PubMed Health⌕ Search

Biomedical subjects

M Chami

Publications and source records attributed to M Chami.

At least 19 recordsLinked to original sources
PubMed2003

Membrane protein reconstitution and crystallization by controlled dilution.

Efficient reconstitution of membrane proteins for functional analyses can be achieved by dilution of a ternary mixture containing proteins, lipids and detergents. Once the dilution reaches the point where the free detergent concentration would become lower than the critical micellar concentration, detergent is recruited from the bound detergent pool, and association of proteins and lipids is initiated. Here we show that dilution is also suitable for the assembly of two-dimensional crystals. A device has been designed that allows controlled dilution of a protein-lipid-detergent mixture to induce formation of densely packed or crystalline proteoliposomes. Turbidity is used to monitor the progress of reconstitution on-line, while dilution is achieved by computer-controlled addition of buffer solution in sub-microliter steps. This system has mainly been tested with porin OmpF, a typical beta-barrel protein, and aquaporin-1, a typical alpha-helical protein. The results demonstrate that large, highly ordered two-dimensional crystals can be produced by the dilution method.

Aquaporin 1↗
PubMed2003

Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications.

Corynebacteria belong to a distinct Gram-positive group of bacteria including mycobacteria and nocardia, which are characterized by the presence of mycolic acids in their cell wall. These bacteria share the property of having an unusual cell envelope structural organization close to Gram-negative bacteria. In addition to the inner membrane, the cell envelope is constituted of a thick arabinogalactan-peptidoglycan polymer covalently linked to an outer lipid layer, which is mainly composed of mycolic acids and probably organized in an outer membrane like structure. In some species, the cell is covered by a crystalline surface layer composed of a single protein species, which is anchored in the outer membrane like barrier. An increasing number of reports have led to a better understanding of the structure of the cell wall of Corynebacterium glutamicum. These works included the characterization of several cell wall proteins like S-layer protein and porins, genetic and biochemical characterization of mycolic acids biosynthesis, ultrastructural description of the cell envelope, and chemical analysis of its constituents. All these data address new aspects regarding cell wall permeability towards macromolecules and amino acids but also open new opportunities for biotechnology applications.

Biotechnology↗
PubMed2002

[Association of ovarian juvenile granulosa cell tumor with Maffucci's syndrome].

The authors report a rare case of the ovarian juvenile granulosa cell tumor associated with Maffucci's syndrome (enchondromathosis + hemangiomas), no heriditary mesodermal dysplasia. Sarcomatous changes of chondromas are encountered most frequently; however other various typed neoplasma have been reported: ovarian juvenile granulosa cell tumor may occur not infrequently in female patients with Maffucci's syndrome in the first or second decades. Sarcomatous changes of choromas established prognosis of the Maffucci's syndrome.

Adolescent↗
PubMed2001

Identification of human cancer-related genes by naturally occurring Hepatitis B Virus DNA tagging.

Proviral tagging has been used in animals as a powerful tool for cancer genetics. We show that a similar approach is possible in patients with hepatocellular carcinoma (HCC) infected by Hepatitis B Virus (HBV), a human pararetrovirus which may act by insertional mutagenesis. In this work, the HBV genome is used as a probe to identify cancer-related genes. By using HBV-Alu-PCR, we obtained 21 HBV/cellular DNA junctions from 18 different patients. In six of 21, we found the HBV DNA integrated into a cellular gene: (1) Sarco/Endoplasmic Reticulum Calcium ATPase1 Gene; (2) Thyroid Hormone Receptor Associated Protein 150 alpha Gene; (3) Human Telomerase Reverse Transcriptase Gene; (4) Minichromosome Maintenance Protein (MCM)-Related Gene; (5) FR7, a new gene expressed in human liver and cancer tissues; and (6) Nuclear Matrix Protein p84 Gene. Seven junctions contained unique cellular sequences. In the remaining eight, the HBV DNA was next to repetitive sequences, five of them of LINE1 type. The cellular genes targeted by HBV are key regulators of cell proliferation and viability. Our results show that studies on HBV-related HCCs allow to identify cellular genes involved in cancer. We therefore propose this approach as a valuable tool for functional cancer genomic studies in humans.

Base Sequence↗
PubMed2001

Two-dimensional crystallization of membrane proteins: the lipid layer strategy.

Due to the difficulty to crystallize membrane proteins, there is a considerable interest to intensify research topics aimed at developing new methods of crystallization. In this context, the lipid layer crystallization at the air/water interface, used so far for soluble proteins, has been recently adapted successfully to produce two-dimensional (2D) crystals of membrane proteins, amenable to structural analysis by electron crystallography. Besides to represent a new alternative strategy, this approach gains the advantage to decrease significantly the amount of material needed in incubation trials, thus opening the field of crystallization to those membrane proteins difficult to surexpress and/or purify. The systematic studies that have been performed on different classes of membrane proteins are reviewed and the physico-chemical processes that lead to the production of 2D crystals are addressed. The different drawbacks, advantages and perspectives of this new strategy for providing structural information on membrane proteins are discussed.

Air↗
PubMed2001

hH-Rev107, a class II tumor suppressor gene, is expressed by post-meiotic testicular germ cells and CIS cells but not by human testicular germ cell tumors.

By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene. In this study, two transcripts (1 and 0.8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12-q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a ninefold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors.

Amino Acid Sequence↗
PubMed2001

SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

Adult↗
PubMed2001

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins.

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.

Bacterial Outer Membrane Proteins↗
PubMed2000

Use of detergents in two-dimensional crystallization of membrane proteins.

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.

Crystallization↗
PubMed2000

Hepatitis B virus-related insertional mutagenesis implicates SERCA1 gene in the control of apoptosis.

We have used the Hepatitis B Virus DNA genome as a probe to identify genes clonally mutated in vivo, in human liver cancers. In a tumor, HBV-DNA was found to be integrated into the gene encoding Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA), which pumps calcium, an important intracellular messenger for cell viability and growth, from the cytosol to the endoplasmic reticulum. The HBV X gene promoter cis-activates chimeric HBV X/SERCA1 transcripts, with splicing of SERCA1 exon 11, encoding C-terminally truncated SERCA1 proteins. Two chimeric HBV X/SERCA1 proteins accumulate in the tumor and form dimers. In vitro analyses have demonstrated that these proteins localize to the ER, determine its calcium depletion and induce cell death. We have also shown that these biological effects are related to expression of the SERCA, rather than of the viral moiety. This report involves for the first time the expression of mutated SERCA proteins in vivo in a tumor cell proliferation and in vitro in the control of cell viability. Oncogene (2000).

Aged↗
PubMed1997

Reconstitution of FhuA, an Escherichia coli outer membrane protein, into liposomes. Binding of phage T5 to Fhua triggers the transfer of DNA into the proteoliposomes.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of ferrichrome and is the receptor of bacteriophage T5. Purified FhuA was reconstituted into liposomes. The size of the proteoliposomes and the distribution of the proteins in the vesicles were determined by freeze fracture electron microscopy. Unilamellar vesicles with a diameter larger than 200 nm were observed frequently. FhuA was symetrically oriented in the proteoliposomes. Reconstituted FhuA was functional as binding of phage T5 induced the release of phage DNA and its transfer inside the vesicles.

Bacterial Outer Membrane Proteins↗
PubMed1997

The S-layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C-terminal hydrophobic domain.

PS2 is the S-layer protein of Corynebacterium glutamicum. The S-layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70 degrees C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S-layer, which remains organized. Because we show that trypsin cleaves the C-terminal part of the protein, we conclude that this domain is involved in the association of the S-layer to the cell but is not essential in the interaction between individual PS2 proteins within the S-layer. A modified form of PS2, deleted of its C-terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S-layer and is mainly released into the medium. We suggest that PS2 is anchored via its C-terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed1997

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

ATP-Binding Cassette Transporters↗
PubMed1997

[Imaging of AIDS in children].

In children, AIDS is mainly related to materno-foetal transmission. Due to antiviral therapy and prevention of infections, the initially very poor prognosis has improved and the length of survival has increased. There are two groups of children: the first (25%) in which the disease occurs early and is very severe, a second one in which the disease develops later after an asymptomatic period which can last several years. Manifestations of AIDS in children are mainly pulmonary and digestive infections, central nervous system infections are much rarer than in adults, neurologic disorders are mainly due to the HIV itself. Tumors are also rarer than in adults but may occur, including lymphomas and smooth muscle tumors.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Ultrasound contribution in the analysis of the newborn and infant normal and clubfoot: a preliminary study.

The aim of this study was to evaluate the potential use of ultrasound in the preoperative assessment of clubfoot and follow-up growth. We first studied 50 normal newborns and infants (aged 1 day to 1 year). The normal values established were reliable, dynamic and reproducible measurements allowing the assessment of osteo-articular relationships and cartilage morphology. Ultrasound appears to be more useful than radiography because it allows delineation of cartilaginous structures (which are not visible on conventional radiographs) and dynamic study of foot positions, and has no danger from radiation. A future paper will contain preliminary results of cases of clubfoot studied by ultrasound.

Clubfoot↗
PubMed1995

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: a combined freeze-etch electron microscopy and biochemical study.

The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze-fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimensions as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.

Bacterial Proteins↗
PubMed1991

[MRI and chronic osteoarticular infections. Contribution of inversion-recuperation sequence with fat signal cancellation in the follow-up of chronic osteitis under medical treatment. Apropos of 14 cases].

MR Imaging was performed with T1 weighted, Rho T2* (Grass) and S.T.I.R. sequences on medically treated chronic osteomyelitis patients. MR Imaging is definitely the best tool available in thorough evaluation of the extent of lesions. It allows the evolution or remission of focal inflammatory lesions to be traced for follow up. Bone sequestra needing surgery can be identified and soft tissue abcesses differentiated from cellulitis. In future, MR Imaging could replace tomography and fistulography, while scintigraphy would be more useful in cases of recent acute infections and their differential diagnoses.

Adolescent↗