Human adipocyte function is impacted by mechanical cues.

Fibrosis is a hallmark of human white adipose tissue (WAT) during obesity-induced chronic inflammation. The functional impact of increased interstitial fibrosis (peri-adipocyte fibrosis) on adjacent adipocytes remains unknown. Here we developed a novel in vitro 3D culture system in which human adipocytes and decellularized material of adipose tissue (dMAT) from obese subjects are embedded in a peptide hydrogel. When cultured with dMAT, adipocytes showed decreased lipolysis and adipokine secretion and increased expression/production of cytokines (IL-6, G-CSF) and fibrotic mediators (LOXL2 and the matricellular proteins THSB2 and CTGF). Moreover, some alterations including lipolytic activity and fibro-inflammation also occurred when the adipocyte/hydrogel culture was mechanically compressed. Notably, CTGF expression levels correlated with the amount of peri-adipocyte fibrosis in WAT from obese individuals. Moreover, dMAT-dependent CTGF promoter activity, which depends on ß1-integrin/cytoskeleton pathways, was enhanced in the presence of YAP, a mechanosensitive co-activator of TEAD transcription factors. Mutation of TEAD binding sites abolished the dMAT-induced promoter activity. In conclusion, fibrosis may negatively affect human adipocyte function via mechanosensitive molecules, in part stimulated by cell deformation.

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.

Curvularia Blight on Lolium perenne on Turfgrasses in Argentina.

Lolium perenne L. is commonly used alone or in association with blue-grass and fescues in sport fields, parks, and gardens. During 2003, symptoms of an unknown disease were observed on L. perenne turfgrass in western Buenos Aires. Initial symptoms were indefinite yellow and green dappled spots that extended downward from the leaf tip, turned brown and finally gray, causing leaf death. Segments of symptomatic leaf tissues were surface sterilized and placed on 2% potato dextrose agar in petri dishes. After 4 days at room temperature, blackish brown colonies developed with dark brown septate conidiophores. Conidia were 21 to 29 × 9 to 13 µm, 3-septa, curved at the third cell from the base that is longer and darker than the others. Cells at each end are subhyaline and intermediate cells are medium brown. These characteristics are consistent with Curvularia lunata (Wakker) Boedijng (1). Pathogenicity tests were performed in five plastic trays with substrate of natural soil and sand (1:1 [v/v]) where the turfgrass (L. perenne cv. El Cencerro) was seeded. Plants were inoculated by spraying a suspension of 2 × 106 conidia per ml of sterile distilled water. Controls were sprayed with sterile distilled water. The trays were covered with transparent plastic bags, sprayed periodically with water, and incubated at 25°C in a greenhouse for 20 days. The first symptoms were observed 3 days later. After 9 days, 24% of the grass surface area showed blight lesions. C. lunata was consistently reisolated from affected tissues. Control plants remained symptomless. To our knowledge, this is the first report of C. lunata affecting L. perenne in Argentina. Reference: (1) M. B. Ellis and I. A. S. Gibson Cochliobolus lunatus (conidial state: Curvularia lunata). Page 474 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1975.

Occurrence of Blight Caused by Sclerotium rolfsii on Lolium perenne in Argentina.

A perennial ryegrass (Lolium perenne L.) lawn located at Castelar (Buenos Aires Province) showed disease symptoms during the summer of 2003. Chlorotic patches as much as 15 cm in diameter appeared on the lawn. Later, dead plants with white mycelia developing on the crown and surrounding soil occurred at the periphery of the rings. Plants showed leaf chlorosis and crown and root rot. No sclerotia developed on plant organs. Diseased plants were collected, washed with running tap water for 4 h, and disinfested in 5% NaOCl for 2 min. Pieces, 3 to 5 mm long from symptomatic leaves, crowns, and roots, were incubated on 2% potato dextrose agar (PDA) at 22 to 25°C with a 12-h light/dark cycle. Mycelia growing on the soil surface was transferred to PDA and incubated under the same conditions. After 3 to 4 days, white, conspicuous mycelia that produced sclerotia grew from diseased tissue pieces and soil mycelial samples. Sclerotia were nearly spherical, 1 to 2 mm in diameter, white but turning brown with age, and produced in large numbers over the entire colony surface. Primary hyphae showed clamp connections at the septa. A pathogenicity test was performed with 20 1-month-old plants of L. perenne grown in a 1:1 (v/v) mixture of sand and soil contained in 24- × 17- × 4-cm plastic trays. Seven-day-old fungal cultures grown on PDA were cut into 1- cm2 pieces and placed among the plants on the substrate. Each tray was inoculated with seven inoculum pieces. Five trays of plants were inoculated with the fungus, and plants in five trays that served as controls had only sterile pieces of PDA placed on the substrate. All plants were maintained at 25°C and watered frequently. First symptoms, consisting of chlorosis, were observed 4 days after inoculation. Of the plants, 34, 59, 60, 65, and 70% developed symptoms 6, 9, 14, 17, and 21 days after inoculation, respectively. Control plants remained healthy. The fungus was reisolated from diseased plants and identified as Sclerotium rolfsii Sacc. (teleomorph Athelia rolfsii (Curzi) C.C. Tu & Kimbr.) on the basis of morphological and cultural characteristics (3,4). The disease has been observed causing stalk rot on perennial ryegrass in the United States (1) and Australia (2). To our knowledge, this is the first report of S. rolfsii causing disease on L. perenne in Argentina. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society. St. Paul, MN. 1989. (2) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory. Online publication. ARS, USDA, 2007. (3) J. E. M. Mordue. No. 410 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, UK, 1974. (4) Z. K. Punja and A. Damiani. Mycologia 88:694, 1996.

Monensin and forskolin inhibit the transcription rate of sucrase-isomaltase but not the stability of its mRNA in Caco-2 cells.

Treatment of Caco-2 cells with forskolin (25 microM) or monensin (1 microM) has previously been shown to cause a marked decrease in the level of sucrase-isomaltase (SI) mRNA, without any effect on the expression of dipeptidylpeptidase IV (DPP-IV). In the present work, we report that there is no significant difference in the stability of SI mRNA between control and treated cells. On the other hand, we demonstrate a decrease in the transcription rate of SI mRNA which is sufficient to account for the decrease in the steady-state level of SI mRNA both in forskolin- and monensin-treated Caco-2 cells.

Glucose-dependent transcriptional regulation of the human sucrase-isomaltase (SI) gene.

We have previously shown that the transcription of the human sucrase-isomaltase (SI) gene was negatively regulated by glucose. Using two clonal metabolic variants of the human colon adenocarcinoma cell line Caco-2 we demonstrate here that: 1) although similar growth-related variations of phosphoenolpyruvate carboxykinase (PEPCK), frutose 1,6-diphosphatase (F1, 6-dPase), pyruvate kinase (PK) and SI mRNA levels are observed, only F1,6-dPase, PK and SI mRNA levels vary in the same way in response to modifications of glucose utilization; and 2) regulatory elements responsible for the glucose-dependent transcription of the SI gene are located within the -370/+30 region of the promoter.

Modulation of rat preadipocyte adipose conversion by androgenic status: involvement of C/EBPs transcription factors.

Androgenic status affects rat preadipocyte adipose conversion from two deep intra-abdominal (epididymal and perirenal) fat depots differently. The aim of this study was to establish whether these site-specific alterations of adipogenesis are related to altered expressions of the transcriptional factors regulating proliferation and differentiation of preadipocytes, c-myc and CCAAT/enhancer binding proteins (C/EBPs: C/EBPalpha and beta). The increased proliferation of epididymal and perirenal preadipocytes from castrated rats was not linked to variations in c-myc mRNA and protein levels. The expression of the early marker of adipogenesis, lipoprotein lipase (LPL), was decreased by androgenic deprivation in epididymal cells but remained insensitive to the androgenic status in perirenal preadipocytes. In contrast, LPL expression increased in subcutaneous preadipocytes from castrated rats, an effect which was partly corrected by testosterone treatment. Expression of C/EBPbeta was unaffected by androgenic status whatever the anatomical origin of the preadipocytes. In contrast, the mRNA and protein levels of C/EBPalpha were greatly decreased by androgenic deprivation in epididymal cells, an alteration which could not be corrected by in vivo testosterone administration. Altogether these results demonstrated that in preadipocytes androgenic deprivation affects site-specifically the expression of LPL, an early marker of adipogenesis and of C/EBPalpha, a master regulator of adipogenesis. These observations contribute to an explanation of why castration induces defective adipose conversion in rat epididymal preadipocytes specifically.

Structure of the phosphatidylcholines of the lung surfactant at birth in normal full term infants.

1. This investigation was undertaken for the purpose of determining the structure of the phosphatidylcholines of lung surfactant system present at birth in normal full term newborn infants. 2. The procedure, using tracheal aspirates as lung secretions, combines a cold-acetone precipitation and a two-dimensional thin-layer chromatography of the lipid extract. 3. Different species of phosphatidylcholines were isolated and found to account together for over 60% of the total phospholipids in tracheal aspirates. Analysis of the fatty acids esterifying the alpha- and beta-carbon of these different phosphatidylcholines showed palmitic acid as the major component with little myristic acid. 4. This fatty acid analysis revealed furthermore that the major phosphatidylcholine fraction was almost exclusively alpha, beta-dipalmitoylphosphatidylcholine. 5. This study shows that the procedure described provides a useful and simple method for the extraction, isolation and characterisation of the functional components of lung surfactant in living human newborns.

Studies on the phospholipids in tracheal aspirate from normal full term newborn infants. Comparison with amniotic fluid.

The phospholipids in tracheal aspirates from normal full term newborn infants were studied and compared to the phospholipids present in the corresponding amniotic fluids. The same classes of phospholipids were recovered from both fluids, i.e. lysophosphatidylcholine (LPC), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (S), phosphatidyldimethylethanolamine (PDME), phosphatidylethanolamine (PE), polyglycerophosphatides (PGP), phosphatidic acids (PA) and two different species of phosphatidylcholine (PC-1 and PC-2). Except for PA, the proportion as well as the fatty acid composition of each of these phospholipids was similar in tracheal aspirates and in amniotic fluids. The predominant fraction (PC-1) was the most saturated one. PI, PDME, LPC and S were, like PC-1, predominantly constituted by saturated fatty acids and accounted together for a concentration equivalent to that of PC-1. Since tracheal aspirate originates from the lung, these data suggest that the pulmonary surfactant activity in normal full term newborn infants results not only from the presence of PC-1, but also from that of PI, PDME, LPC and S.

[Susceptibility of Streptococcus pyogenes isolates from pharyngeal exudates in Cordoba (Spain)]. / Sensibilidad de aislamientos faríngeos de Streptococcus pyogenes en la provincia de Córdoba (España).

Streptococcus pyogenes is an important human pathogen. Betalactams are still the drug of choice for the treatment of infections caused by this microorganism. In recent years an increase in the use of macrolides for initial treatment in respiratory infections has been observed; consequently, the number of macrolide-resistant isolates has also increased. We investigated the susceptibility of S. pyogenes to penicillin, erythromycin, clarithromycin and clindamycin in Cordoba during 2000, 2001 and the first 6 months of 2002. We obtained 100 isolates of S. pyogenes from 1232 pharyngeal exudates, all of which were susceptible to penicillin and 39 of which were resistant to erythromycin and clarithromycin. Twenty-six of these 39 isolates were susceptible to clindamycin.

[Ludwig's angina, its evolution and treatment]. / Angina de Ludwig, evolución y tratamiento.

An anaerobic neck infection located in the submandibular layer is described in this paper. We report a case of satisfactory evolution and make a bibliographic review of this problem. All the AA. agree that the key to successful management of such infections is early diagnosis, prompt surgery and associated antibiotic therapy.

[Update of surgical criteria in thyroid pathology]. / Actualización de los criterios quirúrgicos en la patología tiroidea.

Review of an aleatory sample composed by 100 patients suffering some thyroid disorder, operated at ENT Department of the Hospital, during the last 5 years. Statistical evaluation of the significance and reliability of several parameters, clinical items, diagnostic tests and surgical judgements contrasted with the corresponding nowadays bibliography.

[Neuroendocrine carcinoma of the larynx. Review of literature in reference to one case report]. / Carcinoma neuroendocrino de laringe. Revisión de la literatura a propósito de un caso.

Neuroendocrin tumors are unusual neoplasies, even more within the ENT-sphere. Report of 1 case treated in our Service. Review of immunohistochemical criteria with the aim of achieving the diagnosis. Some bibliographical series are analyzed with the target to pick up the treatment of these uncommon growths.

[Anomalies of the first branchial cleft]. / Anomalías de la primera hendidura branquial.

Account of the case of a little girl of four and a half years brought to the consulting otologist because of her earache, otorrhoea, retroauricular inflammatory swelling and oedema of the external ear canal on left side. Frequency, evolution, diagnostic means and management measures for first branchial cleft are reviewed.

Physical Map of the Channel Catfish Virus Genome: Location of Sites for Restriction Endonucleases EcoRI, HindIII, HpaI, and XbaI.

The overall arrangement of nucleotide sequences in the DNA of channel catfish virus has been studied by cleavage with four restriction endonucleases. Physical maps have been developed for the location of sites for EcoRI, HindIII, HpaI, and XbaI. The sum of the molecular weights of fragments generated by each restriction enzyme indicates a molecular weight of approximately 86 x 10(6) for the channel catfish virus genome. Fragments corresponding to the molecular ends of channel catfish virus DNA have been identified by their sensitivity to exonuclease treatment. The distribution of restriction sites in the genome shows that sequences included in a 12 x 10(6)-molecular weight region at one end are repeated with direct polarity at the other end, and that the overall genomic sequence order is nonpermuted.

Activity of hepatocyte nuclear factor 1alpha and hepatocyte nuclear factor 1beta isoforms is differently affected by the inhibition of protein phosphatases 1/2A.

Phosphorylation/dephosphorylation processes are known to control the activity of several transcription factors. The nutrition-dependent expression of sucrase-isomaltase and Na+/glucose co-transporter 1, two proteins implicated in the intestinal absorption of glucose, has been shown to be closely related to modifications of hepatocyte nuclear factor 1 (HNF1) activity. This study was conducted to determine whether phosphorylation/dephosphorylation processes could control HNF1 activity. We show that expression of the gene encoding sucrase-isomaltase is inhibited in the enterocytic Caco-2 clone TC7 by okadaic acid at a concentration that is known to inhibit protein phosphatases 1/2A and that does not affect cell viability. At the same concentration, phosphorylation of the HNF1alpha and HNF1beta isoforms is greatly enhanced and their DNA-binding capacity is decreased. The phosphorylation state of HNF1beta isoforms directly affects their DNA-binding capacity. In contrast, the decreased DNA-binding activity of the HNF1alpha isoforms, which was observed after the inhibition of protein phosphatases 1/2A, is due to a net decrease in their total cellular and nuclear amounts. Such an effect results from a decrease in both the HNF1alpha mRNA levels and the half-life of the protein. This is the first evidence for the implication of protein phosphatases 1/2A in the control of the activity of HNF1 isoforms. Moreover, these results emphasize a physiological role for the balance between phosphatases and kinases in the nutrition-dependent regulation of HNF1-controlled genes.

Transcriptional activity in 3T3, F9, and PCC4 embryonal carcinoma cells: a systematic deletion and linker-scanning study of the polyomavirus enhancer.

The polyomavirus (PyV) genome is not expressed in undifferentiated embryonal carcinoma (EC) cells such as PCC4 or F9 EC cells. All the viral mutants that have been selected for their expression in these cells harbor mutations or rearrangements within a region which is important for early and late transcription (as transcriptional enhancer) as well as for viral DNA replication. We have studied the role of the different parts of this enhancer on the transcription driven by early PyV promoter. Two series of constructions containing progressive deletions starting from the ends of the region and one series containing linker-scanning mutations were tested in a luciferase assay in three cell lines: 3T3, F9, or PCC4. The results revealed that some regions that have not yet been shown to bind transcription factors are nevertheless important for transcriptional activity. Two subregions between nucleotides 5179 and 5187 and between 5220 and 5227 were found to be inhibitory for the activity of the enhancer in EC cells. The PEA1/AP1 binding site was also unexpectedly shown to be important in F9 EC cells.

Two HNF-1 binding sites govern the glucose repression of the human sucrase-isomaltase promoter.

We have previously shown, using the Caco-2 clone PF11, that glucose represses transcription of the human sucrase-isomaltase (SI) gene and that the -370/+30 fragment of the SI gene conferred glucose-regulated expression on a heterologous gene. Different fragments beginning at the already characterized SI footprint (SIF) 1 (-53/-37), SIFR (-153/-129) or SIF3 (-176/-156) elements [Wu, Chen, Forslund and Traber (1994) J. Biol. Chem. 269, 17080-17085] were tested, in comparison with the -370/+30 fragment, for their capacity to inhibit reporter gene expression under high-glucose (25 mM) conditions. Unlike SIF1 and SIFR, the addition of the HNF (hepatocyte nuclear factor)-1-binding element SIF3 to the promoter fragment was required for repression under high-glucose conditions. This effect was enhanced when the SI promoter was extended to position -370, indicating that the -370/-176 region contains elements that may co-operate with SIF3 to increase the metabolic control of the SI promoter. We have characterized an additional HNF-1-binding site near to and upstream from SIF3; SIF4. By mutagenesis of the three HNF-1-binding elements we show that the two distal HNF-1-recognition sites are the most important for the glucose regulation of the SI gene. Moreover, this glucose regulation was abolished in PF11 cells overexpressing vHNF-1C (variant HNF, an isoform of the HNF-1 family). We thus propose that the differential binding of HNF-1-family proteins to their DNA targets on the SI promoter constitutes the molecular mechanism that controls the glucose regulation of the SI gene transcription.

A limited upstream region of the human sucrase-isomaltase gene confers glucose-regulated expression on a heterologous gene.

We have previously shown that glucose can exert a repressive effect on the transcription of the sucrase-isomaltase (SI) gene in the differentiated enterocyte-like human colon carcinoma cell lines HT-29 and Caco-2. To characterize the region through which glucose exerts this effect, three different-length fragments of the 5'-flanking region of the human SI gene were linked to the reporter gene luciferase in an episomal vector carrying a hygromycin resistance gene. These fragments were used for transfection into a clone of the Caco-2 cell line, PF11, which has high glucose consumption and only expresses SI at high levels when cultured in the presence of a low supply of glucose. By using the stably transformed PF11 cells grown either in standard high glucose (25 mM) or in low glucose (1 mM) it was possible to show that the smallest fragment of the SI promoter, extending from bases -370 to +30, contains all the information required for the glucose repression of the reporter gene luciferase.

Molecular cloning and characterization of endogenous SV40 DNA from human HBL-100 cells.

The human HBL-100 cell line harbours SV40 DNA integrated in tandem at a unique site. The SV40 T-antigen expressed in these cells is defective in a function essential to the replication of the viral genome. The integrated SV40 sequences were molecularly cloned in a bacteriophage, and a subclone (plasmid pSVHBI) containing a complete SV40 DNA was isolated. As compared to SV40 wild-type strain 776, sequence analysis of pSVHBI early region revealed the presence of several DNA alterations. Among these, a point mutation at position 3199, predicting a change at amino-acid 540 of arginine to isoleucine, was shown by marker rescue to be responsible for the deficiency of T-antigen. This novel mutation further delimits one of the T-antigen domains involved in SV40 DNA replication. Transfection experiments demonstrated that the transforming activity of the SV40 genome from HBL-100 cells is still preserved. Moreover, several transformed human cell clones thus obtained could be permanently established in culture.