Gene expression profiling of 3T3-L1 adipocytes exposed to phloretin

Adipocyte dysfunction plays a major role in the outcome of obesity, insulin resistance and related cardiovascular complications. Thus, considerable efforts are underway in the pharmaceutical industry to find molecules that target the now well-documented pleiotropic functions of adipocyte. We previously reported that the dietary flavonoid phloretin enhances 3T3-L1 adipocyte differentiation and adiponectin expression at least in part through PPARγ activation. The present study was designed to further characterize the molecular mechanisms underlying the phloretin-mediated effects on 3T3-L1 adipocytes using microarray technology. We show that phloretin positively regulates the expression of numerous genes involved in lipogenesis and triglyceride storage, including GLUT4, ACSL1, PEPCK1, lipin-1 and perilipin (more than twofold). The expression of several genes encoding adipokines, in addition to adiponectin and its receptor, is positively or negatively regulated in a way that suggests a possible reduction in systemic insulin resistance and obesity-associated inflammation. Improvement of insulin sensitivity is also suggested by the overexpression of genes associated with insulin signal transduction, such as CAP, PDK1 and Akt2. Many of these genes are PPARγ targets, confirming the involvement of PPARγ pathway in the phloretin effects on adipocytes. In light of these microarray data, it is reasonable to assume that phloretin may be beneficial for reducing insulin resistance, in a similar way to the thiazolidinedione class of antidiabetic drugs.     

Genome Sequence of Streptococcus gallolyticus: Insights into Its Adaptation to the Bovine Rumen and Its Ability To Cause Endocarditis

Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is an increasing cause of endocarditis among streptococci and frequently associated with colon cancer. S. gallolyticus is part of the rumen flora but also a cause of disease in ruminants as well as in birds. Here we report the complete nucleotide sequence of strain UCN34, responsible for endocarditis in a patient also suffering from colon cancer. Analysis of the 2,239 proteins encoded by its 2,350-kb-long genome revealed unique features among streptococci, probably related to its adaptation to the rumen environment and its capacity to cause endocarditis. S. gallolyticus has the capacity to use a broad range of carbohydrates of plant origin, in particular to degrade polysaccharides derived from the plant cell wall. Its genome encodes a large repertoire of transporters and catalytic activities, like tannase, phenolic compounds decarboxylase, and bile salt hydrolase, that should contribute to the detoxification of the gut environment. Furthermore, S. gallolyticus synthesizes all 20 amino acids and more vitamins than any other sequenced Streptococcus species. Many of the genes encoding these specific functions were likely acquired by lateral gene transfer from other bacterial species present in the rumen. The surface properties of strain UCN34 may also contribute to its virulence. A polysaccharide capsule might be implicated in resistance to innate immunity defenses, and glucan mucopolysaccharides, three types of pili, and collagen binding proteins may play a role in adhesion to tissues in the course of endocarditis.Several studies have reported that the proportion of infective endocarditis due to Streptococcus gallolyticus has increased during the last decades, concomitantly with a decrease of cases due to oral streptococci (35). S. gallolyticus is now becoming the first cause of infectious endocarditis among streptococci in Europe (16). Furthermore, S. gallolyticus endocarditis is associated with rural residency, suggesting transmission from animals (29). However, the reasons for the emergence of this pathogen remain poorly understood. S. gallolyticus belongs to the Streptococcus bovis group known for more than 60 years to cause endocarditis (45). Recently, the former species S. bovis has been divided into four major species (50, 53). S. gallolyticus corresponds to S. bovis biotype I (mannitol fermentation positive), the closely related species S. pasteurianus to biotype II/2 (mannitol negative and β-glucuronidase positive), and the more distantly related species S. infantarius to biotype II/1 (mannitol negative and β-glucuronidase negative). S. macedonicus, the fourth species, commonly found in cheese, is nonpathogenic and also considered a S. gallolyticus subspecies (53, 62). A majority of endocarditis cases was due, among the formerly S. bovis group, to S. gallolyticus strains (4).Multiple studies have shown that endocarditis due to S. gallolyticus as well as positive blood culture for this species is often associated with gastrointestinal malignancy (4, 6). This association has led to a strong indication for gastrointestinal investigation and endoscopic follow-up in the case of S. gallolyticus infections (66). The association of S. gallolyticus infection with colon cancer is a major but still unsolved issue. It may be just incidental, as the alteration of the digestive mucosa may favor the translocation of the bacteria into the bloodstream. Alternatively, the tumor may contribute to the proliferation of S. gallolyticus in close proximity to the gut epithelium, increasing its probability of translocating through the gut barrier. It has also been suggested that the bacterium itself contributes to carcinogenesis (60, 69). In addition to human disease, S. gallolyticus may also cause diseases in animals, like septicemia in pigeons (19), outbreaks in broiler flocks (11), or bovine mastitis (28).Independent from its association to disease, S. gallolyticus has been isolated as a tannin-resistant bacterium from the feces of different mammalian herbivores, including the koala (48) or the Japanese large wood mouse (52), and it is also a normal inhabitant of the rumen (39). Its resistance to tannins is linked to its tannase activity, a characteristic which also led this bacterium to be named “gallolyticus” as it is able to decarboxylate gallate, an organic acid derived from tannin degradation. S. gallolyticus is also known to express other degradative functions unique among streptococci, like a bile salt hydrolase or an amylase. These properties allow its multiplication outside the animal host, as S. gallolyticus was isolated from a digester fed with shea cake (derived from the nuts of the African tree Vitellaria paradoxa) rich in tannins and aromatic compounds (12). S. gallolyticus is a commensal of the human intestinal tract but remains a rarely detected (2.5 to 15%) low-abundance species (10, 40). In herbivores, overgrowth of S. bovis may become deleterious. For example, ingestion of large amounts of rapidly fermented cereal grains leads to a destabilization of the rumen flora and to the proliferation of acid-tolerant bacteria, including S. gallolyticus. This is accompanied by the overproduction of mucopolysaccharides that stabilize the foam, resulting in feedlot bloat, a significant cause of economical loss (14).Virulence and colonization factors of S. gallolyticus in humans are largely unknown. Studies of the bird host have shown that this Streptococcus species expresses a capsular polysaccharide, and five different serotypes have been described (19). In addition, electron microscopy studies have revealed the presence of fimbria-like structures on the surface of S. gallolyticus. It was hypothesized that capsules and/or fimbriae are involved in virulence (63). S. gallolyticus isolates responsible for endocarditis exhibited heterogeneous patterns of adherence to extracellular matrix (ECM) proteins, which suggests that they produce different surface components (55). Recently, a collagen binding adhesin together with 10 putative ECM binding proteins were identified in the draft genome sequence of a human isolate of S. gallolyticus (54).Here we describe the sequence and analysis of the genome of S. gallolyticus strain UCN34 isolated from a human case of endocarditis associated with colon cancer. Analysis of the predicted proteins revealed unique metabolic and cell surface features among streptococci, which contribute to its adaptation to the rumen and to its ability to cause endocarditis. We showed by comparative genomics that many of the corresponding genes were probably acquired by lateral gene transfer (LGT) from other Firmicutes of the gut microbiota.     

Loss of Multi-Epitope Specificity in Memory CD4+ T Cell Responses to B. Pertussis with Age

Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4⁺ T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4⁺ T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4⁺ T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.     

Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly

One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies. Received: 22 November 1995 / Accepted: 23 February 1996     

Angiogenesis in the pathogenesis of inflammatory joint and lung diseases

This paper reviews hypotheses about roles of angiogenesis in the pathogenesis of inflammatory disease in two organs, the synovial joint and the lung. Neovascularisation is a fundamental process for growth and tissue repair after injury. Nevertheless, it may contribute to a variety of chronic inflammatory diseases, including rheumatoid arthritis, osteoarthritis, asthma, and pulmonary fibrosis. Inflammation can promote angiogenesis, and new vessels may enhance tissue inflammation. Angiogenesis in inflammatory disease may also contribute to tissue growth, disordered tissue perfusion, abnormal ossification, and enhanced responses to normal or pathological stimuli. Angiogenesis inhibitors may reduce inflammation and may also help to restore appropriate tissue structure and function.     

The role of MHC class I heterodimer expression in mouse ankylosing enthesopathy

 Ankylosing enthesopathy (ANKENT) is a spontaneous mouse joint disease with strikingly similar pathology to human HLA-B27-associated enthesopathies such as ankylosing spondylitis. In C57Bl/10 mice, transgenic HLA-B^*2702 as well as H2 genes have been shown to be relative risk factors for ANKENT. To investigate the role of major histocompatibility complex (MHC) class I expression in disease pathogenesis, ANKENT occurrence was compared among β₂-microglobulin (β₂m) knockout littermates with or without transgenes for HLA-B^*2702 and human β₂m. In the knockout phenotype lacking β₂m, ANKENT occurrence is significantly reduced (P = 0.016). In the absence of β₂m, B^*2702 is not detected on the cell membrane, nor does it increase the risk for ANKENT. This means that the previous finding that HLA-B^*2702 increases susceptibility to ANKENT in C57Bl/10 mice cannot be ascribed to a transgene insertion effect. Rather, in order to increase disease susceptibility, B^*2702 must be coexpressed with mouse β₂m (mo-β₂m). In contrast, when HLA-B^*2702 is expressed with β₂m of human origin, disease susceptibility is not affected. Thus, both H2^b-derived class I heterodimers and HLA-B^*2702/mo-β₂m heterodimers contribute to ANKENT susceptibility.     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.