Application of MALDI-TOF mass spectrometry in screening and diagnostic research.

During the last years, mass spectrometry has revolutionised protein biochemistry and has advanced to a superior tool for the identification and detailed analysis of peptides and proteins. The high throughput allowed by some mass spectrometry platforms has enabled the important step from analysis of individual proteins to proteomics. Recently, an additional field of mass spectrometry applications has emerged - namely screening and diagnostic research. In contrast to protein identification, screening applications have to detect analyte molecules of defined molecular weights which can be calculated beforehand, for example by means of chemical structures. Here, the accuracy and sensitivity of mass spectrometry has to be combined with the requirements of high-throughput analyses, in particular speed and automation. These criteria are especially fulfilled by state of the art matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) instruments. The first high throughput screening (HTS) application proved to be genotyping of single nucleotide polymorphisms. The same principle was later applied for several quality control issues, for example for oligonucleotides, peptide or compound libraries. This development has culminated in the screening and profiling of complex biomarker patterns in clinical proteomics to detect a molecular fingerprint for specific diseases in biological samples. Thus, mass spectrometry based methods are expected to enable a very early diagnosis of diseases with minimally invasive methods of investigation. This type of high end screening application has the potential to revolutionise the early diagnosis of many diseases. Here, we give an overview of the application of mass spectrometry in the fields of screening and diagnostic research.

Glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha-1 and GFRalpha-2 in the human testis.

Glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha-1 and GFRalpha-2 were found in the human testis during fetal development (15-34 weeks of gestation) and in adult men (51-86 years of age) by means of RT-PCR, immunohistochemistry and Western blot techniques. Gene expression of GDNF could be established in the human testis and immunoreactivity (IR) for GDNF was detectable in Leydig cells, Sertoli cells, some spermatocytes and round spermatids as well as in smooth muscle cells of the wall of arterioles and small arteries. In the adult human testis, Sertoli and Leydig cells showed GFRalpha-1-IR, whereas GFRalpha-2-IR was located exclusively in Leydig cells. Different to man, in the rat GDNF-IR in Sertoli cells was detectable only until postnatal day10, providing evidence for species related variability in the expression of GDNF. These findings suggest a critical role for GDNF during the differentiation of testicular structures and provide evidence for an additional important function in the adult human and rodent testis.

Genotools SNP manager: a new software for automated high-throughput MALDI-TOF mass spectrometry SNP genotyping.

Analysis of single nucleotide polymorphisms (SNPs) is a rapidly growing field of research that provides insights into the most common type of differences between individual genomes. The resulting information has a strong impact in the fields of pharmacogenomics, drug development, forensic medicine, and diagnostics of specific disease markers. The technique of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a highly suitable tool for the analysis of DNA. It supplies a very versatile method for addressing a high-throughput SNP genotyping approach. Here, we present the Bruker genotools SNP MANAGER, a new software tool suitable for highly automated MALDI-TOF MS SNP genotyping. The genotools SNP MANAGER administers the sample preparation data, calculates masses of allele-specific primer extension products, performs genotyping analysis, and displays the results. In the current study, we have used the genotools SNP MANAGER to perform an automated duplex SNP analysis of two biallelic markers from the promoter of the gene encoding the inflammatory mediator interleukin-6.

Progressive inactivation of the haploid expressed gene for the sperm-specific endozepine-like peptide (ELP) through primate evolution.

The endozepine-like peptide (ELP) is a novel intracellular molecule which is expressed in high amounts at both mRNA and protein levels very specifically in late haploid male germ cells. It is closely related to the ubiquitous acyl-CoA binding protein, is highly conserved, shares a similar ability to bind mid-long chain acyl-CoA, and is thus likely to be involved in mature sperm metabolism. While it has been characterized from diverse mammals, it has so far not been possible to identify an equivalent molecule in the primate testis. Using a PCR approach, combined with cDNA cloning and Northern hybridization, testicular transcripts and/or genomic DNA were analysed for different primate species, including human. In the marmoset and cynomolgus macaque normally structured transcripts appear to be expressed, though at a low level. In the human testis, two rare transcripts were characterized, hELP1 and hELP2, the products of independent duplicated genes. Both transcripts were longer than in non-mammalian species, included frame-shift mutations and substantial sequence insertions, preventing the translation of a sensible protein. Genomic PCR analysis of three anthropoid species, chimpanzee, gorilla and orangutan, showed the presence of a similarly mutated hELP1 gene. Only in the gorilla was a hELP2 gene identified, apparently lacking the frame-shift mutation, and thus potentially able to give rise to a functional ELP protein. Taken together, these results show that during primate evolution there has been a progressive inactivation of the ELP gene, initially with a down-regulation in lower primates, and subsequently with inactivating mutations in the open reading frame. At some time during simian evolution prior to these mutations there has been a gene duplication, though this second gene has also become inactivated in humans. In its pattern of evolution the ELP gene shows similarities with the MDC/fertilin family, whose members are also considered essential components of haploid sperm in non-primates, but which are progressively inactivated in anthropoids and humans. We should like to speculate that the established subfertility of the human male may not be a recent event, but the consequence of a longer evolutionary process whereby primates have traded off absolute fertility against social or sexual advantages.

Structure and expression of the mouse gene encoding the endozepine-like peptide from haploid male germ cells.

The endozepine-like peptide (ELP) represents a testis-specific isoform of the ubiquitous acyl-CoA binding protein (ACBP) and is highly expressed in late haploid stages of male germ cell development. The genomic sequence of the functional ELP gene as well as that of a pseudogene were analysed from independent bacteriophage clones of a 129sv mouse genomic library. Unlike the ACBP gene, which comprises four exons, the ELP gene has only a single intron within the region of the 5' untranslated region, suggesting that, like some other haploid expressed genes, the ELP gene might have evolved by retroposon-mediated gene duplication. Primer extension analysis was used to define the start site for transcription and hence the 5' promoter region. Electrophoretic mobility shift analysis was carried out on this region comparing nuclear extracts from adult mouse testis with those from mouse liver. Several testis-specific DNA-protein complexes were observed throughout 700 bp upstream of the transcription start site. One of these could be identified as corresponding to a steroidogenic factor-1 (SF-1) binding element. Further analysis using pure transcription factors showed that this element at position -340 was able to bind specifically to both SF-1 and to the germ cell nuclear factor (GCNF). Immunohistochemical analysis using an ELP-specific antibody showed that expression was very restricted within the testis to the postmeiotic germ cells, and in the ovary to interstitial/luteal cells, cell-types known to express GCNF and SF-1, respectively. Testes of CREM-tau knockout mice, lacking all spermatogenic stages later than round spermatids, were devoid of ELP immunoreactivity, whereas in RAD6 knockout mice the few remaining elongated spermatids were clearly defined by this excellent late haploid marker product. The ELP gene and its product thus offer an ideal system with which to investigate the differentiation of late haploid stages of spermatogenesis.

The rat endozepine-like peptide gene is highly expressed in late haploid stages of male germ cell development.

The structure of the endozepine-like peptide (ELP) gene is closely related to the intracellular acyl-CoA binding protein (ACBP), but unlike the generalized distribution of the latter, it is restricted to the male germ cells of the testis. In the present study, a combination of nonradioactive in situ mRNA hybridization and immunohistochemistry was used to precisely determine the cellular expression patterns of ELP mRNA and protein in control and methoxyacetic acid (MAA)-treated rat testes. ELP transcripts are first detectable in late stages (step 6) of round spermatids, with transcription increasing through late-elongating steps. Translation of the ELP mRNA is delayed, with first immunohistochemical staining occurring in elongated spermatids at step 16, and protein accumulating through step 19. ELP immunoreactivity proves to be an excellent marker for late spermatid stages and highlights the presumably clonal recovery of spermatids following MAA treatment.

Rat endozepine-like peptide (ELP): cDNA cloning, genomic organization and tissue-specific expression.

A cDNA encoding the rat homolog of the previously characterized murine endozepine-like peptide (ELP) was isolated by a PCR cloning strategy. Sequence comparison with the murine cDNA sequence revealed a conservation of the ELP primary structure between both rodent species with minor amino acid exchanges. We investigated the genomic organization of the rat ELP gene by genomic PCR. This indicated the presence of a single short intron of 451bp interrupting the 5' untranslated region. Tissue-dependent ELP expression was determined by Northern hybridization and semiquantitative RT-PCR. Northern hybridization showed an ELP specific transcript in both the male and the female gonad, but the level of ovarian ELP transcription was considerably lower than in the testis. RT-PCR analysis demonstrated a low and varying level of ELP background expression in all examined tissues. In contrast to the closely related ACBP gene, ELP shows a different genomic organization and a more regulated expression pattern, and may exert a specific function as a gonadal acyl-CoA pool former and transporter.

Sertoli and Leydig cells of the human testis express neurofilament triplet proteins.

Using RT-PCR, western blot and enzyme and fluorescence immunocytochemical techniques, the three isoforms of neurofilament proteins (NFPs), namely NF-L (NFP-68 kDa), NF-M (NFP-160 kDa) and NF-H (NFP-200 kDa) were found in Sertoli and Leydig cells of human testes. RT-PCR showed specific for the three NFP fragments in testicular tissue, in isolated seminiferous tubules and in isolated Leydig cells. In protein preparations from the same testicular components, western blot analysis detected bands with molecular weights characteristic for NF-H, NF-M and NF-L. Application of immunofluorescence and immunoenzyme methods on cryostat and paraffin sections resulted in differences in the staining pattern in Sertoli cells and Leydig cells. In these cells, the NFPs showed predominantly a perinuclear location from which bundles emerge that were directed towards the basal, apical and lateral extensions of the Sertoli cells as well as the periphery of Leydig cells. NF-H coexists with vimentin-type filaments as seen by dual staining and staining of consecutive serial sections of material embedded in paraffin. In Sertoli cells, vimentin and NF-H showed distinct dynamic changes depending on the stage of spermatogenesis and some structural variations of seminiferous tubules. Although in some tubules both vimentin and NF-H immunoreactivity was present at high levels, in the Sertoli cells from most individuals an inverse relationship in the staining intensity of vimentin and NF-H was observed. The strongest NF-H immunoreactivity was detected in Sertoli cells associated with stage 3 spermatids, whereas vimentin immunoreactivity was most abundant in association with stage 5 spermatids. The leydig cells did not show functional changes of the NFP immunoreactivity. The results obtained provide new evidence for the heterogeneous phenotype of human Sertoli cells and raise the question of their exact nature and origin.

Sorption Isotherms of Gases by Polymer Membranes in the Glassy State: An Explanation Based on the Nonequilibrium Thermodynamics.

Sorption isotherms of glassy polymers are concave to the pressure axis, and the absolute sorption levels are almost an order of magnitude higher than that of rubbery polymers on the relatively low-pressure side. There are several models to interpret this behavior, and the dual sorption model is the most widely accepted one among them. In the present work, sorption isotherms were first derived from permeation data with several gases for four polymer membranes which might be representative of the dual sorption model: two of them were in a glassy state (cellulose acetate and polyamide), the third one was a block copolymer which was composed of a glassy polymer and a rubbery polymer at the measuring temperature, and the fourth one was a Nafion membrane which was taken as a model membrane because of its channel structure with adsorption sites of charged groups supported by a rubbery polymer. These results did not necessarily support the dual sorption model. Subsequently, the validity of the underlying assumptions of two other sorption models for glassy polymers, that is, the matrix model and the deformation model, were examined and a new equation for the sorption isotherm with an ordering parameter was derived, which implied that the glassy polymer was in a nonequilibrium state and changed from the glassy to the rubbery state by absorbing the gas. Copyright 1998 Academic Press.

Molecular cloning and testicular expression of the gene transcripts encoding the murine multiubiquitin-chain-binding protein (Mcb1).

A murine cDNA encoding a homolog of the human multiubiquitin-chain-binding protein Mcb1 was isolated and sequenced from a mouse testis cDNA library. The encoded Mcb1 protein is highly conserved between mouse and human. Northern hybridisation showed expression of Mcb1 transcripts in all examined mouse tissues. In the testis, however, there is additionally a second, longer Mcb1 transcript in wild-type mice that is absent in the azoospermic W/Wv mutant mice, suggesting expression of this transcript in association with germ cell differentiation.

A novel endozepine-like peptide (ELP) is exclusively expressed in male germ cells.

A cDNA clone encoding a novel endozepine-like peptide (ELP) was isolated from mouse testes, sequenced, and its mRNA expression characterized by northern and in situ hybridization. ELP mRNA was found exclusively in the late spermatid stages of spermatogenesis in the testes of sexually mature mice and in no other tissue or cell type examined. It was also expressed in rat, bovine, porcine and sheep testes. Mouse ELP-encoding cDNA was used to construct expression vectors for the production of ELP in bacteria, and the purified bacterial protein used to raise polyclonal antibodies in rats. These antibodies identified the predicted endogenous ELP in extracts of mouse testis and epididymis and in no other tissue. Immunohistochemistry confirmed that the ELP antigen was present only in late spermatids and spermatozoa, particularly within the cytoplasmic droplet which is retained by the mature spermatozoa during their transit into the epididymis. We conclude that ELP is an intracytoplasmic peptide exclusively expressed in post-meiotic spermatozoa and which may be involved in the energy metabolism of the mature sperm.

Molecular cloning and expression of the relaxin-like factor from the mouse testis.

A complementary DNA encoding the mouse homolog of the testicular relaxin-like factor was cloned from a subtractive complementary DNA library, comprising clones preferentially expressed in the testes of mutant w/wv mice. The predicted amino acid structure conforms with that for other members of the insulin, insulin-like growth factor, and relaxin hormone family, with A, B, and C (connecting) domains. Northern and in situ transcript hybridization showed that the gene is exclusively expressed at a high level in the Leydig cells of both mutant and wild type testes and is up-regulated at puberty. The level of the specific messenger RNA is such as to imply that this novel member of the insulin family represents a major secretory product of Leydig cells, with a potential to be a part of a novel systemic feedback loop to other organs, including the brain.