Erytrocyte membrane anionic charge in type 2 diabetic patients with retinopathy

BACKGROUND: The Steno hypothesis states that changes in basement membrane anionic charge leads to diabetic microvascular complications. In diabetic nephropathy, loss of basement membrane glycosaminoglycans and the association between glomerular basement membrane heparan sulphate and proteinuria has been documented. A correlation between erythrocyte surface and the glomerular capillary wall charges has also been observed. The aim of this study is to evaluate the relationship between retinopathy and erythrocyte anionic charge and urinary glycosaminoglycan excretion in type 2 diabetic patients. METHODS: 49 subjects (58 ± 7 yrs, M/F 27/22) with type 2 diabetes with proliferative retinopathy (n = 13), nonproliferative retinopathy (n = 13) and without retinopathy (n = 23) were included in the study. 38 healthy subjects were selected as control group (57 ± 5 yrs, M/F 19/19). Erythrocyte anionic charge (EAC) was determined by the binding of the cationic dye, alcian blue. Urinary glycosaminoglycan and microalbumin excretion were measured. RESULTS: EAC was significantly decreased in diabetic patients with retinopathy (255 ± 30 ng alcian blue/10(6 )RBC, 312 ± 30 ng alcian blue/10(6 )RBC for diabetic and control groups respectively, p < 0.001). We did not observe an association between urinary GAG and microalbumin excretion and diabetic retinopathy. EAC is found to be negatively corralated with microalbuminuria in all groups. CONCLUSIONS: We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients

Mechanism of action for N-substituted benzamide-induced apoptosis

We have analysed the mechanism of action for induction of apoptosis by N-substituted benzamides using declopramide as a lead compound. We show here that declopramide at doses above 250 μM in the mouse 70Z/3 pre-B cell line or in the human promyeolocytic cancer cell line HL60 induced cytochrome c release into the cytosol and caspase-9 activation. The broad spectrum caspase inhibitor zVADfmk and caspase-9 inhibitor zLEDHfmk inhibited apoptosis and improved cell viability when administrated to cells 1 h before exposure to declopramide, whereas the caspase-8 inhibitor zIEDHfmk had less effect. Also, the over expression of Bcl-2 by transfection in 70Z/3 cells inhibited declopramide-induced apoptosis. Prior to the induction of apoptosis, a G2/M cell cycle block was induced by declopramide. The cell cycle block was also observed in the presence of broad spectrum caspase inhibitor zVADfmk and in a transfectant expressing high levels of Bcl-2. Furthermore, while p53 was induced in 70Z/3 cells by declopramide, neither the apoptotic mechanism nor the G2/M cell cycle block were dependent on p53 activation since both effects were also seen in p53 deficient HL60 cells after addition of declopramide

N-substituted benzamides inhibit NFκB activation and induce apoptosis by separate mechanisms

Benzamides have been in clinical use for many years in treatment against various disorders. A recent application is that as a sensitizer for radio- or chemotherapies. We have here analysed the mechanism of action of N-substituted benzamides using an in vitro system. We found that while procainamide was biologically inert in our system, the addition of a chloride in the 3′ position of the benzamide ring created a compound (declopramide) that induced rapid apoptosis. Furthermore, declopramide also inhibited NFκB activation by inhibition of IκBβ breakdown. An acetylated variant of declopramide, N-acetyl declopramide, showed no effect with regard to rapid apoptosis induction but was a potent inhibitor of NFκB activation. In fact, the addition of an acetyl group to procainamide in the 4′ position was sufficient to convert this biologically inactive substance to a potent inhibitor of NFκB activation. These findings suggest two potential mechanisms, induction of early apoptosis and inhibition of NFκB mediated salvage from apoptosis, for the biological effect of N-substituted benzamides as radio- and chemo-sensitizers. In addition it suggests that N-substituted benzamides are potential candidates for the development of anti-inflammatory compounds using NFκB as a drug target. © 1999 Cancer Research Campaig

Regulation of Pathologic Retinal Angiogenesis in Mice and Inhibition of VEGF-VEGFR2 Binding by Soluble Heparan Sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis

Risk of adenocarcinomas of the oesophagus and gastric cardia in patients hospitalized for asthma

In the first cohort study of the question we followed 92 986 (42 663 men and 50 323 women) adult patients hospitalized for asthma in Sweden from 1965 to 1994 for an average of 8.5 years to evaluate their risk of oesophageal and gastric cardia adenocarcinoma. Standardized incidence ratio (SIR) adjusted for gender, age and calendar year was used to estimate relative risk, using the Swedish nationwide cancer incidence rates as reference. Asthmatic patients overall had a moderately elevated risk for oesophageal adenocarcinoma (SIR = 1.5, 95% confidence interval CI, 0.9–2.5) and gastric cardia cancer (SIR = 1.4, 95% CI, 1.0–1.9). However, the excess risks were largely confined to asthmatic patients who also had a discharge record of gastro-oesophageal reflux (SIR = 7.5, 95% CI, 1.6–22.0 and SIR = 7.1, 95% CI, 3.1–14.0, respectively). No significant excess risk for oesophageal squamous-cell carcinoma or distal stomach cancer was observed. In conclusion, asthma is associated with a moderately elevated risk of developing oesophageal or gastric cardia adenocarcinoma. Special clinical vigilance vis-à-vis gastro-esophageal cancers seems unwarranted in asthmatic patients, but may be appropriate in those with clinically manifest gastro-oesophageal reflux.   http://www.bjcancer.com © 2001 Cancer Research Campaig

Scintigraphic evaluation of oesophageal transit during radiotherapy to the mediastinum

Background: To quantitatively evaluate radiation-induced impaired oesophageal transit with oesophageal transit scintigraphy and to assess the relationships between acute oesophagitis symptoms and dysmotility.\ud \ud Methods: Between January 1996 and November 1998, 11 patients affected by non-small-cell carcinoma of the lung not directly involving the oesophagus, requiring adjuvant external beam radiotherapy (RT) to the mediastinum were enrolled. Oesophageal transit scans with liquid and semisolid bolus were performed at three pre-defined times: before (T0) and during radiation at 10 Gy (T1) and 30 Gy (T2). Two parameters were obtained for evaluation: 1) mean transit time (MTT); and 2) ratio between peak activity and residual activity at 40 seconds (ER-40s). Acute radiation toxicity was scored according to the joint EORTC-RTOG criteria. Mean values with standard deviation were calculated for all parameters. Analysis of variance (ANOVA) tests and paired t-Tests for all values were performed.\ud \ud Results: An increase in the ER-40s from T0 to T1 or T2 was seen in 9 of 11 patients (82%). The mean ER-40s value for all patients increased from 0.8306 (T0) to 0.8612 (T1) and 0.8658 (T2). These differences were statistically significant (p < 0.05) in two paired t-Tests at T0 versus T2 time: overall mean ER-40s and upright ER-40s (p = 0.041 and p = 0.032, respectively). Seven patients (63%) showed a slight increase in the mean MTT value during irradiation but no statistically significant differences in MTT parameters were found between T0, T1 and T2 (p > 0.05).\ud \ud Conclusion: Using oesophageal scintigraphy we were able to detect early alterations of oesophageal transit during the third week of thoracic RT

Testing an Emerging Paradigm in Migration Ecology Shows Surprising Differences in Efficiency between Flight Modes

To maximize fitness, flying animals should maximize flight speed while minimizing energetic expenditure. Soaring speeds of large-bodied birds are determined by flight routes and tradeoffs between minimizing time and energetic costs. Large raptors migrating in eastern North America predominantly glide between thermals that provide lift or soar along slopes or ridgelines using orographic lift (slope soaring). It is usually assumed that slope soaring is faster than thermal gliding because forward progress is constant compared to interrupted progress when birds pause to regain altitude in thermals. We tested this slope-soaring hypothesis using high-frequency GPS-GSM telemetry devices to track golden eagles during northbound migration. In contrast to expectations, flight speed was slower when slope soaring and eagles also were diverted from their migratory path, incurring possible energetic costs and reducing speed of progress towards a migratory endpoint. When gliding between thermals, eagles stayed on track and fast gliding speeds compensated for lack of progress during thermal soaring. When thermals were not available, eagles minimized migration time, not energy, by choosing energetically expensive slope soaring instead of waiting for thermals to develop. Sites suited to slope soaring include ridges preferred for wind-energy generation, thus avian risk of collision with wind turbines is associated with evolutionary trade-offs required to maximize fitness of time-minimizing migratory raptors

The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeleto ns. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology

Membrane-Associated Heparan Sulfate Proteoglycan Is a Receptor for Adeno-Associated Virus Type 2 Virions

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector