Detection of human papillomavirus gene sequences in cell lines derived from laryngeal tumors

The role of Human Papillomaviruses (HPV) in laryngeal carcinomas has been studied with conflicting results. To evaluate the etiologic relationship between HPV infection and epithelial malignancy of the larynx we studied five laryngeal carcinoma cell lines obtained from patients undergoing surgery for laryngeal tumors. The paraffin embedded biopsy samples of the original tumor and different passages of the new established cell lines were investigated by PCR with consensus primers specific for HPV DNA. The findings indicate that HPV infection is associated with some larynx carcinomas. The positive association has been enhanced when a method of enrichment of epithelial cells from fresh tumor samples was used. All tumor cells enriched smears were positive for HPV DNA not only by PCR but also by in situ hybridization (ISH). Investigated by PCR, different passages of larynx tumor cell lines maintained expression of HPV DNA. At subsequent passages ISH gives constantly no signals suggesting a minimal amount of viral harbored sequences. In one cell line propagated more than 60 population doublings, the chromosomal frequency distribution shifted from modal number 46 at the 5^th passage to 63 at the 60^th passage. The mechanisms by which persistent HPV infection maintains continuous cell proliferation were discussed.     

Demonstration of sensory-motor innervation of postmetamorphic forelimb regenerates of Triturus alpestris (Urodela) on cryotome serial sections using horseradish peroxidase

A method has been developed to obtain horseradish peroxidase-treated serial sections containing spinal cord as well as bilateral ventral and dorsal roots, dorsal root ganglia and spinal nerves. Young postmetamorphic newts (Triturus alpestris) served as experimental animals. After cryotome cross sectioning the forelimb region of the trunk, slices 80 microns in thickness were mounted serially with up to 15 sections per slide. This facilitated subsequent staining manipulations and made partial loss of sections less likely.     

<Emphasis Type="Italic">Alona alsafadii</Emphasis> n. sp. from Yemen,a primitive,groundwater-dwelling member of the <Emphasis Type="Italic">A. karua</Emphasis>-group

The effect of algae on the production of musty-smelling compounds by actinomycetes was studied. Streptomyces spp., causing intensive musty odor, were isolated from hypertrophic Lake Kasumigaura and cultured in association with algae from the same lake. Isolate E and I effectively utilized the cyanobacteria, Microcystis aeruginosa and Anabaena spiroides, and the diatom, Synedra acus, as a carbon source and produced a musty-smelling 2-methylisoborneol in the shaken sediment cultures. High populations of algae and actinomycetes, and aerobic condition in the sediment seem responsible for the occurrence of musty odor in Lake Kasumigaura.     

Thiophene synthesis and distribution in young developing plants of Tagetes patula and Tagetes erecta

Thiophene synthesis and accumulation were investigated in organsof Tagetes patula and T. erecta. Thiophene accumulation startedrapidly in germinating seedlings of both species. Roots andhypocotyls were the major thiophene accumulating organs and5-(3-buten-1-ynyl)-2, 2-bithienyl (BBT) and 5-(4-acetoxy-1-butynyl)-2,2 -bithienyl (BBTOAc) were the major accumulated compounds.Higher thiophene concentrations were reached in Tagetes patulathan in T. erecta. Accumulation patterns for individual thiopheneswere different within organs, between organs and between bothspecies. Within hypocotyls of Tagetes patula, thiophene concentrationswere high in the epidermis and the vascular tissue and low inthe parenchymatic tissues of cortex and pith. Synthesis of thiopheneswas high in the roots and hypocotyls and very low in the leaves.Transport of thiophenes from the roots into the shoot occurred,but the rate of transport was too low to explain the high concentrationsin the hypocotyl. It is concluded that for the main part thiophenesare accumulated where they are synthesized. Key words: Tagetes, hiophenest, synthesis, accumulation, secondary metabolites     

Lignin-Degrading Enzymes of the Commercial Button Mushroom, Agaricus bisporus

Agaricus bisporus, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by A. bisporus. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in A. bisporus suggests significant roles for these two enzymes in lignin degradation by this fungus.     

Expression of active spinach glycolate oxidase in Aspergillus nidulans

The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity. (c) 1996 John Wiley & Sons, Inc.     

Expression of CYP4F2 in human liver and kidney: assessment using targeted peptide antibodies

P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61-74 (WGHQGMVNPTEEG) and 65-77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly variable CYP4F2 expression in liver (16.4+/-18.6pmol/mg microsomal protein; n=29) and kidney cortex (3.9+/-3.8 pmol/mg; n=10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r> or =0.63; p<0.05) with leukotriene B4 and arachidonate omega-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate omega-hydroxylase in human liver.     

Anti-proliferative effect of cytohesin inhibition in gefitinib-resistant lung cancer cells

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been proven to efficiently inhibit the proliferation of a subset of non small-cell lung cancers (NSCLC). Unfortunately, the majority of NSCLC expressing wild type EGFR is primarily resistant to EGFR-TKI treatment. Here, we show that the proliferation of the gefitinib-resistant NSCLC cell lines H460 and A549 is reduced by the small molecule SecinH3 which indirectly attenuates EGFR activation by inhibition of cytohesins, a class of recently discovered cytoplasmic EGFR activators. SecinH3 and gefitinib showed a synergistic antiproliferative effect, which correlated with a profound inhibition of Akt activation and survivin expression. Treating mice bearing H460 xenografts with SecinH3 showed the antiproliferative and pro-apoptotic effect of SecinH3 in vivo. Our data suggest that targeting the EGFR indirectly by inhibiting its cytoplasmic activators, the cytohesins, has the potential to improve the treatment of primarily EGFR-TKI resistant lung cancers.     

Wolframin deficiency is accompanied with metabolic inflexibility in rat striated muscles

The protein wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. Mutations in Wfs1 gene cause autosomal recessive disorder Wolfram syndrome (WS). The first symptom of the WS is diabetes mellitus, so accurate diagnosis of the disease as WS is often delayed. In this study we aimed to characterize the role of the Wfs1 deficiency on bioenergetics of muscles. Alterations in the bioenergetic profiles of Wfs1-exon-5-knock-out (Wfs1KO) male rats in comparison with their wild-type male littermates were investigated using high-resolution respirometry, and enzyme activity measurements. The changes were followed in oxidative (cardiac and soleus) and glycolytic (rectus femoris and gastrocnemius) muscles. There were substrate-dependent alterations in the oxygen consumption rate in Wfs1KO rat muscles. In soleus muscle, decrease in respiration rate was significant in all the followed pathways. The relatively small alterations in muscle during development of WS, such as increased mitochondrial content and/or increase in the OxPhos-related enzymatic activity could be an adaptive response to changes in the metabolic environment. The significant decrease in the OxPhos capacity is substrate dependent indicating metabolic inflexibility when multiple substrates are available.