Nuclear factor 90 (NF90) and its C-terminally extended isoform, NF110, have been isolated as DNA-and RNA-binding proteins together with the less-studied protein NF45. These complexes have been implicated in gene regulation, but little is known about their cellular roles and whether they are redundant or functionally distinct. We show that heterodimeric core complexes, NF90-NF45 and NF110-NF45, exist within larger complexes that are more labile and contain multiple NF90/110 isoforms and additional proteins. Depletion of the NF45 subunit by RNA interference is accompanied by a dramatic decrease in the levels of NF90 and NF110. Reciprocally, depletion of NF90 but not of NF110 greatly reduces the level of NF45. Coregulation of NF90 and NF45 is a posttranscriptional phenomenon, resulting from protein destabilization in the absence of partners. Depletion of NF90-NF45 complexes retards cell growth by inhibition of DNA synthesis. Giant multinucleated cells containing nuclei attached by constrictions accumulate when either NF45 or NF90, but not NF110, is depleted. This study identified NF45 as an unstable regulatory subunit of NF90-NF45 complexes and uncovered their critical role in normal cell division. Furthermore, the study revealed that NF90 is functionally distinct from NF110 and is more important for cell growth.Human nuclear factor 90 (NF90) and nuclear factor 45 (NF45) were originally purified as a sequence-specific DNA binding complex regulating the interleukin-2 (IL-2) promoter (10, 17). NF90 is the founder member of a family of proteins generated from differentially spliced transcripts of the ILF3 gene (12). NF90 and NF110, which differ at their C termini, are the two most prominent ILF3 isoforms in cells (12,33,42,55). Both have been repeatedly isolated in diverse studies and have been given a variety of names. For example, MPP4 (M-phase phosphoprotein 4) is similar, if not identical, to NF90 and is phosphorylated during M phase (23), and closely related proteins 4F.1 and 4F.2 were characterized in Xenopus as doublestranded RNA (dsRNA)-binding proteins (3). NF90 is also known as DRBP76, NFAR1, and TCP80 (34, 43, 55), and NF110 is also known as ILF3, NFAR2, TCP110, and CBTF 122 (4,43,53,55). Underlining the importance of these proteins, knockout of the mouse ILF3 gene led to muscle degeneration, respiratory failure, and death soon after birth (44).NF90 and NF110 contain two dsRNA binding motifs (dsRBMs) which are responsible for their ability to interact with structured RNA. They also have an RGG domain that is capable of nucleic acid binding, and NF110 has an additional GQSY region that can interact with nucleic acids. Although characterized as DNA-binding proteins (17,36,40,41), NF90 and NF45 do not contain a recognized sequence-specific DNAbinding domain and the complex containing NF90 and NF45 does not appear to interact with DNA directly. NF90 and NF45 have been purified in complexes containing the Ku proteins and DNA-protein kinase (PK), as well as eukaryotic initiation factor 2 (eIF2), and it is likely ...

A 450-kDa human epidermal autoantigen was originally identified as a protein that reacted with the serum from an individual with a subepidermal blistering disease. Molecular cloning of this protein has now shown that it contains 5065 amino acids and has a molecular mass of 552 kDa. As reported previously this protein, which we call epiplakin, belongs to the plakin family, but it has some very unusual features. Epiplakin has 13 domains that are homologous to the B domain in the COOH-terminal region of desmoplakin. The last five of these B domains, together with their associated linker regions, are particularly strongly conserved. However, epiplakin lacks a coiled-coil rod domain and an aminoterminal domain, both of which are found in all other known members of the plakin family. Furthermore, no dimerization motif was found in the sequence. Thus, it is likely that epiplakin exists in vivo as a single-chain structure. Epitope mapping experiments showed that the original patient's serum recognized a sequence unique to epiplakin, which was not found in plectin. Immunofluorescence staining revealed the presence of epiplakin in whole sheets of epidermis and esophagus, in glandular cells of eccrine sweat and parotid glands and in mucous epithelial cells in the stomach and colon.Clarification of the basic structure of desmoplakin has been followed by the identification of many related proteins, such as BPAG1, 1 plectin, envoplakin, and periplakin (1-7). These proteins form a family known as the "plakin family" (8). Almost all members of this family have a common structure, with predicted globular amino-terminal and COOH-terminal domains that are separated by a central rod domain. Some homologous domain structures have been identified in both globular domains of many plakins, while the central domain is rich in heptad repeats and is believed to form a parallel ␣-helical coiled-coil structure with a dimerization partner (9). As suggested by this model, it has been demonstrated that desmoplakin I can form homodimers in vitro (10). Early investigations revealed that the COOH-terminal domains of plakins are involved in binding to intermediate filaments (11)(12)(13). The amino-terminal domains of desmoplakin and of BPAG1 are believed to bind to desmosomes or hemidesmosomes. Furthermore, some splicing variants of plectin and BPAG1 have actinor microtubule-binding domains at their amino termini, and it has been proposed that these domains form cross-links between microfilaments and/or microtubules and intermediate filaments (14 -16). Studies of a few inheritable diseases that appear to involve plectin or desmoplakin and of a BPAG1 null mouse have shown that each plakin plays a critical role in the tissue integrity in specific tissues (5,(17)(18)(19). Moreover, it seems likely that, in many autoimmune blistering diseases, plakins, located in the epidermis, might be target antigens, and these plakins are used for markers of specific diseases (2, 20 -22). However, their pathological roles remain to be clarified. Several years ago ...

The heterodimer of Tas1R2 and Tas1R3 is a broadly acting sweet taste receptor, which mediates mammalian sweet taste toward natural and artificial sweeteners and sweet-tasting proteins. Perception of sweet taste is a species selective physiological process. For instance, artificial sweeteners aspartame and neotame taste sweet to humans, apes and Old World monkeys but not to New World monkeys and rodents. Although specific regions determining the activation of the receptors by these sweeteners have been identified, the molecular mechanism of species-dependent sweet taste remains elusive. Using human/squirrel monkey chimeras, mutagenesis and molecular modeling, we reveal that the different responses of mammalian species towards the artificial sweeteners aspartame and neotame are determined by the steric effect of a combination of a few residues in the ligand binding pocket. Residues S40 and D142 in the human Tas1R2, which correspond to residues T40 and E142 in the squirrel monkey Tas1R2, were found to be the critical residues for the species dependent difference in sweet taste. In addition, human Tas1R2 residue I67, which corresponds to S67 in squirrel monkey receptor, modulates the higher affinity of neotame than that of aspartame. Our studies not only shed light on the molecular mechanism of species dependent sweet taste toward artificial sweeteners, but also provide guidance for designing novel effective artificial sweet compounds.

Fibrillar networks are intimately involved in several morphogenetic processes which underlie the harmonious development of the vertebrate embryo. Recent genetic evidence has demonstrated that the minor types V and XI collagen are key regulators of types I and XI fibrillogenesis in non-cartilaginous and cartilaginous matrices, respectively. A comprehensive understanding of the expression and regulation of the genes coding for the chains of the minor collagen types is therefore relevant to animal morphogenesis and development. The present study was undertaken to elucidate the embryonic pattern of expression of the gene coding for the mouse a1 chain of type XI colagen ( C o l l l a l ) using the technique of in situ hybridization. Transcripts of the C o l l l a l gene were detected as early as 11 days of gestation. The oll(X1) transcripts were found to accumulate mostly in cartilaginous tissues, such as the chondrocranium and the developing limbs. Like the major cartilage-specific collagen (type XI), Colllal expression was also noted in the neuro-epithelium of the brain. However, al(X1) transcripts accumulated in several other non-cartilaginous sites. They include odontoblasts, trabecular bones, atrioventricular valve of the heart, the tongue, the intestine, and the otic vesicle. Altogether, the data confirm that C o l l l a l has a broader spectrum of expression than previously thought. This finding raises the possibility that the (ul(X1) chain may participate in the formation of stage-and tissue-specific trimers with distinct functional properties.

Background. Clostridium difficile infection is often considered to result from recent acquisition of a C difficile isolate in a healthcare setting. However, C difficile spores can persist for long periods of time, suggesting a potentially large community environmental reservoir. The objectives of this study were to assess community environmental contamination of toxigenic C difficile and to assess strain distribution in environmental versus clinical isolates.Methods.From 2013 to 2015, we collected community environmental swabs from homes and public areas in Houston, Texas to assess C difficile contamination. All positive isolates were tested for C difficile toxins A and B, ribotyped, and compared with clinical C difficile isolates obtained from hospitalized patients in Houston healthcare settings.Results.A total of 2538 environmental samples were collected over the study period. These included samples obtained from homes (n = 1079), parks (n = 491), chain stores (n = 225), fast food restaurants (n = 123), other commercial stores (n = 172), and hospitals (n = 448). Overall, 418 environmental isolates grew toxigenic C difficile (16.5%; P < .001) most commonly from parks (24.6%), followed by homes (17.1%), hospitals (16.5%), commercial stores (8.1%), chain stores (7.6%), and fast food restaurants (6.5%). A similar distribution of ribotypes was observed between clinical and environmental isolates with the exception that ribotype 027 was more common in clinical isolates compared with environmental isolates (P < .001).Conclusions.We identified a high prevalence of toxigenic C difficile from community environs that were similar ribotypes to clinical isolates. These findings suggest that interventions beyond isolation of symptomatic patients should be targeted for prevention of C difficile infection.