Differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate.

Thin-layer chromatography (TLC) is a simple, fast and inexpensive separation method. Unambiguous identification of the TLC spots is, however, often a problem. Here we show for the first time that oligosaccharides (derived from dextran, alginate, hyaluronan and chondroitin sulfate) can be characterized by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) directly on a TLC plate. The applied oligosaccharides were either commercially available or obtained from the polysaccharides by HCl-induced hydrolysis. Normal phase TLC was followed by MALDI-TOF MS subsequent to matrix deposition. It will be shown that high quality mass spectra can be obtained that enable unequivocal assignments. It will also be shown that the high content of formic acid in the solvent system does not confer major problems but is responsible for the partial formylation of the analyte and minor N-acetyl loss from hyaluronan and chondroitin sulfate.

Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells.

PURPOSE: Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. MATERIALS AND METHODS: The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy. RESULTS: Both pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. CONCLUSIONS: These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.

An ultrasonographic method to study reproductive seasonality in ewes isolated from rams.

In studies concerning the seasonality in sheep, the effect of the teaser-ram, which can by itself induce oestrus as well as the development of ovary function bodies, has not been considered. In this study, a method is developed to determine ovarian activity during the year while excluding a possible influence of the ram. The study was carried out on 10 German Blackhead Mutton ewes (GBM) and 10 German Mutton Merino ewes (GMM) and lasted for 14 months. The ewes were kept without contact with any ram, and once a week a transrectal ultrasonography was conducted to determine ovary function bodies (corpora lutea, follicles) and a blood sample was taken to analyse progesterone (P4) concentration. In both breeds, the number of ovulating ewes, the ovulation rate (OR), the size of the corpora lutea (CL) and P4-concentrations showed significant seasonal fluctuations. During the main breeding period (September-February), the OR was higher (2.0 +/- 0.4) than during the rest of the year (1.6 +/- 0.5). Outside the main breeding season (March-August), approximately 40% GBM ewes and only approximately 15% GMM ewes produced CL. The cycle and the related seasonal changes in the P4-concentration coincide with different ultrasonically diagnosed CL (developing or regressing, insufficient, no CL). Between the volume of fully developed CL and P4-concentration, correlations of 0.57 (GMM) and 0.45 (GBM), respectively, were found. On average, P4-concentration in September to February was by approximately 1.60-1.66 nmol/l in the GMM and by approximately 1.80-1.86 nmol/l in the GBM higher than in spring and summer. Considering the OR, the P4-values corresponded with the CL volumes, i.e. in the months from August to February the CL values were significantly larger than in the months March to August. The ultrasonic ovarian diagnosis is a useful method to determine the ram-uninfluenced seasonality of reproduction in sheep.

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research.

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.

Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidylcholines.

Spermatozoa with plasma membranes that lost their asymmetry or permeability for larger molecules can be identified by binding of annexin V to membrane phosphatidylserine (PS). Paramagnetic annexin-V-conjugated microbeads (AN-MB) can be used to eliminate these spermatozoa by magnetic activated cell sorting (MACS). Semen samples of six healthy volunteers with normal spermiogram parameters were divided into two sperm fractions by MACS as a function of bound AN-MB, and their individual lipid compositions were examined by matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS). As a model system, liposomes composed of phosphatidylcholines (PC) from egg yolk were digested by phospholipase A2 (PLA2). The MALDI-TOF mass spectra of organic extracts of both sperm subpopulations differed significantly. The ratio between lyso-phosphatidylcholine LPC 16 : 0 and PC 16 : 0/22 : 6 was approximately 2.5-4.7-fold higher (median 2.9) in the sperm group binding AN-MB than in spermatozoa with intact membrane unable to bind AN-MB. The ratio between LPC 22 : 6 and PC 16 : 0/22 : 6 was also enhanced in the spermatozoa with impaired membrane structure (factor in the range: 1.9-3.9; median 2.6). These alterations corresponded to the effects of PLA2 on artificial phospholipids. It is concluded that spermatozoa with deteriorated membrane and exposed PS are characterized by an increased lyso-phosphatidylcholine content that is likely generated by phospholipases.

CsCl as an auxiliary reagent for the analysis of phosphatidylcholine mixtures by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is mainly used for protein and peptide analysis. However, there is growing evidence that also phospholipids like phosphatidylcholines (PC) can be easily analyzed by MALDI-TOF MS. In MALDI-TOF methodology, the sample is cationized by the addition of inorganic ions. This process is strongly dependent on the corresponding ion concentration. In biological samples various cations are present (mainly H+, Na+ and K+) and, therefore, a mixture of different adducts is formed. Since phospholipids exhibit a wide distribution of different fatty acid residues a considerable peak overlap may occur. This is a major problem since the peak assignment in a mixture will be often unclear. In this paper we demonstrate that this problem can be easily overcome by mixing the analyte with caesium chloride (CsCl). This yields naturally non-occurring Cs+ adducts that are apparent due to the large shift of the molecular mass. The proposed method facilitates the clear assignment of most peaks. Besides that, we will show that CsCl can also be used for the determination of the relative fatty acid composition of a given PC sample. For this purpose naturally occurring mixtures of PCs as well as organic extracts of human lipoproteins-that are mainly composed of PC and sphingomyeline-are used.

Parameters influencing the determination of liposome lamellarity by 31P-NMR.

The lamellarity of liposomes influences to a great extent the encapsulation efficiency, the efflux rate of liposomally encapsulated material, and the fate of a drug after cellular uptake. 31P-NMR in combination with the use of chemical shift reagents has been described for the determination of lamellarity of liposomes and this study was performed to evaluate the applicability of 31P-NMR analysis as published in the past. To date, very few details about the required conditions throughout the measurements are known. In this study the influence of various parameters on the measurement, such as different buffers with changing ion concentrations, varying pH and different shift reagents at increasing concentrations was investigated. Results were discussed by using cryo-electronmicroscopy as a reference method. The data of this study show that 31P-NMR might not result in the correct determination of liposome lamellarity, depending on the experimental settings and the shape of the liposomes.

Inhibition of inositol uptake in astrocytes by antisense oligonucleotides delivered by pH-sensitive liposomes.

An oligonucleotide of 20 bases, complementary to a region of the sodium/myo-inositol cotransporter (SMIT) mRNA, was used to investigate the uptake efficiency and activity of transferred antisense oligonucleotides with regard to substrate uptake. We compared the efficiency of oligonucleotide delivery after application of either free or liposome-encapsulated material. Delivery of liposome-encapsulated material (marker or oligonucleotides) into astrocytoma cells and primary astrocyte cultures was more effective with pH-sensitive liposomes [dioleoylphosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate (CHEMS)] than with non-pH-sensitive liposomes (soy lecithin) or free material in solution. Antisense activity was evaluated by determination of myo-inositol uptake and detection of SMIT transcripts by RT-PCR. Encapsulation of oligonucleotides in pH-sensitive liposomes increased the inhibition of inositol uptake at least 50-fold compared with application of free oligonucleotides in solution.

St Louis encephalitis: a review of 11 cases in a 1995 Dallas, Tex, epidemic.

OBJECTIVE: To update some of the clinical features of St Louis encephalitis (SLE), a common arboviral infection that occurs in epidemic patterns in the south-central and midwestern United States. METHODS: Eleven patients with SLE from a 1995 epidemic in Dallas, Tex, were studied clinically, radiologically, neurophysiologically, and neuropathologically (in 1 case). RESULTS: The electroencephalograms and magnetic resonance imaging (MRI) scans of our patients revealed features that have received little attention in previous studies. Of the 9 patients who were examined with electroencephalography, all 9 had seizures or other abnormalities, and 1 had nonconvulsive status epilepticus. Two of 6 patients who had MRIs showed substantia nigra edema. Finally, 2 (18%) of our patients had coinfection with the human immunodeficiency virus. CONCLUSIONS: The MRI findings of substantia nigra edema in patients with SLE have not been previously reported. Nonconvulsive status epilepticus can occur in patients with SLE and should be considered in patients with prolonged encephalopathy. Finally, human immunodeficiency virus coinfection may be a risk factor for symptomatic SLE infection.

[Real-time ultrasonographic (B-mode) pregnancy diagnosis in sheep. 4. Results of a field study in Germany]. / Die ultrasonographische Trächtigkeitsdiagnostik (B-Mode) beim Schaf. Teil 4: Ergebnisse einer Feldstudie in Deutschland.

Between January 1993 and April 1998 in a total of 27,378 sheep an ultrasonographic early pregnancy diagnosis was done. The determined pregnancy rates (mean = 82.2%) were related to the mating systems, duration of mating season and time of the year. The benefits of scanning are described. If the pregnancy rate is lower than 97% the early pregnancy diagnosis is economical to flock management. Cases of uterine and fetal pathology (fetal mortality, maceration, hydrometra, pyometra) are presented.

[Ultrasonic pregnancy diagnosis (B-mode) in sheep. 2. Comparative studies using transcutaneous and transrectal pregnancy diagnosis]. / Die ultrasonographische Trächtigkeits-diagnostik (B-Mode) beim Schaf. Teil 2: Vergleichende Untersuchungen zur transkutanen und transrektalen Trächtigskeitsdiagnostik.

The aim of this study was to check the earliest time for the ultrasonic pregnancy diagnosis in flock management of sheep after finishing the mating season. Therefore a transcutaneous pregnancy diagnosis (5 MHz) took place on a total of 1060 German merino-mutton sheep between day 10 and 90 after mating (a.m.) and a transrectal diagnosis (5 MHz) took place on a total of 1442 German merino-mutton sheep between day 10 and 70 a.m. At the beginning of the third week a.m. the accuracy of transrectal diagnosis was over 95% and increased to 100% on day 35 a.m. At that time the accuracy of transcutaneous diagnosis was 80% and reached 100% on day 60 a.m. Considering the reached accuracies the pregnancy diagnosis should not take place before day 35 after the end of the breeding season. After day 35 a.m. it is advisable to combine the transcutaneous and the transrectal pregnancy diagnosis. That means that at first all animals are examined by transcutaneous sonography and in the second step the so-called "non-pregnant sheep" are checked by transrectal ultrasound. From the beginning of the third month a.m. a single transcutaneous pregnancy diagnosis is sufficient.

[Purpura fulminans with extensive skin necroses]. / Purpura fulminans mit ausgedehnten Hautnekrosen.

Purpura fulminans must be treated as an emergency in internal medicine and dermatology. Its characteristic features are the sudden development of progressively enlarging haemorrhagic skin necrosis, severe disseminated intravascular coagulation with consumption of anticoagulant factors, and signs of shock. Purpura fulminans can be classified into a neonatal form with inherited protein C deficiency and an acquired type for which multiple causes are known. Clinically it is characterized by massive ecchymosis with haemorrhagic blebs and acral necrosis. Histologically the lesions show widespread extravasation of erythrocytes and thrombosis of small vessels. Thrombocytopenia, decrease of coagulation factors, the presence of fibrinogen split products and fragmented erythrocytes in the blood smear help to confirm the diagnosis. The therapy includes fresh-frozen plasma, heparin, antibiotics and surgical debridement of necrotic areas. It is important to recognize the disease promptly because the mortality rate is about 30-40% and only quick intervention helps to save the life of the patient.

[Hematogenous contact eczema cause by phytogenic drugs exemplified by kava root extract]. / Hämatogenes Kontaktekzem durch pflanzliche Medikamente am Beispiel des Kavawurzel-extraktes.

The increasing promotion of herbal drugs may lead to allergic problems. A case of systemic contact-type dermatitis after oral administration of kava extract illustrates this special problem. The kava plant is a member of the black pepper family; an intoxicant beverage prepared from the roots of this plant is used ceremonially by many traditional societies of the Southern Pacific. The beverage induces relaxation, enhances a sense of sociability and promotes sleep. These effects are utilized in herbal drugs containing kava, which are sold for insomnia, nervousness and depression. The ichthyosiform kava dermopathy is a well-known side effect of excessive use of kava; in this case report we describe an acute allergic side-effect of kava extract.

Characteristic but unfamiliar--the cowpox infection, transmitted by a domestic cat.

An 11-year-old girl had been suffering from 2 recently developed reddish ulcerated nodules on the right side of her neck and, concomitantly, from a very hard swelling as well as from painless lymphadenopathy. Subsequently, thick black eschars appeared on the surface of the ulcers. Using the negative staining technique, an orthopox virus infection could be identified by electron microscopy. The patient's characteristic history as well as her close contact with a cat frequently hunting nice indicated the diagnosis of a cowpox infection, which was unequivocally confirmed by identification of both the viral culture and specific antibodies in the serum of the patient.

[Kaposi-Irgang lupus erythematosus profundus. Lupus erythematosus panniculitis]. / Lupus erythematodes profundus Kaposi-Irgang. Lupus erythematosus panniculitis.

Lupus panniculitis is a rare variant of cutaneous lupus erythematosus. Two typical cases demonstrate the association of lupus panniculitis with signs of discoid lupus erythematosus and systemic lupus erythematosus. Clinically it is characterised by subcutaneous nodules or plaques, which may ulcerate leaving depressed atrophic scars. Histologically the lesions show lobular panniculitis. A positive immunofluorescent band test with deposition of IgG, IgM and C3 at the dermal-epidermal junction and blood vessel walls helps to confirm the diagnosis. The knowledge of the clinical features and histopathology of the disease is important because lupus panniculitis may precede systemic lupus erythematosus by some years.

Increased expression of epidermal IL-8 receptor in psoriasis. Down-regulation by FK-506 in vitro.

IL-8 is a chemotactic cytokine with proinflammatory and growth-promoting activities. Recently it has been shown to influence several functions of keratinocytes, including HLA-DR expression, chemotaxis, and proliferation by binding to a specific receptor. Because psoriasis vulgaris is characterized by epidermal hyperproliferation and infiltration of inflammatory cells, we investigated the expression of IL-8 and its receptor in normal and psoriatic epidermis using semiquantitative reverse-transcriptase-polymerase chain reaction. In addition the mRNA levels of the proto-oncogenes c-ras, c-raf, c-myc, and HER-2 were also investigated as potential growth-promoting stimuli in psoriatic epidermis. IL-8 mRNA was only detected in lesional psoriatic epidermis, and IL-8R-specific mRNA was found to be 10 times increased in lesional psoriatic epidermis. There was no significant difference in the protooncogene mRNA levels. In order to test the relevance of the massively increased IL-8R levels in psoriatic epidermis, we investigated the effect of the antipsoriatic drug FK-506 on specific IL-8 and IL-8R mRNA expression. FK-506 dose dependently inhibited IL-8R expression and function. Our data suggest that in psoriatic skin, elevated IL-8 levels and markedly increased IL-8R expression may act in concert to induce the cardinal signs of psoriasis--epidermal hyperproliferation and leukocyte infiltration. IL-8R may prove a molecular target for antipsoriatic drugs such as FK-506.