Biomedical subjects

A Pawlak

Publications and source records attributed to A Pawlak.

At least 19 recordsLinked to original sources

PubMed2004

Identification of transcripts by macroarrays, RT-PCR and in situ hybridization in human ejaculate spermatozoa.

Round spermatids contain high levels of extremely varied mRNAs that are synthesized either throughout early spermatogenesis or during spermiogenesis from the haploid genome. Concomitantly, with major changes in the chromatin organization, arrest of transcription occurs at midspermiogenesis. However, previous investigations using RT-PCR have revealed the persistence of numerous and different transcripts in ejaculated spermatozoa. In the present study, a step-by-step analysis by means of macroarray hybridization, RT-PCR and in situ hybridization was performed to identify more accurately the different mRNA species found in the human ejaculated spermatozoa. The data showed an extended pattern of various transcripts encoding a diverse range of proteins involved in signal transduction and cell proliferation. For the first time, they demonstrated that mRNAs coding for the transcription factors NFkappaB, HOX2A, ICSBP, protein kinase JNK2, growth factor HBEGF and receptors RXRbeta and ErbB3 accumulate within the sperm nucleus. The origin and fate of the sperm transcripts remain subject to discussion.

Ejaculation↗

PubMed2001

Human testis expresses a specific poly(A)-binding protein.

In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids.

Amino Acid Sequence↗

PubMed2001

Mapping the distribution of emissive molecules in human ocular lipofuscin granules with near-field scanning optical microscopy.

Several high resolution imaging techniques are utilized to probe the structure of human ocular lipofuscin granules. Atomic force microscopy reveals typical granule sizes to be about one micrometre in diameter and hundreds of nanometres in height, in agreement with previous electron microscopy results. For issues concerning the role of lipofuscin in age-related macular degeneration, recent attention has focused on the orange-emitting fluorophore, A2E. Confocal microscopy measurements are presented which reveal the presence of a highly emissive component in the granules, consistent with the presence of A2E. It is shown, however, that the interpretation of these results is complicated by the lack of structural details about the particles. To address these issues, near-field scanning optical microscopy (NSOM) measurements are presented which measure both the lipofuscin fluorescence and topography, simultaneously. These measurements reveal distinct structure in the fluorescence image which do not necessarily correlate with the topography of the granules. Moreover, direct comparison between the NSOM fluorescence and topography measurements suggests that A2E is not the major component in lipofuscin. These measurements illustrate the unique capabilities of NSOM for probing into the microstructure of lipofuscin and uncovering new insights into its phototoxicity.

Aged↗

PubMed2001

Structure of the 5' sequences of the human gamma-glutamyltransferase gene.

In humans, five distinct mRNAs code for gamma-glutamyltransferase (GGT). Their coding regions are identical and their 5' untranslated regions exhibit both common and type-specific sequences. To elucidate the mecanisms that generate these different mRNAs, we cloned and determined the structure of the 5' region of the human GGT gene. The common regions of the 5' UTR are encoded by five exons, localized within a 2.4-kb region of the genomic DNA. Three of them are separated only by intron-donor or intron-acceptor sites at their boundaries. Alternative splicing of these exons may determine the unique pattern of the different GGT mRNA 5' UTRs in a tissue-specific manner. In addition, we have isolated a genomic fragment containing the most distal 5' sequences of the major GGT mRNA in HepG2 cells. Primer extension analysis revealed one major transcription initiation site while 5' RACE indicated that one more distal initiation site could be present. In the putative promoter sequence neither classical TATA or CAAT boxes were found. However, sites for AP1, AP2, CREB, GRE and SP1 transcription factors were identified. Chimeric plasmids, containing this genomic region fused to the luciferase gene, were transiently expressed in three cell lines of different origin: HeLa cells, ovarian carcinoma A2780 cells and V79 lung fibroblasts. The significant promoter activities obtained indicate a transcription start within this region. However, differences in the level of expression were found between the different cell lines used. These data suggest that the human GGT gene employs regulatory sequences and alternative splicing, and gene expression may therefore be regulated in tissue specific and cell-type-specific manners.

5' Untranslated Regions↗

PubMed2001

Meiotic human sperm cells express a leucine-rich homologue of Caenorhabditis elegans early embryogenesis gene, Zyg-11.

We cloned a human protein (Hzyg) homologue to Caenorhabditis elegans Zyg-11, an essential protein for cell division at the initial developmental stages of this species, and to a Drosophila melanogaster gene product (Mei-1) which is likely to be involved in meiosis. Hzyg mRNA encodes a protein of 766 amino acids (88 kDa), 14% of which are leucine residues, with some being arranged in four leucine rich repeat motives usually involved in protein-protein interactions. Hzyg is encoded by a single gene, located on chromosome 9q32-q34.1, and transcribed as two mRNA: a 5 kb transcript strongly expressed in testis and skeletal muscle and barely detectable in other human tissues, and an abundant 3.1 kb mRNA detected only in the testis. By using in-situ hybridization and immunohistochemistry, we clearly established the presence of Hzyg expression in pachytene spermatocytes (stage V) and spermatids (stage I and/or II) around the time of meiosis. The cell specific expression of Hzyg transcripts in testis, and the conservation of this gene among distant species, suggest that this protein may have an important role during meiosis.

Adenosine Triphosphatases↗

PubMed2001

Expression of the TAR RNA binding protein in human testis.

In testis, several RNA binding proteins have been shown to play a role in the translational regulation of specific transcripts. The human protein TRBP (TAR RNA binding protein) is the homologue of the mouse Prbp (Prm-1 RNA binding protein) involved in the protamine mRNA translational delay. TRBP is known to activate the HIV-1 long terminal repeat but this protein has never been investigated during spermatogenesis. The aim of this work was to analyse the TRBP expression in human testis. By Northern blot analysis, we demonstrated a major 1.5 kb transcript present at a high level in human testis and, to a lesser extent, in some other tissues. In-situ hybridization revealed that this transcript was present only in elongating spermatids. Antibodies raised against a 27 amino acid TRBP-specific peptide revealed a single protein of 43 kDa expressed in the cytoplasm of elongated spermatids. At the ultrastructural level, quantitative analysis of both TRBP mRNA and protein, using electron microscopy in-situ hybridization and immunocytochemistry, showed that TRBP is expressed mainly in spermatids at steps 3-4 of spermiogenesis. These results are in agreement with the probable role of TRBP in the control of human protamine mRNA translation.

Gene Expression↗

PubMed2001

Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy.

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale approximately 150 nm.

Humans↗

PubMed1999

Chromosomal localization of three human poly(A)-binding protein genes and four related pseudogenes.

In humans, the poly(A)-binding proteins (PABPs) comprise a small nuclear isoform and a conserved gene family that displays at least three functional proteins: PABP1, inducible PABP (iPABP), and PABP3, plus four pseudogenes (1, 2, 3, and PABP4). In situ hybridization of PABP3 cDNA as the probe on metaphasic chromosomes have revealed five possible loci for this gene family at 2q21-q22, 13q11-q12, 12q13.3-q15, 8q22, and 3q24-q25. Amplifications of specific DNA fragments from a human-rodent somatic cell hybrid panel have allowed us to associate PABP1 and PABP3 with 8q22 and 13q11-q12, respectively. The iPABP gene has been assigned to chromosome 1. This result, compared with radiation hybrid database information, strengthens the location of this gene to 1p32-p36. The pseudogenes PABP4, 1, and 2 have been assigned to chromosomes 15, 4, and 14, respectively. Three loci detected on chromosome spreads are not associated with any amplified fragment. They might represent other related PABP genes not yet identified.

Animals↗

PubMed1998

Identification and mapping of 26 human testis mRNAs containing CAG/CTG repeats.

Various types of pathologies, including neurodegenerative diseases, as well as different types of neoplasia, are related to genes exhibiting simple tandem repeat instabilities. In order to seek for new candidate genes for such disorders, we screened 4.10(6) human testis cDNAs for CAG- and CTG-containing clones. Among 910 positive clones, we characterized 109 cDNAs corresponding to 26 independent mRNAs. Fourteen of these mRNAs represent new genes. The corresponding clones contain between 3 and 19 consecutive CAG or CTG triplets. We assigned 15 out of these 26 genes to 14 different human chromosomes. These genes represent new potential candidates for diseases associated with CAG or CTG repeat mutations.

Chromosome Mapping↗

PubMed1997

Molecular cloning and characterization of a mono(ADP-ribosyl)transferase from human testis.

A human homologue of the rodent T cell mono(ADP-ribosyl)transferase RT6 mRNA was identified by a systematic partial sequencing of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono(ADP-ribosyl)transferase and a C terminal part which contains three repeated motives (GEKNQKLEDH) and a region characteristic of glycophosphatidyl inositol anchored proteins. This mRNA is transcribed from a gene localized in 4q13-q21. Surprisingly, it is not expressed in human white blood cells but it exhibits a very specific testis expression in which it is likely to correspond to a new ADP-ribosyl transferase.

ADP Ribose Transferases↗

PubMed1997

The intermediate layer: a morphologic study of the elastin and hyaluronic acid constituents of normal human vocal folds.

The lamina propria of vocal folds are important in voice production. We evaluated the morphologic features of elastin and hyaluronic acid, two important constituents of the lamina propria. Thirty normal human vocal folds were obtained from patients dying of traumatic causes without vocal fold injury. These tissues were immediately prepared for histologic and ultrastructural examination by standard methods. For specific study of the ultrastructure of the layers of the lamina propria, six vocal folds were divided horizontally through the midplane of the lamina propria. We found that the elastin composition of the vocal folds is variable, the largest amount being seen in the midportion on elastin-van Gieson (EVG) staining and ultrastructural evaluation. The superficial layer of the lamina propria contains fewer large elastin fibers. In this region, we found that elastin was predominantly composed of elaunin and oxytalan, which stain poorly with EVG. Using computer-assisted image analysis, we quantified the differences in elastin composition between the layers. The amount of elastin varied between men and women, and these differences could not be accurately measured by the methods employed. Hyaluronic acid was abundant especially in the midportion of the lamina propria and was significantly more abundant in men than women on quantification. The significance of these observations in normal vocal folds is discussed.

Adult↗

PubMed1997

Human PEX1 is mutated in complementation group 1 of the peroxisome biogenesis disorders.

Human peroxisome biogenesis disorders (PBDs) are a group of genetically heterogeneous autosomal-recessive disease caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. These lethal diseases include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum's disease (IRD), three phenotypes now thought to represent a continuum of clinical features that are most severe in ZS, milder in NALD and least severe in IRD2. At least eleven PBD complementation groups have been identified by somatic-cell hybridization analysis compared to the eighteen PEX complementation groups that have been found in yeast. We have cloned the human PEX1 gene encoding a 147-kD member of the AAA protein family (ATPases associated with diverse cellular activities), which is the putative orthologue of Saccharomyces cerevisiae Pex1p (ScPex1p). Human PEX1 has been identified by computer-based 'homology probing' using the ScPex1p sequence to screen databases of expressed sequence tags (dbEST) for human cDNA clones. Expression of PEX1 rescued the cells from the biogenesis defect in human fibroblasts of complementation group 1 (CG1), the largest PBD complementation group. We show that PEX1 is mutated in CG1 patients.

Adenosine Triphosphatases↗

PubMed1997

Identification and chromosomal localization of human genes containing CAG/CTG repeats expressed in testis and brain.

Human genes containing triplet repeats have been demonstrated to be involved in several neurodegenerative diseases by expansion of the repeat in succeeding generations. To identify novel genes involved in such pathologies, we have isolated transcripts containing (CAG/CTG)n repeats using two approaches. First, we screened 4 x 10(6) clones representing 10 copies of a human testis cDNA library using a (CAG)14 oligonucleotide probe. Among the 910 clones identified, the 243 clones with the strongest hybridization signal were sequenced partially from 3' or 5' ends. This provided us with 251 partial sequences that grouped into clusters corresponding to 39 genes, of which 19 represent unknown species. Second, we selected 203 additional ESTs containing (CAG/CTG)n repeats representing 121 clusters from the IMAGE consortium infant brain cDNA library. From these two series of sequences, we have localized 95 genes on human chromosomes using a panel of whole genome radiation hybrid (Genebridge 4). These genes are located on all of the chromosomes except for chromosome X, the highest density being observed on chromosome 19.

Brain↗

PubMed1996

Human testis specifically expresses a homologue of the rodent T lymphocytes RT6 mRNA.

A human homologue of the rodent T cell mono ADP-ribosyl transferase RT6 mRNA was identified by a systematic analysis of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono ADP-ribosyl transferase and a C-terminal part characteristic of glycophosphatidyl inositol anchored protein. This mRNA, transcribed from a gene localized in 4q13-q21, is not expressed in white blood cells but is specific for human testis in which it is likely to correspond to a new ADP-ribosyl transferase.

ADP Ribose Transferases↗

PubMed1996

A 94 kb genomic sequence 3' to the murine Xist gene reveals an AT rich region containing a new testis specific gene Tsx.

X chromosome inactivation in both mouse and human requires the presence of a cis acting locus, the X inactivation centre. This locus is thought to be involved in the initiation and spreading of the inactivation signal in early development. In order to increase our understanding of the mouse X inactivation centre, a 94 kb region immediately distal to the Xist gene has been sequenced and analysed for the presence of transcription units and/or potential cis acting regulatory elements. We have identified a novel gene, Tsx, lying 40 kb 3' from Xist. Tsx is expressed specifically in the testis and shows no convincing homology to proteins currently in the databases. A rat homologue, also X linked, has been isolated. The mouse and rat Tsx sequences are highly divergent, suggesting that part of the X inactivation centre, including both Xist and Tsx are subject to relatively weak evolutionary constraints.

Animals↗

PubMed1996

High-level expression of functional human gamma-glutamyl transpeptidase using the baculovirus system.

The understanding of the structure and function of gamma-glutamyl transpeptidase (GGT) has been hindered by the difficulty of obtaining large quantities of functional enzyme. A recombinant baculovirus, encoding the human hepatoma cell (Hep G2) GGT, was easily purified using a histochemical procedure to reveal GGT activity. Infected insect cells synthesized a large amount of enzymatically active GGT representing up to 10% of the total cell extract protein. The GGT specific activity of the infected cells was 13 units per mg of protein which is the highest GGT expression level reported to date, 260-times more than in Hep G2 cells. The recombinant protein displayed an apparent molecular mass (M(r), 58,000 for the heavy subunit), immunoreactivity and catalytic features similar to those of the native protein. The high-level expression of functional GGT should provide an excellent tool to further study the structure-function relationships of the protein.

Animals↗

PubMed1996

Identification of a cat eye syndrome using DNA sequence dosage analysis.

We report here the detection of a Cat Eye Syndrome (CES) in a woman who does not exhibit the related phenotype, due to intensive surgery. The analysis of her karyotype reveals a small supernumerary bisatellited chromosome likely to correspond to a fragment of chromosome 13, 15, 21 or 22 on banding analysis. Southern blot of genomic DNA of this patient and her parents hybridized with probes specific of these chromosomes, revealed a DNA amplification of the 22q11 region for the patient, likely to correspond to a CES.

Abnormalities, Multiple↗

PubMed1995

Characterization of a large population of mRNAs from human testis.

We present the results of single-pass sequencing of 779 expressed sequence tags from normal human testis cDNA clones. Of the sequences generated, 319 (41%) appeared to be completely unknown and are likely to represent new genes, and 289 (37%) were identified based on exact or nonexact matches to sequences in public databases. In analyses of hybridization of four tissues, testis, brain, liver, and kidney, 6 of 12 cDNAs clones revealed testis-specific expression. This argues for the value of the combination of random sequencing and analysis of cellular expression for large-scale characterization of gene expression in the testis.

Adult↗