The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.     

Major internal nuclear matrix proteins are common to different human cell types

Cancer invasion and metastasis are associated with matrix degradation. We describe a novel in vivo model of invasion by squamous epithelial neoplastic cells derived from transgenic mice grown on acellular human dermis. Human dermis was subjected to multiple freeze-thaw cycles to render it acellular, maintaining the basement membrane of the former dermal-epidermal junction. Cells representing discrete stages of a multistep transgenic mouse model of epidermal carcinogenesis (neonatal transgenic keratinocytes, moderately/poorly differentiated squamous cell carcinoma, and lymph node metastasis) were seeded onto the basement membrane surface, grown in culture for 4 days, grafted in a subpannicular pocket of athymic mice, and harvested after 3 weeks. Histological analysis demonstrated that neonatal transgenic keratinocytes did not degrade the basement membrane or invade the underlying dermis. In contrast, malignant cells derived from both a moderately differentiated squamous carcinoma and a lymph node metastasis were highly invasive. Immunohistochemical analysis revealed collagenase only in nests of invading malignant cells in contact with the dermal matrix, but not in the tumor mass remaining above the basement membrane, suggesting that this proteinase may be required for stromal invasion. This novel model recapitulates the events seen in malignant invasion: transgenic keratinocytes are unable to penetrate the dermis while cells from a moderately differentiated carcinoma and from lymph node metastasis consistently invade.     

Inhibiting and deactivating effects of water on the selective catalytic reduction of nitric oxide with ammonia over MnOx/Al2O3

The effect of water on the selective catalytic reduction (SCR) of nitric oxide with ammonia over alumina supported with 2–15 wt.-% manganese oxide was investigated in the temperature range 385–600 K, with the emphasis on the low side of this temperature window. Studies on the effect of 1–5 vol.-% water vapour on the SCR reaction rate and selectivity were combined with TPD experiments to reveal the influence of water on the adsorption of the single SCR reactants. It turned out that the activity decrease due to water addition can be divided into a reversible inhibition and an irreversible deactivation. Inhibition is caused by molecular adsorption of water. TPD studies showed that water can adsorb competitively with both ammonia and nitric oxide. Additional kinetic experiments revealed that adsorbed ammonia is present in excess on the catalyst surface, even in the presence of water. Reduced nitric oxide adsorption is responsible for the observed reversible decrease in the reaction rate; the fractional reaction order changes from 0.79 in the absence of water to 1.07 in its presence. Deactivation is probably due to the dissociative adsorption of water, resulting in the formation of additional surface hydroxyls. As the amount of surface hydroxyls formed is limited to a saturation level, the deactivating effect on the catalyst is limited too. The additional hydroxyls condense and desorb in the temperature range 525–775 K, resulting in a lower degree of deactivation at higher temperature. A high temperature treatment at 775 K results in a complete regeneration. The amount of surface hydroxyls formed per unit surface area decreases at increasing MnO_x-loading. The selectivity to the production of nitrogen is enhanced significantly by the presence of gas phase water.     

The region Ser333-Arg356 of the alpha-chain of human C4b-binding protein is involved in the binding of complement C4b

Human C4b-binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the alpha-chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the alpha-chain of C4BP that interacts with C4b, tryptic fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333-Arg356 of the alpha-chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the alpha-chain of C4BP is likely to be involved in the interaction with C4b.     

Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.     

High-resolution HLA typing for the DQB1 gene by sequence-based typing

BACKGROUND: Monocytic tissue factor (TF), initiating the extrinsic blood coagulation pathway, is often upregulated under septic or inflammatory conditions. The complex activating mechanism remains largely unclear and no effective strategy has been firmly established. In this study, we used a model monocytic cell line (human leukemic THP-1 promonocytes) to address (1) the nature of TF activation in response to bacterial endotoxin and (2) the application of anti-inflammatory cytokines in relieving monocytic hypercoagulation. RESULTS: TF in THP-1 cells was substantially activated by exposure to bacterial endotoxin (LPS; 5 micrograms/ml) for 6 h. Human recombinant IL-4 (500 ng/ml) and IL-10 (500 ng/ml) inhibited TF activation induced by LPS. To determine if these cytokines depressed LPS recognition resulting in such inhibition, we employed an anti-CD14 mAb (UCHM-1; Sigma Chemical) to address the role of CD14 in LPS transmembrane signaling. LPS-induced TF activation was depressed by 35% upon inclusion of the anti-CD14 mAb (1:10 dilution). This antibody alone mimicked TF activation which accounted for 35% of the LPS-induced TF activation, suggesting the activating role of CD14 ligation. In addition, the anti-CD14 mAb elicited the production of nitric oxide (NO) which was found to be independent of TF activation. NO production could serve as an independent index for monitoring LPS recognition. IL-4 depressed the anti-CD14 mAb-induced TF activation as well as NO elicitation, indicating the blockade of CD14 ligation. In contrast, IL-10 showed differential inhibitory activities. TF activation induced by either LPS or anti-CD14 mAb was inhibited by IL-10 which did not show any inhibition on NO elicitation under these conditions. In a separate approach, neither IL-4 nor IL-10 inhibited phorbol ester-induced NO elicitation. More direct evidence came from an epifluorescent demonstration showing that IL-4 blocked binding of FITC-conjugated LPS and anti-CD14 mAb to THP-1 cells. CONCLUSIONS: Taken together, the results suggest that LPS action in relation to TF activation consists of CD14-independent and -dependent signaling including CD14 ligation. We also showed that anti-inflammatory cytokines (IL-4 and -10) significantly depressed TF activation. IL-4 antagonized CD14-dependent LPS recognition leading to the depression in TF activation.     

Morphological and functional polymorphism within clonal thyroid nodules

Thirty-nine thyroid nodules, removed because of recent growth, were analyzed morphologically by serial histological sections for the classical histomorphological hallmarks of follicular cell replication and for immunohistochemically demonstrable overexpression of the growth-associated ras-gene product p21ras. Clonal analysis was performed using the highly informative probe M27 beta that detects polymorphisms on the locus DXS255 of the X-chromosome. Twenty-four nodules were of clonal and 15 nodules were of poly-clonal origin. Only 3 out of the 24 clonal nodules were histomorphologically uniform. In all others, the structural hallmarks of active growth and the P21ras growth-marker expression were remarkably heterogeneous throughout the tumors. There were no histomorphological characteristics distinguishing these clonal tumors from polyclonal nodules. Even if a clonal thyroid tumor may be originally homogeneous in respect to the parameters studied here, mechanisms must exist that create wide heterogeneity of growth and of morphogenetic potential among the individual follicular cells during further expansion of the nodule. Thus, clonal nodules are much more common in nodular goiters than hitherto assumed on grounds of the classical morphological criteria. The diagnosis of a true monoclonal nodule can no longer rely on morphological and functional criteria alone but requires molecular or cytogenetic analysis of clonality.