Tailoring the properties of (catalytically)-active inclusion bodies.

BACKGROUND: Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. RESULTS: We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. CONCLUSION: We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors.

BACKGROUND: Conventional experiments in small scale are often performed in a 'Black Box' fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. RESULTS: The same 'mastermix' (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects). CONCLUSIONS: The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.

Myelodysplastic syndromes: a community hospital-based study of prognostic factors and International Prognostic Scoring System.

Various prognostic factors and the International Prognosis Scoring System (IPSS) were assessed in our community hospital-based retrospective study of 55 cases of myelodysplastic syndromes (MDS). All cases were reviewed for clinical, hematologic, histopathologic, and cytogenetic data. The median follow-up was 1.61 years. Twenty patients (36%) were classified as refractory anemia (RA); seven (13%) as refractory anemia with ringed sideroblasts (RARS); 13 (24%) as chronic myelomonocytic leukemia (CMML); 11 (20%) as refractory anemia with excess blasts (RAEB); and four (7%) as refractory anemia with excess blasts in transformation (RAEB-t). Twenty-seven (49%) died during the follow-up period, seven with acute myelogenous leukemia (AML). The median survival was 2.8 years. The variables that showed association with survival by univariate analysis included the absolute neutrophil count, French-American-British (FAB) subtype, percentage of blasts, number of cytopenias, abnormal localization of immature precursors, and IPSS score. When entered into a regression model, IPSS showed a trend towards an association with survival (P 0.09). We conclude that the IPSS can prognostically stratify MDS patients. However, no independent prognostic factor was confirmed in our analysis. Further studies are needed to assess the utility of IPSS.

Spastic disorder in patients with hereditary multiple exostoses, but without spinal cord compression: a new syndrome?

We describe a 37 year old man with a history of a gait disorder which had been worsening over a period of three years. Clinical examination showed the typical signs of a spastic tetraparesis with increased tone of all the extremities. Sensation, autonomic and cerebellar functions were not disturbed. Multiple exostoses had been present since early childhood, but none had been found in the spine or the cranium to cause the tetraspastic disorder. MRI scan was normal. Pedigree analysis of four generations showed that other family members were affected by both disorders. Chromosomal analysis was normal. We consider this to be a previously unknown hereditary syndrome transmitted as an autosomal dominant and manifesting a combination of spastic tetraparesis and multiple exostoses.

Elevated serum CK-BB levels in patients with cerebral transtentorial herniation after ischemic stroke.

We examined the time course of CK and its isoenzymes in 15 patients with severe ischemic stroke. Patients with cerebral transtentorial herniation (n = 7) had the highest CK-BB activity during herniation (1.54 +/- 0.6 U/L, mean +/- SD; range: 1.0-2.6 U/L). These values were distinctly above the values of a control group of 20 patients with non-neurological diseases (0.39 +/- 0.2 U/L, mean +/- SD). In patients with smaller lesions without herniation (n = 8) the maximum CK-BB increase was lower (0.56 +/- 0.26 U/L, mean +/- SD).

[Intracranial pressure-controlled treatment of brain edema with glycerin and sorbitol in intracerebral hemorrhage]. / ICP-gesteuerte Hirnödembehandlung mit Glyzerin und Sorbit bei intrazerebralen Blutungen.

The continuous epidural registration of intracerebral pressure showed that the pronounced brain edema which develops during the 4th to 14th day of an intracerebral hemorrhage could lead to an increase in intracerebral pressure (ICP greater than 25 mmHg) requiring treatment. During the therapy extensive ICP crises (ICP greater than 35 mmHg), lasting for 1 to 3 days and only controllable through high doses of glycerol and sorbitol, developed. Glycerol (50 g orally) and sorbitol (50 g i.v.) lowered the pressure during this phase for approximately 3 h and 1.5 h respectively. These time intervals were in accordance with the changes in plasma osmolality through the administration of both substances. Due to its longer efficacy, glycerol provides an important supplement or alternative to sorbitol therapy, especially as the permitted maximum dosage would have to have been exceeded in a treatment consisting exclusively of sorbitol. The duration of the decrease in intracerebral pressure lasted longer during the remainder of the treatment in the case of both substances, being decisively dependent on the intracerebral pressure intensity. The relatively harmless epidural measurement of intracerebral pressure allowed an optimal control of the brain edema therapy as the dosage of the hyperosmolar substances could be given exactly in accordance with the intracerebral pressure intensity and subsequently varying efficacy.

[Creatine kinase BB activity in the serum of patients with cerebrovascular diseases]. / Kreatinkinase-BB-Aktivität im Serum bei Patienten mit cerebrovaskulären Erkrankungen.

There are conflicting reports of an increase in the activity of creatine kinase BB isoenzyme (CK-BB) in the serum of patients with cerebrovascular disease. The serum CK-BB activity of 33 patients with ischemic brain infarction, subarachnoid hemorrhage or intracerebral hemorrhage was measured with a bioluminescence method (CK-B Kit, LKB-Wallac) in combination with immunoprecipitation. The results were compared with lesions determined by computed tomography. In the control group (N = 19) there was a mean activity of 0.35 +/- 0.26 U/l (means +/- SE). In patients with small lesions (N = 11) the activity was 0.41 +/- 0.21 U/l, which was not significantly elevated when compared to the control group (Mann/Whitney U test). Therefore, patients with more extensive lesions (N = 12) and the group with severe lesions (N = 10) showed a significant elevation, with a mean activity of 0.61 +/- 0.34 U/l and 1.12 +/- 0.52 U/l, respectively. The group with severe lesions had a maximum activity on the first day after the initial symptoms.

[Diagnostics in islet cell tumours (author's transl)]. / Zur Diagnostik bei Inseizelltumoren.

With modern imaging systems like sonography and computer-tomography it is mostly not possible to identify adenomas of the islet-cell-apparatus of the pancreas. These tumours are mostly too small to deform the contours of the pancreas, but because of their intense vascularisation they can be well diagnosed by angiography in form of selective coeliacography, mesentericography or superselective angiography of the pancreatico-duodenal artery. This invasive method is not only necessary to confirm the tentative diagnosis of an adenoma, but also important for its localisation, which is essential for surgery.

[Pringle's disease with skeletal changes]. / Morbus Pringle mit Skelettveränderungen.

A 45 year old woman with Pringle's disease (adenoma sebaceum), gingival and digital fibromas is reported, who had also characteristical skeletal lesions. Three of five children have cutaneous lesions, one of them also skeletal lesions.

[Instrumental esophageal perforations--diagnosis and treatment (author's transl)]. / Instrumentelle Osophagusperforationen

In the last 6 years 16 patients with an iatrogenic esophageal lesion have been treated. Most frequently this lesion results from esophagoscopies and bouginages. Laying of a gastric tube, extraction of a foreign body or dilatation of a cardiospasm are rare reasons for a lesion. Only three patients suffered from cancer. The early diagnosis and immediately operation is decisive for the therapy's success. The operation with primary closure of the defect and drainage should be aspired. After this the best results are levelled. If the surgical operation is carried on after the 24-hours limit, the operation itself is of minor importance as the lethality of these patients is very high and does not depend on the kind of therapy.

Late results following to side-to-side anastomosis of the small intestine in infancy and childhood.

1. Malabsorptions and anemia can be used by ulceration or stenosis of the anastomosis, also well known in other anastomotic techniques. 2. Long-term problems are seldom seen in children secondary to side-to-side anastomosis if the technique is satifactory. Blind pouches may result from unsatisfactory technique, or may develop just as in the adult. They may or may not be symptomatic. We surely have to ask whether the blind loop will really grow with the growing child. Nevertheless this study only has historical character because since 1965 we only use end-to-end anastomosis and its modifications with the well known good results.