Expression of human genes for adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase after genetic transformation of mouse cells with purified human DNA

Human DNA purified from HeLa cells and from three strains of skin fibroblasts was precipitated with calcium phosphate and added to mouse cells that were deficient in adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT). Selection for cells possessing either of the phosphoribosyltransferases was imposed by blocking de novo synthesis of purine nucleotides with azaserine in a medium supplemented with adenine and hypoxanthine. The frequency of colony formation after selection was 1.7×10^–7–3.3×10^–6. Excepting some azaserine-resistant colonies that appeared only in the first experiment and infrequent revertants expressing mouse APRT, all characterized clones expressed the human forms of APRT or HPRT according to the criteria of specific immunoprecipitation and electrophoretic mobility. The frequency of transfer of the human APRT gene was much greater than that of HPRT. Transfer efficiency was not significantly reduced when HeLa DNA was sheared to 6.5–13.5 kb size or when the donor DNA was isolated from a transferent that expressed human APRT.     

In search of candidate genes critically expressed in the human endometrium during the window of implantation

BACKGROUND: In this prospective randomized blinded clinical trial, we examined gene expression profiles of the human endometrium during the early and mid-luteal phases of the natural cycle. METHODS: An endometrial biopsy was performed on day 16 (LH +3) or on day 21 (LH +8), followed by RNA extraction and microarray analysis using an Affymetrix HG-U95A microchip. Data analysis was carried out using pairwise multiple group comparison with the significance analysis of microarrays (SAM) software. RESULTS: With a false discovery rate of 0, the analysis revealed that 107 genes were significantly and differently expressed (> or =2-fold) during the early versus the mid-luteal phase of the cycle. Forty-five of these genes have not been previously linked to endometrial receptivity. Validation of the microarray data was accomplished using semiquantitative RT-PCR. We demonstrated the presence of estrogen and progesterone response elements (ERE and PRE) by analysis of the 5'-flanking regions of a subset of differentially regulated genes. CONCLUSIONS: Using a strict bioinformatics approach of microarray data, we demonstrated significant changes in candidate genes during the transition of the early to the mid-luteal phase of the human endometrium that may have functional significance for the opening and maintenance of the window of implantation.     

Number of germ cells and somatic cells in human fetal ovaries during the first weeks after sex differentiation

BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal ovaries during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the gonads. METHODS: One ovary with attached mesonephros from each of 11 individual legal abortions was used for estimation of cell numbers. After recovery of the fetus, the ovary-mesonephric complexes were immediately isolated, fixed and processed for histology. A stereological method was utilized to estimate the total number of oogonia in all ovaries and somatic cells in seven of them. RESULTS: The number of oogonia per ovary increased from approximately 26,000 in week 6 to approximately 250,000 in week 9 and somatic cells from approximately 240,000 to approximately 1.4 x 10(6). The ratio of oogonia to somatic cells tended to increase throughout the period. The concentration of oogonia was similar in the cranial (mesonephric connected) part and the caudal part of the ovaries. CONCLUSIONS: This is the first stereological estimation of the number of oogonia and somatic cells in human fetal ovaries, and the first estimation of germ cells and somatic cells in ovaries aged <9 weeks. The number of oogonia in week 9 is comparable to the numbers previously published based on non-stereological estimations. We found early stages of meiosis in fetal ovaries from week 9.     

Number of germ cells and somatic cells in human fetal testes during the first weeks after sex differentiation

BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal testes during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the testes. METHODS: One testis with attached mesonephros from each of 10 individual legal abortions was used. After recovery of the fetus, the testes were immediately isolated, fixed and processed for histology. The optical fractionator technique, a stereological method, was utilized to estimate the total number of germ cells in ten testes and somatic cells in six of them. RESULTS: The number of germ cells per testis increased from approximately 3000 in week 6 to approximately 30000 in week 9. The ratio of germ cells to Sertoli cells was approximately 1:11 and the ratio of germ cells to somatic cells was approximately 1:44 throughout this period. CONCLUSIONS: For the first time, germ cell and somatic cell number have been determined during early human fetal testis development. Knowledge of the number of germ cells in this period may be very important, because several environmental pollutants are suspected to result in decreased semen quality in men born of mothers exposed to these pollutants during pregnancy.     

Expression profiling of IL-10-regulated genes in human monocytes and peripheral blood mononuclear cells from psoriatic patients during IL-10 therapy

Interleukin-10 (IL-10), originally identified as an inhibitor of pro-inflammatory cytokine production, exerts multiple immunomodulatory functions. Its ability to inhibit a Th1 response has been used in clinical trials for the treatment of inflammatory diseases including psoriasis. However, little is known about the molecular mechanisms of IL-10 functions. We aimed at identifying possible mediators of in vitro IL-10 treatment in monocytes by gene chip technology using Hu95a Affymetrix mRNA arrays with 12,000 genes. To prove relevance of the identified genes for the clinical situation we compared these in vitro results with genes being regulated by IL-10 in peripheral blood mononuclear cells from psoriatic patients undergoing IL-10 therapy. A high proportion of the 1,600 genes up-regulated and 1,300 genes down-regulated in vitro was found to be similarly regulated in vivo. Some genes, which were previously unknown to be regulated by IL-10, can be assigned to known IL-10 functions like e.g. the increase of pathogen clearance. Other new potentially immunomodulating genes have been identified to be regulated by IL-10, but their impact needs to be experimentally evaluated. We could confirm a recently reported up-regulation of heme oxygenase-1 (HO-1). However, we demonstrate that the anti-inflammatory mechanisms of IL-10 remain functional even when HO-1 is irreversibly inhibited.     

The evolution of the search for novel genes in mammalian sex determination: from mice to men

Disorders of sex determination are a genetically heterogeneous group of rare disorders, presenting with sex-specific phenotypes and variable expressivity. Prior to the advent of the Human Genome Project, the identification of novel mammalian sex determination genes was hindered by the rarity of disorders of sex determination and small family sizes that made traditional linkage approaches difficult, if not impossible. This article reviews the revolutionary role of the Human Genome Project in the history of sex determination research and highlights the important role of inbred mouse models in elucidating the role of identified sex determination genes in mammalian sex determination. Next generation sequencing technologies has made it possible to sequence complete human genomes or exomes for the purpose of providing a genetic diagnosis to more patients with unexplained disorders of sex determination and identifying novel sex determination genes. However, beyond novel gene discovery, these tools have the power to inform us on more intricate and complex regulation-taking place within the heterogeneous cells that make up the testis and ovary.     

Expression survey of genes critical for tooth development in the human embryonic tooth germ.

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks.     

Expression of immunomodulatory genes, their protein products and specific ligands/receptors during the window of implantation in the human endometrium

We have demonstrated up-regulation of the immunomodulatory genes decay accelerating factor (DAF), interleukin 15 (IL-15) and osteopontin (OPN) during the window of implantation (WOI). Here, we characterized gene expression and determined the localization of their protein products and respective ligands at the opening and closure of the WOI. In addition, we used laser capture microdissection (LCM) to analyze the cell type-specific gene expression. Human endometrial biopsies from cycle Days 16, 21 and 24 were evaluated by real-time RT-PCR. Purified epithelial and stromal cells were obtained by LCM. Localization of the proteins and their ligands was assessed by immunohistochemistry. mRNA expression of DAF, IL-15 and OPN was significantly increased throughout the WOI. DAF, OPN and alpha(v)beta(3) integrin were strongly immunolocalized to the glandular compartment by Days 21 and 24, whereas C3, IL-15 and IL-15Ralpha were highly stained in both glandular and stromal compartments. After LCM, gene expression of DAF was 4.8-fold increased in epithelium versus stroma, whereas for OPN there was a 2-fold increase. For IL-15, the expression in stroma was 8.7-fold higher than in epithelial cells. The progressive increase of the expression of these immunomodulatory genes, proteins and ligands during the WOI, support a critical role at the time of endometrial receptiveness.     

Evidence for an increase in presumed somatic mutation during the ageing of human cells in culture.

A method has been developed for the direct measurement of genetic variants in mammalian cells in culture which does not require cell division and is therefore suitable for ageing studies. Previous evidence suggests that these variants are mutations. The variant frequency was measured during the lifespan of populations of MRC-5 human embryo lung fibroblasts. The frequency increased substantially during the lifespan of the culture and regression analysis shows that the increase is exponential. No increase invariant frequency was found in a immortal transformed line cultured for over 100 passages. Evidence is presented that the variants are regulatory mutations. This is the first direct evidence for the involvement of presumed somatic mutations in the ageing of mammalian cells. 5-Fluorouracil but not p-fluorophenylalanine shortened the life of the cells and increase the variant frequency. The data lend support to a model in which mutations are an expected consequence of errors in protein synthesis.     

Analysis of clones from a human cartilage cDNA library provides insight into chondrocyte gene expression and identifies novel candidate genes for the osteochondrodysplasias

To begin to define the gene expression pattern in fetal cartilage and to identify uncharacterized candidate genes for the osteochondrodysplasias, we analyzed clones from a fetal cartilage cDNA library. Sequence analysis of 420 cDNA clones identified 210 clones derived from established genes but, for many of them, expression in cartilage had not been previously reported. Among the established genes were 14 genes known to produce skeletal abnormalities in either humans or mice when mutated. Thirty-two uncharacterized genes and their respective chromosomal positions were also identified. To further understand the expression profile of these genes in fetal cartilage, we constructed a cDNA microarray utilizing the clones. The microarray was used to determine which genes had higher expression in cartilage as compared with dedifferentiated, cultured chondrocytes. Many of the established genes, as well as five of the uncharacterized genes, had increased expression in cartilage, suggesting an important role for these genes in the differentiated state of chondrocytes. These data provide new candidate genes for the osteochondrodysplasias and demonstrate the usefulness of cartilage cDNA microarrays in expanding our understanding of the complexity of fetal cartilage gene expression.     

Expression of a candidate marker for progenitor cells, Musashi-1, in the proliferative regions of human antrum and its decreased expression in intestinal metaplasia

AIM: Reliable makers for progenitor cells in the human stomach have not been elucidated. The aim of the present study was to clarify whether Musashi-1 (Msi-1), which has recently been proposed as a stem cell marker in mouse intestine, serves as a marker for progenitor cells in human stomach. METHODS AND RESULTS: Immunohistochemistry revealed that Msi-1+ cells were detected especially in the isthmus/neck region (the putative position of stem cells) of the adult antrum, but were limited to the basal regions of fetal pyloric glands during the early stages of development. These results suggest that Msi-1 expression occurs specifically in the stem cell-containing regions. Msi-1+ cells were intermingled with proliferating cell nuclear antigen (PCNA)+ cells in the isthmus/neck region of the adult antrum, but did not coexpress PCNA or Ki 67. Msi-1 expression overlapped partly with expression of MUC 5 AC and MUC 6, indicating that Msi-1+ cells retain some features of both foveolar and pyloric gland cell differentiation phenotypes. In contrast, Msi-1 expression in gastric glands showing intestinal metaplasia (IM) became weaker than that in the glands without IM. CONCLUSION: The specific expression of Msi-1 within the proliferative regions suggests that Msi-1 is a marker of cells with progenitor characteristics before active proliferation in human antrum.     

Expression of the W6/32 HLA epitope by cells of rat, mouse, human and other species: critical dependence on the interaction of specific MHC heavy chains with human or bovine beta 2-microglobulin

The HLA class I epitope W6/32 is conformationally dependent on both heavy chain and beta 2-microglobulin (beta 2M). Previously, the W6/32 epitope has been detected in humans and other primates as well as from bovine sources. Two controversial reports suggest the W6/32 epitope is constitutively expressed by either normal or transformed murine cells expressing the Db allele. Here we show that the appearance of the W6/32 epitope in murine cells results from the association of either the Db or Kd gene products with either bovine or human beta 2M. We use congenic mouse strains and hybrid H-2 class I genes between Db and Kb to map the W6/32 epitope to particular amino acid residues in the alpha 2 domain. Subsequently, we show that beta 2M exchange is not confined to murine or human cells in vitro but can be detected after beta 2M injection into a mouse. The data presented suggests that beta 2M exchange takes place at the cell surface under physiological conditions and indicates that MHC class I heavy chains are in an equilibrium between the bound and unbound form of beta 2M.     

Molecular Ig gene analysis reveals that monocytoid B cell lymphoma is a malignancy of mature B cells carrying somatically mutated V region genes and suggests that rearrangement of the kappa-deleting element (resulting in deletion of the Ig kappa enhancers) abolishes somatic hypermutation in the human

Five cases of monocytoid B cell lymphoma (MBCL) were analyzed for somatic mutations in the rearranged V region genes. Somatic mutations were found in four of the five cases, whereas one unusual CD5⁺ lymphoma harbored unmutated V region genes. Since somatic mutations are introduced into V regiongenes of antigen-activated B cells in the course of T cell-dependent immune responses, these results suggest a derivation of the tumor B cells in MBCL from antigen-experienced mature B cells. An analysis of the ϰ-deleting element in two of the cases in which mutated V_H but unmutated and nonfunctional V_ϰ gene rearrangements were found suggests that somatic hypermutation does not take place in human rearranged V_ϰ region genes when the C_ϰ gene and the ϰ enhancers have been deleted in cis by rearrangement of the ϰ-deleting element.     

Expression of a novel secreted splice variant of the receptor for advanced glycation end products (RAGE) in human brain astrocytes and peripheral blood mononuclear cells

The engagement of the receptor for advanced glycation end products (RAGE) on the cell surface induces cellular dysfunction in a number of pathophysiological situations of vascular dysfunction, tumor cell invasion, inflammatory response, and T cell infiltration. The administration of truncated, soluble RAGE can modulate RAGE-mediated perturbations. Here, we report a novel splice variant (delta8-RAGE) of RAGE mRNA, which lacks exon 8 of the genomic RAGE gene and contains an early stop codon in exon 10 due to a frame shift mutation. delta8-RAGE mRNA was found in human primary astrocytes and peripheral blood mononuclear cells (PBMCs). Transient transfection experiments demonstrated that delta8-RAGE mRNA was translated into a secretory protein as deduced. Moreover, two different segments of the spliced variant were identified in PBMCs by RT-PCR. The findings of this study suggest that the diverse splicing variants of RAGE are possible in many tissues and their products may influence the RAGE-mediated pathogenesis and immune modulation.