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Aims: Type 2 diabetes (T2D) is a risk factor for ischemic stroke (IS) and associated with an adverse prognosis. Both stroke and diabetes care has evolved substantially during the last decade. This study aimed to determine the prevalence of T2D among IS patients along with time trends in the risk profile, use of glucose-lowering medications, quality-of-care and clinical outcomes, including stroke severity; length-of-stay; mortality, readmission and recurrent stroke in a large national cohort., Methods: Registry-based cohort study including all IS events in Denmark from 2004 to 2020. IS with co-morbid T2D were compared to IS without diabetes while adjusting for age, sex, stroke severity, co-morbidity and socio-economic factors., Results: The study included 169,262 IS events; 24,479 with co-morbid T2D. The prevalence of T2D in IS increased from 12.0% (2004-2006) to 17.0% (2019-2020). The adjusted absolute 30-day mortality risk in IS with T2D decreased from 9.9% (2004-2006) to 7.8% (2019-2020). The corresponding adjusted risk ratios (aRR) were 1.22 95% confidence interval (1.09-1.37) and 1.29 (1.11-1.50), respectively. The aRR of 365-day mortality was in 2004-2006: 1.20 (1.12-1.29) and in 2019-2020: 1.34 (1.22-1.47). The 30- and 365-day readmissions rates were also consistently higher in IS with T2D., Conclusions: The prevalence of T2D in IS increased over time. The 30- and 365-day mortality rates decreased over the time-period but were consistently higher in IS with co-morbid T2D. Readmissions were also higher in IS with T2D. This highlights an urgent need for strategies to further improve the prognosis in IS patients with co-morbid T2D., (© 2024 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)

Background and Purpose: Cardiovascular outcome trials demonstrate that glucagonlike peptide-1 receptor agonists (GLP-1RAs) reduce the risk of major adverse cardiovascular events in patients with type 2 diabetes (T2D), whereas dipeptidyl peptidase-4 inhibitors (DPP-4is) have not shown cardiovascular benefits. We compared acute ischemic stroke (AIS) with T2D treated with either a GLP-1RA or DPP-4i prior to the index stroke., Methods: This national cohort study included AIS patients with T2D from 2017 to 2020 in Denmark who were users of a GLP-1RA or DPP-4i. To be categorized as a user, we required at least 12 months of exposure and no concurrent treatment with another newer glucose-lowering medication during the last 3 months prior to the index stroke. GLP-1RA users were compared to users of DPP-4i while adjusting for the calendar year of index stroke, age, sex, comorbidity, and socioeconomic factors., Results: The study included 1567 AIS events with T2D; 593 were users of GLP-1RA and 974 of DPP-4i. The absolute risk of a very severe stroke was 2.4% (95% confidence interval [CI] = 1.2-3.7) in GLP-1RA users and 6.1% (95% CI = 4.6-7.7) in DPP-4i users. The corresponding adjusted risk ratio (aRR) of GLP-1RA versus DPP-4i was 0.49 (95% CI = 0.24-1.00). The aRRs of 30-day and 365-day mortality were 0.55 (95% CI = 0.32-0.94) and 0.72 (95% CI = 0.53-0.98), respectively., Conclusions: The risk of a very severe stroke as well as the 30-day and 365-day poststroke mortality rates were lower among the AIS patients with comorbid T2D receiving GLP-1RA prior to the index stroke compared to those receiving DPP-4i. Hence, GLP-1RA may improve stroke outcomes in comparison with DPP-4i., (© 2024 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Background: The INSPiRE-TMS trial (Intensified Secondary Prevention Intending a Reduction of Recurrent Events in Transient Ischemic Attack and Minor Stroke Patients) investigated effects of a multicomponent support program in patients with nondisabling stroke or transient ischemic attack. Although secondary prevention targets were achieved more frequently in the intensified care group, no significant differences were seen in rates of recurrent major vascular events. Here, we present the effects on prespecified patient-centered outcomes., Methods: In a multicenter trial, we randomized patients with modifiable risk factors either to the intensified or conventional care alone program. Intensified care was provided by stroke specialists and used feedback and motivational interviewing strategies (≥8 outpatient visits over 2 years) aiming to improve adherence to secondary prevention targets. We measured physical fitness, disability, cognitive function and health-related quality of life by stair-climbing test, modified Rankin Scale, Montreal Cognitive Assessment, and European Quality of Life 5 Dimension 3 Level during the first 3 years of follow-up., Results: Of 2072 patients (mean age: 67.4years, 34% female) assessed for the primary outcome, patient-centered outcomes were collected in 1,771 patients (877 intensified versus 894 conventional care group). Physical fitness improved more in the intensified care group (mean between-group difference in power (Watt): 24.5 after 1 year (95% CI, 5.5-43.5); 36.1 after 2 years (95% CI, 13.1-59.7) and 29.6 (95% CI, 2.0-57.3 after 3 years). At 1 year, there was a significant shift in ordinal regression analysis of modified Rankin Scale in favor of the intensified care group (common odds ratio, 1.23 [95% CI, 1.03-1.47]) but not after 2 (odds ratio, 1.17 [95% CI, 0.96-1.41]) or 3 years (odds ratio, 1.16 [95% CI, 0.95-1.43]) of follow-up. However, Montreal Cognitive Assessment and European Quality of Life 5 Dimension scores showed no improvement in the intensified intervention arm after 1, 2, or 3 years of follow-up., Conclusions: Patients of the intensified care program group had slightly better results for physical fitness and modified Rankin Scale after 1 year, but none of the other patient-centered outcomes was significantly improved., Registration: URL: https://www., Clinicaltrials: gov; Unique identifier: NCT01586702.

Background: Patients with recent stroke or transient ischaemic attack are at high risk for a further vascular event, possibly leading to permanent disability or death. Although evidence-based treatments for secondary prevention are available, many patients do not achieve recommended behavioural modifications and pharmaceutical prevention targets in the long-term. We aimed to investigate whether a support programme for enhanced secondary prevention can reduce the frequency of recurrent vascular events., Methods: INSPiRE-TMS was an open-label, multicentre, international randomised controlled trial done at seven German hospitals with acute stroke units and a Danish stroke centre. Patients with non-disabling stroke or transient ischaemic attack within 2 weeks from study enrolment and at least one modifiable risk factor (ie, arterial hypertension, diabetes, atrial fibrillation, or smoking) were included. Computerised randomisation was used to allocate patients (1:1) either to the support programme in addition to conventional care or to conventional care alone. The support programme used feedback and motivational interviewing strategies with eight outpatient visits over 2 years aiming to improve adherence to secondary prevention targets. The primary outcome was the composite of major vascular events consisting of stroke, acute coronary syndrome, and vascular death, assessed in the intention-to-treat population (all patients who underwent randomisation, did not withdraw study participation, and had at least one follow-up). Outcomes were assessed at annual follow-ups using time-to-first-event analysis. All-cause death was monitored as a safety outcome. This trial is registered with ClinicalTrials.gov, NCT01586702., Findings: From Aug 22, 2011, to Oct 30, 2017, we enrolled 2098 patients. Of those, 1048 (50·0%) were randomly assigned to the support programme group and 1050 (50·0%) patients were assigned to the conventional care group. 1030 (98·3%) patients in the support group and 1042 (99·2%) patients in the conventional care group were included in the intention-to-treat analysis. The mean age of analysed participants was 67·4 years and 700 (34%) were women. After a mean follow-up of 3·6 years, the primary outcome of major vascular events had occurred in 163 (15·8%) of 1030 patients of the support programme group and in 175 (16·8%) of 1042 patients of the conventional care group (hazard ratio [HR] 0·92, 95% CI 0·75-1·14). Total major vascular event numbers were 209 for the support programme group and 225 for the conventional care group (incidence rate ratio 0·93, 95% CI 0·77-1·12; p=0·46) and all-cause death occurred in 73 (7·1%) patients in the support programme group and 85 (8·2%) patients in the conventional care group (HR 0·85, 0·62-1·17). More patients in the support programme group achieved secondary prevention targets (eg, in 1-year-follow-up 52% vs 42% [p<0·0001] for blood pressure, 62% vs 54% [p=0·0010] for LDL, 33% vs 19% [p<0·0001] for physical activity, and 51% vs 34% [p=0·0010] for smoking cessation)., Interpretation: Provision of an intensified secondary prevention programme in patients with non-disabling stroke or transient ischaemic attack was associated with improved achievement of secondary prevention targets but did not lead to a significantly lower rate of major vascular events. Further research is needed to investigate the effects of support programmes in selected patients who do not achieve secondary prevention targets soon after discharge., Funding: German Federal Ministry of Education and Research, Pfizer, and German Stroke Foundation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: To investigate the effects of centralizing the acute stroke services in the Central Denmark Region (CDR)., Methods: The CDR (1.3 million inhabitants) centralized acute stroke care from 6 to 2 designated acute stroke units with 7-day outpatient clinics. We performed a prospective "before-and-after" cohort study comparing all strokes from the CDR with strokes in the rest of Denmark to discover underlying general trends, adopting a difference-in-differences approach. The population comprised 22,141 stroke cases hospitalized from May 2011 to April 2012 and May 2013 to April 2014., Results: Centralization was associated with a significant reduction in length of acute hospital stay from a median of 5 to 2 days with a length-of-stay ratio of 0.53 (95% confidence interval 0.38-0.75, data adjusted) with no corresponding change seen in the rest of Denmark. Similarly, centralization led to a significant increase in strokes with same-day admission (mainly outpatients), whereas this remained unchanged in the rest of Denmark. We observed a significant improvement in quality of care captured in 11 process performance measures in both the CDR and the rest of Denmark. Centralization was associated with a nonsignificant increase in thrombolysis rate. We observed a slight increase in readmissions at day 30, but this was not significantly different from the general trend. Mortality at days 30 and 365 remained unchanged, as in the rest of Denmark., Conclusions: Centralizing acute stroke care in the CDR significantly reduced the length of acute hospital stay without compromising quality. Readmissions and mortality stayed comparable to the rest of Denmark., (© 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Objective: To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates., Methods: EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation., Results: Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins. Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate. By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation. ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion., Conclusions: Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates., (Copyright © 2017 Novo Nordisk A/S. Published by Elsevier GmbH.. All rights reserved.)

Background and Purpose: We designed and validated a simple prehospital stroke scale to identify emergent large vessel occlusion (ELVO) in patients with acute ischemic stroke and compared the scale to other published scales for prediction of ELVO., Methods: A national historical test cohort of 3127 patients with information on intracranial vessel status (angiography) before reperfusion therapy was identified. National Institutes of Health Stroke Scale (NIHSS) items with the highest predictive value of occlusion of a large intracranial artery were identified, and the most optimal combination meeting predefined criteria to ensure usefulness in the prehospital phase was determined. The predictive performance of Prehospital Acute Stroke Severity (PASS) scale was compared with other published scales for ELVO., Results: The PASS scale was composed of 3 NIHSS scores: level of consciousness (month/age), gaze palsy/deviation, and arm weakness. In derivation of PASS 2/3 of the test cohort was used and showed accuracy (area under the curve) of 0.76 for detecting large arterial occlusion. Optimal cut point ≥2 abnormal scores showed: sensitivity=0.66 (95% CI, 0.62-0.69), specificity=0.83 (0.81-0.85), and area under the curve=0.74 (0.72-0.76). Validation on 1/3 of the test cohort showed similar performance. Patients with a large artery occlusion on angiography with PASS ≥2 had a median NIHSS score of 17 (interquartile range=6) as opposed to PASS <2 with a median NIHSS score of 6 (interquartile range=5). The PASS scale showed equal performance although more simple when compared with other scales predicting ELVO., Conclusions: The PASS scale is simple and has promising accuracy for prediction of ELVO in the field., (© 2016 American Heart Association, Inc.)

Objective: In large-vessel occlusion, endovascular therapy is superior to medical management alone in achieving recanalisation. Reducing time delays to revascularisation in patients with large-vessel occlusion is important to improving outcome., Patients and Methods: A campaign was implemented in the Central Denmark Region targeting the identification of patients with large-vessel occlusion for direct transport to a comprehensive stroke centre. Time delays and outcomes before and after the intervention were assessed., Results: A total of 476 patients (153 pre-intervention and 323 post-intervention) were included. They were treated with either intravenous tissue plasminogen activator or endovascular treatment (alone or in combination with intravenous tissue plasminogen activator). Endovascular therapy patients' median system delay was reduced from 234 to 185 min (adjusted relative risk delay 0.79 (95% confidence interval: 0.67-0.93)). The in-hospital delay was the main driver with an adjusted relative risk delay of 0.76 (confidence interval: 0.62-0.94), while pre-hospital delay was almost significantly reduced with an adjusted relative delay of 0.86 (confidence interval: 0.71-1.04). This was achieved without increasing the intravenous tissue plasminogen activator-treated patients' delay. Significantly more patients treated with endovascular therapy in the post-interventional period achieved functional independence (62% versus 43%), corresponding to an adjusted odds ratio of 3.08 (95% confidence interval: 1.08-8.78)., Conclusion: Direct transfer of patients with suspected large-vessel occlusion to a comprehensive stroke centre leads to shorter treatment times for endovascular therapy patients and is, in turn, associated with an increase in functional independence. We recorded no adverse effects on intravenous tissue plasminogen activator treatment times or outcome., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands., (Copyright © 2011 Elsevier B.V. All rights reserved.)

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

The main purpose of this study was to examine the effects of a selective histamine H(3) receptor antagonist, NNC 38-1202, on caloric intake in pigs and in rhesus monkeys. The compound was given intragastrically (5 or 15 mg/kg), to normal pigs (n=7) and subcutaneously (1 or 0.1mg/kg) to obese rhesus monkeys (n=9). The energy intake recorded following administration of vehicle to the same animals served as control for the effect of the compound. In addition, rhesus monkey and pig histamine H(3) receptors were cloned from hypothalamic tissues and expressed in mammalian cell lines. The in vitro antagonist potencies of NNC 38-1202 at the H(3) receptors were determined using a functional GTPgammaS binding assay. Porcine and human H(3) receptors were found to have 93.3% identity at the amino acid level and the close homology between the monkey and human H(3) receptors (98.4% identity) was confirmed. The antagonist potencies of NNC 38-1202 at the porcine, monkey and human histamine H(3) receptors were high as evidenced by K(i)-values being clearly below 20 nM, whereas the K(i)-value on the rat H(3) receptor was significantly higher (56+/-6.0 nM). NNC 38-1202, given to pigs in a dose of 15 mg/kg, produced a significant (p<0.05) reduction (55%) of calorie intake compared with vehicle alone, (132.6+/-10.0 kcal/kgday versus 59.7+/-10.2 kcal/kgday). In rhesus monkeys administration of 0.1 and 1mg/kg decreased (p<0.05) average calorie intakes by 40 and 75%, respectively. In conclusion, the present study demonstrates that antagonistic targeting of the histamine H(3) receptor decreases caloric intake in higher mammalian species.

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor., (Copyright 2002 Elsevier Science B.V.)

cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

The hypothalamic satiety peptide CART (cocaine and amphetamine regulated transcript) is expressed at high levels in anorectic rat glucagonomas but not in hypoglycemic insulinomas. However, a non-anorectic metastasis derived from the glucagonoma retained high CART expression levels and produced circulating CART levels comparable to that of the anorectic tumors. Moreover, distinct glucagonoma lines derived by stable HES-1 transfection of the insulinoma caused severe anorexia but retained low circulating levels of CART comparable to that of insulinoma bearing or control rats. Islet tumor associated anorexia and circulating CART levels are thus not correlated, and in line with this peripheral administration of CART (5-50 mg/kg) produced no effect on feeding behavior. In the rat two alternatively spliced forms of CART mRNA exist and quantitative PCR revealed expression of both forms in the hypothalamus, in the different islet tumors, and in the islets of Langerhans. Immunocytochemistry as well as in situ hybridization localized CART expression to the somatostatin producing islet D cell. A potential endocrine/paracrine role of islet CART remains to be clarified.

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.

The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure. We have introduced specific mutations (T71I, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase from Bacillus lentus. This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel. The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule. The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process.

The trypsin from Fusarium oxysporum is equally homologous to trypsins from Streptomyces griseus, Streptomyces erythraeus and to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibited form of the enzyme has been crystallized from 1.4 M Na2SO4, buffered with citrate at pH 5.0-5.5. The crystals belong to space group P2(1) with cell parameters a = 33.43 A, b = 67.65 A, c = 39.85 A and beta = 107.6 degrees. There is one protein molecule in the asymmetric unit. X-ray diffraction data to a resolution of 1.8 A were collected on film using synchrotron radiation. The structure was solved by molecular replacement using models of bovine and S. griseus trypsins and refined to an R-factor of 0.141. The overall fold is similar to other trypsins, with some insertions and deletions. There is no evidence of the divalent cation binding sites seen in other trypsins. The covalently bound inhibitor molecule is clearly visible.

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].