DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

Haematological findings in cats naturally infected with feline immunodeficiency virus

The types and prevalence of haematological abnormalities occurring in FIV infected cats were determined. In addition, the role of FIV infection per se in influencing haematological values was examined by analysing results between infected and non-infected cats which had been allocated to similar clinical disease groups. FIV-positive cats were grouped as asymptomatic carriers (AC), cats with AIDS-related complex (ARC) or AIDS. FIV-negative cats were placed into matched groups using the same criteria and designated as healthy, ARC or AIDS. Healthy FIV-negative cats also formed the reference ranges for peripheral blood and bone marrow. Anaemia was no more frequent in sick (ARC and AIDS) FIV-positive cats than sick (ARC and AIDS) FIV-negative cats. However, it was observed more frequently in FIV-positive cats than FIV-negative cats in the absence of concurrent disease, suggesting a direct effect of FIV infection. Bone marrow was affected by FIV infection; as evidenced by anaemic FIV-positive cats having proportionally less Type I reticulocytes than anaemic FIV-negative cats. In addition, FIV-positive cats demonstrated proportionally fewer mature erythroid cells in their marrow. This implied that FIV may cause a decreased life span or maturation arrest of the erythroid cell line. Lymphopenia and eosinopenia were seen more frequently in AC FIV-positive cats than healthy FIV-negative cats, suggesting the direct involvement of FIV. Thus, although FIV infection affected some haematological findings in AC cats, it appeared that haematological abnormalities in sick FIV-positive cats may be due as much to the disease state as to the virus specifically. Apart from the subjective assessment that bone marrow of FIV-positive cats appeared hypercellular, there were no pathognomonic features for FIV infection.     

Prostate specific antigen and prostate specific acid phosphatase in adenocarcinoma of Skene's paraurethral glands and ducts

An autopsy case of adenocarcinoma of Skene's paraurethral gland co-incident with renal cell carcinoma is described. The adenocarcinoma showed distinct prostate specific antigen and prostate specific acid phosphatase pointing to the equivalence between the male prostate and Skene's paraurethral glands and ducts. Skene's gland are the homologue of the prostate in females and tumours arising from them are immunohistochemically similar to male prostate carcinoma.In the title and text the authors used the official term of Nomina Anatomica paraurethral (Skene's) glands and ducts. Nevertheless recently published data on cross-antigenicity between the male prostate and Skene's glands and the newly discovered exocrine and neuroendocrine parameters of the prostate homologue in the female, comparable with the male prostate (Zaviai 1987), support the use of the same term — the prostate — for prostatic tissue in both sexes (Zaviai 1987, Zaviai et al. 1985). The designations female prostate homologue or female prostate equivalent are a compromise between terms the female prostate and Skene's paraurethral glands.     

TRP channels: a TR(I)P through a world of multifunctional cation channels

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC (Canonical) group, the TRPV (Vanilloid) group, the TRPM (Melastatin) group, the TRPP (Polycystin), the TRPML (Mucolipin), the TRPA (Ankyrin) and the TRPN (NOMP) family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a readers guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.     

Comparative Characterization of Cytokine Production by Concanavalin A-Activated Splenocytes from BALB/c and C57Bl/6 Mice after Cold Exposure

The level of cytokines produced by ConA activated splenocytes was studied in male BALB/c and C57Bl/6 mice after single and repeated cold exposure (–20°C, 3 min). Single cold exposure significantly decreased IL-2, -3, -4, -5, -10, -12, IFN- production in BALB/c mice and decreased IL-2 content and increased TNF- level in C57Bl/6 mice. Repeated cold exposure normalized the content of IL-2, -4, -10, -12, and IFN- in BALB/c mice, which reflects the development of adaptive immune reactions. In C57Bl/6 mice IL-2, -3, -5, -10, -12, and IFN- production remained significantly decreased, which attested to dysadaptive processes.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 188–190, February, 2005     

Adjuvant-induced inflammatory disease in the rat: Plasma levels of peptide hydrolases and protease inhibitors reflect disease activity

Both during the primary (localized) inflammation and the development of the secondary (generalized) inflammation in adjuvant-treated rats, the plasma level of functional -macroglobulins increases while proteases (measured as peptide hydrolases) sharply decrease. The decreased peptide hydrolase levels during episodes when protease spillage into the bloodstream is elevated, suggests a more rapid clearance of -macroglobulin-protease complexes associated with inflammation.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Adrenerge α- und β-Receptoren und ihre spezifischen Hemmstoffe

Zusammenfassung Die wichtigsten pharmakologischen Befunde, die zur Klassifizierung der adrenergen - und -Receptoren geführt haben, werden besprochen. Hemmstoffe der -Receptoren werden bereits seit längerer Zeit zu therapeutischen Zwecken verwandt. Ausführlicher werden die Hemmstoffe der -Receptoren behandelt, die erst in den letzten Jahren Eingang in die Klinik gefunden haben. Die pharmakologischen Wirkungen, Nebenwirkungen und therapeutischen Indikationen der Receptorenhemmstoffe werden besprochen.

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Summary The pharmacological evidence for the classification of adrenergic - and -receptors is presented. Drugs blocking -receptors have been used therapeutically for many years. -adreneric receptor blocking agents were introduced only recently; they are discussed in more detail. The pharmacological actions, side effects and therapeutic uses of the receptor blocking drugs are summarized.

     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Structural and immunocytochemical studies on α-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminidase deficiency (Schindler/Kanzaki disease)

-N-Acetylgalactosaminidase (-NAGA) deficiency (Schindler/Kanzaki disease) is a clinically and pathologically heterogeneous genetic disease with a wide spectrum including an early onset neuroaxonal dystrophy (Schindler disease) and late onset angiokeratoma corporis diffusum (Kanzaki disease). In -NAGA deficiency, there are discrepancies between the genotype and phenotype, and also between urinary excretion products (sialyl glycoconjugates) and a theoretical accumulated material (Tn-antigen; Gal NAc1--Ser/Thr) resulting from a defect in -NAGA. As for the former issue, previously reported genetic, biochemical and pathological data raise the question whether or not E325K mutation found in Schindler disease patients really leads to the severe phenotype of -NAGA deficiency. The latter issue leads to the question of whether -NAGA deficiency is associated with the basic pathogenesis of this disease. To clarify the pathogenesis of this disease, we performed structural and immunocytochemical studies. The structure of human -NAGA deduced on homology modeling is composed of two domains, domain I, including the active site, and domain II. R329W/Q, identified in patients with Kanzaki disease have been deduced to cause drastic changes at the interface between domains I and II. The structural change caused by E325K found in patients with Schindler disease is localized on the N-terminal side of the tenth -strand in domain II and is smaller than those caused by R329W/Q. Immunocytochemical analysis revealed that the main lysosomal accumulated material in cultured fibroblasts from patients with Kanzaki disease is Tn-antigen. These data suggest that a prototype of -NAGA deficiency in Kanzaki disease and factors other than the defect of -NAGA may contribute to severe neurological disorders, and Kanzaki disease is thought to be caused by a single enzyme deficiency.     

Differential Production of Chemokines by Phagocytosing Rat Neutrophils and Macrophages

In this study, rat neutrophils and macrophages produced cytokine-induced neutrophil chemoattractants (CINCs) and rat macrophage inflammatory protein-1 (MIP-l) in different patterns during phagocytosis of heat-killed yeast cells in vitro. The cultured supernatants of the phagocytosing rat neutrophils and macrophages had chemotactic activities toward neutrophils, and the chemotactic potencies were markedly inhibited by anti-CINCs IgGs or/and anti-MIP-l IgG, suggesting that CINCs and MIP-l are major neutrophil chemoattractants produced by the phagocytosing neutrophils and macrophages. Dexamethasone suppressed the production of CINCs and MIP-1 by the phagocytosing cells in a dose-dependent manner. Our results demonstrate significant differences in the production of CINCs and MIP-1 by neutrophils and macrophages during phagocytosis of yeast cells and thus may suggest the different contribution of each chemokine to neutrophil recruitment in the processes of inflammation in rats.     

Three methods to elicit sigma-optokinetic nystagmus in Java monkeys

Summary Sigma-optokinetic nystagmus (-OKN) can be elicited in awake Java monkeys (Macaca fascicularis) when stationary periodic visual patterns (grid of black white stripes, row of equally spaced dots) are illuminated stroboscopically. Three methods were found to be useful in inducing the -OKN: postrotatory nystagmus, optokinetic afternystagmus (OKAN) following normal OKN and a gradual transition from -movement (-OKN) to -OKN. The properties found for -OKN in man are also present in monkeys with the one exception that monkeys have a long-lasting -OKAN in darkness which is not present in man. The average angular speed ¯V_e of -OKN slow phases was related to the flash frequency f_s and the spatial period P_s of the stripe pattern according to the following equation: {ie519-01} The constant k was 1 or close to 1.     

Exposure to Topical Bovine Thrombin During Surgery Elicits a Response Against the Xenogeneic Carbohydrate Galactose α1-3Galactose

Exposure of humans to topical bovine thrombin has been associated with development of antibodies against bovine and human coagulation factors and blood coagulation abnormalities. However, the nature of this humoral response is unknown. In this study, numerous glycoproteins in the topical bovine thrombin were found to contain the Gal1-3Gal epitope, which is known to be highly immunogenic. More importantly, Gal1-3Gal is recognized by natural antibodies that are found in all normal individuals and are known to effectively mediate complement activation and subsequent destruction of xenogeneic tissues. Thus, primary exposure of normal individuals to topical bovine thrombin is expected to result in an immediate immune reaction against that reagent. Further, following exposure to topical bovine thrombin, the levels of anti-Gal1-3Gal IgG rose to levels tenfold greater than the average level of natural anti-Gal1-3Gal IgG in naive individuals. Thus, Gal1-3Gal in topical bovine thrombin accounts for, at least in part, the highly immunogenic nature of this reagent.     

Characterization of an isogenic set of methicillin-resistant and susceptible mutants ofStaphylococcus aureus

A low affinity penicillin binding protein (PBP2) present in methicillin resistant strains but absent in isogenic sensitive strains of Staphylococcus aureus is responsible for the phenotypic expression of the methicillin resistance. An additional factor controlling methicillin resistance is known to map on the chromosome at the insertion site 22003 (chr::TnS51). The influence of this factor on PBP2 synthesis was analysed. Isogenic methicillin sensitive and resistant mutants derived from a Staphylococcus aureus strain carrying methicillin resistance factors and the 22003 (chr::Tn551) insertion were characterized by population analysis and restriction analysis of the DNA surrounding the Tn551 insertion site, and their PBP2 content was determined. Both methicillin resistant and sensitive mutants produced levels of PBP2 similar to those of the original methicillin resistant parent. The mere presence of this PBP2 could therefore not be solely responsible for the phenotypic expression of methicillin resistance. The nature and site of action of the additional factor determined by the 22003 (chr::Tn551) site controlling expression of methicillin resistance still remains unknown.     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.     

The tubular vacuolation process in amphibian skeletal muscle

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes vacuolation in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface (detubulation), particularly if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for the first time two types of vacuole, open and closed, whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed close to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. Open vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. Closed vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca2+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by open vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole closure took place well after formation of open vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of open vacuole formation. These findings identify electrophysiological detubulation of striated muscle with closure of initially open vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.This revised version was published online in September 2005 with corrections to the Cover Date.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Spatial EEG Synchronisation Over Sensorimotor Hand Areas in Brisk and Slow Self-Paced Index Finger Movements

The changes of spatial EEG synchronisation during brisk and slow voluntary self-paced movements of the right and left index finger were analysed in 12 right-handed and 11 left-handed subjects. EEG was recorded from the left and right sensorimotor area using 24 closely spaced electrodes. A novel measure of spatial EEG synchronisation, -complexity, was computed separately for the left and right sensorimotor area in 64 overlapping one-second epochs representing 4.5 s of the pre-movement and 3.5 s of the post-movement period. -complexity was higher, hence spatial synchronisation was lower, in slow than in brisk movements, especially in the right-handed. A sustained increase of -complexity was observed during execution of a slow movement. A decrease of -complexity which was often associated with a brief burst of spatially synchronised 10-Hz oscillations occurred at the onset of extensor muscle contraction. We suggest that increased spatial EEG synchronisation at movement onset may prevent spillover of excitation from the sensorimotor hand area to other cortical regions. During movement, the cortical neuronal assemblies subserve distinct, specialised functions manifesting in increased -complexity.     

Reconstitution of chemokine-induced actin polymerization in undifferentiated human leukemia cells (HL-60) by heterologous expression of interleukin-8 receptors

The chemokines interleukin-8 (IL-8) and GRO bind in neutrophils to the interleukin-8 receptor and (IL-8R and ) triggering reorganization of the actin cytoskeleton and activation of phospholipase C (PLC). Reconstitution of chemokine-induced activation of PLC indicated coupling of IL-8R and to pertussis toxin-insensitive or . To identify the signal transduction mechanisms of chemokine-induced actin response, undifferentiated human leukemia cells (HL-60 cells) constitutively expressing , and were chosen for reconstitution studies. Expression of recombinant receptors after transfection of the cells with the cDNA of IL-8R and was confirmed by binding studies with radiolabeled ligands. IL-8R bound IL-8 with high affinity (Kd1 nM) and GRO with low affinity (Kd 1 M), whereas IL-8R bound both IL-8 and GRO with high affinity (Kd1 nM). Flow cytometric actin measurements indicated that high affinity ligand-receptor interactions in both receptor transfectants displayed inducible responses. Pretreatment of transfectants with pertussis toxin caused ADP-ribosylation of G-proteins and blocked chemokine-induced polymerization, indicating involvement of or , but not in this response.accepted by M.J. Parnham     

The excretion of highly soluble gases by the lung in man

The excretion (E) of inert gases by the lung depends on, among other things, their blood-gas partition coefficients (). According to conventional gas exchange models, E should increase with increasing However, recent models that take into account the tidal character of breathing and the buffering capacity of lung tissue predict that E will show a minimum in the range of large values (>10). Further, this local minimum should shift to larger values in exercise conditions as compared to rest conditions. The aim of this study is to verify this predicted behaviour of E. The experiments were carried out with seven healthy subjects at rest and at three work loads (50 W, 100 W and 150 W) on a bicycle ergometer. The behaviour of E was determined from the results of a simultaneous washin of four tracer gases: ethyl acetate (75), acetone (330), ethanol (2000) and acetic acid (20000). The washin lasted 4 min, and E was calculated from E = 1–p /p _I, where p _I and P are the partial pressures of the tracer gas in inspired and mixed expired gas determined from the recordings obtained during the last minute of washin. p _I and P were measured with a mass spectrometer. Comparison of the E values of the four gases shows that at rest a minimum value for E is found for acetone. In exercise conditions, however, the smallest E value is found for the more soluble ethanol or acetic acid. Further, under exercise conditions the E values for ethyl acetate and acetone are larger than their respective values at rest. In general, the experimental findings are consistent with the predicted behaviour of E. This means that gas exchange in the airways between gaseous and dissolved tracer gas in the airway lumen and in the airway tissue, respectively, cannot be ignored for highly soluble tracer gases. In addition, the observed differences between the E values of the four highly soluble tracer gases imply that the dead space ventilation ( ) depends on , i.e. the value of is not unique. Therefore, the result for obtained from a highly soluble tracer gas will, in general, not apply to other tracer gases.