The expression of the epitope H recognized by the monoclonal antibody H is higher in astrocytomas compared to anaplastic astrocytomas and glioblastomas

The epitope H contains an O-linked N-acetylglucosamine residue in a specific conformation and/or environment recognized by the monoclonal antibody H (mAbH). mAbH stains two bands with Mr x0.001 of 209 and 62 in lysates of cultured rat astrocytes. In normal human brains epitope H is absent from the overwhelming majority of normal astrocytes and only sparse reactivity is observed, confined mostly to fibrous astrocytes. Upregulation of the epitope H takes place in reactive astrocytes. In the present study we used the mAbH to investigate the immunohistochemical expression of the epitope H in 41 cases of astrocytic tumors including 19 cases of astrocytomas, 8 cases of anaplastic astrocytomas and 14 cases of glioblastomas. Seven out of 19 cases (37%) of astrocytomas showed weak staining, 10 cases (53%) moderate staining and 2 cases (10%) intense staining. Two out of 8 cases (25%) of anaplastic astrocytomas appeared negative, 3 cases (37.5%) showed weak staining and 3 cases (37.5%) moderate staining. Four out of 14 cases (28.5) of glioblastomas appeared negative, 7 cases (50%) showed weak staining, 2 cases (14%) showed moderate staining and only one case (7.5%) showed intense staining. There was a statistically significant elevation of the expression of the epitope H in astrocytomas compared to anaplastic astrocytomas and glioblastomas (p=0.047). These results indicate that the expression of the epitope H decreases in parallel with the increase of the grade of astrocytic tumors from low to higher grade neoplasms. This could be of interest for predicting the progression of an astrocytic tumor since it is documented that astrocytomas progress to tumors of higher grade of malignancy. Further investigation of the antigens bearing the epitope H might help to gain further insight into the mechanisms which regulate the progression of astrocytic tumors and to examine the relevance of the mAbH staining with respect to the prognosis of these neoplasms.     

Genomic rearrangements on VCAM1, SELE, APEG1and AIF1 loci in atherosclerosis

The inflammatory nature of atherosclerosis has been well established. However, the initial steps that trigger this response in the arterial intima remain obscure. Previous studies reported a significant rate of genomic alterations in human atheromas. The accumulation of genomic rearrangements in vascular endothelium and smooth muscle cells may be important for disease development. To address this issue, 78 post-mortem obtained aortic atheromas were screened for microsatellite DNA alterations versus correspondent venous blood. To evaluate the significance of these observations, 33 additional histologically normal aortic specimens from age and sex-matched cases were examined. Loss of heterozygosity (LOH) was found in 47,4% of the cases and in 18,2% of controls in at least one locus. The LOH occurrence in aortic tissue is associated to atherosclerosis risk (OR 4,06, 95% CI 1,50 to 10,93). Significant genomic alterations were found on 1p32-p31, 1q22-q25, 2q35 and 6p21.3 where VCAM1, SELE, APEG1 and AIF1 genes have been mapped respectively. Our data implicate somatic DNA rearrangements, on loci associated to leukocyte adhesion, vascular smooth muscle cells growth, differentiation and migration, to atherosclerosis development as an inflammatory condition.     

Characterization and replication properties of the Zymomonas mobilis ATCC 10988 plasmids pZMO1 and pZMO2

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2.     

The lipid composition of isolated cytoplasmic lipid droplets from a human cancer cell line, BE(2)M17

(1)H nuclear magnetic resonance spectroscopy (NMR) resonances from lipids in tumours are associated with tumour grade and treatment response. The origin of these NMR signals is mainly considered to be cytoplasmic lipid droplets (LDs). Techniques exist for isolating LDs but little is known about their composition and its relationship to NMR signals. In this work, density-gradient ultracentrifugation was performed on homogenised human cancer cells to isolate LDs. (1)H NMR was performed on whole cells, isolated LDs and their extracts. Heteronuclear single quantum coherence spectroscopy (HSQC) and liquid chromatography mass spectroscopy (LC-MS) were performed on lipid extracts of LDs. Staining and microscopy were used to characterize isolated LDs. An excellent agreement in chemical shift and relative signal intensity was observed between lipid resonances in cells and isolated LD spectra supporting that NMR-visible lipids originate primarily from LDs. Isolated LDs showed high concentrations of unsaturated lipids, a oleic-to-linoleic acid ratio greater than two and a cholesteryl ester (ChE)-to-cholesterol (Ch) ratio close to unity. These ratios were several-fold greater than respective ratios in whole cells, demonstrating isolation is important to characterize LD composition. LDs contain a specific group of lipid species that are likely to contribute to the (1)H NMR spectrum of cells.     

Histidine-rich Ca-binding protein interacts with sarcoplasmic reticulum Ca-ATPase

Depressed cardiac Ca cycling by the sarcoplasmic reticulum (SR) has been associated with attenuated contractility, which can progress to heart failure. The histidine-rich Ca-binding protein (HRC) is an SR component that binds to triadin and may affect Ca release through the ryanodine receptor. HRC overexpression in transgenic mouse hearts was associated with decreased rates of SR Ca uptake and delayed relaxation, which progressed to hypertrophy with aging. The present study shows that HRC may mediate part of its regulatory effects by binding directly to sarco(endo)plasmic reticulum Ca-ATPase type 2 (SERCA2) in cardiac muscle, which is confirmed by coimmunostaining observed under confocal microscopy. This interaction involves the histidine- and glutamic acid-rich domain of HRC (320-460 aa) and the part of the NH(2)-terminal cation transporter domain of SERCA2 (74-90 aa) that projects into the SR lumen. The SERCA2-binding domain is upstream from the triadin-binding region in human HRC (609-699 aa). Specific binding between HRC and SERCA was verified by coimmunoprecipitation and pull-down assays using human and mouse cardiac homogenates and by blot overlays using glutathione S-transferase and maltose-binding protein recombinant proteins. Importantly, increases in Ca concentration were associated with a significant reduction of HRC binding to SERCA2, whereas they had opposite effects on the HRC-triadin interaction in cardiac homogenates. Collectively, our data suggest that HRC may play a key role in the regulation of SR Ca cycling through its direct interactions with SERCA2 and triadin, mediating a fine cross talk between SR Ca uptake and release in the heart.     

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha- mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.      

The association among herbivory tolerance,ploidy level,and herbivory pressure in <Emphasis Type="Italic">cardamine pratensis</Emphasis>

We tested whether differences in ploidy level and previous exposure to herbivory can affect plant tolerance to herbivory. We conducted a common garden experiment with 12 populations of two ploidy levels of the perennial herb Cardamine pratensis (five populations of tetraploid ssp. pratensis and seven populations of octoploid ssp. paludosa). Earlier studies have shown that attack rates by the main herbivore, the orange tip butterfly Anthocharis cardamines, are lower in populations of octoploids than in populations of tetraploids, and vary among populations. In the common garden experiment, a combination of natural and artificial damage significantly reduced seed and flower production. We measured tolerance based on four plant-performance metrics: survival, growth, seed production and clonal reproduction. For three of these measurements, tolerance of damage did not differ between ploidy levels. For clonal reproduction, the octoploids had a higher tolerance than the tetraploids, although they experience lower herbivore attack rates in natural populations. Populations from sites with high levels of herbivory had higher tolerance, measured by seed production, than populations with low levels of herbivory. We did not detect any significant costs of tolerance. We conclude that high intensity of herbivory has selected for high tolerance measured by seed production in C. pratensis.