Isolation of Aspergillus caninus (Synonym: Phialosimplex caninus) from a Canine Iliac Lymph node

Mycopathologia - Aspergillus caninus (synonym: Phialosimplex caninus) is an anamorphic fungus species associated with systemic infections in dogs that has been transferred from the genus...     

Cloning of a new aquaporin (AQP10) abundantly expressed in duodenum and jejunum

A new aquaporin (AQP10) was identified in human small intestine. This gene encoded a 264-amino-acid protein with high sequence identity with AQP3 (53%), 9 (52%), and 7 (43%). These AQPs constitute one subfamily of AQP family that is differentiated from the other subfamily of AQP (AQP0, 1, 2, 4, 5, 6, and 8) by sequence homology. Ribonuclease protection assay and Northern blotting demonstrated almost exclusive expression of AQP10 mRNA in the duodenum and jejunum. In situ hybridization localized it in absorptive jejunal epithelial cells. Xenopus oocytes expressing AQP10 exhibited an increased osmotic water permeability in a mercury-sensitive manner. Although AQP10 belongs to the AQP subfamily, which has been characterized by permeability to water and neutral solutes such as urea and glycerol, it was not permeable to urea nor glycerol. The specific expression of AQP10 suggests its contribution to the water transport in the upper portion of small intestine.     

Gene structures and regulation of the alkane hydroxylase complex in Acinetobacter sp. strain M-1

In the long-chain n-alkane degrader Acinetobacter sp. strain M-1, two alkane hydroxylase complexes are switched by controlling the expression of two n-alkane hydroxylase-encoding genes in response to the chain length of n-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively.     

PDZ interaction sites in integrin alpha subunits. T14853, TIP/GIPC binds to a type I recognition sequence in alpha 6A/alpha 5 and a novel sequence in alpha 6B

We used published peptide library data to identify PDZ recognition sequences in integrin alpha subunit cytoplasmic domains and found that the alpha(6)A and alpha(5) subunits contain a type I PDZ binding site (TSDA*) (asterisk indicates the stop codon). The alpha(6)A cytoplasmic domain was used for screening a two-hybrid library to find interacting proteins. The bulk of the captured cDNAs (60%) coded for TIP-2/GIPC, a cytoplasmic protein with one PDZ domain. The interaction of TIP-2/GIPC with different integrin subunits was tested in two-hybrid and in vitro binding assays. Surprisingly, TIP-2/GIPC bound strongly to the C terminus of both alpha(6)A and alpha(6)B, although the alpha(6)B sequence (ESYS*) is not suggestive of a PDZ binding site because of its polar C-terminal residue. For high affinity interaction with TIP-2/GIPC, at least one of the residues at positions -1 and -3 must be negatively charged. An aliphatic residue at position 0 increases the affinity of but is not required for this interaction. The alpha(5) integrin subunit also bound to TIP-2/GIPC. The alpha(6) integrin and TIP-2/GIPC co-localize in retraction fibers in carcinoma cells plated on laminin, a finding suggesting a functional interaction in vivo. Our results demonstrate that both splice variants of alpha(6) integrin contain a conserved PDZ binding site that enables interaction with TIP-2/GIPC. The binding site in alpha(6)B defines a new subclass of type I PDZ interaction site, characterized by a non-aliphatic residue at position 0.     

Serum amyloid P component is the Shiga toxin 2-neutralizing factor in human blood

It has been suggested that some factor present in human plasma binds to Shiga toxin 2 (Stx2) and neutralizes it in vitro (Bitzan, M., Klemt, M., Steffens, R., and Muller-Wiefel, D. E. (1993) Infection 21, 140-145). This factor does not exist in other species (Caprioli, A., Luzzi, I., Seganti, L., Marchetti, M., Karmali, M., Clarke, I., and Boyd, B. (1994) Recent Adv. VTEC Infect. 353-356). Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it. Purification was carried out by serially subjecting human plasma to Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration high performance liquid chromatography (HPLC), using Stx2-neutralizing activity as the indicator. The gel-filtration HPLC fraction yielded a single band on SDS-polyacrylamide gel electrophoresis. Twenty N-terminal amino acid residues of this fraction were analyzed and found to correspond perfectly to human serum amyloid P component (HuSAP). Because commercially available HuSAP also showed Stx2 binding and neutralizing activity, we identified this factor as HuSAP.     

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.     

Chlamydocin analogues from the soil fungus Peniophora sp.: structures and plant growth-retardant activity

Three chlamydocin analogues that show activity for reducing the height of rice seedlings without blotch and wilting were isolated from the culture filtrate of the soil fungus Peniophora sp. and were characterized by spectral data and chemical conversion. For two of these, it is their first isolation as natural products. The proposed role of the functional groups of the 2-amino-8-oxodecanoic acid moiety of these analogues is discussed in terms of growth retardant activity.     

17beta-estradiol induces apoptosis in the preosteoclastic FLG 29.1 cell line

Although compelling data have demonstrated the effectiveness of estrogen replacement therapy for the treatment of accelerated bone loss in postmenopausal osteoporosis and ovariectomized animals, the mechanisms by which estrogens reduce bone resorption remain to be elucidated. To address this issue, in the present study we investigated whether estrogens were able to induce programmed cell death or apoptosis in osteoclast precursors. To this purpose, a preosteoclastic cell line (FLG 29.1) was cultured in the absence or presence of nanomolar concentrations of 17beta-estradiol (17betaE2). Using time-lapse videomicroscopy, it was shown that 17betaE2 induced FLG 29.1 cell apoptosis in a dose- and time-dependent manner. Furthermore, a significant increase in the activity of caspase 3 enzyme and in the number of nuclei undergoing DNA fragmentation was observed in FLG 29.1 cells treated with 17betaE2 compared to untreated cells. Finally, transmission electron microscopy of the treated cells showed typical apoptotic morphology. These data indicate that 17betaE2 is able to promote in vitro apoptosis in preosteoclastic cells and suggest that estrogenic molecules may exert in vivo a direct role in negatively modulating the pool of undifferentiated bone marrow cells capable ultimately of maturing into osteoclasts.     

Characterization of an outer membrane protein gene, pgmA, and its gene product from Porphyromonas gingivalis

A gene upstream from fimA, the gene encoding fimbrilin, on the chromosome of Porphyromonas gingivalis was sequenced and shown to be the gene encoding an outer membrane protein in this organism based on homology and biochemical analyses. Therefore, the gene (formerly ORF5) was designated pgmA, the P. gingivalis outer membrane protein A gene. The gene product, PgmA, was sensitive to protease, and was detected as a 60-kDa protein from wild-type strains with trichloroacetic acid treatment, which was carried out to destroy intrinsic proteases, and from protease-deficient mutants without this treatment prior to electrophoresis. PgmA was indeed present in the membrane fraction. Its nature was determined to be that of outer membrane proteins in gram-negative bacteria based on attempts at differential extraction of inner membrane proteins with detergents. No evidence has been found thus far from functional analyses that this protein is related to fimbrial morphogenesis and functions or to serum resistance of this organism.