatment of rats on cysteamine-induced duodenal ulcers and to correlate them with changes in zinc serum and tissue levels. Atomic absorption spectrophotometry was used to determine zinc serum and tissue concentrations in all animal groups. Cysteamine produced marked duodenal ulceration in control animals 24 h after application, with an increase in endogenous zinc tissue concentrations and a marked decrease in serum concentrations. Zinc sulphate (20, 40 or 80 mg kg-1) applied per os one hour prior to cysteamine application inhibited the development of duodenal lesions in a dose-related manner. The application of zinc sulphate in a single intraperitoneal (i.p.) application (80 mg kg-1) did not, however, prevent the formation of duodenal lesions. In order to assess zinc absorption from the gastrointestinal tract, one group of rats received a single oral dose of zinc sulphate (80 mg kg-1) without cysteamine application. The observations of this study seem to indicate that zinc plays an important cytoprotective role in duodenal ulcer disease.

Parathyroid hormone inhibits collagen synthesis and the activity of rat col1a1 transgenes mainly by a cAMP-mediated pathway in mouse calvariae.

We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.

Pentagastrin cytoprotection in ethanol-induced gastric mucosal lesions in rats.

To establish pentagastrin cytoprotection, the effectiveness of various doses of pentagastrin on ethanol induced gastric mucosal lesions was investigated in Wistar rats. Significant protection was obtained only after parenteral pretreatment with the exception of the lowest dose (1 microgram/kg b.w.). Pentagastrin cytoprotection is not mediated either by a dopamine, muscarinic or gastrin/CCK receptor or by prostaglandin synthesis. However, the protective effect of pentagastrin is abolished by prior vagotomy, although this procedure alone or sham operation is ineffective to influencing control-ethanol lesions. In secretory studies pentagastrin increased both the volume of gastric juice and total acid output. Unlike cytoprotection, these were reversed by vagotomy, but also with atropine and problumide, whereas domperidone and indomethacin were ineffective.

Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation.

Msx2 is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels of Msx2 protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression of Msx2 prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreased proliferation and accelerated differentiation. In other studies, we showed that the Msx2 promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture.

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.