Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D

Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processing. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc7O pre-mRNA intron. Competition studies indicate that a trans-acting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this factor is common to both intronic and non-intronic snoRNAs possessing nucleotide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region containing boxes C and D does not bind fibrillarin. Collectively, our results indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, thereby stabilizing the intronic snoRNA sequence in an RNP complex during processing.     

Distinct regions of U3 snoRNA interact at two sites within the 5' external transcribed spacer of pre-rRNAs in Trypanosoma brucei cells

U3 snoRNA is required for early pre-rRNA processing events that include cleavage of the 5' external transcribed spacer (5'ETS) and 18S rRNA maturation. Herein, psoralen RNA crosslinking has been used to indicate novel in vivo interactions between the minimally-sized Trypanosoma brucei U3 snoRNA and pre-rRNAs. Two discrete U3 crosslinks were mapped to 5'ETS sequences, then individually isolated by hybrid selection following digestion of pre-rRNAs. Crosslink positions within these U3-site1 and U3-site2 complexes were resolved by RNaseH digestion and primer extension analyses. Hinge bases of U3 contacted site1 bases U140 and U142 just 3' of the processed primary site. This is the first experimental evidence of a U3 RNA interaction adjacent to a major 5'ETS cleavage site and supports a critical role for U3 in its processing. Highly conserved box A bases contacted site2 base U945, 187 nt upstream of 18S-like rRNA sequences. Site2 sequences are not required for primary processing, thus, a U3 interaction here might have roles in subsequent downstream processing events. These results clearly demonstrated that distinct U3 snoRNA sequences crosslinked different regions of the 5'ETS and support a model for U3 as a multifunctional snoRNA.     

Variations in the subunit content and catalytic activity of the cytochrome c oxidase complex from different tissues and different cardiac compartments

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra-threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri-threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.     

Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus

We have raised specific antibodies to the second immunoglobulin-like domain of fibroblast growth factor receptors (FGFRs) and used these to investigate the expression and subcellular localization of FGFR-1, -2, -3, and -4 in breast epithelial cells. All four receptors classes could be detected in breast cell lines; however, FGFR-4 and FGFR-2 appeared to be expressed at a higher level in breast cancer cell lines than in normal epithelial cells. Surprisingly, FGFR-3 localized in the cell nucleus by immunofluorescence. A second antibody to a separate epitope confirmed this finding and showed that the form of FGFR-3 present must contain an intact kinase domain as well as the growth factor binding domain. Western analysis of fractionated cells revealed the presence of two forms of FGFR-3 of 135 and 110 kDa. The 110-kDa form was predominantly found in the nucleus, whereas the 135 kDa form was sometimes found in the nucleus. RT-PCR analysis of FGFR-3 mRNA showed the presence of a splice variant in which exons 7 and 8 are deleted. This results in the translation of FGFR-3 missing the transmembrane domain but with an intact kinase domain, which could be a soluble, intracellular receptor. Transfection experiments showed that FGFR-3 containing this deletion and no signal peptide gave an identical nuclear staining pattern to that seen in breast epithelial cells. We conclude that two forms of FGFR-3 are present in breast epithelial cells; a full-length 135-kDa receptor, which has a conventional membrane localization, and a novel soluble form of 110 kDa.     

Determination of antibiotics in different water compartments via liquid chromatography-electrospray tandem mass spectrometry

The mechanisms by which BCG exerts its antitumour activity remain unclear. Attachment of BCG to the bladder via FN has been shown to be an important step in initiating its antitumorigenic activity. The mechanism(s) by which BCG operates requires LAK cells, BCG-activated killer cells, T lymphocytes (CD4) helper cells and CD8 suppressor/cytotoxic cells) and monocytes. The optimal route of administration is intravesical. The efficacy of a BCG vaccine depends on the viability, dose and strain. Differences in efficacy and side-effects have not been shown between different strains. Low-dose regimens successfully protect from recurrences, with fewer side-effects. The initial schedule of BCG is a course of six instillations in 6 weeks; when the patient fails this course, two possibilities arise. The first is maintenance therapy; response rates improve but there is more local and systemic toxicity. The second is a further 6-week course, and this seems most useful in those with a sustained response to the initial treatment. The clinical response to BCG therapy can be monitored using cytokine measurements or p53 determinations. Toxicity remains a major problem in BCG treatment and triple antituberculosis combination therapy should be given for 3 months in those with severe systemic side-effects. The use of prophylactic isoniazid is not recommend to decrease side-effects. The clinical results of BCG have been good, with success rates of 58-100%, with a minimal follow-up of one year in prophylaxis. BCG seems superior to intravesical therapy, but at the cost of inducing more adverse effects. BCG is not indicated for low- and intermediate-risk patients, in whom chemotherapy is the first choice. BCG can also be used to eliminate tumour after an incomplete TUR, or in patients who are unfit for surgery, with a 60-70% success rate. The primary and best treatment for CIS is intravesical BCG; encouraging results have been reported, with success rate of 42-83% after a minimal follow-up of one year. Although currently BCG seems to be the choice for high-risk superficial TCC, many questions remain unanswered, especially about the mechanism(s) of action, the optimal dose and clinical schedule.     

Laboratory testing of UV transmission through fabrics may underestimate protection

Laboratory testing of the ultraviolet protection factor (UPF) provided by fabrics normally utilizes a collimated source of UV radiation and either a broadband sensor or spectroradiometer to detect the radiation, both unattenuated and after passing through the fabric sample held in a flat tensionless state. We report the results of an in vivo study of UV transmission through various T-shirts at several sites on the trunk and arms of a life-size mannequin irradiated with diffuse radiation and using UV sensitive polymer films as the sensor. We found a variation in UPF by a factor of two or more at different anatomical sites for a given T-shirt, with lower UPFs seen at sites where the fabric is stretched; and found that at every site and for each T-shirt this in vivo UPF was higher than the conventional in vitro UPF determined using collimated radiation.     

Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments

OBJECTIVE: To examine the immunohistochemical and ultrastructural features of the rare pleomorphic adenocarcinomas of the ciliary epithelium (CE). DESIGN: Retrospective case series. PARTICIPANTS: The study materials included 12 cases of adenocarcinoma of the ciliary epithelium: 9 cases of CE hyperplasia and 3 cases of CE adenomas. INTERVENTION: Histologic sections were stained with hematoxylin-eosin, alcian blue, periodic acid-Schiff, and occasionally with Masson trichrome. Additionally, the following immunohistochemical markers were used: Kermix (ae1/ae3 + ck1), cytokeratin 7 (CK7), cytokeratin 20 (CK20), epithelial membrane antigen, CAM 5.2, S-100 protein, neuron-specific enolase, glial fibrillary acid protein, smooth muscle actin, and vimentin. Five lesions were studied ultrastructurally. Clinical data were available in all cases, and follow-up was obtained in 9 of the 12 patients. RESULTS: Nine tumors occurred in phthisical eyes in adults. The tumor cells were arranged in tubular and solid patterns and surrounded by thick basement membrane (BM) material and fibrous stroma. Immunohistochemistry (IM) of adenocarcinomas showed positivity with kermix (8 of 12 lesions), CAM 5.2 (7 of 12), and CK7 (5 of 12). Ultrastructurally, the tumor cells were surrounded by a thick, homogeneous, and/or multilaminar BM and attached to each other by junctional complexes. CONCLUSIONS: Clinically, this intraocular neoplasm should be considered in adults with a longstanding blind eye with an epibulbar mass and/or proptosis of recent duration. Fatal cases only occurred in tumors with extraocular extension. Adenocarcinomas of CE should be differentiated from amelanotic melanoma and metastatic lesions by the presence of a thick BM material around the tumor cells and intraocular fibrosis. Immunohistochemistry is helpful in differentiating from melanomas but not helpful in cases of metastatic carcinomas.     

3T3-L1 adipocytes express two isoforms of phospholipase D in distinct subcellular compartments

Phospholipase D has been implicated as an important enzyme in a range of cellular responses, including regulated secretion and the formation of secretory vesicles, cell proliferation and control of cell morphology. As insulin treatment of adipocytes has been shown to stimulate a phosphatidylcholine-specific phospholipase D and also modulates membrane trafficking, we wished to determine which isoform(s) of phospholipase D were present within adipocytes, to identify their subcellular distribution, and examine how this distribution may change in response to insulin. Using RT-PCR, 3T3-L1 adipocytes were found to express two isoforms of phospholipase D, specifically PLD1b and PLD2a. Using isoform-specific antibodies, PLD1 and PLD2 were found to be present predominantly in intracellular membranes, unlike the situation reported in other cells. Detailed analysis of the intracellular localisation of PLD1 and PLD2 revealed that these isoforms are differentially localised within adipocytes, implying functionally distinct roles for PLD activity in distinct subcellular compartments.     

Functional morphology of nucleolus organizer regions of chromosomes and nucleoli in human multiple myeloma cell lines. I. Variation of the morphology and silver staining of nucleolus organizer regions of chromosomes in RMPI 8226 and U 266 cell lines with different level of differentiation of during 7 days after cell passage

The morphology and Ag-staining of nucleoli in human multiple myeloma cell lines RPMI 8226 and U 266, distinguished from each other in the differentiation degree, were quantitatively studied, and the production of immunoglobulins or their fragments by the line cells was evaluated throughout 7 days after cell seeding. The less differentiated cell line RPMI 8226 and the high differentiated cell line U 266 were revealed to differ in both the initial level of immunoglobulin production and dynamics of immunoglobulin accumulation in culture medium. The total number of Ag-stained nucleolar-organizer regions (AgNORs) per nucleus in cells RPMI 8226 was significantly higher than in cells U 266 in all times after seeding of the cells. In both cell lines changes in the quantity and shape of nucleoli and also in the total number of AgNORs per nucleus and pattern of AgNORs distribution within nucleoli correlated with the cell cycle phase. Relationships between morphofunctional changes in nucleoli and the differentiation degree and proliferative activity of the cells, and also between the number of Ag-positive nucleolar-organizing metaphase chromosomes and the functional activity of interphase AgNORs are discussed.     

Anatomy of the upper extremity muscle compartments

Schizophrenia, a devastating disease characterized by a combination of various types of disturbed behaviors, thoughts, and feelings, may likewise be heterogeneous in etiology. Recent advances in neuroscience and psychopharmacology have suggested a wide array of competing mechanisms that may be involved in schizophrenia, including but not limited to deficits in one or more neurotransmitters and second messenger systems (e.g., dopamine, serotonin, gamma-aminobutyric acid, glutamate, and noradrenaline), neurodevelopmental defects in brain circuitry, and viral infection. Psychiatric genetic studies indicate that schizophrenia is a disorder with multifactorial inheritance. Since cerebral metabolic activity reflects regional brain work for all neurotransmitter systems, imaging metabolism directly with fluorodeoxyglucose and indirectly with blood flow and hemoglobin oxygen saturation can provide information about the functional neuroanatomy of a deficit in individual patients and allow patients to be grouped into more homogeneous subgroups for intensive study. This review summarizes metabolic imaging studies in schizophrenia over the past decade.     

Cooperative interactions between adjacent troponin-tropomyosin complexes may be transmitted through the actin filament

Recent analyses of the assembly of thin filaments containing altered forms of troponin (or no troponin) suggested that the strongly cooperative nature of troponin-tropomyosin binding to actin might be primarily caused by indirect interactions involving the actin lattice, rather than by direct contacts between neighboring troponin-tropomyosin molecules. To test this hypothesis, thin filament assembly was examined using either cardiac tropomyosin digested with carboxypeptidase A (cbpTm) or a tropomyosin with defective function at both amino and carboxyl termini (unacetylated cbpTm). Compared to intact troponin-tropomyosin, both troponin-cbpTm and troponin-unacetylated cbpTm had much weaker binding to actin; however, cooperative interactions were only slightly reduced. These data support the implication that the primary source of the cooperativity involves troponin-tropomyosin-promoted conformational changes within the actin polymer. Surprisingly, the effects of tropomyosin amino- and carboxyl-terminal structural defects on troponin-tropomyosin binding to actin were not additive. In the presence of troponin, tropomyosin molecules with either defect had the same diminution in actin affinity as molecules with both defects. Finally, the Ca2+ sensitivity of troponin-tropomyosin binding to actin was increased by alteration of either end of tropomyosin.     

Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation

The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing. Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome. Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation. Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state. Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage. In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner. Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome.     

Integrin alphavbeta3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways

Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.     

Extraction of noble metals from the recycle slag of refining production

Results of investigations of concentration of metallurgical slag using electrical separators, jugs and flotation machines, sluices, concentration table, and Itomack centrifugal separator are presented. As a result, two production flowsheets are suggested, wet and dry, which provide recovery of ～95% silver and ～89% gold mixed with a ～15.5% metal content.