Recent developments of collagen-based materials for medical applications and drug delivery systems

In this review, an attempt was made to summarize some of the recent developments in the application of collagen as a biomaterial and in drug delivery systems. The main applications covered include: collagen for burn/wound cover dressings; osteogenic and bone filling materials; antithrombogenic surfaces; and immobilization of therapeutic enzymes. Recently, collagen used as a carrier for drug delivery has attracted many researchers throughout the world. The use of collagen for various drug delivery systems has also been reviewed in this article. Collagen-based drug delivery systems include: injectable microspheres based on gelatin (degraded form of collagen); implantable collagen-synthetic polymer hydrogels; interpenetrating networks of collagen; and synthetic polymers collagen membranes for ophthalmic delivery. Recent efforts to use collagen-liposomal composites for controlled drug delivery, as well as collagen as controlling membranes for transdermal delivery, were also reviewed. In this review, the main emphasis was on the work done in our laboratory.     

Factitious insulinoma

A 32-year-old woman was admitted with signs of recurrent hypoglycaemia. Within 72 hours hypoglycaemia was successfully provoked by prolonged fasting. Also, blood samples demonstrated high levels of serum insulin and C-peptide and the insulin-glucose ratio was abnormally high. An insulinoma was strongly suspected. However, extensive imaging displayed no tumour in the pancreas. The patient also had extensive psychological and social problems. The psychiatrist suggested a factitious disorder. High serum concentrations of insulin and C-peptide in combination with the psychiatric disorder led to the suspicion of abuse of sulfonylurea derivatives by the patient. This was confirmed by toxicological screening. A patient with unexplained hypoglycaemia, especially if an insulinoma cannot be detected, should be suspected of abusing sulfonylurea derivatives.     

Inactivation in vitro of the Escherichia coli outer membrane protein FhuA by a phage T5-encoded lipoprotein

The toxicity on three human tumor cell lines (A431, HeLa and MCF7) of five phenanthroperylenequinones (hypericin and derivatives) and two perylenequinones (cercosporin and calphostin C) was investigated after photosensitization (4 J/cm2). Furthermore, the antiproliferative effect on HeLa cells was studied for the phenanthroperylenequinones. Hypericin, 2,5-dibromohypericin, 2,5,9,12-tetrabromohypericin and perylenequinones displayed a potent cytotoxic and antiproliferative effect in the nanomolar range. Hypericin dicarboxylic acid exhibited no photoactivity. In general, the antiproliferative activity correlated well with the photocytotoxicity. However, the nonphotocytotoxic compound hexamethylhypericin showed potent antiproliferative activity in the nanomolar range, probably exerting its action by protein kinase C inhibition. Without light irradiation, no cytotoxic and antiproliferative effect was observed for any photocytotoxic phenanthroperylenequinone compound. Furthermore, confocal laser microscopy revealed that the subcellular localization in A431 cells was similar for the photoactive compounds; the photosensitizers were mainly concentrated in the perinuclear region, probably corresponding with the Golgi apparatus and the endoplasmic reticulum. In addition, the accumulation of the photosensitizers in HeLa cells was investigated. All compounds except hypericin dicarboxylic acid were found to concentrate to a large extent in the cells. The compound 2,5,9,12-tetrabromohypericin seemed intrinsically more effective than hypericin since the intracellular concentration of the bromoderivative was a magnitude of order lower than that of hypericin although both compounds showed similar photobiological activity.     

Changes in cerebral perfusion pressure in puerperal women with preeclampsia

OBJECTIVE: To determine the variation in the estimated maternal cerebral perfusion and cerebrovascular resistance (the resistance area product) in the puerperium. METHODS: The maternal middle cerebral artery was evaluated by transcranial Doppler ultrasound in ten women 2 days before labor, in 21 women in early labor and at 24 and 48 hours postpartum, and in 6 women at 1 week postpartum. Cerebral blood flow velocities were determined. Women were diagnosed initially with mild preeclampsia. Estimated cerebral perfusion pressure was Vmean/[Vmean - Vdiastolic] [BPmean - BPdiastolic]. Because the diameter of the vessels could not be measured directly, an index of resistance was calculated: the resistance area product = BPmean/velocitymean. We calculated an index of cerebral blood flow to be estimated cerebral perfusion pressure divided by resistance area product. Our study had a power of 80% to detect a 16-cm/second increase in middle cerebral blood flow velocity. RESULTS: Estimated maternal cerebral perfusion was maintained for up to 1 week postpartum. Cerebrovascular resistance did not change in the puerperium. Cerebral blood flow index (+/-standard deviation) was significantly increased at 1 week postpartum compared with early labor levels (28.3 +/-6.9 versus 46.7+/-15.6, respectively) (P < .05). CONCLUSION: Cerebral blood flow 1 week postpartum increased significantly over early labor values. These persistent changes in the cerebral vasculature might put patients at risk for seizures up to 1 week postpartum.     

Calnexin association is not sufficient to protect T cell receptor alpha proteins from rapid degradation in CD4+CD8+ thymocytes

During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRalpha molecules, having a half-life of approximately 20 min in immature CD4+CD8+ thymocytes compared with >75 min in mature T cells. The molecular basis for TCRalpha instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRalpha proteins in all other T cell types examined. Here, we compared the processing of TCRalpha glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+ thymocytes and splenic T cells. These studies show that TCRalpha glycoproteins synthesized in CD4+CD8+ thymocytes were processed in a similar manner as those made in splenic T cells and that TCRalpha proteins stably associated with calnexin in both cell types. Interestingly, however, TCRalpha association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+ thymocytes compared with splenic T cells. Finally, TCRalpha degradation in CD4+CD8+ thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin.TCRalpha complexes. These data demonstrate that calnexin association is not sufficient to protect TCRalpha proteins from rapid degradation in CD4+CD8+ thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRalpha glycoproteins.     

Prevalence of ethanol consumption may be higher in women than men in a university health service population as determined by a biochemical marker: whole blood-associated acetaldehyde above the 99th percentile for teetotalers

To estimate ethanol consumption by university students attending a student health facility, a biochemical marker of alcohol intake [whole blood associated acetaldehyde (WBAA)] was quantified by fluorimetric HPLC. Over a two year period we studied blood samples, coded by date and sex, from 645 females and 332 males, and compared the results to previously established reference ranges for teetotalers by sex. Men had higher absolute values for WBAA than women (9.9 versus 9.5 microM in the present study). However, significantly greater numbers of women (74%) than men (44%) had WBAA levels above the 99th percentile for teetotalers. Variations occurred during the academic year, with significant elevations occurring in the late fall and winter months. Testing of WBAA levels in a student health service may be important especially for women to facilitate counseling on the dangers of alcohol abuse.     

Standardized ultrasound examination for evaluation of instability of the acromioclavicular joint

Anteroposterior X-ray views of both acromioclavicular (AC) joints with 10-kg weights held in each hand are the generally accepted procedure for diagnosis of Tossy I-III grades of AC joint separation. An analogous diagnosis can be made by standardized ultrasound examination. Ten individuals with Tossy-I, 11 with Tossy-II and 8 with Tossy-III instability were examined both radiographically and by B-mode ultrasound. The degree of AC joint separation was uniformly determined on the basis of a calculated index (AC Index = AC joint width of uninjured side/AC joint width of injured side). The mean AC Index for Tossy-I instability determined by ultrasound was 1.0; mean indices of 0.49 and 0.5 were determined for Tossy-II injury by ultrasound and X-ray, respectively, and of 0.21 and 0.2, respectively, for Tossy-III instability. Statistical analysis showed significant differences between the mean AC indices of all three groups (P < 0.0001). We conclude that the reliability of ultrasound examination of AC joint instability is equal to that of radiographic measurement. Standard X-rays of the shoulder remain mandatory only to exclude fracture. The indication for operative stabilization of the AC joint can be established on the basis of the grade of AC joint instability measured by the side-effect-free and cost-effective method of ultrasound examination (AC Index < 0.3 equivalent to Tossy-III instability).     

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.