beta-Arrestin 1 and 2 stabilize the angiotensin II type I receptor in distinct high-affinity conformations.

BACKGROUND AND PURPOSE: The angiotensin II type 1 (AT(1)) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or beta-arrestins often induces higher binding affinity for agonists. Here, we examined interactions between AT(1A) receptors and beta-arrestins to look for differences between the AT(1A) receptor interaction with beta-arrestin1 and beta-arrestin2. EXPERIMENTAL APPROACH: Ligand-induced interaction between AT(1A) receptors and beta-arrestins was measured by Bioluminescence Resonance Energy Transfer 2. AT(1A)-beta-arrestin1 and AT(1A)-beta-arrestin2 fusion proteins were cloned and tested for differences using immunocytochemistry, inositol phosphate hydrolysis and competition radioligand binding. KEY RESULTS: Bioluminescence Resonance Energy Transfer 2 analysis showed that beta-arrestin1 and 2 were recruited to AT(1A) receptors with similar ligand potencies and efficacies. The AT(1A)-beta-arrestin fusion proteins showed attenuated G protein signalling and increased agonist binding affinity, while antagonist affinity was unchanged. Importantly, larger agonist affinity shifts were observed for AT(1A)-beta-arrestin2 than for AT(1A)-beta-arrestin1. CONCLUSION AND IMPLICATIONS: beta-Arrestin1 and 2 are recruited to AT(1A) receptors with similar ligand pharmacology and stabilize AT(1A) receptors in distinct high-affinity conformations. However, beta-arrestin2 induces a receptor conformation with a higher agonist-binding affinity than beta-arrestin1. Thus, this study demonstrates that beta-arrestins interact with AT(1A) receptors in different ways and suggest that AT(1) receptor biased agonists with the ability to recruit either of the beta-arrestins selectively, would be possible to design.

Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression.

Excess collagen IV expression by mesangial cells contributes to diabetic glomerulosclerosis. We hypothesized that in high glucose reactive oxygen species (ROS) generation by NADPH oxidase is PKC dependent and required for collagen IV expression by mesangial cells. In rat mesangial cells cultured in 5 mM (NG) or 25 mM d-glucose (HG), RT-PCR and Western immunoblotting detected p22(phox) and p47(phox) mRNA and protein, respectively. Quantitative real-time RT-PCR analyzed collagen IV mRNA. With the use of confocal microscopy, ROS were detected with dichlorofluorescein and intracellular collagen IV by immunofluorescence. In HG, ROS were generated within 1 h, sustained up to 48 h, and prevented by a NADPH oxidase inhibitor, diphenylenechloride iodonium (DPI), or a conventional PKC isozyme inhibitor, Gö6976. In NG, phorbol myristate acetate stimulated ROS generation that was inhibited with DPI. In HG, expression of p22(phox) and p47(phox) was increased within 3 to 6 h and inhibited by Gö6976. In HG, Gö6976 or transfection with antisense against p22(phox) reversed the 1.8-fold increase in collagen IV mRNA. In HG, the antioxidants Tempol or Tiron, or transfection with antisense against p22(phox) or p47(phox), prevented ROS generation and the 2.3-fold increase in collagen IV protein. Increased mitochondrial redox potential in HG was unaffected by transfection with antisense against p22(phox). We conclude that in HG, mesangial cell ROS generation by upregulated NADPH oxidase is dependent on conventional PKC isozymes and also required for collagen IV expression.

Stretch-induced mesangial cell ERK1/ERK2 activation is enhanced in high glucose by decreased dephosphorylation.

Glomerular hypertension and hyperglycemia are major determinants of diabetic nephropathy. We sought to identify the mechanisms whereby stretch-induced activation of mesangial cell extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) is enhanced in high glucose (HG). Mesangial cells cultured on fibronectin Flex I plates in normal glucose (NG; 5.6 mM) or HG (30 mM), were stretched by 15% elongation at 60 cycles/min for up to 60 min. In HG, a 5-min stretch increased ERK1/ERK2 phosphorylation by 6.4 +/- 0.4/4.3 +/- 0.3-fold (P < 0.05 vs. NG stretch). In contrast, p38 phosphorylation was increased identically by stretch in NG and HG. Unlike many effects of HG, augmentation of ERK activity by HG was not dependent on protein kinase C (PKC) as indicated by downregulation of PKC with 24-h phorbol ester or inhibition with bisindolylmaleimide IV. In both NG and HG, pretreatment with arginine-glycine-aspartic acid peptide (0.5 mg/ml) to inhibit integrin binding or with cytochalasin D (100 ng/ml) to disassemble filamentous (F) actin, significantly reduced phosphorylation of ERK1/ERK2 and p38. To determine whether the rate of mitogen-activated protein kinase dephosphorylation is affected by HG, cellular kinase activity was inhibited by depleting ATP. Post-ATP depletion, phosphorylation of ERK1/ERK2 was reduced to 36 +/- 9/51 +/- 14% vs. 9 +/- 5/7 +/- 6% in NG (P < 0.05, n = 5). Thus stretch-induced ERK1/ERK2 and p38 activation in both NG and HG is beta(1)-integrin and F-actin dependent. Stretch-induced ERK1/ERK2 is enhanced in high glucose by diminished dephosphorylation, suggesting reduced phosphatase activity in the diabetic milieu. Enhanced mesangial cell ERK1/ERK2 signaling in response to the combined effects of mechanical stretch and HG may contribute to the pathogenesis of diabetic nephropathy.

Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-alpha, -delta, and -epsilon.

In endothelin-1 (ET-1)-stimulated mesangial cells, to identify the independent roles of calcium and protein kinase C (PKC) causing contraction, the changes in planar surface area in response to ET-1, ionomycin, or phorbol 12-myristate 13-acetate (PMA) were compared. ET-1, PMA, and ionomycin reduced planar area to 49 +/- 3%, 56 +/- 3%, and 78 +/- 2% of basal (means +/- SE, n = 40-50 cells), respectively. ET-1 or ionomycin increased cytosolic calcium from 80 +/- 7 to 220 +/- 30 nM or 97 +/- 10 to 192 +/- 10 nM, respectively. The myosin light chain kinase inhibitor, ML-7, blunted ET-1- but not PMA-stimulated contraction (82 +/- 3% and 48 +/- 6% of time 0, respectively). Cells pretreated with 10 microM chelerythrine for 1 h or PMA for 24 h failed to contract to either ET-1 or PMA. To identify the specific PKC isoform response to ET-1, cytosolic, membrane, and particulate fractions of mesangial cell lysates were immunoblotted with PKC isoform-specific polyclonal antibodies. ET-1 increased membrane PKC-alpha, -delta, and -epsilon to 173 +/- 30%, 162 +/- 26%, and 166 +/- 11% of basal (P < 0.05 vs. basal), respectively, and decreased PKC-delta and PKC-epsilon in the cytosol to 56 +/- 11% and 37 +/- 6% of basal, respectively (P < 0.05). ET-1 increased particulate PKC-delta and PKC-epsilon to 172 +/- 15% and 187 +/- 33% of basal (P < 0.05), respectively. PKC-alpha in the cytosol and particulate fractions was not altered by ET-1, but translocation to the nucleus and cell periphery was observed by confocal immunofluorescence imaging. Ionomycin did not change PKC isoform distribution. PKC-zeta was expressed but unaltered by ET-1. Therefore, mesangial cell ET-1-stimulated contraction not only involves a calcium-dependent pathway but also includes the activation of one or more PKC-alpha, -delta, and -epsilon, but not PKC-zeta.

Chelation of intracellular calcium prevents mesangial cell proliferative responsiveness.

Mesangial cell transformation into a proliferative phenotype, observed in many glomerular diseases, occurs in response to growth factors and cytokines. This study tests the hypothesis that intracellular calcium is necessary for stimulation of mesangial cell proliferative responsiveness to a variety of growth factors. Furthermore, these experiments tested whether nonspecific calcium entry via a calcium ionophore was sufficient to elicit the same response. Rat primary mesangial cells (passages 5 to 10) were growth-arrested for 48 h in 0.5% fetal bovine serum (FBS), then stimulated with 0.1 microM endothelin-1, 1.9 microM platelet-derived growth factor (PDGF)-BB, 0.5% FBS, or 0.1 microM ionomycin, with or without the intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Calcium signaling was measured in Fura-2-loaded cells on coverslips by dual-wavelength spectrofluorometry and in Fluo-3-loaded cells by confocal fluorescence laser microscopy. [3H]-Thymidine incorporation was measured after 12 to 24 h of stimulation with each test agent. Expression of c-fos mRNA was analyzed by Northern blot. All test agents stimulated a significant increase in cytosolic and nuclear calcium, which were both effectively inhibited with BAPTA. All agents stimulated a significant increase in [3H]-thymidine incorporation and enhanced c-fos mRNA expression (no detectable c-fos mRNA was observed in quiescent cells). BAPTA prevented the enhanced [3H]-thymidine incorporation stimulated by endothelin-1 and PDGF, and partial inhibition of FBS-stimulated incorporation with BAPTA was observed. BAPTA inhibited c-fos expression observed in response to these agents. Phorbol ester induction of c-fos mRNA in the absence of raised cytosolic or nuclear calcium was also suppressed by BAPTA. Cell viability as measured by thiazolyl blue and trypan blue was not altered by BAPTA. It is concluded that normal regulation of intracellular calcium is necessary for mesangial cell proliferative responsiveness.

A proposed mechanism for p-aminoclonidine allergenicity based on its relative oxidative lability.

p-Aminoclonidine (apraclonidine) is a selective alpha 2 adrenergic agonist used to reduce intraocular pressure in the treatment of glaucoma. Use of apraclonidine is frequently associated with severe local allergic effects which warrant discontinuation of the drug in affected patients. We have assessed the oxidative lability of apraclonidine relative to a panel of adrenergic agonists and/or known allergens; amodiaquine, epinephrine, clonidine, and brimonidine. These compounds were compared by their electrochemical potentials as well as their oxidative lability in the presence of several oxidative enzyme systems (i.e., horseradish peroxidase, lactoperoxidase, myeloperoxidase, and diamine oxidase). The half-lives for enzymatic oxidation of these compounds were found to parallel the electrochemical oxidation potentials in the order: amodiaquine approximately epinephrine < apraclonidine << clonidine approximately brimonidine. The production of a reactive electrophilic intermediate of apraclonidine was demonstrated through the formation of two glutathione apraclonidine adducts from the horseradish peroxidase/H2O2-mediated oxidation of apraclonidine in the presence of glutathione. A mechanism for apraclonidine allergenicity in vivo is proposed wherein apraclonidine is bioactivated through oxidation to the bis-iminoquinone followed by protein conjugation to form an apraclonidine-protein hapten that elicits the immune response.

Mesangial cell actin disassembly in high glucose mediated by protein kinase C and the polyol pathway.

High glucose alters mesangial cell cytoskeletal structure and function. We postulated that high glucose causes mesangial cell filamentous (F) actin disassembly through a protein kinase C (PKC) mechanism involving the polyol pathway. Rat mesangial cells (passage < 10, N = 60/group) were growth-arrested and then cultured in glucose 5.6 mM (NG), 15 mM (MG) or 30 mM (HG) for 48 hours, with or without the aldose reductase inhibitor Tolrestat 0.3 mM. F and globular (G) actin were labeled with rhodamine-phalloidin and FTTC-DNase-1, respectively. Both fluorescence probes were imaged simultaneously in each cell using dual-channel confocal laser microscopy. In HG, F-actin disassembly was observed and measured by a 40% decrease in F-/G-actin fluorescence intensity ratio (no change in NG + mannitol 24.4 mM). In separate experiments, cells were labeled with BODIPY FL-bisindolylmaleimide, specific for most PKC isoforms, and fluorescence intensity/cell was measured. In NG, exposure to phorbol 12-myristate 13-acetate (PMA) 0.1 microM for 15 minutes caused perinuclear and nuclear translocation of PKC, and F-actin disassembly identical to observations in HG alone. In HG, total PKC fluorescence increased by 50% and PMA exposure for 24 hours normalized both the total PKC and F-/G-actin fluorescence ratio. In NG and HG, exposure (15 min) to PMA 0.1 microM increased PKC activity three to four times, measured by in situ 32P-phosphorylation of EGF-receptor substrate. By immunofluorescence and confocal imaging, diacylglycerol-sensitive PKC-delta was localized to the cytosol in NG, and after 15 minutes exposure to PMA, translocated to the perinuclear region and plasma membrane. In HG. PKC-delta immunofluorescence was significantly increased/cell, distributed in a cytoskeletal pattern and the intensity was glucose-concentration dependent (30 > 15 > 5.6 mM). In HG, exposure to PMA for 24 hours returned the PKC-delta fluorescence to the intensity and cytosolic pattern observed in NG, and simultaneously prevented F-actin disassembly. Tolrestat significantly reduced the total PKC and PKC-delta fluorescence intensity and F-actin disassembly observed in HG. Immunoblot confirmed increased PKC-delta in HG, which was normalized by Tolrestat. The immunofluorescence pattern of diacylglycerol-insensitive PKC-delta was unchanged in HG, with PMA or Tolrestat. We conclude that mesangial cell F-actin disassembly in high glucose is likely mediated through diacylglycerol-sensitive PKC isoforms, including PKC-delta and involves the polyol pathway.

Mechanisms of adrenergic agonist induced allergy bioactivation and antigen formation.

Reduction of elevated intraocular pressure with alpha 2 agonists has proved to be an exciting new therapeutic approach for the treatment of glaucoma. We have studied the chemical reactivities of several alpha 2 agonists and known allergens to elucidate the origin of the observed ocular allergic response to the alpha 2 agonist apraclonidine. The oxidation potentials of clonidine, apraclonidine, brimonidine, and two known allergens, amodiaquine, and epinephrine, were measured vs. a standard calomel electrode.. Agents that were oxidatively labile were treated with both chemical and enzymatic oxidants. Clonidine and brimonidine proved to be oxidatively stable in sharp contrast to apraclonidine which had an oxidation potential similar to that observed with epinephrine and amodiaquine, two known allergy-inducing agents. In addition, two glutathione-apraclonidine conjugates formed by the in-situ reaction of glutathione with an enzymatically oxidized apraclonidine intermediate were isolated and their structures determined using spectroscopic methods. The structures were shown to be analogous to those obtained with amodiaquine and epinephrine. Apraclonidine, like amodiaquine and epinephrine, possesses a hydroquinone-like subunit and can be readily oxidized and conjugated with thiols modeling well known hapten-forming reactions. Brimonidine, like clonidine, lacks the hydroquinone subunit and does not undergo the thiol conjugation reactions.

Synthesis and evaluation of 2-(arylamino)imidazoles as alpha 2-adrenergic agonists.

A series of 2-(arylamino)imidazoles was synthesized and evaluated for activity at alpha 1- and alpha 2-adrenoceptors. This class of agents has been shown to have potent and selective agonist activity at the alpha 2-adrenoceptors. The most potent member of this class, 2-[(5-methyl-1,4-benzodioxan-6yl)amino]imidazole, proved efficacious for the reduction of intraocular pressure upon topical administration and for the reduction of blood pressure upon intravenous administration. During the course of our studies, we developed a new reagent that allowed rapid assembly of the target compounds. This reagent, N-(2,2-diethoxyethyl)carbodiimide, was convenient to prepare and was stable under low-temperature storage conditions.

Characterization of brimonidine metabolism with rat, rabbit, dog, monkey and human liver fractions and rabbit liver aldehyde oxidase.

1. In vitro metabolism of 14C-brimonidine by the rat, rabbit, dog, monkey and human liver fractions was studied to assess any species differences. In vitro metabolism with rabbit liver aldehyde oxidase and human liver slices, and in vivo metabolism in rats were also investigated. The hepatic and urinary metabolites were characterized by liquid chromatography and mass spectrometry. 2. Up to seven, six, 11 and 14 metabolites were detected in rat liver S9 fraction, human liver S9 fraction, human liver slices and rat urine respectively. Rabbit liver aldehyde oxidase catalysed the metabolism of brimonidine to 2-oxobrimonidine and 3-oxobrimonidine, and further oxidation to the 2,3-dioxobrimonidine. Menadione inhibited the liver aldehyde oxidase-mediated oxidation. 3. Hepatic oxidation of brimonidine to 2-oxobrimonidine, 3-oxobrimonidine and 2,3-dioxobrimonidine was a major pathway in all the species studied, except the dog whose prominent metabolites were 4',5'-dehydrobrimonidine and 5-bromo-6-guanidinoquinoxaline. 4. These results indicate extensive hepatic metabolism of brimonidine and provide evidence for aldehyde oxidase involvement in brimonidine metabolism. The species differences in hepatic brimonidine metabolism are likely related to the low activity of dog liver aldehyde oxidase. The principal metabolic pathways of brimonidine are alpha(N)-oxidation to the 2,3-dioxobrimonidine, and oxidative cleavage of the imidazoline ring to 5-bromo-6-guanidinoquinoxaline.

Synthesis and evaluation of 2-[(5-methylbenz-1-ox-4-azin-6-yl)imino]imidazoline, a potent, peripherally acting alpha 2 adrenoceptor agonist.

We have synthesized 2-[(5-methylbenz-1-ox-4-azin-6-yl)imidazoline, 3, a potent, peripherally acting alpha 2 adrenoceptor agonist. The agent is conveniently prepared in five steps from 2-amino-m-cresol. The agent has demonstrated good selectivity for alpha 2 adrenoceptors in binding and functional studies. When applied topically to eyes, the agent is efficacious for the reduction of intraocular pressure. The agent does not penetrate the blood-brain barrier and, as a consequence, does not lower blood pressure or induce sedation when administered topically or intravenously. We have determined the pKa and log P in water versus both octanol and dodecane of 3 and a set of related agents. The best physical parameter to explain its lack of central nervous system penetration appears to be log P measured in octanol versus water.

Computers in case management. Advancing health care delivery through technology.

Fast efficient access to patient information is an essential management tool in the current healthcare environment. The clinical case management computer module at Stanford Health Services provides and integrates essential patient data elements to facilitate clinical case coordination and assist the nurse case managers with documentation of clinical data for discharge planning and utilization review. Information systems such as this one are critical to the provision of comprehensive, cost-effective patient care management.

Influence of endothelin 1 on human atrial myocardium--myocardial function and subcellular pathways.

The influence of endothelin 1 on isometrically contracting human atrial muscle strip preparations was investigated under physiological conditions (37 degrees C, 1 Hz, Ca2+ 2.5 mM). Endothelin dose-dependently increased isometric tension from 3 x 10(-10) M to 1 x 10(-7) M. At 1 x 10(-7) M the inotropic effect of endothelin was maximum with isometric tension being increased by 32 +/- 6% (n = 11, p < 0.05). At 1 x 10(-7) M endothelin the positive inotropic effect was preceded by a transient negative inotropic effect with a decline in tension by -5 +/- 1% (n = 11, p < 0.05). Endothelin prolonged time from peak tension to 50% relaxation (RT50) by 29 +/- 5%. With BQ123 a competitive antagonist of the ETA receptor positive inotropic effect and the prolongation of relaxation was significantly reduced and initial negative a inotropic effect was abolished, indicating a ETA receptor mediated effect. Preincubation with phorbolmyristateacetate (10(-5) M) to downregulate proteinkinase C (PKC) eliminated the positive inotropic effect of endothelin. Similarly, N-5,5-dimethylamiloride (10(-5) M) which inhibits Na+/H(+)-exchanger activity, abolished the positive inotropic effect of ET. However, with either PMA or DMA the initial transient negative inotropic effect was still present (-13 +/- 7%, n = 9, p < 0.05 and -3 +/- 1%, n = 6, p < 0.05). Furthermore, both substances did not abolish the prolongation of twitch time parameters observed under endothelin. After preincubation with PMA, endothelin prolonged RT50 by 18 +/- 6% and with DMA by 11 +/- 2%. Using the photoprotein aequorin as an indicator for intracellular calcium concentrations showed that the positive inotropic effect was mainly mediated by an increase of systolic intracellular calcium concentrations. Thus, the present data indicate that the positive inotropic effect of endothelin in human atrial myocardium results from activation of PKC with a subsequent activation of the Na+/H(+)-exchanger. However, the initial negative inotropic effects as well as the prolongation of relaxation seem to result from a different intracellular mechanism of endothelin.

Glomerular mesangial cell altered contractility in high glucose is Ca2+ independent.

In diabetes, loss of renal arteriolar smooth-muscle cell contractility leads to intraglomerular hypertension. In glomeruli isolated from streptozotocin (STZ)-induced diabetic rats, the mesangial cells (smooth muscle-like) display loss of contractile responsiveness to angiotensin II. This study examines the mechanistic relationship between altered mesangial cell contractility and vasopressor hormone-stimulated Ca2+ signaling in high glucose. Glomeruli were isolated from normal or STZ-induced diabetic rats to observe ex vivo mesangial cell contractile function. Also, rat mesangial cells were cultured (10-20 passages) in normal (5.6 mmol/l) or high (10-25.6 mmol/l) glucose for 1-5 days. Reduction of glomerular volume and decreased planar surface area of cultured mesangial cells in response to vasoconstrictor stimulation over 60 min were measured by videomicroscopy and personal computer-based morphometry. Contraction of glomeruli isolated from STZ-administered rat in response to endothelin (ET)-1 (0.1 mumol/l) or the Ca2+ ionophore A23187 (5 mumol/l) was impaired significantly compared with that in normal glucose. In the presence of arginine vasopressin (AVP) (1.0 mumol/l) or ET-1 (0.1 mumol/l), mesangial cells demonstrated a dose-dependent loss of contractile response to increasing glucose concentrations (5.6-25.6 mmol/l) within 24 h of high-glucose exposure, which was sustained for 5 days. Mesangial cells in high glucose were consistently smaller in size compared with those in normal glucose. Mesangial cells were preloaded with myo-[2-3H]inositol and intracellular [3H] inositol phosphate release in response to AVP (1.0 mumol/l) was analyzed by Dowex chromatography. Comparing cells in normal (5.6 mmol/l) verus high (25.6 mmol/l) glucose, we observed no significant difference in stimulated inositol phosphate levels from 10 to 60 s.(ABSTRACT TRUNCATED AT 250 WORDS)

[Knee arthroscopy under local anesthesia with or without anesthesiologic assistance. A cost-benefit analysis]. / Knaeledsartroskopi i lokalanaestesi med eller uden anaestesiologisk assistance. En omkostnings-effekt-analyse.

The benefit of anaesthesiological assistance during arthroscopy of the knee in local anaesthesia was evaluated in a cost-effectiveness analysis. One hundred consecutive patients had arthroscopy of the knee performed in local anaesthesia without anesthesiological assistance. In 15% of the cases the arthroscopy was insufficient because of pain reaction. Sixteen percent of the patients indicated that they would prefer general anaesthesia for a similar procedure in the future. The costs for arthroscopy of the knee in local anaesthesia without anaesthesiological assistance were calculated to Dkr. 2055. The amount includes costs for rearthroscopy in local anaesthesia with anaesthesiological assistance for 15% of the patients. Thirty-three patients had arthroscopy of the knee done in local anaesthesia with anaesthesiological assistance. General anaesthesia was needed for twelve percent of the patients. The cost for this procedure, including the costs of possible general anaesthesia were calculated to Dkr. 2458. Any significant difference in the sensation of pain during the arthroscopy could not be demonstrated between the two groups. Based on this study it is recommended that arthroscopy of the knee in local anaesthesia is planned without anaesthesiological assistance.

Podocytic cytoskeletal disaggregation and basement-membrane detachment in puromycin aminonucleoside nephrosis.

Puromycin aminonucleoside--(PAN) treated rats develop acute nephrotic syndrome, mimicking human minimal lesion disease. In PAN nephrosis, podocyte detachment from the glomerular basement membrane (GBM) is the most likely cause of massive proteinuria in this model. To elucidate further the mechanisms of PAN-induced cellular dysfunction, new methods were employed to visualize podocyte cytoskeletal aggregation and to measure fibrillar attachment to the GBM. Adult Sprague-Dawley rats (n = 4/group) received a single tail-vein injection of PAN (75 mg/kg). On days 1, 2, 3, and 5 following injection, 24-hour urine collections were obtained for creatinine clearance, albuminuria, and total proteinuria. Then kidneys from each group were fixed by perfusion. Podocytic cytoskeleton was visualized by scanning electron microscopy. Subepithelial GBM staining and attachment fiber number, observed on digitized images of transmission electron micrographs, were quantitated with computer-based density analysis. A significant reduction in attachment fiber number in the GBM lamina rara externa occurred by day 5. On scanning electron micrographs, the secondary and tertiary podocytic processes were observed to be formed by highly aggregated cytoskeleton, which became partially disaggregated by day 3, was totally absent by day 5, and normalized by day 20. Immunogold staining revealed that actin and vinculin localized to the tertiary podocytic processes in the normal state were dispersed into the cell body following PAN. Podocyte cytoskeletal disaggregation precedes, and detachment from the GBM occurs simultaneously with, the onset of massive proteinuria in the PAN model.

[Total hip alloplasty. The long-term economical consequences]. / Total hoftealloplastik. Langtidsøkonomiske konsekvenser.

A cost-effectiveness analysis concerning total hip arthroplasty was undertaken in 1984. The analysis included 86 patients submitted to operation in Odense and Svendborg Hospitals. After a follow-up period of five years, revision had proved necessary in one patient but no re-operations were undertaken on account of deep infection or aseptic loosening of the implants. The annual accumulated netto cost of total hip arthroplasty including the costs of readmission was calculated to be approximately 23,000 Danish crowns (2,000 pounds). This must be compared with a lasting marked improvement in the quality of life resulting from operation.

Demonstration of a pronounced effect of noncovalent binding selectivity on the (+)-CC-1065 DNA alkylation and identification of the pharmacophore of the alkylation subunit.

Studies on the structural origin of the DNA alkylation selectivity of the antitumor antibiotic (+)-CC-1065 are detailed. The sites of alkylation of double-stranded DNA were examined for simple derivatives of 7-methyl-1,2,8,8a-tetrahydrocycloprop[1,2-c]pyrrolo[3,2-e]indol- 4(5H)-one (CPI), (+)-CC-1065, and agents incorporating the parent 1,2,7,7a-tetrahydrocycloprop[1,2-c]indol-4-one (CI) left-hand subunit. The CI subunit of the agents is a much more reactive alkylating agent than the natural CPI alkylation subunit of CC-1065. Consequently, simple derivatives of CI were found to alkylate double-stranded DNA under milder conditions than were simple derivatives of CPI, and the marked similarities in the CI and CPI DNA alkylation profiles illustrate that CI represents the minimum pharmacophore of CPI. Comparisons of the DNA alkylation profiles of (+)-N-butyloxycarbonyl-CPI, (+)-N-acetyl-CPI, and (+)-CC-1065 revealed distinctions in the CPI and (+)-CC-1065 sites of alkylation, whereas the incorporation of the reactive CI electrophile into an analog of CC-1065 (CI-CDPI2) (CDPI, N3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylic acid) provided an agent that possesses the characteristic CC-1065 DNA alkylation profile (site selectivity and relative site intensity). These observations suggest that the noncovalent binding selectivity of the agents may restrict the number of available DNA alkylation sites and play a productive role in controlling the sequence-selective alkylation by effectively delivering the electrophile to A + T-rich minor groove regions of DNA possessing accessible adenine N-3 alkylation sites. In turn, the noncovalent binding selectivity may be derived from preferential binding within the narrower, sterically more accessible A + T-rich minor groove of double-stranded DNA.