Gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors.

Exposure of mouse resident and thioglycollate-elicited peritoneal macrophages to IFN-gamma leads to a marked increase in the TNF-alpha (tumor necrosis factor/cachectin), IL-1 and u-PA (urokinase-type plasminogen activator) mRNA levels. Nuclear run-on experiments show that IFN-gamma acts by enhancing the transcription of these three genes. Transcription of these three genes is also rapidly and transiently induced by cycloheximide, an inhibitor of protein synthesis, indicating that they are under the control of short-lived repressors.

Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis.

The role of TNF-alpha/cachectin in the pneumopathy elicited by bleomycin has been investigated. After a single intratracheal bleomycin instillation, an increase of the lung TNF-alpha mRNA level was evident, from days 5 to 15, as shown by Northern gel analysis of whole lung RNA. In contrast, lung IL-1-alpha and GM-CSF mRNA were not detectable. In mice passively immunized with rabbit anti-mouse TNF-alpha IgG, the bleomycin-induced collagen deposition, evaluated by the total lung hydroxyproline assay on day 15, was prevented. Depletion of the CD4 and CD8 T lymphocytes by an in vivo treatment with mAb prevented the bleomycin-induced increase of TNF mRNA level and fibrosis. After an administration of bleomycin in continuous intraperitoneal perfusion, the diffuse alveolar damage observed by light and electron microscopy was almost completely prevented by anti-TNF antibody. These results indicate that in response to bleomycin, the T lymphocytes induce, by an undefined mechanism, an increase of the pulmonary TNF production, which leads to alveolar damage, growth of fibroblast, and collagen deposition.

Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages.

We have explored the cis-acting elements necessary for the LPS-mediated activation of the mouse TNF-alpha promoter by transfecting a set of 5' deletion mutants linked to the CAT reporter gene into primary bone marrow-derived macrophages. A major drop in inducibility by LPS was seen upon deletion of a region mapping between nt -655 and nt -451. Gel retardation assays revealed that LPS induced the appearance in this region of several specific DNA-protein complexes mapping to sequence motifs with strong homology to the kappa B enhancer. Constructs containing two or more copies of one of the kappa B enhancer motifs linked to a heterologous promoter were inducible by LPS. Additional deletion of a region between nt -301 and nt -241, which contains a MHC class II-like "Y box" and formed a Y box-specific complex with a protein whose concentration was increased by LPS, caused a nearly complete loss of inducibility by LPS. We speculate that NF-kappa B and/or related proteins are involved in the LPS-induced transcriptional activation of the TNF-alpha gene, and that factors interacting with the Y box can additionally modulate the activity of the gene in macrophages.

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function.

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.

The NOT, SPT3, and MOT1 genes functionally interact to regulate transcription at core promoters.

Previous studies demonstrated that mutations in the Saccharomyces cerevisiae NOT genes increase transcription from TATA-less promoters. In this report, I show that in contrast, mutations in the yeast MOT1 gene decrease transcription from TATA-less promoters. I also demonstrate specific genetic interactions between the Not complex, Mot1p, and another global regulator of transcription in S. cerevisiae, Spt3p. Five distinct genetic interactions have been established. First, a null allele of SPT3, or a mutation in SPT15 that disrupts the interaction between Spt3p and TATA-binding protein (TBP), allele specifically suppressed the not1-2 mutation. Second, in contrast to not mutations, mutations in MOT1 decreased HIS3 and HIS4 TATA-less transcription. Third, not mutations suppressed toxicity due to overexpression of TBP in mot1-1 mutants. Finally, overexpression of SPT3 caused a weak Not- mutant phenotype in mot1-1 mutants. Collectively, these results suggest a novel type of transcriptional regulation whereby the distribution of limiting TBP (TFIID) on weak and strong TBP-binding core promoters is regulated: Mot1p releases stably bound TBP to allow its redistribution to low-affinity sites, and the Not proteins negatively regulate the activity of factors such as Spt3p that favor distribution of TBP to these low-affinity sites.

Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B.

This study characterizes the interaction of murine macrophage nuclear proteins with the tumor necrosis factor alpha (TNF-alpha) promoter. Gel retardation and methylation interference assays showed that stimulation of TNF-alpha gene transcription in peritoneal exudate macrophages was accompanied by induction of DNA-binding proteins that recognized with different affinities four elements related to the kappa B consensus motif and a Y-box motif. We suggest that the basal level of TNF-alpha expression in macrophages is due to the binding of a constitutive form of NF-kappa B, present at low levels in nuclei from resting thioglycolate exudate peritoneal macrophages, to some if not all of the kappa B motifs; we postulate that this constitutive form contains only the 50-kilodalton (kDa) DNA-binding protein subunits of NF-kappa B, not the 65-kDa protein subunits (P. Baeuerle and D. Baltimore, Genes Dev. 3:1689-1698, 1989). Agents such as glucocorticoids, which decrease TNF-alpha transcription, diminished the basal level of nuclear NF-kappa B. Stimulation of Stimulation of TNF-alpha transcription in macrophages by lipopolysaccharide, gamma interferon, or cycloheximide led to an increased content of nuclear NF-kappa B. This induced factor represents a different form of NF-kappa B, since it generated protein-DNA complexes of slower mobility; we propose that this induced form of NF-kappa B contains both the 50- and 65-kDa protein subunits, the latter ones being necessary to bind NF-kappa B to its cytoplasmic inhibitor in uninduced cells (Baeuerle and Baltimore, Genes Dev., 1989). In resting cells, this inducible form of NF-kappa B was indeed detectable in the cytosol after deoxycholate treatment. UV cross-linking experiments and gel retardation assays indicated that the inducible form of NF-kappa B is in a higher-order complex with other proteins.

c-fos gene transcription in murine macrophages is modulated by a calcium-dependent block to elongation in intron 1.

Cultured mouse thioglycolate-elicited peritoneal macrophages exhibit a strong block to transcriptional elongation beyond the end of the c-fos gene first exon. This block is absent in freshly isolated peritoneal cells, appears slowly during culture, and does not require adherence of the cells. The extent of this block is largely responsible for the levels of c-fos mRNA in cultured macrophages, even after modulation by agents such as the tumor promoter phorbol myristate acetate and increased intracellular cyclic AMP, which also increase the activity of the c-fos promoter. When macrophages are cultured in the absence of mobilizable calcium, the block can no longer be relieved by any inducing agent. Conversely, upon calcium influxes, there is little alteration in the level of transcriptional initiation, but transcription proceeds efficiently through the entire c-fos locus. These results suggest the presence of an intragenic calcium-responsive element in the c-fos gene and illustrate its key role in the control of c-fos gene transcription.

The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5.

The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes. The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other. In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4. These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins. ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations. These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins.

Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits.

The yeast CCR4-NOT protein complex is a global regulator of RNA polymerase II transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern. A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells. Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially com-plement corresponding not mutations in yeast. Interestingly, human CALIF is highly homologous to, although clearly different from, a recently described human CAF1 protein. Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4-NOT complex.

Balance quality assessment as an early indicator of physical frailty in older people.

Frailty is an increasingly common geriatric condition that results in an increased risk of adverse health outcomes such as falls. The most widely-used means of detecting frailty is the Fried phenotype, which includes several objective measures such as grip strength and gait velocity. One method of screening for falls is to measure balance, which can be done by a range of techniques including the assessment of the Centre of Pressure (CoP) during a balance assessment. The Balance Quality Tester (BQT) is a device based on a commercial bathroom scale that can evaluate balance quality. The BQT provides instantaneously the position of the CoP (stabilogram) in both anteroposterior (AP) and mediolateral (ML) directions and can estimate the vertical ground reaction force. The purpose of this study was to examine the relationship between balance quality assessment and physical frailty. Balance quality was compared to physical frailty in 186 older subjects. Rising rate (RR) was slower and trajectory velocity (TV) was higher in subjects classified as frail for both grip strength and gait velocity (p<;0.05). Balance assessment could be used in conjunction with functional tests of grip strength and gait velocity as a means of screening for frailty.

Receptor-mediated phagocytosis by macrophages induces a calcium-dependent transient increase in c-fos transcription.

The transcription of the c-fos gene and the level of c-fos mRNA in mouse peritoneal macrophages are rapidly, strongly and transiently increased after Fc- and C3b-mediated phagocytosis, but not after phagocytosis of latex particles. In order to induce both phagocytosis and a rise in c-fos mRNA, binding to receptors must be followed by mobilization of Ca++ from intracellular Induction of c-fos transcription in macrophages by other agents acting through different intracellular "messengers', i.e. phorbol esters (protein kinase C), cholera toxin (cAMP) and dexamethasone (glucocorticoid receptor) also depends on intracellular Ca++. In all these conditions, induction of c-fos transcription is inhibited by the calmodulin antagonist W7, suggesting a common Ca++-dependent pathway for c-fos gene activation in macrophages.

The essential function of Not1 lies within the Ccr4-Not complex.

The five Saccharomyces cerevisiae Not proteins are associated with the Ccr4 and Caf1 proteins in 1.2 MDa and 2 MDa complexes. The Not proteins have been proposed to repress transcription of promoters that do not contain a canonical TATA sequence, while the Ccr4 and Caf1 proteins are required for non-fermentative gene expression. The mechanism of transcriptional regulation by the Ccr4-Not complex is unknown and the role of its different components is unclear. Only Not1p is essential for yeast viability.Here, we show that most strains carrying combinations of two null alleles of the non-essential CCR4-NOT genes are non-viable. This would suggest that the Ccr4-Not complex is essential. We find that Not1p consists of at least two domains, a C-terminal domain that is essential for yeast viability, and a N-terminal domain that is dispensable but required for yeast wild-type growth. The essential C-terminal domain of Not1p can associate with Not5p, and both proteins are present in 1.2 and 2 MDa complexes in the absence of the N-terminal Not1p domain. In contrast, in the absence of the N-terminal domain of Not1p, Ccr4p does not efficiently associate in large complexes nor with the C-terminal domain of Not1p. Healthy growth is observed when both domains of Not1p are expressed in trans, and is correlated with their physical association, together with Ccr4p, in large complexes. These results are consistent with the essential function of Not1p lying within the Ccr4-Not complex.

TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes.

The hect-domain has been characterized as a conserved feature of a group of E3 ubiquitin ligases. Here we show that the yeast hect-domain protein TOM1p regulates transcriptional activation through effects on the ADA transcriptional coactivator proteins. Null mutations of tom1 result in similar defects in transcription from ADH2 and HIS3 promoters, and enhanced transcription from the GAL10 promoter as do null mutations in ngg1/ada3. Strains with disruptions of both ngg1 and tom1 have the same phenotype as strains with a disruption of only ngg1 implying that these genes are acting through the same pathway. In the absence of TOM1p, the normal associations of the ADA proteins with SPT3p and the TATA-binding protein are reduced. The action of TOM1p is most likely mediated through ubiquitination since mutation of Cys3235 to Ala, corresponding residues of which are required for thioester bond formation with ubiquitin in other hect-domain proteins, results in similar changes in transcription as the null mutation. A direct role for TOM1p in regulation of ADA-associated proteins is further supported by the finding that SPT7p is ubiquitinated in a TOM1p-dependent fashion and that TOM1p coimmunoprecipitates with the ADA proteins.

Characterization of NOT5 that encodes a new component of the Not protein complex.

The yeast HIS3 gene has two core promoters: TC, a TATA-less element and TR, a canonical TATA element. Four genes encode global negative regulators of transcription that preferentially repress TC-dependent transcription: NOT1 (CDC39), NOT2 (CDC36), NOT3 and NOT4 (SIG1, MOT2). Genetic and biochemical experiments suggest that the products of these genes are associated in a complex and regulate TFIID function. In this paper, we describe a new gene, NOT5, that also represses transcription of the HIS3 TATA-less promoter preferentially and encodes a protein whose N-terminal region is 44% identical to that of Not3p. Our results indicate that NOT5 is involved in Not function and encodes a product that is physically associated with the other Not proteins. First, overexpression of NOT3 or NOT4 suppresses mutations in NOT5. Secondly, mutations in NOT4 are synthetically lethal with mutations in NOT5. Thirdly, NOT5 interacts with NOT1 and NOT3 in the two-hybrid assay. Finally, Not1p, Not3p and Not4p co-immunoprecipitate with Not5p.

Enteral nutrition in French institutionalized patients: a multicentric study.

BACKGROUND: No previous studies have demonstrated either a nutritional improvement, or a survival benefit from tube placement in an institutionalized population. OBJECTIVE: The aim of this study was to determine current indications for tube feeding in French geriatric centers and to evaluate clinical outcome and mortality rates in these frail very old patients. DESIGN: Between November 1, 2000 and April 31, 2001, we prospectively recruited all hospitalized or institutionalized patients who received enteral nutrition (EN) in 7 Departments of Geriatric Medicine in France. Nutritional parameters and main indications of EN were recorded at the time of feeding tube placement. Pneumonia and mortality rates were observed over a period of one year. RESULTS: 57 patients of mean age 81.6 7.8 yrs underwent placement of a feeding tube. Mean BMI value was 20.7 4.8 and mean serum albumin level 26.1 6.1 g/L. The most frequent indications for EN included stoke (39%) and other neurologic diseases (42%). Fourteen patients (25%) died within 30 days, and 27 (47%) died over the 12-month follow-up period. During the first month, an episode of pneumonia was noted in 26 cases (55%). CONCLUSION: The similarity between rates of early mortality reported in our study and those reported in several previous studies involving younger, ambulatory subjects is surprising because we might expect poorer survival in our frail elderly patients. We can think that French geriatric teams have changed their attitudes toward EN in recent years, EN being less frequently used in patients with advanced dementia and at the end-stage of life.