Effectiveness of intensification therapies in Danes with Type 2 diabetes who use basal insulin: a population-based study.

AIMS: To examine the usage and real-life effectiveness of intensification therapies in people with Type 2 diabetes treated with basal insulin. METHODS: We used population-based healthcare databases in Denmark during 2000-2012 to identify all individuals with a first basal insulin prescription (with or without oral drugs), and evaluated subsequent intensification therapy with bolus insulin, premixed insulin or glucagon-like peptide-1 (GLP-1) receptor agonists. Poisson regression was used to compute the adjusted relative risks of reaching glycaemic control targets. RESULTS: We included 7034 initiators of basal insulin (median age 64 years, diabetes duration 5.3 years, 84% with oral co-medication and median (interquartile range) pre-insulin HbA1c level 77 (65-92) mmol/mol [9.2% (8.1-10.6%)]. Of these, 3076 (43.7%) received intensification therapy after a median of 11 months: 58.5% with premixed insulin, 29.0% with bolus insulin, 10.6% with GLP-1 receptor agonists, and 1.9% with more than one add-on. Overall, 22% had attained an HbA1c level of < 53 mmol/mol (< 7%) by 3-6 months after intensification, while 38% attained an HbA1c < 58 mmol/mol (< 7.5%). Compared with premixed insulin intensification, attainment of HbA1c < 53 and < 58 mmol/mol was similar with bolus insulin add-on [adjusted relative risk 1.03 (95% CI 0.86-1.24) and 1.02 (95% CI 0.91-1.15), and higher for GLP-1 receptor agonist add-on [adjusted relative risk 1.56 (95% CI 1.27-1.92) and 1.27 (1.10-1.47)]. CONCLUSIONS: Among people with Type 2 diabetes, 22 and 38% reached a target HbA1c < 53 mmol/mol (< 7%) or < 58 mmol/mol (< 7.5%), respectively, after intensification of their basal insulin therapy. Compared with premixed insulin, target attainment was similar with bolus insulin and higher with GLP-1 receptor agonists.

Evaluating antibiotic stewardship programs in patients with bacteremia using administrative data: a cohort study.

When introducing new antibiotic guidelines for empirical treatment of bacteremia, it is imperative to evaluate the performance of the new guideline. We examined the utility of administrative data to evaluate the effect of new antibiotic guidelines and the prognostic impact of appropriate empirical treatment. We categorized 2,008 adult patients diagnosed with bacteremia between 2010 and 2012 according to whether they received cephalosporins or fluoroquinolones (old regimen) or not (new regimen). We used administrative data to extract individual level data on mortality, readmission, and appropriateness of treatment, and computed adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for 30-day mortality and post-discharge readmission by regimen and appropriateness of treatment. In total, 945 (47.1%) were treated by the old regimen and 1,063 (52.9%) by the new. The median length of stay (8 days) did not differ by regimen and neither did the proportion of those receiving appropriate empirical treatment (84.1% vs. 85.5%). However, fewer patients with the new regimen were admitted to the intensive care unit (ICU; 3.8% vs. 12.0%) and they had lower 30-day mortality (16.4% vs. 23.4%). The adjusted 30-day mortality HR for appropriate versus inappropriate treatment was 0.79 (95% CI 0.62-1.01) and 0.83 (95% CI 0.66-1.05) for the new versus the old regimen. The HR for 30-day readmission for appropriate versus inappropriate treatment was 0.91 (95% CI 0.73-1.13) and 1.05 (95% CI 0.87-1.25) for the new versus the old regimen. This study demonstrates that administrative data can be useful for evaluating the effect and quality of new bacteremia treatment guidelines.

Staphylococcus aureus skin and soft tissue infections in primary healthcare in Denmark: a 12-year population-based study.

A rise in community-onset Staphylococcus aureus infections has been observed in European countries. To ascertain secular trends of S. aureus infections in primary healthcare in Denmark, we conducted this register-based study in the North Denmark region, during the period 1997-2008. We identified all skin and mucosa specimens obtained by general practitioners and all prescriptions for the preferred oral anti-staphylococcal antibiotic, dicloxacillin. Repeat observations within a 12-month period were excluded prior to the calculation of age and gender standardised incidence rates per 100,000 person-years. We included 108,758 specimens, of which 42,778 (39%) yielded S. aureus. The annual incidence rate of specimens doubled during the study period, reaching 2,399 in 2008. The overall rate of S. aureus isolates increased 2-fold to a stable rate at about 850, but for isolates from children and for impetigo specimens, the increase was steeper, with a peak in 2002. A total of 156,462 dicloxacillin prescriptions had been redeemed and the annual prescription rate increased 2.5-fold, peaking at 3,714 in 2007. In conclusion, the annual rates of specimens, S. aureus infections and dicloxacillin prescriptions more than doubled in primary healthcare during the 12-year study period. A major impetigo epidemic and calls for antibiotic stewardship with increased utilisation of specimens were contributing factors.

Changes in blood culture methodology have an impact on time trends of bacteraemia: a 26-year regional study.

The incidence of bacteraemia has increased considerably during the last decades. This may be related to population ageing, increased use of invasive procedures, and increased ascertainment of bacteraemia. Generalized additive and generalized linear models were used to analyse the impact of four successive improvements in blood culture methodology on the recovery of prevalent blood culture isolates while simultaneously controlling for underlying time trend and seasonal variation. Between 1981 and 2006, 20 091 bacteraemias comprising 22 800 blood culture isolates were diagnosed. The changes in methodology increased the recovery of some bacterial groups; the greatest impact was observed for Enterobacteriaceae, pneumococci and Staphylococcus aureus whereas recovery of ß-haemolytic streptococci, Pseudomonas aeruginosa, other Gram-negative aerobes, and fungi was not affected. Changes in blood culture methodology should be taken into account when assessing time trends of bacteraemia.

High incidence of candidaemia in a nationwide cohort: Underlying diseases, risk factors and mortality.

BACKGROUND: Denmark has a high incidence rate of candidaemia. A Nordic study suggested a higher Danish prevalence of haematological malignancies as an underlying reason. This nationwide study ascertained clinical characteristics of Danish candidaemia patients and investigated potential factors contributing to the high incidence and mortality. METHODS: Microbiological and clinical data for candidaemia patients in 2010-2011 were retrieved. 30-day mortality was estimated by hazard ratios (HR) with 95% confidence intervals (CI, Cox regression). RESULTS: Data were available for 912/973 candidaemia episodes (93.7%). Intensive care unit (ICU) held the largest share of patients (43.2%). Prevalent host factors were multi-morbidity (≥2 underlying diseases, 74.2%) and gastrointestinal disease (52.5%). Haematological disease was infrequent (7.8%). Risk factors included antibiotic exposure (90.5%), CVC (71.9%) and Candida colonisation (66.7%). 30-day mortality was 43.4%, and 53.6% in ICU. Mortality was lower for patients with recent abdominal surgery (HR 0.70, 95% CI: 0.54-0.92). CONCLUSION: A substantial prevalence of multi-morbidity and a high 30-day mortality was found. We hypothesise, that an increasing population of severely ill patients with prolonged supportive treatment and microbiological testing may in part explain the high candidaemia incidence in Denmark. Nationwide studies are warranted to clarify this issue.

Long-term mental wellbeing of adolescents and young adults diagnosed with venous thromboembolism: results from a multistage mixed methods study.

Essentials Long-term mental wellbeing of adolescents and young adults with venous thromboembolism is unclear. This multistage mixed methods study was based on Danish nationwide registry data and interviews. Mental wellbeing is negatively impacted in the long-term and uncertainty of recurrence is pivotal. The perceived health threat is more important than disease severity for long-term mental wellbeing. SUMMARY: Background Critical and chronic illness in youth can lead to impaired mental wellbeing. Venous thromboembolism (VTE) is a potentially traumatic and life-threatening condition. Nonetheless, the long-term mental wellbeing of adolescents and young adults (AYAS) with VTE is unclear. Objectives To investigate the long-term mental wellbeing of AYAS (aged 13-33 years) diagnosed with VTE. Methods We performed a multistage mixed method study based on data from the Danish nationwide health registries, and semistructured interviews with 12 AYAS diagnosed with VTE. An integrated mixed methods interpretation of the findings was conducted through narrative weaving and joint displays. Results The integrated mixed methods interpretation showed that the mental wellbeing of AYAS with VTE had a chronic perspective, with a persistently higher risk of psychotropic drug purchase among AYAS with a first-time diagnosis of VTE than among sex-matched and age-matched population controls and AYAS with a first-time diagnosis of insulin-dependent diabetes mellitus. Impaired mental wellbeing was largely connected to a fear of recurrence and concomitant uncertainty. Therefore, it was important for the long-term mental wellbeing to navigate uncertainty. The perceived health threat played a more profound role in long-term mental wellbeing than disease severity, as the potential life threat was the pivot which pointed back to the initial VTE and forward to the perception of future health threat and the potential risk of dying of a recurrent event. Conclusion Our findings show that the long-term mental wellbeing of AYAS diagnosed with VTE is negatively affected, and highlights these patients' need for adequate support.

Risk factors for extended-spectrum ß-lactamase-producing Escherichia coli urinary tract infection in the community in Denmark: a case-control study.

OBJECTIVE: To verify the role of proton pump inhibitors (PPI) and nitrofurantoin, which have appeared as novel risk factors for carriage of extended-spectrum ß-lactamase (ESBL) -producing Escherichia coli, as risk factors for ESBL E. coli urinary tract infection (UTI). We included known risk factors to ascertain whether our findings are comparable with those of previous studies. METHODS: Population-based case-control study including 339 cases with community-onset ESBL E. coli UTI in 2007-2012, 3390 non-ESBL E. coli UTI controls and 3390 population controls. We investigated potential risk factors by estimating ORs and 95% CIs adjusting for sex, age and co-morbidity. RESULTS: Comparing cases with non-ESBL E. coli UTI, PPI use yielded an OR of 1.6 (95% CI 1.2-2.0) and antibiotic exposure gave an OR of 1.4 (95% CI 1.1-1.8); these were driven by nitrofurantoin (OR 1.8; 95% CI 1.3-2.6) and macrolides (OR 1.7; 95% CI 1.2-2.3). Other risk factors included previous hospitalization with one or two and more than two hospitalizations versus none yielding ORs of 1.9 (95% CI 1.4-2.5) and 4.6 (95% CI 3.2-6.8), recent surgery (OR 2.0; 95% CI 1.5-2.8), renal disease (OR 2.2; 95% CI 1.4-3.4), chronic pulmonary disease (OR 1.4; 95% CI 1.0-2.0) and cancer (OR 1.5; 95% CI 1.1-2.1). Comparing cases with population controls, we found that most risk factors were also risk factors for non-ESBL UTI. CONCLUSIONS: ESBL E. coli UTI were associated with previous hospitalization and surgery. Nitrofurantoin and macrolides augmented the risk. PPIs had a moderate effect but may be important facilitators of ESBL carriage due to their widespread use.

Gender differences in the outcome of community-acquired Staphylococcus aureus bacteraemia: a historical population-based cohort study.

OBJECTIVES: Female gender has been suggested to be associated with poor outcome in patients with Staphylococcus aureus bacteraemia (SAB), but existing data remain sparse and conflicting. We investigated clinical outcomes in female and male patients with community-acquired (CA-) SAB. METHODS: Population-based medical registers were used to conduct a cohort study of all adult patients with CA-SAB in northern Denmark, 2000-2011. Thirty-day mortality after CA-SAB for female and male patients was estimated by the Kaplan-Meier method. Using Cox proportional hazards regression, we computed hazard ratios (HRs) of death according to gender, overall and stratified by age groups, co-morbidity level, and selected major diseases while adjusting for potential confounders. Moreover, we estimated 30-day prevalence proportions for SAB-associated infective endocarditis and osteomyelitis by gender. RESULTS: Among 2638 patients with CA-SAB, 1022 (39%) were female. Thirty-day mortality was 29% (n = 297) in female patients and 22% (n = 355) in male patients, yielding an adjusted HR (aHR) of 1.30 (95% CI, 1.11-1.53). This association appeared robust across age groups, whereas no consistent pattern was observed according to co-morbidity level. Compared with male patients, the prognostic impact of gender was most pronounced among female patients with diabetes (aHR 1.52; 95% CI 1.04-2.21)), and among female patients with cancer (aHR 1.40; 95% CI 1.04-1.90). The 30-day prevalence of infective endocarditis or osteomyelitis did not differ according to gender. CONCLUSION: Female patients with CA-SAB experienced increased 30-day mortality compared with male patients. Gender should be considered in the triage and risk stratification of CA-SAB patients.

Prevalence and predictors of distress in women taking part in surgical continuity of care for breast cancer: A cohort study.

PURPOSE: Women with breast cancer often experience distress. This cohort study investigated the prevalence of distress, predictors of distress, and changes in distress during surgical continuity of care for breast cancer (from diagnosis to commencement of adjuvant treatment). METHODS: The participants were 1079 women with breast cancer who were recruited between April 2013 and May 2014 from 11 breast surgery departments in Denmark. Distress was evaluated using the Distress Thermometer (DT) and predictors of distress were assessed with a self-administered questionnaire at the time of diagnosis (T1), at discharge (T2), and by the start of adjuvant treatment or follow-up (T3). Repeated measures ANOVA, simple and multiple linear regression, and mixed effects regression models were used to identify predictors and estimate changes in distress. RESULTS: At T1, 249 (24.3%) women reported no or minimal distress, 298 (29.1%) moderate distress, and 407 (39.8%) severe distress. The mean distress was 5.5 points on the DT, which decreased by 0.70 (95% confidence interval (CI) -0.80, -0.54) points from T1 to T3. Predictors of distress were time since diagnosis, age, prior or concurrent intake of antidepressants or sedative medicine, prior emotional status, children living at home, feelings regarding femininity and attractiveness, and hospital. CONCLUSIONS: More than two-thirds of women with breast cancer experienced moderate or severe distress. Mean distress decreased slightly during surgical continuity of care. However, for some women, distress remained unchanged or even worsened. These findings highlight the need to identify the individual women with distress and offer them adequate support and care.

Antibiotic treatment and mortality in patients with Listeria monocytogenes meningitis or bacteraemia.

Invasive Listeria monocytogenes infections carry a high mortality despite antibiotic treatment. The rareness of the infection makes it difficult to improve antibiotic treatment through randomized clinical trials. This observational study investigated clinical features and outcome of invasive L. monocytogenes infections including the efficacy of empiric and definitive antibiotic therapies. Demographic, clinical and biochemical findings, antibiotic treatment and 30-day mortality for all episodes of L. monocytogenes bacteraemia and/or meningitis were collected by retrospective medical record review in the North Denmark Region and the Capital Region of Denmark (17 hospitals) from 1997 to 2012. Risk factors for 30-day all-cause mortality were assessed by logistic regression. The study comprised 229 patients (median age: 71 years), 172 patients had bacteraemia, 24 patients had meningitis and 33 patients had both. Significant risk factors for 30-day mortality were septic shock (OR 3.0, 95% CI 1.4-6.4), altered mental state (OR 3.6, 95% CI 1.7-7.6) and inadequate empiric antibiotic therapy (OR 3.8, 95% CI 1.8-8.1). Cephalosporins accounted for 90% of inadequately treated cases. Adequate definitive antibiotic treatment was administered to 195 patients who survived the early period (benzylpenicillin 72, aminopenicillin 84, meropenem 28, sulfamethoxazole/trimethoprim 6, and piperacillin/tazobactam 5). Definitive antibiotic treatment with benzylpenicillin or aminopenicillin resulted in a lower 30-day mortality in an adjusted analysis compared with meropenem (OR 0.3; 95% CI 0.1-0.8). In conclusion, inadequate empiric antibiotic therapy and definitive therapy with meropenem were both associated with significantly higher 30-day mortality.

Precision medicine in the age of big data: The present and future role of large-scale unbiased sequencing in drug discovery and development.

High throughput molecular and functional profiling of patients is a key driver of precision medicine. DNA and RNA characterization has been enabled at unprecedented cost and scale through rapid, disruptive progress in sequencing technology, but challenges persist in data management and interpretation. We analyze the state-of-the-art of large-scale unbiased sequencing in drug discovery and development, including technology, application, ethical, regulatory, policy and commercial considerations, and discuss issues of LUS implementation in clinical and regulatory practice.

Molecular structure of a barley alpha-amylase-inhibitor complex: implications for starch binding and catalysis.

alpha-Amylases are widely occurring, multidomain proteins with a catalytic (beta/alpha)8-barrel. In barley alpha-amylase, insight into the catalytic mechanism is gained from the X-ray crystal structure of its molecular complex with acarbose, a pseudotetrasaccharide that acts like a transition-state analogue and which is shown to bind at two specific regions of the enzyme. The structure of the complex has been refined to an R-factor of 15.1% for all observations with Fo>sigma(Fo) between 10 and 2.8 A resolution. A difference Fourier map produced after refinement of the native structure against the data of the acarbose complex clearly revealed density corresponding to two oligosaccharide-binding sites. One of these is defined as the surface-located starch granule-binding site characteristic of cereal alpha-amylases. It involves stacking of two acarbose rings on Trp276 and Trp277. The other binding region is the active site covering subsites -1, +1 and +2. Here, Glu204 is positioned to act in general acid/base catalysis protonating the glucosidic oxygen atom assisted by Asp289. A water molecule that bridges Glu204 and Asp289 is found at the entrance cavity containing a total of five water molecules. This water molecule is proposed to reprotonate Glu204 and supply the hydroxyl ion for nucleophilic attack on the glucosyl C1 atom. Asp 179 acts as the nucleophile that can bind covalently to the substrate intermediate after bond cleavage. The present complex structure together with the conservation of active-site residues among alpha-amylases and related enzymes, are consistent with a common catalytic mechanism for this class of retaining carbohydrases.

Development of a questionnaire encompassing indicators of distress: a tool for use with women in surgical continuity of care for breast cancer.

PURPOSE: Women with breast cancer often experience significant distress. Currently, there are no questionnaires aimed at identifying women's unique and possible changing indicators for distress in surgical continuity of care for breast cancer. We developed and tested three questionnaires specifically for this use. METHODS AND SAMPLE: We first searched PubMed, CINAHL and PsycINFO to retrieve information on previously described indicators. Next, we conducted a focus group interview with 6 specialised nurses, who have extensive experience about consequences of breast cancer for women in surgical continuity of care. The questionnaire was tested on 18 women scheduled for breast cancer surgery. Subsequently, the women were debriefed to gain knowledge about comprehensibility, readability and relevance of items, and the time needed to complete the questionnaire. After adjustment, the questionnaires were field-tested concomitantly with a clinical study, which both consisted of a survey and an interview study. KEY RESULTS: Three multi-item questionnaires were developed specific to different time points in surgical continuity of care. The questionnaires share a core of statements divided into seven sub-scales: emotional and physical situation, social condition, sexuality, body image, religion and organisational factors. Besides the core of statements, each questionnaire has different statements depending on the time point of surgical continuity of care when it was to be responded to. CONCLUSION: The questionnaires contain comprehensive items that can identify indicators for distress in individual women taking part in surgical continuity of care. The items were understandable and the time used for filling in the questionnaires was reasonable.

The experience of distress in relation to surgical treatment and care for breast cancer: An interview study.

PURPOSE: A diagnosis of breast cancer is a key turning point in a woman's life that may lead to her experiencing severe and persistent distress and potentially presaging a psychiatric disorder, such as major depression. In Denmark an increased standardization of care and a short hospital stay policy minimize the time of medical and nursing surveillance. Consequently, there is the potential risk that distress goes unnoticed, and therefore, untreated. Therefore, the purpose of this study was to explore the experience of distress in Danish women taking part in surgical continuity of care for breast cancer. METHODS AND SAMPLE: A phenomenological-hermeneutic approach inspired by the French philosopher Paul Ricoeur was conducted to explore the experience of distress in relation to surgical treatment and care for breast cancer. Semi-structured interviews were conducted with 12 women who recently had surgery for breast cancer at six departments of breast surgery in Denmark from May 2013 to November 2013. KEY RESULTS: The understanding of the experience of distress in the period of surgical continuity of care for breast cancer is augmented and improved through a discussion related to four identified themes: A time of anxiety, loss of identities, being treated as a person and being part of a system, drawing on theory and other research findings. CONCLUSION: Distress experienced by women in the period following diagnosis arises from multiple sources. Support and care must be based on the woman's individual experience of distress.

A novel CO2- and SO2-tolerant dual phase composite membrane for oxygen separation.

A novel dual phase composite oxygen transport membrane (Al0.02Ga0.02Zn0.96O1.02-Gd0.1Ce0.9O1.95-δ) was successfully prepared and tested. This membrane shows chemical stability against CO2 and SO2, and stable oxygen permeation over 300 hours in CO2 was demonstrated. ZnO is cheap and non-toxic and is therefore highly advantageous compared to other common materials used for this purpose.

Trends in length of stay, mortality and readmission among patients with community-acquired bacteraemia.

In patients hospitalized with severe infection, premature discharge may lead to increased risk of readmission and death. We conducted this population-based cohort study to examine trends in length of stay (LOS) and 30-day mortality and hospital readmission rates after bacteraemia from 1994 through 2013. We used Cox regression to compute hazard ratios (HRs) for 30-day mortality and 30-day postdischarge readmission rates by calendar period and quintiles of LOS, adjusting for age, sex and comorbidity. Among 7618 patients hospitalized with community-acquired bacteraemia during the study period, median LOS decreased from 12 days (quartiles 7-21 days) in 1994-1998 to 9 days (quartiles 6-16 days) in 2009-2013 (25% relative reduction). The 30-day mortality fell from 16.7% to 15.0%, yielding an adjusted 30-day HR of 0.80 (95% confidence interval (CI) 0.68-0.95). Almost one fifth (19.4%) of patients discharged alive were readmitted within 30 days. Concurrently, the adjusted HR of readmission tended to increase (adjusted HR 1.09, 95% CI 0.93-1.28) in 2009-2013 compared with 1994-1998. Compared with the middle quintile of LOS (9-12 days), the risk of readmission was slightly higher for patients discharged within 5 days (adjusted HR 1.12, 95% CI 0.92-1.37), especially for readmission due to infection (adjusted HR 1.38, 95% CI 1.03-1.85). Readmission risk was lowest for 6 to 8 days LOS (adjusted HR 0.80, 95% CI 0.67-0.95) and highest for LOS ≥23 days (adjusted HR 1.30, 95% CI 1.11-1.53). The declining LOS after community-acquired bacteraemia between 1994 and 2013 was not accompanied by increased 30-day mortality but by slightly increased readmission rates.

Expression of cDNAs encoding barley alpha-amylase 1 and 2 in yeast and characterization of the secreted proteins.

Amylolytic strains of the yeast, Saccharomyces cerevisiae, were constructed by transformation with expression plasmids containing cDNAs encoding either AMY1 (clone E) or AMY2 (clone pM/C). The alpha-amylases were efficiently secreted into the culture medium directed by their own signal peptides. When clone E without its 5'-noncoding region was expressed from the yeast PGK promoter, AMY1 was produced as 1% of total cell protein and was thus the major protein secreted, whereas a similar construct derived from pM/C produced much less AMY2. This level is the highest reported for a plant protein secreted by yeast as mediated by the endogenous signal peptide. Production of AMY1 increased 25-fold when the 5'-noncoding part of clone E which contains a 12-bp dG.dC homopolymer tail had been removed. Moreover, expression was one to two orders of magnitude higher when genes encoding AMY1 or AMY2 were inserted between promoter and terminator of the yeast PGK gene in comparison to expression directed from the ADC1 or GAL1 promoters. Recombinant AMY1 and AMY2 had the same Mr and N-terminal sequence as the corresponding barley malt enzymes. Furthermore, none of the enzymes were found to be N-glycosylated. Isoelectric focusing indicated that transformed yeast cells secreted one major form of AMY2 and four dominant forms of AMY1. One AMY1 form corresponded to one of the major forms found in malt while the others, having either low activity or unusually high pI, probably reflect inefficient/incorrect processing. Enzyme kinetic properties and pH activity-dependence of recombinant AMY2 were essentially identical to those of malt AMY2.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of barley malt alpha-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast.

Germinating barley produces two alpha-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMY1) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMY1 is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMY1::AMY2 hybrid.

Gos1p, a Saccharomyces cerevisiae SNARE protein involved in Golgi transport.

Specific transport between secretory compartments requires that vesicular carriers contain targeting proteins known as SNAREs. Ten v-SNAREs have been identified in the genome of the yeast Saccharomyces cerevisiae by sequence analysis. We report here the characterization of Gos1p, a v-SNARE localized to the Golgi compartment and likely homolog of the mammalian protein GOS-28/GS28. Gos1p is a type II membrane protein with characteristic SNARE sequence hallmarks and is functionally a SNARE protein. Gos1p was originally identified as a 28 kDa protein in an immunoprecipitate of the cis-Golgi t-SNARE Sed5p. This interaction between Sed5p and Gos1p is direct as demonstrated by in vitro binding with recombinant proteins. Deletion of GOS1 results in viable haploids with modest growth and secretory defects. Close examination of the secretory phenotype of GOS1-disrupted cells suggests that Gos1p may play a role in multiple transport steps, specifically ER-Golgi and/or intra-Golgi transport.

Treatment with tumor-reactive Fab-IL-2 and Fab-staphylococcal enterotoxin A fusion proteins leads to sustained T cell activation, and long-term survival of mice with established tumors.

C215Fab-IL-2 fusion protein, with full IL-2 and antigen binding activity, was produced in E. coli at high level (>50 mg/l). When co-administered with Fab-superantigen fusion protein (C215Fab-SEA) in mice strong and sustained T cell activation was observed. Combination treatment of mice carrying B16 melanoma transfected with C215 antigen was also more efficient than using C215Fab-SEA (p<0.01) or C215Fab-IL-2 alone (p<0.001). In a long-term survival experiment 5/12 mice having received combination treatment 5 days after i.v. inoculation of B16 cells survived >85 days. Improved therapeutic efficacy correlated with increased tumor infiltration by activated CD25+ T cells, indicating a T cell mediated mechanism.