Optimized Ki-67 staining in murine cells: a tool to determine cell proliferation.

The reliable analysis of the cell cycle status has become increasingly relevant for scientific and clinical work, especially for the determination of tumor cell growth. One established method to characterize the proliferation activity of cells is the analysis of the Ki-67 protein. Ki-67 is expressed in the nucleus during the whole cell cycle except for the G0 phase. Several different protocols exist for the examination of the Ki-67 protein in tissue and cell culture, but most of them are defined for human cells. For the analysis of the Ki-67 protein in murine tissue and cell culture there is a variety of protocols existing which recommend different fixation and permeabilization reagents or special kits. In this study, we established a reliable protocol for Ki-67 staining in murine cells and tissue based on PFA fixation, which can be used not only for flow cytometry but also for immunofluorescence microscopy analysis. We tested our protocol successfully with three different Ki-67 anti-mouse antibodies in cell culture, regenerating liver tissue and mouse melanoma tumor to demonstrate the general applicability.

[Treatment of carbon monoxide poisoning in Germany : A retrospective single center analysis]. / Behandlung von Kohlenmonoxidvergiftungen in Deutschland : Eine retrospektive Single-Center-Analyse.

BACKGROUND: The symptoms of acute carbon monoxide (CO) poisoning are unspecific, ranging from headaches to unconsciousness and death. In addition to acute symptoms, delayed severe neurological sequelae may occur. While a total of 440 deaths by CO poisoning were registered in Germany in 1999, a total of 594 patients died (0.73 per 100,000 inhabitants) in 2014 and in 2015 the number even increased to 648 deaths. A national database on clinical symptoms, course of illness or quality of care concerning CO poisoning does not yet exist. METHODS: The treatment data of patients admitted to the Hyperbaric Emergency Centre Wiesbaden (HEC) from 2013 to 2017 with CO poisoning formed the basis of the study. This was a comparative evaluation of patient demographics, poisoning sources, symptom spectrum, course of treatment and time intervals registered on the preclinical and clinical levels. RESULTS: From 2013 to 2017 a total of 476 patients (282 men and 194 women) with an average non-invasively measured CO-Hb of 15% (Q0.25 = 7.6%, Q0.75 = 22.3%) were treated with hyperbaric oxygen. Heaters (n = 131), charcoal barbecues (n = 93), fires (n = 90), hookahs (n = 78) and combustion engines (n = 37) were the most frequent CO sources identified. Headaches, vertigo, nausea and syncope were the most prevalent symptoms. A median of 91 min (Q0.25 = 53 min; Q0.75 = 147 min) passed between first medical contact and BGA-validated diagnosis. In total, 151 patients were transferred directly to the HEC, whereas 325 patients were secondarily transferred. The delay in this subgroup took 183 min (median Q0.25 = 138 min; Q0.75 = 248 min). After receiving the first hyperbaric treatment, 80% were free of symptoms. Remaining symptoms included headache (10%), fatigue (8%), vertigo (5%) and nausea (3%) and 45 patients terminated further treatment. Of the patients 417 received a second hyperbaric treatment and 370 patients were treated 3 times. After the third treatment, 89% were free of symptoms and 5% still reported headaches, 3% vertigo and 2% fatigue. In total, 6 patients died and 430 patients were symptom-free after treatment. CONCLUSION: Commonly known sources (fire, charcoal grills) aside, many poisonings by smoking a hookah were observed. This study highlights the importance of considering CO poisoning as a differential diagnosis when encountering patients, especially of younger age, with non-specific neurological symptoms, as well as the importance of early initiation of treatment. A direct correlation between CO-Hb values (whether measured noninvasively or by invasive BGA) and the initial symptoms could not be demonstrated. In total, substantial time expired between the diagnosis and start of treatment of patients transported to a primary care hospital compared to those transported directly to the HEC. Although analysis showed adequate treatment with oxygen in the preclinical interval, administration of oxygen during primary hospital stay showed room for improvement. Introducing a national CO poisoning register and uniform treatment guidelines could improve in-house clinical processes. Multicenter studies are needed to close the gaps identified in the quality of care in Germany.

Cardiovascular magnetic resonance assessment of acute cardiovascular effects of voluntary apnoea in elite divers.

BACKGROUND: Prolonged breath holding results in hypoxemia and hypercapnia. Compensatory mechanisms help maintain adequate oxygen supply to hypoxia sensitive organs, but burden the cardiovascular system. The aim was to investigate human compensatory mechanisms and their effects on the cardiovascular system with regard to cardiac function and morphology, blood flow redistribution, serum biomarkers of the adrenergic system and myocardial injury markers following prolonged apnoea. METHODS: Seventeen elite apnoea divers performed maximal breath-hold during cardiovascular magnetic resonance imaging (CMR). Two breath-hold sessions were performed to assess (1) cardiac function, myocardial tissue properties and (2) blood flow. In between CMR sessions, a head MRI was performed for the assessment of signs of silent brain ischemia. Urine and blood samples were analysed prior to and up to 4 h after the first breath-hold. RESULTS: Mean breath-hold time was 297 ± 52 s. Left ventricular (LV) end-systolic, end-diastolic, and stroke volume increased significantly (p < 0.05). Peripheral oxygen saturation, LV ejection fraction, LV fractional shortening, and heart rate decreased significantly (p < 0.05). Blood distribution was diverted to cerebral regions with no significant changes in the descending aorta. Catecholamine levels, high-sensitivity cardiac troponin, and NT-pro-BNP levels increased significantly, but did not reach pathological levels. CONCLUSION: Compensatory effects of prolonged apnoea substantially burden the cardiovascular system. CMR tissue characterisation did not reveal acute myocardial injury, indicating that the resulting cardiovascular stress does not exceed compensatory physiological limits in healthy subjects. However, these compensatory mechanisms could overly tax those limits in subjects with pre-existing cardiac disease. For divers interested in competetive apnoea diving, a comprehensive medical exam with a special focus on the cardiovascular system may be warranted. TRIAL REGISTRATION: This prospective single-centre study was approved by the institutional ethics committee review board. It was retrospectively registered under ClinicalTrials.gov (Trial registration: NCT02280226 . Registered 29 October 2014).

[Patient Satisfaction as a Measure of Quality of Patient Care - Comparison between a University Hospital and a General Hospital]. / Patientenzufriedenheit als Maßeinheit im Qualitätsmanagement ­ ein Vergleich zwischen einem Universitätsklinikum und einem Krankenhaus der Regelversorgung.

Objective: The aim of the study was to compare the satisfaction of patients of a general hospital with a university hospital. A patient satisfaction index was calculated based on the assessed levels of satisfaction. In general, higher satisfaction could lead to increased competitiveness and improved cost efficiency for achieving profitability. Methods: The authors developed and administered a questionnaire on distinct parameters presumed to reflect the quality of the hospital stay to patients of a university hospital (University Hospital of Bonn, UKB, 1 224 beds) and a general hospital (Johanniterkrankenhaus, Bonn, JKH, 364 beds). Data were collected anonymously. Patient satisfaction and the relative importance of each parameter were assessed. The quotient of both parameters yields the patient satisfaction index (PZI). In order to account for possible differences in patient demands, statistical analysis was performed. Results: The demands and wishes, satisfaction and importance of the retrieved parameters did not differ significantly between the patients of the hospitals in any of aspects assessed (information, participation, contact and comfort during the hospital-stay). The study showed that communication and the contact to physicians and nurses was significantly more important for the patients than comfort (each p<0.001). The highest PZI were found in the categories entertainment (UKB 1.02; JKH 1.25) and contact to the nursing personnel (PZI UKB 0.94; PZI JKH 0.96). The standard of medical-technical care (UKB 0.93; JKH 0.95) was also highly ranked by patients of both hospitals. Needs for improvement related especially to the communication of errors (UKB 0.33; JKH 0.31). Discussion: Surveillance of patient wishes and criticism may result in a more patient-oriented care on a daily basis. Scrutinizing the resources employed may lead to more efficient use of resources and personnel and thus help cut costs and improve the attractiveness of hospitals.

[Carbon monoxide intoxication-New aspects and current guideline-based recommendations]. / Kohlenmonoxidintoxikation ­ neue Aspekte und aktuelle leitlinienbasierte Empfehlungen.

Carbon monoxide poisoning is a common and potentially life-threatening intoxication, showing an interindividual variety of unspecific symptoms as well as late neurological and other sequelae. Two new German guidelines (S2k guidelines diagnosis and treatment of carbon monoxide poisoning as well as S3 guidelines oxygen therapy in the acute care of adult patients) focus on current evidence-based information on diagnostics as well as therapeutic options with considerable uncertainty remaining. This review summarizes current information and presents a flow scheme for daily practical use.

Changes in hormones after apneic hypoxia/hypercapnia - An investigation in voluntary apnea divers.

BACKGROUND: Prolonged apnea is characterized by hypoxia/hypercapnia. Hypoxia can be associated with hormonal dysfunction. We raised the question as to whether steroid hormonal and gonadotropin levels could be influenced by short-term hypoxia/hypercapnia in a model of dry apnea in trained apnea divers. METHODS: Adrenal, sex steroid and pituitary hormones were measured in ten trained voluntary apnea divers before, immediately after, 0.5 h and 4 h after a maximal breath-hold. Apnea was carried out under dry conditions. RESULTS: Corticosterone, progesterone, cortisol, 17-OH-progesterone, dehydroepiandrosterone and androstenedione showed a significant continuous increase with a maximum at 0.5 h after apnea, followed by a decrease back to or below baseline at 4 h after apnea. Testosterone, estradiol, cortisone and dihydrotestosterone showed a decrease 4 h after apnea. Dehydroepiandrosteronesulfate, luteinizing hormone (LH) and follicle stimulating hormone (FSH) showed no significant changes. CONCLUSION: Even a single apnea resulted in two different patterns of hormone response to apnea, with increased adrenal and reduced sex steroid levels, while LH/FSH showed no clear kinetic reaction. Apnea divers might be a suitable clinical model for hypoxic disease.

[Mucormycosis due to Mycocladus corymbiferus in a fattening pig]. / Mucormykose durch Mycocladus corymbiferus bei einem Mastschwein.

A fattening pig with enlarged head and abdominal lymph nodes was examined. An aspirate of the abscesses did not produce a conclusive diagnosis. Only an excision with subsequent histological and bacteriological examination showed the mould Mycocladus corymbiferus (syn. Absidia corymbifera) to be present. Similar abscesses should be examined as actinomycosis and leucosis are the main differential diagnoses.

DHA Supplementation Attenuates MI-Induced LV Matrix Remodeling and Dysfunction in Mice.

OBJECTIVE: Myocardial ischemia and reperfusion (I/R) injury is associated with oxidative stress and inflammation, leading to scar development and malfunction. The marine omega-3 fatty acids (ω-3 FA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are mediating cardioprotection and improving clinical outcomes in patients with heart disease. Therefore, we tested the hypothesis that docosahexaenoic acid (DHA) supplementation prior to LAD occlusion-induced myocardial injury (MI) confers cardioprotection in mice. METHODS: C57BL/6N mice were placed on DHA or control diets (CD) beginning 7 d prior to 60 min LAD occlusion-induced MI or sham surgery. The expression of inflammatory mediators was measured via RT-qPCR. Besides FACS analysis for macrophage quantification and subtype evaluation, macrophage accumulation as well as collagen deposition was quantified in histological sections. Cardiac function was assessed using a pressure-volume catheter for up to 14 d. RESULTS: DHA supplementation significantly attenuated the induction of peroxisome proliferator-activated receptor-α (PPAR-α) (2.3 ± 0.4 CD vs. 1.4 ± 0.3 DHA) after LAD occlusion. Furthermore, TNF-α (4.0 ± 0.6 CD vs. 1.5 ± 0.2 DHA), IL-1ß (60.7 ± 7.0 CD vs. 11.6 ± 1.9 DHA), and IL-10 (223.8 ± 62.1 CD vs. 135.5 ± 38.5 DHA) mRNA expression increase was diminished in DHA-supplemented mice after 72 h reperfusion. These changes were accompanied by a less prominent switch in α/ß myosin heavy chain isoforms. Chemokine mRNA expression was stronger initiated (CCL2 6 h: 32.8 ± 11.5 CD vs. 78.8 ± 13.6 DHA) but terminated earlier (CCL2 72 h: 39.5 ± 7.8 CD vs. 8.2 ± 1.9 DHA; CCL3 72 h: 794.3 ± 270.9 CD vs. 258.2 ± 57.8 DHA) in DHA supplementation compared to CD mice after LAD occlusion. Correspondingly, DHA supplementation was associated with a stronger increase of predominantly alternatively activated Ly6C-positive macrophage phenotype, being associated with less collagen deposition and better LV function (EF 14 d: 17.6 ± 2.6 CD vs. 31.4 ± 1.5 DHA). CONCLUSION: Our data indicate that DHA supplementation mediates cardioprotection from MI via modulation of the inflammatory response with timely and attenuated remodeling. DHA seems to attenuate MI-induced cardiomyocyte injury partly by transient PPAR-α downregulation, diminishing the need for antioxidant mechanisms including mitochondrial function, or α- to ß-MHC isoform switch.

Effects of repetitive prolonged breath-hold in elite divers on myocardial fibrosis and cerebral morphology.

BACKGROUND: Prolonged apnea by breath-hold (BH) divers leads to hypoxemia and compensatory mechanisms of the cardiovascular system (i.e. increase of total peripheral resistance, increase of systolic blood-pressure, left-ventricular enlargement) to maintain oxygen supply to oxygen sensitive organs such as the brain. All these changes may result in structural myocardial or subclinical brain alterations. Therefore, the aim of this study was to investigate mid-term effects of repetitive prolonged apnea using cardiac magnetic resonance imaging (CMR) and magnetic resonance imaging of the brain. MATERIALS AND METHODS: 17 elite BH divers (15 males) were investigated at baseline, from whom 9 (7 males) were investigated again at follow-up one year later. CMR included functional imaging and tissue characterization using T1- and T2-mapping as well as late gadolinium enhancement. Results were compared intra-individually and with 50 age matched controls. RESULTS: Mean BH time were 297 ±â€¯52 s (entire cohort) and 315 ±â€¯56 s (sub-cohort) at initial, and 334 ±â€¯104 s at follow-up examination. Apnea resulted in a progressive increase of the left ventricle and impaired function, whichfully resolved after cessation of apnea. At rest, no dilation of the left ventricle was notable (LV-EDV: 106.7 ±â€¯28.8 ml; LV-EDV/BSA: 52.2 ±â€¯12.7 ml/m2). Compared to controls, the apnea group showed significantly lower volumes (LV-EDV: 106.7 ±â€¯28.8 ml vs. 140.9 ±â€¯36.3 ml, p = .008; LV-EDV/BSA: 52.2 ±â€¯12.7 ml/m2 vs. 73.7 ±â€¯12.8 ml/m2). In contrast, LV-EF showed no significant differences between both groups (61.0 ±â€¯7.0% vs. 60.9 ±â€¯3.6%). T1- and T2-mapping revealed no significant differences, neither intra-individually nor in comparison with age matched controls. (T1 pre-contrast: 974.1 ±â€¯12.9 ms vs. 969.4 ±â€¯29.0 ms, p = .2; T1 post-contrast: 368.9 ±â€¯38.5 ms vs. 966.7 ±â€¯40.5 ms, p = .4; ECV: 29.2 ±â€¯1.5% vs. 29.8 ±â€¯1.6%, p = .3; T2. 52 ±â€¯2 ms vs. 52 ±â€¯3 ms; p = .4). Except for one old embolic lesion no structural changes were found in brain imaging. CONCLUSION: Although, prolonged apnea leads to impressive adaptions of the cardiovascular system (i.e. dilation of the left ventricle) and hypertension due to peripheral vasoconstriction no mid-term morphological changes could be observed in both, the myocardium and the brain. BH divers are suitable as a model to investigate acute physiological changes of prolonged apnea and hypoxemia, but not as a model for chronic alterations.

Cerebrovascular Reactivity during Prolonged Breath-Hold in Experienced Freedivers.

BACKGROUND AND PURPOSE: Experienced freedivers can endure prolonged breath-holds despite severe hypoxemia and are therefore ideal subjects to study apnea-induced cerebrovascular reactivity. This multiparametric study investigated CBF, the spatial coefficient of variation as a correlate of arterial transit time and brain metabolism, dynamics during prolonged apnea. MATERIALS AND METHODS: Fifteen male freedivers (age range, 20-64 years; cumulative previous prolonged breath-holds >2 minutes and 30 seconds: 4-79,200) underwent repetitive 3T pseudocontinuous arterial spin-labeling and 31P-/1H-MR spectroscopy before, during, and after a 5-minute breath-hold (split into early and late phases) and gave temporally matching venous blood gas samples. Correlation of temporal and regional cerebrovascular reactivity to blood gases and cumulative previous breath-holds of >2 minutes and 30 seconds in a lifetime was assessed. RESULTS: The spatial coefficient of variation of CBF (by arterial spin-labeling) decreased during the early breath-hold phase (-30.0%, P = .002), whereas CBF remained almost stable during this phase and increased in the late phase (+51.8%, P = .001). CBF differed between the anterior and the posterior circulation during all phases (eg, during late breath-hold: MCA, 57.3 ± 14.2 versus posterior cerebral artery, 42.7 ± 10.8 mL/100 g/min; P = .001). There was an association between breath-hold experience and lower CBF (1000 previous breath-holds reduced WM CBF by 0.6 mL/100 g/min; 95% CI, 0.15-1.1 mL/100 g/min; P = .01). While breath-hold caused peripheral lactate rise (+18.5%) and hypoxemia (oxygen saturation, -24.0%), cerebral lactate and adenosine diphosphate remained within physiologic ranges despite early signs of oxidative stress [-6.4% phosphocreatine / (adenosine triphosphate + adenosine diphosphate); P = .02]. CONCLUSIONS: This study revealed that the cerebral energy metabolism of trained freedivers withstands severe hypoxic hypercarbia in prolonged breath-hold due to a complex cerebrovascular hemodynamic response.

An Outbreak of Porcine Reproductive and Respiratory Syndrome Virus in Switzerland Following Import of Boar Semen.

An outbreak of porcine reproductive and respiratory syndrome virus (PRRSV) occurred in November 2012 in Switzerland (CH), traditionally PRRSV-free. It was detected after a German boar stud informed a semen importer about the detection of PRRSV during routine monitoring. Tracing of semen deliveries revealed 26 Swiss sow herds that had used semen from this stud after its last negative routine monitoring and 62 further contact herds. All herds were put under movement restrictions and examined serologically and virologically. As a first measure, 59 sows from five herds that had previously been inseminated with suspicious semen were slaughtered and tested immediately. Investigations in the stud resulted in 8 positive boars with recent semen deliveries to CH (Seven with antibodies and virus, one with antibodies only). In one boar out of six tested, virus was detected in semen. Of the 59 slaughtered sows, five from three herds were virus-positive. In one herd, the virus had spread, and all pigs were slaughtered or non-marketable animals euthanized. In the remaining herds, no further infections were detected. After confirmatory testings in all herds 3 weeks after the first examination gave negative results, restrictions were lifted in January 2013, and Switzerland regained its PRRSV-free status. The events demonstrate that import of semen from non-PRRS-free countries--even from negative studs--poses a risk, because monitoring protocols in boar studs are often insufficient to timely detect an infection, and infections of sows/herds occur even with low numbers of semen doses. The outbreak was eradicated successfully mainly due to the high disease awareness of the importer and because immediate actions were taken before clinical or laboratory diagnosis of a single case in the country was made. To minimize the risk of an introduction of PRRSV in the future, stricter import guidelines for boar semen have been implemented.

DNA base modifications and membrane damage in cultured mammalian cells treated with iron ions.

We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 microM, 100 microM, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(II) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(II) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.

Formation of DNA-protein cross-links in cultured mammalian cells upon treatment with iron ions.

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydrio-2,4-dioxopyrimidin-5-yl)-methyl]- L-tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.

Polarization-based sensing of glucose using an oriented reference film.

We describe a new approach to glucose sensing using polarization measurements in the presence of a stretch-oriented reference film. The method relies on measurement of the polarized emission from the reference film and of a fluorophore which changes intensity in response to glucose. A glucose-sensitive fluorescent signal was provided by the glucose/galactose binding protein from E. coli. This protein was labeled with an environmentally sensitive fluorophore at a single genetically inserted cysteine residue, and displayed decreased fluorescence upon glucose binding. Using the protein and the reference film we observed glucose-sensitive polarization values for micromolar glucose concentrations. This method of polarization-based sensing is generic and can be used for any sensing fluorophore which displays a change in intensity. In principle, one can construct simple and economical devices for this type of glucose measurement. © 1999 Society of Photo-Optical Instrumentation Engineers.

Steam-sterilizable, fluorescence lifetime-based sensing film for dissolved carbon dioxide.

An autoclavable sensing film was developed for monitoring dissolved CO2. The sensing film, based on fluorescence resonance energy transfer (FRET), consisted of a fluorescent donor, an acceptor, and a quaternary ammonium hydroxide, which were doped in a two-component silicone film. As no aqueous solution was used in the sensing film matrix, the sensing film was unaffected by osmotic pressure. Fluorescence lifetime was selected as the sensing parameter, and measured in frequency domain using phase fluorometry. Upon exposure to 20% CO2-saturated water, a 43 degrees increase in phase angle was observed at 100 MHz. The process was fully reversible when the sensing film was exposed to nitrogen-saturated water. The estimated response and recovery times for 90% signal change were 1 min (for a step change from 0 to 6.7% CO2-saturated water) and 1.5 min (for a step change from 6.7 to 3.3% CO2-saturated water). When used for on-line monitoring of dissolved CO2 produced by a culture of Escherichia coli, the sensing film showed a similar trend to that obtained from off-line measurements using a wet chemistry analyzer.

Green fluorescent protein as a real time quantitative reporter of heterologous protein production.

Since its cloning and commercial availability, applications of green fluorescent protein (GFP) as a reporter gene have become prevalent in many aspects of science. The attributes of GFP could also be applied to the area of heterologous protein production. The work described here represents the first experiments to use GFP as a generic tool to monitor protein production in bioprocess development. We have constructed a plasmid containing an operon fusion of the two reporter genes GFP and chloramphenicol acetyl transferase (CAT). CAT served as a "model" recombinant protein product to demonstrate the in situ quantifiable reporting mechanism of GFP. Our results indicate there is a direct correlation between the fluorescence intensity of GFP and the functional activity of the downstream CAT protein. In addition, there is a quantitative relationship between the level of CAT protein concentration and GFP fluorescence. These experiments provide the groundwork for using GFP as an in situ reporter gene for scale-up and process optimization of recombinant protein production.

Purification of recombinant green fluorescent protein using chromatofocusing with a pH gradient composed of multiple stepwise fronts.

Green fluorescent protein (GFP), which fluoresces in the green region of the visible spectrum and is widely used as a reporter for gene expression and regulation, was overexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP. A two-step chromatofocusing procedure was used to purify GFP starting from cell lysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. The first chromatofocusing step was performed using a low-pressure column in which a retained stepwise pH front formed by adsorbed buffering species was used to capture GFP directly from clarified cell lysate and selectively focus it into a chromatographic band. The second step utilized a high-performance column under mass overloaded conditions where a similar pH front acted as a protein displacer and led to the formation of a highly concentrated rectangular band of GFP. The overall procedure yielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery and purity for GFP of 60% and 80%, respectively. Because the method employs a strong-base ion-exchange column packing and low-cost buffers formed with formic and acetic acids instead of the proprietary column packings and polyampholyte elution buffers more generally used for chromatofocusing, it appears to be a practical alternative for the preparative ion-exchange chromatography of GFP in particular and for the recovery of recombinant proteins from cell lysate in general. A discussion is also given concerning the choice of appropriate buffers for the rational design of pH gradients involving retained, stepwise pH fronts that span a given pH range and of the use of the fluorescence properties of GFP for flow visualization and chromatographic process development.

[Lab-On-A- Chip--systems for biomedical research and diagnosis]. / Lab-On-A-Chip--Systeme für Die Biomedizinische Forschung und Diagnostik.

In today's biomedical research and diagnosis, a number of substances and agents have to be checked. Frequently, plastic micro titer plates are used for this purpose as large-area test platforms. For the first time, plastic micro titer plates with 96 identical microfluidic labon-a-chip structures for simultaneous capillary electrophoresis (CE) have now been produced using microtechnical fabrication methods. Such structures are suited for e.g. the separation of biomolecules. In completely sealed microfluidic channel systems, smallest sample volumes can be processed, separated, mixed with other substances, or detected. Due to the small channel dimensions, these microfluidic systems are characterized by very small sample volumes needed.

Evaluation of 2 hematology analyzers in body fluid mode versus flow cytometry immunophenotyping of mainly neurosurgical cerebrospinal fluid samples.

BACKGROUND: For CSF analysis, exact knowledge of the type and amount of cells is essential, especially for detection of infection or bleeding. The chamber count has been the current reference method to date, yet it is problematic due to its subjectivity depending on the examiner's skill and experience. Therefore, as a reference method, we used an impulse cytophotometric measurement with Epics XL owing to its improved objectify ability and compared this method to the measurement of CSF samples performed with the ADVIA 2120 and XE-5000. MATERIAL AND METHODS: 101 CSF samples were measured with the ADVIA 2120, XE-5000, and Epics XL. For impulse cytophotometric measurement, CD235a was used for identification of erythrocytes; CD45 for the entire leukocyte population; CD56, CD16 and CD14 for monocytes; CD3, CD4 and CD19 for lymphocytes;and CD13, CD15 and CD33 for neutrophile granulocytes. RESULTS: Regarding leukocyte measurements, a strong correlation was obtained between Epics XL and XE-5000 (r = 0.990), with the correlation between Epics XL and ADVIA 2120 not as strong (r = 0.538). This finding is due to the fact that with blood-stained CSF samples (erythrocytes >1,500/µl), no valid results were produced by the ADVIA 2120. In measurements of blood-free CSF samples, correlations between Epics XL, XE-5000, and ADVIA 2120 were almost identical (r = 0.985 and r = 0.964). The same applies to the correlation between polymorphonuclear and mononuclear cells (range 0.920-0.972). In erythrocyte measurements, the correlation between XE-5000 and ADVIA 2120 was excellent (r = 0.945). Impulse cytophotometric measurement of erythrocytes with CD 238 antibodies did not appear to be functional. CONCLUSION: In the measurement of leukocytes in CSF with the ADVIA 2120, no valid results could be obtained in blood-stained CSF samples (erythrocytes >1,500/µl). In blood-free CSF samples (erythrocytes <1,500/µl), measurements of leukocytes, and polymorphonuclear and mononuclear cells performed with the ADVIA 2120 and XE-5000 produced almost identical good results. Determination of CSF cells with the XE-5000 is presently the best automated method for counting leukocytes of blood-stained CSF.