Incident depression in patients diagnosed with multiple sclerosis: a multi-database study.

BACKGROUND AND PURPOSE: Data on rates of newly diagnosed depression after multiple sclerosis (MS) diagnosis are sparse. Here, incident, treated depression in MS patients after diagnosis compared with matched non-MS patients is described. METHODS: A matched cohort study was conducted in two separate electronic medical databases: the US Department of Defense (US-DOD) military healthcare system and the UK's Clinical Practice Research Datalink GOLD (UK-CPRD). The study population included all patients with a first recorded diagnosis of MS and matched non-MS patients. Patients with a history of treated depression were excluded. Incidence rates and incidence rate ratios with 95% confidence intervals for treated depression after MS diagnosis/matched date were estimated. RESULTS: Incidence rate ratios of treated depression amongst MS patients compared with non-MS patients were 3.20 (95% confidence interval 3.05-3.35) in the US-DOD and 1.90 (95% confidence interval 1.74-2.06) in the UK-CPRD. Incidence rate ratios were elevated across age and sex. Rates were higher in females than males but, compared to non-MS patients, males with MS had a higher relative risk than females with MS. CONCLUSIONS: Multiple sclerosis patients in the UK and the USA have a two- to three-fold increased risk of new, treated depression compared to matched non-MS patients.

The risk of treated anxiety and treated depression among patients with psoriasis and psoriatic arthritis treated with apremilast compared to biologics, DMARDs and corticosteroids: a cohort study in the United States MarketScan database.

BACKGROUND: Anxiety and depression are common among psoriasis and psoriatic arthritis (PsA) patients, but rates may differ by treatment. OBJECTIVE: To quantify the risk of incident treated anxiety, depression and mixed anxiety + depression in users of apremilast compared with users of other treatments for psoriasis and PsA. METHODS: We conducted two separate cohort studies of psoriasis and PsA patients treated with apremilast, tumour necrosis factor inhibitor biologics, interleukin-17, -23 or -12/23 inhibitor biologics, conventional DMARDs or systemic corticosteroids in the United States MarketScan database. Cohort entry was date of first study drug after 21 March 2014. We identified cases who had a depression and/or anxiety diagnosis with a prescription for antidepressant/antianxiety medication within 30 days of the diagnosis code. We calculated incidence rates (IRs) and incidence rate ratios with 95% confidence intervals (CIs) for treated anxiety, treated depression and treated anxiety + depression per 1000 patient-years (PY) among patients. RESULTS: Among the psoriasis cohort, IRs for each outcome were similar between exposure categories and highest among users of systemic corticosteroids alone. IRs (95% CI) for apremilast alone were 9.2 (6.6-12.5), 4.6 (2.8-7.1) and 4.6 (2.8-7.1) per 1000 PY for treated anxiety, treated depression and treated anxiety + depression, respectively. In the PsA cohort, the rate of anxiety was highest among users of apremilast alone; rates of depression and anxiety + depression were similar for apremilast compared with other PsA treatments. IRs for each outcome were also high for users of corticosteroids in both the psoriasis and PsA cohorts. CONCLUSIONS: Among patients with psoriasis, users of apremilast had similar rates of anxiety and depression as users of other non-corticosteroid systemic psoriasis treatments. Among PsA patients, users of apremilast had similar rates of depression and anxiety + depression compared with users of other systemic non-corticosteroid PsA drugs; however, the rate of anxiety was slightly higher.

Inducing Hepatitis C Virus Resistance After Pig Liver Transplantation-A Proof of Concept of Liver Graft Modification Using Warm Ex Vivo Perfusion.

Normothermic ex vivo liver perfusion (NEVLP) offers the potential to optimize graft function prior to liver transplantation (LT). Hepatitis C virus (HCV) is dependent on the presence of miRNA(microRNA)-122. Miravirsen, a locked-nucleic acid oligonucleotide, sequesters miR-122 and inhibits HCV replication. The aim of this study was to assess the efficacy of delivering miravirsen during NEVLP to inhibit miR-122 function in a pig LT model. Pig livers were treated with miravirsen during NEVLP or cold storage (CS). Miravirsen absorption, miR-122 sequestration, and miR-122 target gene derepression were determined before and after LT. The effect of miravirsen treatment on HCV infection of hepatoma cells was also assessed. NEVLP improved miravirsen uptake versus CS. Significant miR-122 sequestration and miR-122 target gene derepression were seen with NEVLP but not with CS. In vitro data confirmed miravirsen suppression of HCV replication after established infection and prevented HCV infection with pretreatment of cells, analogous to the pretreatment of grafts in the transplant setting. In conclusion, miravirsen delivery during NEVLP is a potential strategy to prevent HCV reinfection after LT. This is the first large-animal study to provide "proof of concept" for using NEVLP to modify and optimize liver grafts for transplantation.

Infections in patients diagnosed with multiple sclerosis: A multi-database study.

BACKGROUND: Recent data on the rates of infections among patients with multiple sclerosis (MS) are sparse. The objective of this study was to quantify incidence of infections in patients with MS compared with a matched sample of patients without MS (non-MS). METHODS: This study was conducted in two separate electronic medical databases: the United States Department of Defense (US-DOD) military health care system and the United Kingdom's Clinical Practice Research Datalink GOLD (UK-CPRD). We identified patients with a first recorded diagnosis of MS between 2001 and 2016 (UK-CPRD) or 2004 and 2017 (US-DOD) and matched non-MS patients. We identified infections recorded after the MS diagnosis date (or the matched date in non-MS patients) and calculated incidence rates (IRs) and incidence rate ratios (IRRs) with 95% confidence intervals (CIs) by infection site and type. RESULTS: Relative to non-MS patients, MS patients had higher rates of any infection (US-DOD IRR 1.76; 95% CI 1.72-1.80 and UK-CPRD IRR 1.25; 95% CI 1.21-1.29) and a two-fold higher rate of hospitalized infections (US-DOD IRR 2.43; 95% CI 2.23-2.63 and UK-CPRD IRR 2.00; 95% CI 1.84-2.17). IRs of any infection were higher in females compared with males in both MS and non-MS patients, while IRs of hospitalized infections were similar between sexes in both MS and non-MS patients. The IR of first urinary tract or kidney infection was nearly two-fold higher in MS compared with non-MS patients (US-DOD IRR 1.88; 95% CI 1.81-1.95 and UK-CPRD IRR 1.97; 95% CI 1.86-2.09) with higher rates in females compared with males. IRs for any opportunistic infection, candidiasis and any herpes virus were increased between 20 and 52% among MS patients compared with non-MS patients. IRs of meningitis, tuberculosis, hepatitis B and C were all low. CONCLUSION: MS patients have an increased risk of infection, notably infections of the renal tract, and a two-fold increased risk of hospitalized infections compared with non-MS patients.

Formation and intracellular transport of a heterodimeric viral spike protein complex.

We have analyzed the heterodimerization and intracellular transport from the ER to the Golgi complex (GC) of two membrane glycoproteins of a bunyavirus (Uukuniemi virus) that matures by a budding process in the GC. The glycoproteins G1 and G2, which form the viral spikes, are cotranslationally cleaved in the ER from a 110,000-D precursor. Newly synthesized G1 was transported to the GC and incorporated into virus particles about 30-45 min faster than newly synthesized G2. Analysis of the kinetics of intrachain disulfide bond formation showed that G1 acquired its mature form within 10 min, while completion of disulfide bond formation of G2 required a considerably longer time (up to 60 min). During the maturation process, G2 was transiently associated with the IgG heavy chain binding protein for a longer time than G1. Protein disulfide isomerase also coprecipitated with antibodies against G1 and G2. In virus particles, G1 and G2 were present exclusively as heterodimers. Immunoprecipitation with monoclonal antibodies showed that heterodimerization occurred rapidly, probably in the ER, between newly made G1 and mature, dimerization competent G2. Taken together, our results show that these two viral glycoproteins have different maturation kinetics in the ER. We conclude that the apparent different kinetics of ER to GC transport of G1 and G2 is due to the different rates by which these proteins fold and become competent to enter into heterodimeric complexes prior to exit from the ER.

Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment.

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0-10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.

Effects of lifestyle factors on concentrations of salivary cortisol in healthy individuals.

OBJECTIVE: Salivary cortisol is widely used in occupational health research. However, many ordinary daily activities can influence the concentrations of cortisol and the interpretation of field studies. The aim of the present study was to evaluate the effect of lifestyle factors on salivary cortisol in everyday settings. MATERIAL AND METHODS: Healthy employees participated in one or more sub-studies on the effect of eating a vegetable salad versus protein-rich mid-day meal (n = 40), drinking coffee and smoking (n = 12), drinking alcohol (n = 32), awakening at different times (n = 29) and exercising (n = 21). Cortisol in saliva was measured by radioimmunoassay (RIA). RESULTS: When eating a mid-day meal, salivary cortisol was increased by 10 % (CI -1 % to 24 %) 1 h after eating compared to before eating in the case of both types of meal. Salivary cortisol increased by 80 % (CI 9 % to 199 %) after exercising compared to before exercise. The relative awakening response was approximately 100 % when using an alarm clock on both work-days and days off. However, the awakening response was 39 % (CI 10 % to 75 %) on a day off with spontaneous awakening. No effects of alcohol, coffee or smoking were observed. DISCUSSION: In field studies, the biological variation in salivary cortisol may be reduced by restricting physical exercise and in collecting pre-meal samples. However, the protein content of food and moderate consumption of alcohol had no effect on concentrations of cortisol. Differences in relative awakening responses on work-days and days off are related to time and mode of awakening.

Chlorhexidine and preservation of sound tooth structure in older adults. A placebo-controlled trial.

The Trial to Enhance Elderly Teeth Health (TEETH) was designed to test the impact of regular rinsing with a 0.12% chlorhexidine (CHX) solution on tooth loss, and the causes of tooth loss (caries, periodontal disease and trauma) were also investigated. This paper reports on the effectiveness of a 0.12% CHX solution for controlling caries using a tooth surface (coronal and root) survival analysis. A total of 1,101 low income elders in Seattle (United States) and Vancouver (Canada), aged 60-75 years, were recruited for a double-blind clinical trial and assigned to either a CHX (n = 550) or a placebo (n = 551) mouth rinse. Subjects alternated between daily rinsing for 1 month, followed by weekly rinsing for 5 months. All sound coronal and root surfaces at baseline were followed annually for up to 5 years. At each follow-up examination, those tooth surfaces with caries, restored, or extracted were scored as 'carious'. The hazard ratio associated with CHX for a sound surface to become filled, decayed, or extracted was 0.87 for coronal surfaces (95% confidence interval: 0.71-1.14, p = 0.20) and 0.91 for root surfaces (95% confidence interval: 0.73-1.14, p = 0.41). These findings suggest that regular rinsing with CHX does not have a substantial effect on the preservation of sound tooth structure in older adults.

Personality, mental distress, and subjective health complaints among persons with environmental annoyance.

The aim of this study was to assess possible early determinants of idiopathic environmental intolerance (IEI), contributing to an integrated model for the development of IEI. Questionnaires concerning personality traits, current mental distress, subjective health complaints, work load and satisfaction, and options for recovery, were given to 84 persons from the general population attributing annoyance to (i) chemicals/smells (smell-annoyed (SA) n= 29); (ii) electrical equipment (electrically annoyed (EA) n= 16); and (iii) both smells and electricity (generally annoyed (GA) n= 39), but otherwise healthy and in active work. Compared to referents (n= 54), the EA and GA groups showed strongly elevated scores on 5/6 scales within the trait anxiety/neuroticism personality dimension, while the SA group had a slight elevation on only one anxiety scale. Current mental distress and subjective health complaints scores were generally elevated in the EA and GA groups, but only partially in the SA group. Higher proportions of the EA, GA, and SA groups reported low satisfaction with their work situation, including more frequent fatigue after work and a higher, and often unfulfilled, need for recovery. The findings suggest that trait anxiety is prominent already at prodromal stages of IEI, possibly indicating that trait anxiety facilitates the acquisition of attribution of health complaints to environmental factors.

Superantigen-based immunotherapy: a phase I trial of PNU-214565, a monoclonal antibody-staphylococcal enterotoxin A recombinant fusion protein, in advanced pancreatic and colorectal cancer.

PURPOSE: To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas. To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression. PATIENTS AND METHODS: Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg. All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group [ECOG] performance status [PS] = 0 [n = 10]; PS = 1 [n = 11]), 10 had prior radiation, and 18 had prior surgery. RESULTS: Fever and hypotension were the most common toxicities. Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3. Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3. Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity. In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively. Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion. Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients. All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose. The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity. All toxicities were transient and easily managed. No grade 3 toxicity occurred at the higher dose levels. CONCLUSION: PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg. The MTD for a single dose was not determined. The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial.

Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex.

The X-ray structures of dUTPase from equine infectious anaemia virus (EIAV) in unliganded and complexed forms have been determined to 1.9 and 2.0 A resolution, respectively. The structures were solved by molecular replacement using Escherichia coli dUTPase as search model. The exploitation of a relatively novel refinement approach for the initial model, combining maximum likelihood refinement with stereochemically unrestrained updating of the model, proved to be of crucial importance and should be of general relevance.EIAV dUTPase is a homotrimer where each subunit folds into a twisted antiparallel beta-barrel with the N and C-terminal portions interacting with adjacent subunits. The C-terminal 14 and 17 amino acid residues are disordered in the crystal structure of the unliganded and complexed enzyme, respectively. Interactions along the 3-fold axis include a water-containing volume (size 207 A3) which has no contact with bulk solvent. It has earlier been shown that a divalent metal ion is essential for catalysis. For the first time, a putative binding site for such a metal ion, in this case Sr2+, is established. The positions of the inhibitor (the non-hydrolysable substrate analogue dUDP) and the metal ion in the complex are consistent with the location of the active centre established for trimeric dUTPase structures, in which subunit interfaces form three surface clefts lined with evolutionary conserved residues. However, a detailed comparison of the active sites of the EIAV and E. coli enzymes reveals some structural differences. The viral enzyme undergoes a small conformational change in the uracil-binding beta-hairpin structure upon dUDP binding not observed in the other known dUTPase structures.

Superantigen-targeted therapy: phase I escalating repeat dose trial of the fusion protein PNU-214565 in patients with advanced gastrointestinal malignancies.

Antibody-directed, superantigen-induced cytotoxicity has been shown to have potent in vitro and in vivo antitumor effects in preclinical models. In the present study, PNU-214565, a recombinant fusion protein consisting of the Fab of the monoclonal antibody C242 and staphylococcal enterotoxin A (SEA), was used in an escalating repeat dose Phase I clinical trial in patients with advanced gastrointestinal malignancies. A prior single-dose Phase I clinical trial had demonstrated safety at doses of 1.5 ng/kg with toxicities of fever and hypotension that were not dose related. Twenty-seven patients (age range, 36-75 years; median, 62; 14 males and 13 females; 23 colorectal and 4 pancreatic) were treated in the present study with one cycle of four consecutive daily 3-h infusions of PNU-214565 at doses of 0.15 ng/kg (n = 3); 0.5 ng/kg (n = 3), 1.5 ng/kg (n = 4), 2.75 ng/kg (n = 12), and 3.5 ng/kg (n = 5). All patients had a good performance status [Eastern Cooperative Oncology Group: PS = 0 (n = 15), PS = 1 (n = 12)]. As in the single-dose trial, fever and hypotension were the most common toxicities. Dose-limiting toxicity (DLT), consisting of transient hypotension responsive to dopamine, was experienced by one patient treated at the 2.75 ng/kg dose level. One patient with pancreatic cancer metastatic to the liver experienced a partial response of hepatic metastases with stable pancreatic head abnormalities by computed tomography scan. Further dose escalation was suspended when two patients treated in a companion repeat dose Phase I study experienced DLT at the 4 ng/kg dose level. Multiparameter analyses on all patients treated in the two companion single-dose and two-repeated-dose Phase I trials revealed that the levels of patients' pretreatment anti-SEA antibodies protected against toxicity at a given drug dose. By jointly considering weight and the baseline anti-SEA concentration in a patient, it is possible to assign a PNU-214565 dose that will induce systemic cytokine release (a surrogate test to assess for the presence of uncomplexed drug and its ability to induce systemic cellular activation) without DLT. This pharmacodynamically based dosing scheme will be tested in future repeated-dose clinical trials and will define maximally tolerated doses of this powerful new immunotherapy approach.

Capillary blood on filter paper for determination of HbA1c by ion exchange chromatography.

OBJECTIVE: To facilitate HbA1c determination, we evaluated an HbA1c filter paper system enabling capillary blood sampling at home by the patients. RESEARCH DESIGN AND METHODS: Capillary blood (two drops) was applied to a filter paper (HbA1c Via Post) and sent to the laboratory where a small disc was punched out on the filter paper. Hemoglobin was eluted from the disc in a buffer containing cysteine to eliminate the interfering glutathione adduct (HbA3) formed during storage. Analysis was performed by ion-exchange chromatography (Mono S, high-performance liquid chromatography), and the eluate was compared with hemolysate of venous blood from 41 patients. The stability of blood impregnated on filter paper was checked at different temperatures over different periods of time. RESULTS: There was an excellent agreement (r = 0.99) between HbA1c values from capillary blood on filter paper and HbA1c values from venous blood. HbA1c values were constant when stored on filter paper for 5-7 days at 20-21 degrees C (room temperature) or at 4-6 degrees C (refrigerator) for 10 days as well as at -70 degrees C for several months after blood sampling. A new chromatographic-interfering hemoglobin fraction both from venous and capillary samples was identified as free alpha-chain of hemoglobin. CONCLUSIONS: The HbA1c filter paper system enables capillary blood sampling at home, eliminates the need of vein puncture in children and adults, and provides the diabetologist with an HbA1c value when the patient visits the clinic without a need for a previsit phlebotomy.

Effects of gastrin on calcium homeostasis in chickens.

As in the rat, gastrin and an extract of the acid-producing part of the stomach (proventriculus) were found to lower the blood Ca2+ concentration in the chicken. Furthermore, gastrin enhanced the uptake of 45Ca into the femur. It has been suggested previously that gastrin causes hypocalcemia in the rat by releasing gastrocalcin, a hypothetical hormone thought to reside in the acid-producing part of the stomach. The results of the present study in the chicken are in agreement with this concept. Not only exogenous, but also endogenous gastrin lowered blood calcium levels. Thus, the serum gastrin concentration was increased in response to ranitidine-evoked blockade of the gastric acid output; the rise in gastrin was associated with a transient drop in blood calcium. Also, food intake produced a rise in the serum gastrin concentration and a transient drop in blood calcium. However, injection of ranitidine or food intake in proventriclectomized (acid-producing part of the stomach extirpated) chickens failed to lower blood calcium, supporting the view that the gastrin-evoked hypocalcemia depends upon an agent in the gastric (proventriculus) mucosa. We suggest that endogenous and exogenous gastrin evoke hypocalcemia in the chicken by the same mechanism as that which has been postulated in the rat, i.e. by mobilization of the candidate hormone gastrocalcin from endocrine cells in the acid-producing gastric mucosa.

Homotrimeric dUTPases; structural solutions for specific recognition and hydrolysis of dUTP.

Prevention of incorporation of dUTP into DNA is essential for maintenance of the genetic information. Prompt and specific removal of dUTP from the nucleotide pool, as expedited by the ubiquitous enzyme dUTPase, is therefore required for full viability in most biological systems. Conserved structural features perpetuate specificity in choice of substrate, which is crucial as hydrolysis of the structurally closely related nucleotides dTTP, dCTP and UTP would debilitate DNA and RNA synthesis. The most common family of dUTPases is the homotrimeric variety where X-ray structures are available for one bacterial, one mammalian and two retroviral dUTPases. These four enzymes have similar overall structural layouts, but the interactions that stabilise the trimer vary markedly, ranging from exclusively hydrophobic to water-mediated interactions. Trimeric dUTPases contain five conserved sequence motifs, positioned at the subunit interfaces where they contribute to the formation of the active sites. Each of the three identical active sites per trimer is built of residues contributed by all three subunits. One subunit provides residues involved in base and sugar recognition, where a beta-hairpin acts to maintain exquisite selectivity, while a second subunit contributes residues for phosphate interactions. The third subunit supplies a glycine-rich consensus motif located in the flexible C-terminal part of the subunit, known from crystallographic studies to cover the active site in the presence of substrate and certain substrate analogues. All dUTPases studied require the presence of a divalent metal ion, preferably Mg(2+), for optimal activity. The putative position of the essential metal ion has been identified in the structure of one retroviral dUTPase. Structure-function studies are essential if the properties of dUTPases are to be understood fully in relation to their biological role. In this review the structural arrangement of the homotrimeric dUTPases is discussed in the context of active site geometry, achievement of specificity and subunit interactions.

The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase.

The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate (alpha,beta-imido-dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of alpha,beta-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in alpha,beta-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and alpha,beta-imido-dUTP, a difference not observable in C-terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.

The membrane glycoprotein G1 of Uukuniemi virus contains a signal for localization to the Golgi complex.

Members of the Bunyaviridae family acquire their envelopes by budding into the Golgi complex (GC). The accumulation of the membrane glycoproteins G1 and G2 in the GC probably determines the site of maturation. Here we have studied the intracellular transport and targeting to the GC of G1 and G2 of Uukuniemi virus, a member of the Phlebovirus genus, and report on their expression from cloned cDNAs either together or separately by using a T7 RNA polymerase-driven vaccinia virus expression system. When G1 and G2 were expressed together from a full-length cDNA as the p110 precursor, both proteins were localized to the Golgi complex, as evidenced by colocalization with the Golgi marker enzyme mannosidase II. Immunofluorescent staining indicated that G1 expressed alone also localized to the GC. However, pulse-chase experiments showed that G1 remained endoglycosidase H sensitive. G2 expressed alone remained associated with the endoplasmic reticulum (ER). G2 could be rescued from the ER and transported to the GC by coexpression with G1 from separate mRNAs. Coexpression also increased the efficiency of G1 transport to the GC. With none of the constructs could the glycoproteins be observed on the cell surface. These results show that efficient export of G1 and G2 from the ER requires coexpression of both proteins, in conformity with our previous results showing that G1 and G2 form heterodimeric complexes in the ER. Since G1 expressed alone is retained in the GC, we conclude that G1 contains a retention signal for localization to the GC. G2 might thus become associated with the GC indirectly via its interaction with G1.

A long-term cross-over pharmacokinetic study comparing perphenazine decanoate and haloperidol decanoate in schizophrenic patients.

The purpose of the study was to investigate clinical and pharmacokinetic parameters concerning perphenazine decanoate (PD) and haloperidol decanoate (HD) with an interval of 3 weeks during a study period of 51 weeks. This was done by using the available drug preparations in chronic schizophrenic patients in a randomised, double-blind, cross-over, multicentre study. In addition, an elimination phase of 6 weeks was added, when no IM injections of the depot drugs were given. Twenty-nine patients in a stable neuroleptic maintenance phase entered the study. The patients were rated during the trial according to the CPRS-SCHZ and CGI scales, the UKU side effect scale and serum concentrations of the drugs and prolactin were monitored. There was no significant difference between the drugs in antipsychotic efficacy or side effects. Thus, the doses were equipotent with regard to the CPRS-SCHZ scores. However, the patients' global improvement rating was higher for PD (52%) than for HD (39%) (P > 0.05). The elimination of both drugs was very slow. No interaction effects between PD and HD were observed. The serum levels of HD were in most patients lower than those recommended for acute-subacute treatment. The mean doses were 117 mg (0.29 mmol), range 20-313 mg PD and 120 mg (0.32 mmol), range 20-350 mg HD. The serum concentrations in nmol/L of perphenazine and haloperidol (week 24) were 0.8-15.9 and 2.3-46.7, respectively.

Whole-body hyperthermia and ADPRT inhibition in experimental treatment of brain tumors.

Malignant primary brain tumors have hitherto been incurable. One reason for this may be the migrating tumor cells that spread into the surrounding normal brain, creating the basis for inevitable recurrences. Therefore, local therapy may have a temporary effect, but for a cure, the treatment must reach all the tumor cells. Whole-body hyperthermia (WBH) is such a general treatment, which we have studied in a rat glioma model. Cells of the RG2 rat glioma cell line were inoculated in the right caudate nucleus of Fischer-344 rats, which were then either controls or treated with WBH, induced by a radiant heat device for 4-5 sessions of 30 min at body temperature 42 degrees C (rectal), and/or nicotinamide (NAM), an effective radiosensitizer in animal tumor models and an inhibitor of ADPRT (poly adenosine diphosphate ribosyl transferase), a chromatin-bound enzyme suggested to be important in the DNA repair system. We have shown that WBH 42 degrees C alone, or in combination with NAM, has no effect upon tumor growth if a larger number of RG2 cells (5,000) are inoculated. If only 1,000 cells are inoculated, a significant inhibitory effect (p < 0.05) is observed on tumor growth as compared to the untreated control animals. Thus, WBH is feasible, and in some circumstances effective, in a rat glioma model. WBH in combination with other therapies influencing cell metabolism may be of value in future postoperative treatment of human malignant brain tumors.