Citations

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo . However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.

ABSTRACTThe subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. Thetvagenetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility alleletvasencodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chickentvascDNAs had not yet been fully characterized; we cloned the chickentvacDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chickentvascDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 72. Line Ctvarcontains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of TvaRfor the ASLV(A) envelope glycoproteins. Line 72tvar2contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.

ABSTRACT The measles virus (MV) P gene codes for three proteins: P, an essential polymerase cofactor, and V and C, which have multiple functions but are not strictly required for viral propagation in cultured cells. V shares the amino-terminal domain with P but has a zinc-binding carboxyl-terminal domain, whereas C is translated from an overlapping reading frame. During replication, the P protein binds incoming monomeric nucleocapsid (N) proteins with its amino-terminal domain and positions them for assembly into the nascent ribonucleocapsid. The P protein amino-terminal domain is natively unfolded; to probe its conformational flexibility, we fused it to the green fluorescent protein (GFP), thereby also silencing C protein expression. A recombinant virus (MV-GFP/P) expressing hybrid GFP/P and GFP/V proteins in place of standard P and V proteins and not expressing the C protein was rescued and produced normal ratios of mono-, bi-, and tricistronic RNAs, but its replication was slower than that of the parental virus. Thus, the P protein retained nearly intact polymerase cofactor function, even with a large domain added to its amino terminus. Having noted that titers of cell-associated and especially released MV-GFP/P were reduced and knowing that the C protein of the related Sendai virus has particle assembly and infectivity factor functions, we produced an MV-GFP/P derivative expressing C. Intracellular titers of this virus were almost completely restored, and those of released virus were partially restored. Thus, the MV C protein is an infectivity factor.

Recent evidence suggests that alveolar epithelial cells (AECs) may contribute to the development, propagation, and resolution of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Proinflammatory cytokines, pathogen products, and injurious mechanical ventilation are important contributors of excessive inflammatory responses in the lung. In the present study, we used cDNA microarrays to define the gene expression patterns of A549 cells (an AEC line) in the early stages of three models of pulmonary parenchymal cell activation: cells treated with tumor necrosis factor-α (TNFα) (20 ng/ml), lipopolysaccharide (LPS, 1 μg/ml), or cyclic stretch (20% elongation) for either 1 h or 4 h. Differential gene expression profiles were determined by gene array analysis. TNFα induced an inflammatory response pattern, including induction of genes for chemokines, inflammatory mediators, and cell surface membrane proteins. TNFα also increased genes related to pro- and anti-apoptotic proteins, signal transduction proteins, and transcriptional factors. TNFα further induced a group of genes that may form a negative feedback loop to silence the NFκB pathway. Stimulation of AECs with mechanical stretch changed cell morphology and activated Src protein tyrosine kinase. The combination of TNFα plus stretch enhanced or attenuated expression of multiple genes. LPS decreased microfilament polymerization but had less impact on NFκB translocation and gene expression. Results from this study indicate that AECs can tailor their response to different stimuli or/and combination of stimuli and subsequently play an important role in acute inflammatory responses in the lung.

AbstractImmunoglobulin light chain amyloidosis (AL) is characterized by a clonal expansion of plasma cells within the bone marrow. Gene expression analysis was used to identify a unique molecular profile for AL using enriched plasma cells (CD138+) from the bone marrow of 24 patients with AL and 28 patients with multiple myeloma (MM) and 6 healthy controls. Class prediction analysis (PAM) revealed a subset of 12 genes, which included TNFRSF7 (CD27), SDF-1, and PSMA2, that distinguished between these 2 groups with an estimated and observed accuracy of classification of 92%. This model was validated with an independent dataset of 11 patients with AL and 12 patients with MM with 87% accuracy. Differential expression for the most discriminant genes in the 12-gene subset was validated using quantitative real-time polymerase chain reaction and protein expression analysis, which upheld the observations from the micro-array expression data. Functional analyses using a novel network mapping software revealed a number of potentially significant pathways that were dysregulated in patients with AL, with those regulating proliferation, apoptosis, cell signaling, chemotaxis, and migration being substantially represented. This study provides new insight into the molecular profile of clonal plasma cells and its functional relevance in the pathogenesis of light chain amyloidosis.

ABSTRACT Pneumocystis species remain an important cause of life-threatening pneumonia in immunocompromised hosts, including those with AIDS. Responses of the organism to environmental cues both within the lung and elsewhere have been poorly defined. Herein, we report the identification of a cell wall biosynthesis kinase gene ( CBK1 ) homologue in Pneumocystis carinii , isolated by differential display PCR, that is expressed optimally at physiological pH (7 to 8) as opposed to more acidic environments. Expression of Pneumocystis CBK1 was also induced by contact with lung epithelial cells and extracellular matrix. Translation of this gene revealed extensive homology to other fungal CBK1 kinases. Pneumocystis CBK1 expression was equal in the cyst and trophic life forms of the organisms. We further demonstrate that Pneumocystis CBK1 expressed in cbk1Δ Saccharomyces cerevisiae cells restored defective cell wall separation during proliferation. Consistent with this, Pneumocystis CBK1 expression also stimulated transcription of the CTS1 chitinase in cbk1Δ mutant yeast cells, an event necessary for cell wall separation. In addition, Pneumocystis CBK1 cDNA supported normal mating projection formation in response to α-factor in the cbk1Δ yeast cells. Site-directed mutations of serine-303 and threonine-494, potential regulatory phosphorylation sites in Pneumocystis CBK1, abolished mating projection formation, indicating a role for these amino acid residues in CBK1 activity. These findings indicate that Pneumocystis CBK1 is an environmentally responsive gene that may function in signaling pathways necessary for cell growth and mating.

To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele-characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding.