Carmelo B. Bruni

Rab‐interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes

Rab7 is a small GTPase that controls transport to endocytic degradative compartments. Here we report the identification of a novel 45 kDa protein that specifically binds Rab7GTP at its C‐terminus. This protein contains a domain comprising two coiled‐coil regions typical of myosin‐like proteins and is found mainly in the cytosol. We named it RILP (Rab‐interacting lysosomal protein) since it can be recruited efficiently on late endosomal and lysosomal membranes by Rab7GTP. RILP‐C33 (a truncated form of the protein lacking the N‐terminal half) strongly inhibits epidermal growth factor and low‐density lipoprotein degradation, and causes dispersion of lysosomes similarly to Rab7 dominant‐negative mutants. More importantly, expression of RILP reverses/prevents the effects of Rab7 dominant‐negative mutants. All these data are consistent with a model in which RILP represents a downstream effector for Rab7 and both proteins act together in the regulation of late endocytic traffic.

Increased BDNF Promoter Methylation in the Wernicke Area of Suicide Subjects

CONTEXT Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the pathophysiology of suicidal behavior and BDNF levels are decreased in the brain and plasma of suicide subjects. So far, the mechanisms leading to downregulation of BDNF expression are poorly understood. OBJECTIVES To test the hypothesis that alterations of DNA methylation could be involved in the dysregulation of BDNF gene expression in the brain of suicide subjects. DESIGN Three independent quantitative methylation techniques were performed on postmortem samples of brain tissue. BDNF messenger RNA levels were determined by quantitative real-time polymerase chain reaction. SETTING Academic medical center. PATIENTS OR OTHER PARTICIPANTS Forty-four suicide completers and 33 nonsuicide control subjects of white ethnicity. MAIN OUTCOME MEASURES The DNA methylation degree at BDNF promoter IV and the genome-wide DNA methylation levels in the brains Wernicke area. RESULTS Postmortem brain samples from suicide subjects showed a statistically significant increase of DNA methylation at specific CpG sites in BDNF promoter/exon IV compared with nonsuicide control subjects (P < .001). Most of the CpG sites lying in the -300/+500 region, on both strands, had low or no methylation, with the exception of a few sites located near the transcriptional start site that had differential methylation, while genome-wide methylation levels were comparable among the subjects. The mean methylation degree at the 4 CpG sites analyzed by pyrosequencing was always less than 12.9% in the 33 nonsuicide control subjects, while in 13 of 44 suicide victims (30%), the mean methylation degree ranged between 13.1% and 34.2%. Higher methylation degree corresponded to lower BDNF messenger RNA levels. CONCLUSIONS BDNF promoter/exon IV is frequently hypermethylated in the Wernicke area of the postmortem brain of suicide subjects irrespective of genome-wide methylation levels, indicating that a gene-specific increase in DNA methylation could cause or contribute to the downregulation of BDNF expression in suicide subjects. The reported data reveal a novel link between epigenetic alteration in the brain and suicidal behavior.

Role of the small GTPase Rab7 in the late endocytic pathway.

Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.

Helicobacter pylori Up-regulates Cyclooxygenase-2 mRNA Expression and Prostaglandin E2 Synthesis in MKN 28 Gastric Mucosal Cells in Vitro

Helicobacter pylori has been suggested to play a role in the development of gastric carcinoma in humans. Also, mounting evidence indicates that cyclooxygenase-2 overexpression is associated with gastrointestinal carcinogenesis. We studied the effect of H. pylori on the expression and activity of cyclooxygenase-1 and cyclooxygenase-2 in MKN 28 gastric mucosal cells. H. pylori did not affect cyclooxygenase-1 expression, whereas cyclooxygenase-2 mRNA levels increased by 5-fold at 24 h after incubation of MKN 28 cells with broth culture filtrates or bacterial suspensions from wild-type H. pyloristrain. Also, H. pylori caused a 3-fold increase in the release of prostaglandin E2, the main product of cyclooxygenase activity. This effect was specifically related toH. pylori because it was not observed withEscherichia coli and was independent of VacA, CagA, or ammonia. H. pylori isogenic mutants specifically lackingpicA or picB, which are responsible for cytokine production by gastric cells, were less effective in the up-regulation of cyclooxygenase-2 mRNA expression and in the stimulation of prostaglandin E2 release compared with the parental wild-type strain. This study suggests that development of gastric carcinoma associated with H. pylori infection may depend on the activation of cyclooxygenase-2-related events.

Co-operative regulation of endocytosis by three Rab5 isoforms.

Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.

Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons.

We have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels.

Galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas

Galectin‐1 and galectin‐3 are galactoside‐binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional‐cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin‐1 mRNA levels were highly increased in most high‐grade tumors compared with normal bladder or low‐grade tumors. Western blot and immuno‐histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin‐1 in tumors. Galectin‐3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade. Int. J. Cancer (Pred. Oncol.) 84:39–43, 1999.

Control of mRNA processing and decay in prokaryotes

Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5′ to 3′ exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3′ to 5′ exoribonucleases are involved in chemical decay of mRNA. As the 3′ to 5′ exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3′ ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3′ to 5′ exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5′ to 3′ directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5′ to 3′ processive ‘5′ binding nuclease’. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts.

Galectin genes: Regulation of expression

In this review we have summarized the more recent studies on the expression of mammalian galectins. One interesting observation that can be made is that in most of microarrays and/or differential display analysis performed in recent years one or more galectins have been picked up. From a critical evaluation of the pertinent studies the main conclusion that can be drawn is that, although it is not yet clear whether the 14 galectins identified so far have functions in common, a striking common feature of all galectins is the strong modulation of their expression during development, differentiation stages and under different physiological or pathological conditions. This suggests that the expression of different galectins is finely tuned and possibly coordinated. In spite of these observations it is rather unexpected that very few studies have been performed on the molecular mechanisms governing the activity of galectin genes. Published in 2004.

Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells.

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.