Immunohistochemical properties of nerve fibres supplying accessory male genital glands in the pig. A colocalisation study

 Immunohistochemical studies have been performed to investigate the occurrence and coexistence of two catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase, and several neuropeptides, including neuropeptide Y, vasoactive intestinal polypeptide, Leu⁵-enkephalin, somatostatin, calcitonin gene-related peptide and substance P, in nerve fibres supplying porcine accessory genital glands, the seminal vesicles, prostate (body and the disseminated part) and bulbourethral glands. Three major populations of nerve fibres supplying non-vascular elements of the glands have been distinguished (from the largest to the smallest one): (1) noradrenergic fibres, the majority of which contain Leu⁵-enkephalin, neuropeptide Y or, to a lesser extent, somatostatin, (2) non-noradrenergic, putative cholinergic fibres containing vasoactive intestinal polypeptide, neuropeptide Y and/or somatostatin and, (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. Whilst the coexistence patterns within nerves supplying particular glands are similar, the density of innervation varies between the organs. The innervation of the seminal vesicles and prostatic body is more developed than that of the disseminated part of the prostate and bulbourethral glands. The majority of noradrenergic fibres related to blood vessels contain neuropeptide Y only, while the non-noradrenergic nerves contain mainly vasoactive intestinal polypeptide. The possible function and origin of particular nerve fibre populations are discussed. Accepted: 16 November 1998     

The 35 kDa acid metallophosphatase of the frog Rana esculenta liver: studies on its cellular localization and protein phosphatase activity

The cellular localization of the 35 kDa, low molecular mass acid metallophosphatase (LMW AcPase) from the frog (Rana esculenta) liver and its activity towards P-Ser and P-Tyr phosphorylated peptides were studied. This enzyme was localized to the cytoplasm of hepatocytes but did not appear in other cells of liver tissue (endothelium, macrophages, blood cells). This LMW AcPase does not display activity towards (32)P-phosphorylase a under conditions standard for the enzymes of PPP family. Proteins containing P-Ser: rabbit (32)P-phosphorylasea and phosvitin are hydrolysed only at acidic pH and are poor substrates for this enzyme. The frog AcPase is not inhibited by okadaic acid and F(-) ions, the Ser/Thr protein phosphatase inhibitors. Moreover, the frog enzyme does not cross-react with specific antisera directed against N-terminal fragment of human PP2A and C-terminal conserved fragment of the eukaryotic PP2A catalytic subunits. These results exclude LMW AcPase from belonging to Ser/Thr protein phosphatases: PP1c or PP2Ac. In addition to P-Tyr, this enzyme hydrolyses efficiently at acidic pH P-Tyr phosphorylated peptides (hirudin and gastrin fragments). K(m) value for the hirudin fragment (7.55 +/- 1.59 x 10(-6) M) is 2-3 orders of magnitude lower in comparison with other substrates tested. The enzyme is inhibited competitively by typical inhibitors of protein tyrosine phosphatases (PTPases): sodium orthovanadate, molybdate and tungstate. These results may suggest that the LMW AcPase of frog liver can act as PTPase in vivo. A different cellular localization and different response to inhibition by tetrahedral oxyanions (molybdate, vanadate and tungstate) provide further evidence that LMW AcPase of frog liver is distinct from the mammalian tartrate-resistant acid phosphatases.     

Differential expression of Snail1 transcription factor and Snail1-related genes in alveolar and embryonal rhabdomyosarcoma subtypes

Rhabdomyosarcoma (RMS) represents the most common sarcoma of soft tissue among children. Two main RMS subtypes are alveolar (ARMS) and embryonal (ERMS). The major goal of this study was to find differentially expressed genes between RMS subtypes that could explain higher metastatic potential in ARMS and would be useful for the differential diagnosis. Using RQ-PCR analysis we compared expression of Snail1 and Snail-related genes among 7 ARMS and 8 ERMS patients' samples obtained from the primary tumors and among 2 alveolar and 2 embryonal cell lines. Our results show that Snail1 is highly expressed both in ARMS patients' samples and the alveolar cell lines. We also found that the expression of E-Cadherin was downregulated and the expression of Matrix Metalloproteinases 2 and 9 (MMP-2 and MMP-9) was upregulated in ARMS. We assume that, as in many tumors, also in RMS Snail1 acts as a regulator for pathways known for their role in cells' metastasis and that Snail1 activity results in increased MMPs and decreased E-Cadherin expression. Our findings may explain higher ARMS aggressiveness. Moreover, we suggest that further studies should be performed to verify if Snail1 can be considered as a potential target for ARMS therapy.     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K(i)* = 4.5 x 10(-7) mM. The respective inhibition constant of TCoBQ was equal to: K(i)* = 2.4 x 10(-6) mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

DNA methylation of Quercus robur L. plumules following cryo-pretreatment and cryopreservation

Pedunculate oak (Quercus robur) is an ecologically and economically important forest tree species which produces seeds that are classified as recalcitrant. Thus, cryopreservation of seed meristems is a method for long-term preservation of this germplasm in gene banks. During cryopreservation, many factors, such as desiccation, cryoprotection and cooling/rewarming, can induce stress in the frozen meristems. In this study, in vitro survival and the global DNA methylation level of plumules after cryoprotection, desiccation and cryostorage was evaluated. Results indicated that both desiccation and storage in liquid nitrogen have negligible influence on DNA methylation status of Q. robur plumules. These findings support the cryopreservation of plumules as an appropriate method for conservation of Q. robur germplasm.     

Selective recovery of Zn2+ from waste slag from a metal-processing plant by the microscopic fungus Verticillium marquandii

The successful use of hay extract medium for growth of Verticillium marquandii in the presence of toxic black slag, resulted in selective and efficient Zn²⁺ recovery. An alkaline black waste slag originating from short rotary furnace, contained Zn²⁺ and Pb²⁺ (20% and 3.5%, respectively) in a form of compounds only slightly soluble in water. After 90 h of the culturing in bioreactor on hay extract medium with starch (50 g l^–1) and black waste (5 g l^–1) supplements, 80% of zinc and 0.5% of lead were bound by the mycelium. The pH value of the culture medium played a crucial role for limitation of the waste inhibitory effect on mycelium growth and for heavy metal recovery by the fungus.     

Survey of susceptibility of clinical Clostridium diffiicile strains isolated from patients hospitalised in different departments of paediatric hospital to antimicrobial agents

This study was performed to determine the susceptibility of 50 C. difficile strains isolated from faecal samples of children suspected to antibiotic associated diarrhea (AAD) to antimicrobial agents: metronidazole, vancomycin, erythromycin, clindamycin, ciprofloxacin, moxifloksacin, gatifloksacin and imipenem. The all C. difficile strains were sensitived to metronidazole and vancomycin. Twenty six per cent of strains were resistant to erythromycin and clindamycin (MLS(B) type resistance). Resitance to ciprofloxacin, moxifloxacin, gatifloxacin and imipenem was detected in 98%, 8%, 8% and 30% of C. difficile strains, respectively.     

Evaluation of genetic diversity and uniformity of head cabbage DH lines by the use of RAPD markers

DH lines derived from cabbage cvs. Kamienna G?owa, S?awa z Enkhuizen and Langendijker, representing R1 generation, were analysed by the use of RAPD markers for their diversity and uniformity. For the evaluation of genetic diversity, eight primers yielding informative bands were used. Of the total of 83 RAPD bands scored in this study, 16.9% were polymorphic between a set of 13 DH lines. The similarity of the DH lines, estimated by Jaccard's coefficient, was depicted in the UPGMA dendrogram. Fourteen generated informative RAPD bands allowed the identification of DH lines developed from each cultivar. The evaluation of the uniformity for six closely related DH lines was possible by the use of three primers which generate one or two polymorphic bands. The lack of differences among ten plants of the five investigated DH lines manifested their uniformity. One line showed intraline polymorphism with two RAPD primers. The occurrence of the differences at the molecular level among ten plants indicated that their parental R0 plant was probably obtained from somatic cells, not by androgenesis.