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We investigated the incidence, factors affecting referral and outcome of acute renal failure (ARF), in an unselected (predominantly Caucasian) population in the Grampian region of Scotland served by a single renal unit. Case-notes were examined for all patients with a serum creatinine > or = 300 mumol/l. ARF (311 patients) was defined as a temporary rise in serum creatinine > or = 300 mumol/l, or, if the patient died during the acute illness, clinical features indicating acute deterioration of previously normal renal function. Advanced ARF at presentation (51 of the 311 with ARF) was defined as a first recorded serum creatinine > or = 500 mumol/l. Patients were classified into low-, medium-, and high-risk groups according to presence of comorbidity and age. The annual incidence of ARF was 620/million population (pmp), that of advanced ARF 102 pmp. The age-related incidence of ARF ranged from 30 pmp in the age group (0-19 years) to 4266 pmp in the age group > 80 years. Overall, 22% were referred to a nephrologist (34% after excluding those with advanced cancer and age > 80 years). Referral of patients decreased from 100% in the age group 0-19 to 5% in those > 80 years. Referrals in the low-, medium- and high-risk groups were 75%, 30% and 14%, respectively. Patient survival at 2 years was 80%, 42% and 19% for low-, medium-, and high-risk groups, respectively (86%, 44% and 32% for referred patients). Referral and outcome in patients with ARF were significantly influenced by age and presence of comorbidity at presentation.

Connexin43 (Cx43) is a major component of gap junctions. These are widely distributed in the human kidney and are thought to be involved in the inflammatory response and in the regulation of cell growth. Cellular adhesion molecules (CAMs) are also thought to be important in these processes, where they possibly facilitate gap junction formation. The aims of the current study were to define for the first time the expression of Cx43 in inflammatory glomerulonephritis and to compare the localization of this connexin with that of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Human renal biopsies and control sections of normal human kidney were stained using the alkaline phosphatase/anti-alkaline phosphatase immunohistochemical technique, demonstrating that Cx43 was strongly expressed on inflammatory cells, on damaged tubular cells, and on interstitial cells. This pattern of expression was paralleled closely by that of ICAM-1 and, to a lesser extent, by that of VCAM-1. Cx43 is therefore primarily implicated in tubulointerstitial inflammation.

We compared the practice of four orthopaedic consultants as regards the use of MRI and arthroscopy to diagnose problems of the knee. In one year 324 arthroscopies and 66 MR scans were performed for this purpose. We found that MRI is a reliable and cheaper alternative to 'diagnostic arthroscopy'. We consider that patients with definite clinical signs merit an early 'therapeutic arthroscopy', but that all other knees should be investigated by MRI. This policy spares patients from unnecessary and expensive surgery. Arthroscopy for diagnostic purposes should be used only with a specific purpose. Modern MRI can and should replace "having a look".

Aim: To compare the expression of beta 1 integrins in renal biopsies from patients with IgA nephropathy with that found in normal human kidney., Methods: Thirty renal biopsies from patients with IgA disease plus six control specimens were stained with monoclonal antibodies directed against the alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha v, and beta 1 integrin chains using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. The intensity of integrin expression was graded semiquantitatively by a pathologist unaware of the antibody used., Results: Glomerular crescents stained strongly for alpha 3, alpha v, and beta 1, but integrin expression was greatly reduced or absent in fibrotic glomeruli. There were no alterations in the intensity of mesangial cell staining for any of the integrins tested. There was accentuated staining for the alpha 2, alpha 5, alpha v, and beta 1 chains in areas of interstitial scarring plus alpha 2, alpha 3, alpha v, and beta 1 on damaged tubules. Inflammatory cells expressed alpha 4, alpha 5, and beta 1., Conclusions: In IgA nephropathy the interstitium is the main site of altered beta 1 integrin expression. Glomerular crescents also express several beta 1 integrins, but we found no differences in the intensity of integrin expression on mesangial cells. Altered beta 1 integrin expression may play a role in tubulointerstitial scarring in IgA disease. Thus modulation of integrin expression might attenuate this process.

Somatosensory representations occupy parietal postcentral gyral (S1) and lateral sulcal-opercular cortex (S2). To address the issue of possible multiple activation foci in these regions and possible differences due to stimulating skin directly or through an imposed tool, we studied changes in cerebral blood flow with positron emission tomography during passive tactile stimulation of one or two fingertips. Restrained fingers were rubbed with embossed gratings using a rotating drum stimulator in 11 subjects. For different scans, gratings touched the skin directly for optimal stimulation of cutaneous receptors (called skin mode stimulation) or indirectly through an imposed guitar plectrum snugly fitted to the same fingers (called tool mode stimulation). The latter was expected to stimulate deep receptors better. Subjects estimated roughness after each scan. Direct skin contact activated statistically validated foci in both hemispheres. On the contralateral side these foci occurred in the anterior and posterior limbs of the postcentral gyrus and on the ipsilateral side only in the posterior limb. Tool mode stimulation activated one contralateral focus that was in the posterior limb of the postcentral gyrus. These results suggest at least two maps for distal fingertips in S1 with the anterior and posterior foci corresponding, respectively, to activations in area 3b and the junction between areas 1 and 2. In contralateral S2, skin mode stimulation activated a peak that was anterior and medial to a focus associated with tool mode stimulation. The magnitude of PET counts contralateral to stimulation was greater in the anterior S1 and the S2 regions during initial scans but reversed to more activation in the posterior S1 during later scans. These short-term practice effects suggest changes in neural activity with stimulus novelty.

Gap junctions enable intercellular communication and play an important role in a variety of vital cellular functions including differentiation and the control of growth. These junctions are formed by a hexameric of proteins known as connexins. We investigated the distribution of the connexin 43 (Cx43) gap junction protein in renal cells and human kidney using the alkaline phosphatase anti-alkaline phosphatase immunohistochemical technique with a monoclonal antibody directed against the cytoplasmic domain of this antigen. Strong staining was demonstrated on the vascular endothelium, the smooth muscle of larger vessels and on glomerular epithelial cells. In addition, Cx43 was expressed on proximal tubular cells, glomerular endothelial cells and occasional cells infiltrating the interstitium. In areas of tubular atrophy there was increased staining for Cx43. Using reverse transcription-polymerase chain reaction we have also demonstrated that cultured human and rat mesangial cells and human proximal tubular cells express Cx43 messenger RNA. In summary, we have described for the first time the distribution of Cx43 in human kidney and cultured renal cells.

Reconstruction of a full thickness defect of the abdominal or chest wall requires a combination of a rigid or semi-rigid layer and skin cover. The tensor fasciae latae (TFL) flap is unique in that it provides both of these in substantial quantities, but the most difficult aspect of using this flap in the anterior chest and abdomen is finding suitable recipient vessels. We describe a series of nine cases in which full thickness defects of the chest and abdominal wall were repaired using free vascularised TFL flaps. The recipient vessels included the gastroepiploic vessels (n = 2), the deep inferior epigastric artery (n = 3), the internal mammary artery (n = 2), and the superior thyroid and acromiothoracic arteries (n = 1 each). The gastroepiploic and internal mammary vessels are preferred because of their reliability and because the flap pedicle enters the centre of the deep surface of the flap so that if these vessels are used, circumferential tight closure of the fascial layer can be achieved, with consequent decreased risk of vessel kinking and future herniation.

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Brønsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.

Tirana, the only dialysis facility in Albania (pop 4 million), has a stock of 12 patients and three haemodialysis machines. To determine the need for renal services in Albania we studied the incidence and outcome of patients with chronic renal failure (CRF) referred to the renal service in Tirana (pop 300 000) over 1 year. Case-notes of all patients with a serum creatinine concentration > or = 300 mumol/l during the study period (1992) were examined and outcome at 2 years recorded for each patient. In all, 84 patients (mean age 41.6 +/- 17.5 years, 56% male) were referred to nephrologists of whom 35 (42%) came from Tirana, giving an annual incidence of 116 per million pop. 77% were under 40 and had no co-morbid illness. Glomerulonephritis, the most common renal diagnosis, affected 26% patients. 22% patients (mean age 38 +/- 18.1) died within 2 years and only 5% received dialysis. The mean age of those who received dialysis was 29 +/- 8.3 compared with those who were not dialysed (42 +/- 18.0). The 59 patients (24 from Tirana i.e. 80 per million) who were alive with advanced CRF (creatinine > 500) had a mean creatine of 623 +/- 93 mumol/l and would be candidates for dialysis. Patients with progressive renal failure in Albania are regularly followed and treated with antihypertensives and dietary modification. The need for RRT, however, is not being met even for young patients with no co-morbidity.

The benefits of angiotensin-converting enzyme (ACE) inhibition in the management of cardiac failure have been extensively documented. However, little is known about its impact upon the investigation and management of this condition. We assessed how patients diagnosed as having cardiac failure were investigated, which patients were treated with ACE inhibitors and with what dosages. We reviewed the case notes of all 343 patients discharged from Aberdeen Royal Infirmary 1 July-31 December 1992 with a diagnosis of cardiac failure. In addition, a questionnaire was sent to the general practitioners of the 166 patients still alive in October 1994. Only 40% of patients were discharged from hospital on ACE inhibitors. In 58.8%, the diagnosis of cardiac failure was based purely on clinical or radiological grounds. At discharge, 76.1% of patients were on lower doses of ACE inhibitors than those used in the major survival studies; with 68.9% receiving similar doses two years later. The majority of patients with heart failure are under-investigated and under-treated.

Objective: Survival is the ultimate outcome measure in renal replacement therapy (RRT) and may be used to compare performance among centres. Such comparison, however, is meaningless if the influences of comorbidity, age and early deaths are not considered. We therefore studied survival rates on RRT in seven centres in Europe after taking into account the influence of age, early deaths, primary renal diagnoses, and comorbidity., Design: A retrospective survival analysis was carried out on 1407 patients who commenced RRT in seven centres across five European countries during a 7-year period. Patients were stratified into low-, medium- and high-risk groups based mainly on comorbidity and to a lesser extent on age at commencement of RRT. Kaplan-Meier survival and Cox's proportional hazards model were used to compare survival., Results: Before risk stratification overall 2-year survival across the seven centres ranged from 60.2 to 85.3% (69.3-89.9%) after excluding early deaths) masking a range of survivals of 27.4% for the high-risk group with the worst survival to 100% in the low-risk group with the best survival. After excluding early deaths 2-year survival in the low risk groups (n=622) was greater than 90% in all centres. Multivariate analysis showed that the mortality risk increased four fold from low- to medium- and a further 1.6-fold from medium- to high-risk group. However, despite this adjustment for comorbidity and age there still remained a significant difference in survival among some centres, i.e. a 'centre effect' which ranked the centres., Conclusion: Risk stratification diminishes the variance in survival between centres but a centre effect remains despite adjusting for age and comorbidity. Multicentre prospective studies are urgently required to identify the reasons for this apparent centre effect.

The advent of in vitro culture techniques has allowed us to culture homogeneous populations of glomerular mesangial and epithelial cells to aid our understanding of the development of glomerular disease at the cellular level. Advances in our knowledge of the pathogenic mechanisms have made it clear that the response of intrinsic glomerular cells to external stimuli plays an important role in glomerular injury (1, 2). Glomerular cells from several mammalian species have been isolated, propagated, and in some instances, cloned (3-5).

Infiltration of leukocytes into glomerular and interstitial regions of the kidney is a key event in the pathogenesis of human glomerulonephritis. This process is mediated by specific adhesion molecules, some of which are expressed in a coordinated fashion following endothelial cell activation. We have assessed the pattern of expression of the selectins (E, P and L), and the counter-receptors (LFA-1 and ICAM-1, and VLA-4 and VCAM-1 in 119 renal biopsies using sequential sections, and have correlated this with the degree of histological damage (tubular atrophy and interstitial fibrosis) and the intensity of the macrophage infiltrate. Sections were stained with the monoclonal antibodies using a standard alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. There were strong correlations between the following: (1) expression of LFA-1, VLA-4, and L-selectin in the periglomerular region, interstitium and in focal interstitial infiltrates and the presence of macrophages in these regions; (2) de novo tubular expression of ICAM-1 and VCAM-1; (3) staining for ICAM-1 and VCAM-1 on focal cellular infiltrates within the interstitium; and (4) staining for E- and P-selectin on extraglomerular endothelium. These are also strongly correlated with the degree of chronic histological damage. There was, however, no correlation between glomerular expression of adhesion molecules or glomerular macrophage infiltration and chronic histological damage. Although expression of VCAM-1 by the glomerular mesangium was strongly correlated with the presence of cells staining for VLA-4 within the glomerulus, glomerular expression of adhesion molecules correlated poorly with their expression in other sites. These results show that coordinated up-regulation of adhesion molecule expression in the tubulointerstitium is associated with interstitial fibrosis and tubular atrophy and may contribute, therefore, to the progression of renal disease.

These studies have compared the pharmacological profile of two non-peptide human type neurokinin1 (hNK1) receptor selective antagonists, L-741,671 and a quaternised compound L-743,310. In radioligand binding studies L-741,671 and L-743,310 had high affinity for ferret and cloned hNK1 receptors [Ki (nM) ferret 0.7 and 0.1; human 0.03 and 0.06, respectively] but low affinity for rodent NK1 receptors [Ki (nM) 64 and 17, respectively] suggesting that ferret receptors have hNK1-like binding pharmacology. Studies in vivo showed that L-741,671 and L-743,310 had equivalent functional activity in the periphery (ID50s of 1.6 and 2 micrograms/kg i.v., respectively) as measured by inhibition of plasma protein extravasation evoked in the oesophagus of guinea pigs by resiniferatoxin (7 nmol/kg i.v.). Using an in situ brain perfusion technique in anaesthetised rats, L-741,671 was shown to be much more brain penetrant than the quaternary compound L-743,310 which had an entry rate similar to the poorly brain penetrant plasma marker inulin. These compounds thus provided an opportunity to compare the anti-emetic effects of equi-active hNK1 receptor antagonists with and without brain penetration to central NK1 receptor sites. When tested against cisplatin-induced emesis in ferrets, L-741,671 (0.3, 1 and 3 mg/kg i.v.) produced marked dose-dependent inhibition of retching and vomiting but L-743,310 was inactive at 3 and 10 micrograms/kg i.v. In contrast, direct central injection of L-741,671 and L-743,310 (30 micrograms) into the vicinity of the nucleus tractus solitarius or L-743,310 (200 micrograms) intracisternally was shown to inhibit retching and vomiting induced by i.v. cisplatin. L-741,671 and L-743,310 had equivalent functional activity, at the same dose, against cisplatin-induced emesis when injected centrally. These observations indicated that had L-743,310 penetrated into the brain after systemic administration it would have been active in the cisplatin-induced emesis assay and so show that brain penetration is essential for the anti-emetic action of systemically administered NK1 receptor antagonists.

The importance of hospital preadmission programs is increasing in light of the trend toward shorter lengths of stay. New processes are required to prepare patients for same-day admission, to initiate discharge planning at the earliest possible opportunity and to provide education to patients for which there is now less time during hospitalization. This article presents the process and outcomes of one hospital's review of its preadmission program.

Almost all of the existing known antagonists at the glycine site of the N-methyl-D-aspartate (NMDA) receptor have a low propensity for crossing the blood-brain barrier. It has been suggested that in many cases this may be due to the presence of a carboxylic acid which is a common feature of most of the potent full antagonists at this receptor. In this study, 2-aryl-1H-pyrazolo[3,4-c]quinoline-1,4(2H)-diones were found to have high-affinity binding at the glycine receptor. In particular, structure-activity studies identified 7-chloro-3,5-dihydro-2-(4-methoxyphenyl)-1H-pyrazolo[3,4-c]quinoline- 1,4(2H)-dione as the most potent of a series of analogues with an IC50 of 3.3 nM. The measured pKa values in this class of compounds (typically 4.0) indicate they are of equivalent acidity to carboxylic acids. Functional antagonism was demonstrated by inhibition of NMDA-evoked responses in rat cortical slices. Anticonvulsant activity in DBA/2 mice was achieved after dosing by direct injection into the cerebral ventricles, but no activity was seen after systemic administration, suggesting low brain penetration with this class of antagonists.

The 3,5-bis(trifluoromethyl)benzyl ester of N-acetyl-L-tryptophan (3), which was derived from the screening lead N-ethyl-L-tryptophan benzyl ester, has been used as a starting point to identify high-affinity substance P receptor antagonists with improved in vivo activity. Altering the ester moiety to an amide or ether led to a substantial loss in binding affinity, but conversion to a ketone provided compounds with affinity comparable to the equivalent esters. A homochiral synthesis of the key intermediate amino ketone 15 was developed which allows its preparation on a large scale. From this intermediate a range of amine-containing acylamino derivatives were prepared with affinity optimized in the morpholinylbutyramide 161 which has an IC50 of 0.17 nM at the human NK1 receptor. In addition to improving affinity, the amino group also provided aqueous solubility for a number of these derivatives. When tested in vivo the quinuclidine derivative L-737,488 (16i) was found to be an orally active (ID50 = 1.8 mg/kg) inhibitor of substance P-induced dermal extravasation in the guinea pig.

The 3,5-bis(trifluoromethyl)benzyl ester of N-acetyl-L-tryptophan 1 (L-732,138) has been identified previously as a potent and selective substance P receptor antagonist. A series of analogs which introduced a 6-membered heterocyclic ring into the backbone of this structure were prepared for evaluation as bioisosteric replacements of the ester linkage of 1. The 2,5-dioxopiperazine 2 had very weak receptor affinity, but 2-oxopiperazine 5 exhibited modest activity. Examination of the conformations accessible to the substituents on these templates led to exploration of the corresponding 5-membered heterocyclic rings. This study culminated in the identification of oxazolidinedione 14 as a suitable ester mimic in terms of the retention of good NK1 binding affinity.

Comparison of survival data among centers may be used to assess performance, but may be influenced by the number of patients who die during the first 90 days of renal replacement therapy (RRT). Data published by registries in Europe do not detail these deaths, and US data generally exclude them from analysis for financial reasons. To study factors influencing such deaths we compared 42 patients who died within 90 days of first commencing RRT in one Scottish renal unit (group A) between 1971 and 1992 with 42 age- and sex-matched controls who started RRT over the same period and survived longer (group B). Patients who died within 90 days of RRT ranged in age from 25.3 to 83.7 years and had a mean age of 65.2 (SEM, 1.6; 95% confidence interval, 61.9 to 68.4). The proportion of patients who died during the first 90 days of RRT increased from 2% of all patients treated before 1981 to 12% in subsequent years. Thirty-three patients in group A received emergency dialysis via temporary venous access compared with only nine in group B (P < 0.055). There were more patients in group A with a diagnosis of arteriosclerotic renal artery stenosis (14 v 1) and with a history of smoking (15 v 2) than in group B (P < 0.0005). Median renal or nonrenal follow-up before RRT was 1.1 month in group A and 10.6 months in group B (P < 0.0001). Fewer patients in group A had no coexisting disease (1 v 17; P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Patients' perception of their health is an important outcome measure in the management of chronic disease. Comparing that perception from patients receiving different forms of renal replacement therapy (RRT) with data from the general population could be used to monitor the effectiveness of treatment. The short form 36 (SF-36) questionnaire is a general measure of health status which has been validated in the UK and uses eight health scales comprising physical function, social function, role limitation (physical and emotional), mental health, energy, pain and overall health. Using the SF-36 questionnaire, the perception of health of patients receiving RRT was compared with data from healthy control subjects. One hundred and seventy-two of 185 (93%) patients receiving RRT--transplant (n = 102), haemodialysis (n = 43), and peritoneal dialysis (n = 27) completed the questionnaire; scores were compared with those from 542 healthy control subjects. The perception of health of haemodialysis and peritoneal dialysis patients was significantly worse than transplanted patients and controls in six of the eight scales (P < 0.05 dialysis versus transplant and controls). That of transplanted patients was worse in only two and better in one of the eight scales compared with the general population (P < 0.05). Patients were also stratified into low, medium, and high-risk groups based on age and comorbidity and were analysed irrespective of treatment modality. Scores were significantly different across the risk groups in five of the eight scales. We conclude that the SF-36 questionnaire is acceptable to patients on RRT and enables the perception of health of patients receiving RRT to be compared with that of the general population.(ABSTRACT TRUNCATED AT 250 WORDS)

It is believed that human proximal tubular cells may possess immunological function and play an important role in a variety of renal disease states such as interstitial nephritis, allograft rejection and drug induced nephrotoxicity. The role of cytokines and nitric oxide in the human forms of these disease states is not clear. In this study we examined the effect of stimulation with the cytokines IL-1 beta. TNF-alpha and IFN-gamma, individually and in combination, upon primary cultures of human proximal tubular cells. Nitric oxide production increased significantly within 24 hours following cytokine stimulation. This response was inhibited, in a dose dependent manner, by L-NMMA. PCR amplification of mRNA extracted from control and cytokine stimulated human proximal tubular cells revealed a NOS product with a > 97% homology with human hepatocyte inducible nitric oxide synthase. The results of this study clearly show that human proximal tubular cells, in primary culture, are capable of producing nitric oxide in response to an immune challenge secondary to the induction of nitric oxide synthase.

Factors influencing referral of all 304 patients who developed persistent renal failure during one year were studied in the stable Grampian population. The annual incidence of chronic renal failure (CRF) (creatinine > or = 300 mumol/l) was 450/million of the population and of persistent advanced CRF (creatinine > or = 500 mumol/l), 132/million. After excluding those aged > 80 years and those with advanced malignancy, the corresponding incidence figures were 240/million/year and 81/million/year. Only 109 patients (35.8%) were referred to a nephrologist. Patients were divided according to age and coexisting disease into low, medium and high risk groups; 69% of CRF patients in the low, 58% in the medium, and 21% in the high risk group were referred (100%, 88% and 37%, respectively, of the patients with advanced CRF). Two-year patient survival in the low, medium and high risk groups was 100%, 63% and 27%, respectively, in referred patients, and 100%, 48% and 14%, respectively, in non-referred patients. This method of risk stratification identifies patients (particularly those with advanced CRF) likely to have a poor outcome irrespective of referral to a nephrologist. Earlier referral for interventions to delay the progress of the patients' renal and comorbid illnesses has considerable implications for future planning and funding of renal services.

Some of the most dramatic advances in reconstructive surgery have involved microvascular free-flap transfers. Although initial studies stressed the need for the procedures to be restricted to the healthy, with no evidence of vascular disease, it has become apparent that such restrictions need not apply if adequate patient care is available. Refinements of technique and development of new operations have made the free flap available to all. In particular, the procedure has been found to have unique benefits in the elderly and infirm population.

The exoglucanase/xylanase Cex from Cellulomonas fimi hydrolyzes beta-1,4-glycosidic bonds with net retention of anomeric configuration, releasing the disaccharides beta-cellobiose or beta-xylobiose. It uses a double-displacement mechanism involving a glycosyl-enzyme intermediate which is formed and hydrolyzed with general acid/base catalytic assistance. Glu127 was proposed as the acid/base catalyst on the basis of sequence alignments, and mutants at this position were constructed in which the glutamic acid is replaced by alanine or glycine. The following kinetic analysis provides firm support for the assignment of Glu127 as the acid/base catalyst and suggests a more general strategy for identification of this residue in other glycosidases. Substrates which do not require protonic assistance for initial bond cleavage exhibit kcat/Km values similar to those of wild-type enzyme, whereas substrates which do require assistance have kcat/Km values over 6000-fold smaller. Thus rate constants for glycosylation are affected to different degrees by this substitution, depending upon their need for acid catalysis. The deglycosylation rate constant is decreased 200-fold by such substitution, due to the removal of general base catalytic assistance. In the presence of sodium azide a new product, beta-cellobiosyl azide, is formed with these mutants whereas only cellobiose is formed with wild-type enzyme or the Glu127Asp mutant under similar conditions. Addition of azide results in very significant increases in kcat values, ranging from 8-fold for 4''-nitrophenyl cellobioside to over 200-fold for 2'',4''-dinitrophenyl cellobioside, whereas kcat/Km values for these substrates remain essentially constant. No effects on rate upon azide addition are seen with substrates containing aglycons of poor leaving group ability.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycoproteins are proving to be quite common in prokaryotes. Those in S-layers are the best understood in terms of structure. Numerous eubacteria produce non-S-layer glycoproteins about which relatively little is known. The glycans on such proteins and the nature and sites of their linkages to proteins are novel in those glycoproteins which have been examined in any detail. The possible functions of the glycans are mostly not understood. Eubacterial non-S-layer glycoproteins and the glycosylation systems producing them deserve more attention.

As part of a program of screening the Merck sample collection, N-ethyl-L-tryptophan benzyl ester was identified as a weak antagonist at the substance P (NK1) receptor. Structure-activity studies showed that the indole ring system could be replaced by 3,4-dichlorophenyl, alpha- or beta-naphthyl, or benzthiophene with retention or only small loss of affinity. It was found that acylation of the tryptophan nitrogen gave compounds with higher affinity than N-ethyl or other basic amines. Optimization of substitution on the benzyl ester led to the identification of the 3,5-bis-(trifluoromethyl)benzyl ester of N-acetyl-L-tryptophan 26 as a potent and selective substance P receptor antagonist. Compound 26 blocked substance P induced dermal extravasation in vivo and was the most potent compound from this structurally novel class of antagonists which further adds to the diversity of small molecules that bind to the (NK1) receptor.

We have recently shown that a series of N-acyl-L-tryptophan benzyl esters are potent substance P antagonists (Macleod, A. M., Merchant, K. J., Cascieri, M. A., Sadowski, S., Ber, E., Swain, C. J., and Baker, R. (1993) J. Med Chem. 14, 2044-2045). We now report the detailed characterization of the interaction of N-acetyl-L-tryptophan-3,5-bistrifluoromethyl benzyl ester (L-732,138) with the human neurokinin-1 (NK-1) receptor. L-732,138 inhibits the binding of 125I-substance P to the cloned human NK1 receptor expressed in Chinese hamster ovary cells with an IC50 of 2.3 +/- 0.7 nM. In contrast, it has 200-fold lower affinity for the cloned rat NK-1 receptor and has > 1000-fold lower affinity for the human NK-2 and NK-3 receptors. L-732,138 acts as a competitive antagonist of substance P, as shown by functional Schild analysis of the inhibition of substance P-induced inositol phosphate synthesis, by kinetic analysis of the dissociation rate, and by thermodynamic analysis of the equilibrium binding of 125I-substance P to the NK-1 receptor. L-732,138 also competitively inhibits the binding of the quinuclidine amine antagonist, [125I]L-703,606, to the receptor. The compound has 230- and 10-fold reduced affinity for mutant NK-1 receptors in which histidine 265 or histidine 197, respectively, are replaced with alanine. We have previously shown that these residues play key roles in the binding of quinuclidine antagonists to the NK-1 receptor. These results suggest that the tryptophan and quinuclidine series of NK-1 antagonists bind to similar binding sites on the human NK-1 receptor.

In this study, we demonstrate the use of 2H2O, in a manner analogous to 3H2O, to study gluconeogenic flux (deuterium labeling at the carbon-6 position of glucose) relative to overall flux through glucose 6-phosphate (deuterium labeling at the carbon-2 position of glucose) into glucose output and glycogen synthesis during hyperglycemia. Before the study (4 days), jugular and carotid catheters were placed. Rats were fasted for 17 h before the study. 2H2O was infused for 2 h at 3 ml/h, with a subsequent 1-h equilibration period. A hyperglycemic clamp at 180 mg/dl (10 mM) was then performed for 90 min (plasma samples obtained at 10-min intervals). At the end of the experiment, anesthesia was induced and the liver removed. Gas chromatography-mass spectroscopy isotopomer analysis of four different mass clusters from glucose was used to determine deuterium enrichment on the carbon-2 (E2D) and carbon-6 (E6D) positions of plasma glucose and glycogen-glucose. The results show that the labeling pattern in glycogen and plasma glucose was virtually identical. In addition, the E6D-to-E2D ratio in plasma glucose did not change during hyperglycemia. Additional studies were performed to show that the E6D-to-E2D ratio was decreased in the fed state and that the fed animal, compared with the fasted rat, had a marked increase in the ratio when given an epinephrine infusion. Thus it was concluded that this was a robust new technique for analyzing glucose and glycogen metabolism in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Practice of a novel task leads to improved performance. The brain mechanisms associated with practice-induced improvement in performance are largely unknown. To address this question we have examined the functional anatomy of the human brain with positron emission tomography (PET) during the naive and practiced performance of a simple verbal response selection task (saying an appropriate verb for a visually presented noun). As a control state, subjects were asked to repeat the visually presented nouns. Areas of the brain most active during naive performance (anterior cingulate, left prefrontal and left posterior temporal cortices, and the right cerebellar hemisphere), compared to repeating the visually presented nouns, were all significantly less active during practiced performance. These changes were accompanied by changes in the opposite direction in sylvian-insular cortex bilaterally and left medial extrastriate cortex. In effect, brief practice made the cortical circuitry used for verbal response selection indistinguishable from simple word repetition. Introduction of a novel list of words reversed the learning-related effects. These results indicate that two distinct circuits can be used for verbal response selection and normal subjects can change the brain circuits used during task performance following less than 15 min of practice. One critical factor in determining the circuitry used appears to be the degree to which a task is learned or automatic.

A 56-year-old woman developed nephrotic syndrome in association with pulmonary sarcoidosis. Renal biopsy revealed both granulomatous interstitial nephritis and membranous nephropathy. Treatment with steroids resulted in a decrease in proteinuria and there was no deterioration in renal function over a subsequent period of 10 months. This case provides further evidence that secondary membranous nephropathy associated with sarcoidosis should be treated with steroids.

Adequate reconstruction of the mandible when associated with a mucosal deficit requires an osteocutaneous component in which the skin is closely associated with the bone. A curve in the bony framework to simulate the curvature of the mandible is required when the defect is in the anterior segment. The second metatarsal and toe osteocutaneous flap can provide such a mandibular mucosal replacement and has been used successfully for eighteen years.

The social deprivation scores of 169 patients who resided in Grampian region and commenced renal replacement therapy (RRT) in Aberdeen during the period 1 January 1985-30 June 1991 were measured when their serum creatinine concentrations were first > = 300 mumol/l, using the Jarman Underprivileged Area (UPA) and Carstairs indices. The numbers in the population of Grampian for each score were obtained from the Information Services team of Grampian Health Board based on the 1981 census. Comparison of the numbers of patients in each category of both Jarman and Carstairs indices showed no differences between the observed numbers of patients and the numbers in the general population. Thus in this study social deprivation occurred no more frequently in those commencing RRT than in the general population.

Many forms of glomerulonephritis including IgA nephropathy are characterized by mesangial cellular proliferation. Since epidermal growth factor is a potent mitogen for cultured human mesangial cells, we have attempted to localize and quantify the expression of its receptor in normal and abnormal renal biopsies using immunohistochemistry. Using a particular antibody (Amersham, clone EGFR1), the epidermal growth factor receptor (EGF-R) was shown to be predominantly localized in the mesangium of the glomerulus. Visual estimates of intensity of staining suggested that expression of this receptor may be increased in some IgA disease patients with mesangial proliferative glomerular lesions. The neu receptor which has a 50% homology with EGF-R was, however, absent from the glomerulus and cultured mesangial cells did not express detectable levels. Expression of EGF-R by cultured mesangial cells, as assessed by immunostaining, was weak and it was not possible to induce detectable upregulation using different cytokines. The factors leading to increased expression of EGF-R in glomerulonephritis, therefore, remain unknown. Our findings suggest that signalling via EGF-R may play a role in the pathogenesis of proliferative glomerulonephritis. Despite its homology with EGF-R, the neu receptor is unlikely to have similar importance.

To further our understanding of the best way to measure regional CBF with positron emission tomography (PET), we directly compared two candidate tracers ([15O]water and [11C]butanol, administered intravenously) and two popular implementations of the one-compartment (1C) model: the autoradiographic implementation representing a single PET measurement of tissue radioactivity over 1 min and a dynamic implementation representing a sequence of measurements of tissue radioactivity over 200 s. We also examined the feasibility of implementing a more realistic, and thus more complex, distributed-parameter (DP) model by assigning fixed values for all of its parameters other than CBF and tracer volume of distribution (Vd), a requirement imposed by the low temporal resolution and statistical quality of PET data. The studies were performed in three normal adult human subjects during paired rest and visual stimulation. In each subject seven regions of interest (ROIs) were selected, one of which was the primary visual cortex. The corresponding ROI were anatomically equivalent in the three subjects. Regional CBF, Vd, tracer arrival delay, and dispersion were estimated for the dynamic data curves. A total of 252 parameter sets were estimated. With [11C]butanol both implementations of the 1C model provided similar results (r = 0.97). Flows estimated using the 1C models were lower (p < 0.01) with [15O]water than with [11C]butanol. In comparison with the 1C model, the constrained version of the DP used in these studies performed inadequately, overestimating high flow and underestimating low flow with both tracers, possibly as the result of the necessity of assigning fixed values for all of its parameters other than CBF and Vd.

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinant plasmid DNA into Escherichia coli DH5. This in vitro recombination system was used to determine the involvement of eukaryotic topo-isomerases in genetic recombination. Cell-free extracts prepared from a temperature-sensitive topo-isomerase II mutant (top2-1) of S. cerevisiae yielded tetracycline-resistant recombinants, when the recombination assays were performed at both a non-restrictive temperature (30°C) and the restrictive temperature (37°C). This result was obtained whether or not ATP was present in the recombination buffer. Extracts from a non-conditional topo-isomerase I mutant (top1-1) of S. cerevisiae yielded tetracycline-resistant recombinants, as did a temperature-sensitive double mutant (top2-1/top1-8) at the restrictive temperature. The results of this study indicate that neither topo-isomerase I nor topo-isomerase II was involved in the recombinational activity examined.

The immunosuppressive activity and comparative efficacy of rapamycin (RAPA), FK 506 and cyclosporine A (CsA) were investigated in rats following immunization with either xenogeneic sheep red blood cells (SRBC) or allogeneic blood transfusion. RAPA formulated in a polyethylene glycol vehicle, and at a dose of 1.5 mg.kg-1 i.p., was relatively ineffective when compared with FK 506 (1 mg.kg-1) or CsA (15 mg.kg-1) in suppressing antibody production to SRBC. Like FK 506 and CsA however, RAPA proved highly effective in suppressing both the B lymphocytosis and the increase in circulating major histocompatibility complex class II+ cells observed following immunization. All three immunosuppressants caused thymic medullary atrophy, with evidence of epithelial cell damage and increased macrophage phagocytic activity. Administered i.m. (3 mg.kg-1 in olive oil), RAPA was also highly effective in suppressing 1 degree alloantibody responses to MHC class I antigens following blood transfusion. Unlike FK 506 and CsA however, a short (14-day) course of RAPA following 1 degree immunization (transfusion) did no suppress 2 degree alloantibody responses elicited 6 weeks later. Moreover, RAPA did not prevent immunoglobulin isotype switching as observed with FK 506 and CsA. This may reflect the distinct mechanisms of action of RAPA and the latter two agents on T-cell activation/proliferation. Further comparative and combination studies of RAPA with in particular, CsA, are required to further explore to potential of RAPA as an immunotherapeutic agent.

Synthesis of nitric oxide (NO) has been implicated in the development of glomerulonephritis in animal models of the disease. Rat mesangial cells can be stimulated to express an inducible form of NO synthase (NOS) in vitro. Little is known however, about the pathway of induction in human mesangial cells. Here, we report that human mesangial cells require multiple cytokines, unlike rat mesangial cells which require only single stimulants, to produce NO. Our experiments suggest that both interleukin-1 beta (IL-1 beta) and interferon gamma (IFN-gamma) must be present together to elicit a response whilst tumour necrosis factor alpha (TNF-alpha) augments this. The production of nitrite, a stable end product of NO metabolism, was inhibited by NG-monomethyl-L-arginine (L-NMMA), L-nitro-arginine-methyl-ester (L-NAME), cycloheximide and the glucocorticoid dexamethasone.

Survival of patients on renal-replacement therapy (RRT) is no longer improving. Increasingly, such patients are older and have co-morbid conditions affecting organs other than the kidney. In a retrospective study, we calculated actuarial survival of 375 patients starting RRT during a 6 1/2 year period at renal units in Aberdeen and Dundee, UK, after stratification of patients into three risk groups (low, medium, and high) based predominantly on co-morbidity and to a lesser extent on age. 2-year survival differed significantly between low, medium, and high risk groups both before (86%, 60%, and 35%, respectively; p < 0.002 for all comparisons) and after (90%, 70%, 46%; p < 0.004 for all comparisons) excluding early deaths (within 90 days of starting RRT). Overall survival was 61% in Aberdeen and 68% in Dundee (p = 0.04), but 73% and 74%, respectively, when deaths in the first 90 days were excluded (p = 0.73). We conclude that RRT is a highly successful treatment (86% 2-year survival) for patients aged under 70 with no co-morbid conditions (low-risk group); that coexisting non-renal disease has an important influence on survival of patients on RRT; and that risk stratification and analysis of data including and excluding early deaths should allow more valid comparison of data from different centres.