Tissue inhibitors of metalloproteinases 1 and 2 in human seminal plasma and their association with spermatozoa.

A 24-kDa heparin binding protein recently identified as tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in bovine seminal fluid was suggested to play an important role in bull fertility. As no data are present for men, the concentrations of tissue inhibitors of metalloproteinases 1 (TIMP-1) and TIMP-2 were quantified in human seminal plasma of normozoospermic and azoospermic men using enzyme-linked immunosorbent assay methods. TIMP-1 and 2 were not significantly different in both groups and there were no relationships between the concentrations of both TIMPs and other sperm characteristics. It is assumed that TIMPs are released from accessory sex glands.

Quantitative differences in matrix metalloproteinase (MMP)-2, but not in MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2, in seminal plasma of normozoospermic and azoospermic patients.

BACKGROUND: Matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), have been detected in reproductive tissues and seminal plasma. The purpose of this study was to quantify MMP-2, MMP-9, TIMP-1 and TIMP-2 in human seminal plasma and to evaluate their association with sperm. METHODS: Seminal plasma was analysed using ELISA assays for all four analytes in 12 normozoospermic and 12 azoospermic patients and then for MMP-2 only in another 114 men with azoospermia (n = 16), after vasectomy (n = 20) and with sperm counts within the following ranges: 0.3-19 x 10(6)/ml (n = 20), 20-23 x 10(6)/ml (n = 11), 49-57 x 10(6)/ml (n = 12), 96-110 x 10(6)/ml (n = 12), 139-161 x 10(6)/ml (n = 12) and 215-346 x 10(6)/ml (n = 11). Additional zymographic analyses using SDS-PAGE were performed. RESULTS: All investigated MMPs and TIMPs were detected. MMP-9, TIMP-1 and TIMP-2 were not significantly different in normozoospermia and azoospermia. Only the MMP-2 concentration was significantly decreased in azoospermic compared with normozoospermic patients (mean +/- SD: 650.6 +/- 288.9 versus 1677 +/- 910.4 ng/ml respectively; P = 0.0002) and significantly correlated with the number of sperm (r = 0.54; P < 0.0001). CONCLUSION: MMP-2 in seminal plasma was strongly correlated to the sperm count in a linear fashion. Its origin and potential function remain to be elucidated.

Inactivation of the endoplasmic reticulum protein translocation factor, Sec61p, or its homolog, Ssh1p, does not affect peroxisome biogenesis.

Peroxisomes are single membrane-bound organelles present in virtually all eukaryotes. These organelles participate in several important metabolic processes, and defects in peroxisome function and biogenesis are a significant contributor to human disease. Several models propose that peroxisomes arise from the endoplasmic reticulum (ER) in a process that involves the translocation of "group I" peroxisomal membrane proteins into the ER, the exit of these group I peroxisomal membrane proteins from the ER by vesicle budding, and the formation of nascent peroxisomes from vesicles containing the group I peroxisomal membrane proteins. A central prediction of these models is that the formation of nascent peroxisomes requires protein translocation into the ER. Sec61p is an essential component of the ER translocon, and we show here that loss of Sec61p activity has no effect on peroxisome biogenesis. In addition, loss of the SEC61-related gene, SSH1, also has no effect on peroxisome biogenesis. Although some proteins may enter the ER independently of Sec61p or Ssh1p, none are known, leading us to propose that peroxisome biogenesis may not require protein import into the ER, and by extension, transfer of proteins from the ER to the peroxisome.

Mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model for Zellweger syndrome (PEX5 knockout mouse).

Zellweger syndrome (cerebro-hepato-renal syndrome) is the most severe form of the peroxisomal biogenesis disorders leading to early death of the affected children. To study the pathogenetic mechanisms causing organ dysfunctions in Zellweger syndrome, we have recently developed a knockout-mouse model by disrupting the PEX5 gene, encoding the targeting receptor for most peroxisomal matrix proteins (M Baes, P Gressens, E Baumgart, P Carmeliet, M Casteels, M Fransen, P Evrard, D Fahimi, PE Declercq, D Collen, PP van Veldhoven, GP Mannaerts: A mouse model for Zellweger syndrome. Nat Genet 1997, 17:49-57). In this study, we present evidence that the absence of functional peroxisomes, causing a general defect in peroxisomal metabolism, leads to proliferation of pleomorphic mitochondria with severe alterations of the mitochondrial ultrastructure, changes in the expression and activities of mitochondrial respiratory chain complexes, and an increase in the heterogeneity of the mitochondrial compartment in various organs and specific cell types (eg, liver, proximal tubules of the kidney, adrenal cortex, heart, skeletal and smooth muscle cells, neutrophils). The changes of mitochondrial respiratory chain enzymes are accompanied by a marked increase of mitochondrial manganese-superoxide dismutase, as revealed by in situ hybridization and immunocytochemistry, suggesting increased production of reactive oxygen species in altered mitochondria. This increased oxidative stress induced probably by defective peroxisomal antioxidant mechanisms combined with accumulation of lipid intermediates of peroxisomal beta-oxidation system could contribute significantly to the pathogenesis of multiple organ dysfunctions in Zellweger syndrome.

Localization of mRNAs encoding peroxisomal proteins in cell culture by non-radioactive in situ hybridization. Comparison of rat and human hepatoma cells and their responses to two divergent hypolipidemic drugs.

A non-radioactive in situ hybridization (ISH) protocol for localization of mRNAs encoding peroxisomal proteins in hepatoma cell lines from humans (HepG2) and rats (MH1C1) is presented. In comparison to a similar procedure reported for tissue sections, the cell culture preparations require only brief fixation in 4% paraformaldehyde and their permeabilization is achieved by a very low concentration (1 microg/ml) of proteinase K. The exclusive localization of transcripts in the cytoplasm of hepatoma cells with the absence of nuclear staining and the completely negative sense controls confirm the specificity of the method. The marked differences in signal intensity between the results of albumin and beta-actin mRNAs which are of high abundance in contrast to moderate to low abundance of peroxisomal mRNAs show the high sensitivity and the wide range of applicability of our protocol. This is also confirmed by divergent results of treatment of hepatoma cell lines with clofibrate and cetaben on mRNA levels of catalase and acyl-CoA oxidase. The ISH results of drug treatment of cell lines are confirmed also by slot blot analysis of total RNA extracts using 32P-labeled probes. Thus the protocol presented here provides a sensitive tool for ISH localization of mRNAs encoding peroxisomal proteins. In combination with immunocytochemistry it may be useful to monitor intercellular differences in expression levels of specific mRNAs in correlation with the abundance of structurally divergent forms of peroxisomes (tubular versus spherical) and their importance in the biogenesis of peroxisomes.

Complete laparoscopic approach for radical cystectomy and continent urinary diversion (sigma rectum pouch).

Technical and manual progress made in recent years now enables large uro-oncological procedures to be performed by means of laparoscopy. We report the first successful radical laparoscopic cystectomy and laparoscopic construction of a continent urinary diversion. Laparotomy can be avoided completely. The advantages are clear reduction of blood loss and postoperative morbidity with faster convalescence.

Detection of peroxisomal proteins and their mRNAs in serial sections of fetal and newborn mouse organs.

We present a protocol for detection of peroxisomal proteins and their corresponding mRNAs on consecutive serial sections of fetal and newborn mouse tissues by immunohistochemistry (IHC) and nonradioactive in situ hybridization (ISH). The use of perfusion-fixation with depolymerized paraformaldehyde combined with paraffin embedding and digoxigenin-labeled cRNA probes provided a highly sensitive ISH protocol, which also permitted immunodetection with high optical resolution by light and/or fluorescence microscopy. Signal enhancement was achieved by the addition of polyvinyl alcohol (PVA) for ISH color development. For IHC, signal amplification was obtained by antigen retrieval combined with biotin-avidin-HRP and Nova Red as substrate or by the catalyzed reporter deposition of fluorescent tyramide. Using this protocol, we studied the developmental changes in localization of the peroxisomal marker enzymes catalase (CAT) and acyl-CoA oxidase 1 (AOX), the key regulatory enzyme of peroxisomal beta-oxidation, at the protein and mRNA levels in mice from embryonic Day 14.5 to birth (P0.5). The mRNA signals for CAT and AOX were detected in sections of complete fetuses, revealing organ- and cell-specific variations. Here we focus on the localization patterns in liver, intestine, and skin, which showed increasing mRNA amounts during development, with the strongest signals in newborns (P0.5). Immunolocalization of the corresponding proteins revealed, in close correlation with the mRNAs, a distinct punctate staining pattern corresponding to the distribution of peroxisomes. (J Histochem Cytochem 49:155-164, 2001)

Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.

Stiffness--an unknown world of mechanical science?

"Stiffness" is a term used to describe the force needed to achieve a certain deformation of a structure. In the biomechanical world, several different definitions of stiffness are used, but not all of them are explained adequately to those readers who are less familiar with biomechanical terminology. This paper gives examples for specific definitions which are based on the basic definition of stiffness of a loaded structure

Graphical statics a forgotten tool for solving plane mechanical problems.

Graphical statics is an almost forgotten, intuitive drawing method for solving plane mechanical problems. It was already in use in the 19th century for biomechanical problems. It was still a standard method employed by civil engineers in the 1940s. Superceded by modern analytical methods, graphical statics disappeared almost completely. The method is restricted to plane static problems, but still remains a useful tool for visualizing, understanding and checking the actions of force groups occurring in modern biomechanical problems. After defining the basic mechanical terminology (body, motion, forces), the paper is written mainly as a teaching tool for immediate application. Many illustrative examples (sporting activities, functional forces in joints) help to clarify the difficult biomechanical content. For application, it must be assumed that the bodies investigated behave as rigid bodies under the action of the forces, but this does not prevent application of the method to deformable living bodies if specific static configurations of the bodies are considered. The application of the method requires a good anatomical knowledge and experience with the function of the musculoskeletal apparatus of living bodies. If reliable models are used, the method delivers quantitative results of sufficient accuracy. The paper may also help provide a better understanding of publications containing graphical solutions to bio-static problems.

Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells. Absence of peroxisomal reticulum but evidence of close spatial association with the endoplasmic reticulum.

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3'-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.

Current cytochemical techniques for the investigation of peroxisomes. A review.

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorders, with further deepening of our understanding of the metabolic role of this ubiquitous cell organelle. There have been many recent reviews on biochemical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the state-of-the-art cytochemical techniques available for investigation of peroxisomes. After a brief introduction into the use of the 3,3'-diaminobenzidine method for localization of catalase, which is still most commonly used for identification of peroxisomes, the cerium technique for detection of peroxisomal oxidases is discussed. The influence of the buffer used in the incubation medium on the ultrastructural pattern obtained in rat liver peroxisomes in conjunction with the localization of urate oxidase in their crystalline cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeling by automatic image analysis enables quantitative assessment of alterations of proteins in the matrix of peroxisomes. This provides a highly sensitive approach for analysis of peroxisomal responses to metabolic alterations or to xenobiotics. The recent evidence suggesting the involvement of ER in the biogenesis of "preperoxisomes" is mentioned and the potential role of preembedding immunocytochemistry for identification of ER-derived early peroxisomes is emphasized. The use of GFP expressed with a peroxisomal targeting signal for the investigation of peroxisomes in living cells is briefly discussed. Finally, the application of in situ hybridization for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol using perfusion-fixation, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mRNAs encoding peroxisomal proteins. (J Histochem Cytochem 47:1219-1232, 1999)

Effects of fibrin glue and growth factors released from platelets on abdominal hernia repair with a resorbable PGA mesh: experimental study.

INTRODUCTION: The purpose of this study was to investigate if the strength and quality of an abdominal wall repair with a resorbable PGA (polyglycolic acid) mesh can be improved by fibrin glue or releasates from platelets. MATERIALS AND METHODS: An abdominal wall defect in the rat was repaired using a PGA mesh in a sublay technique (CG) alone and either with additional fibrin glue (FG) or with platelet releasates (REL). Endpoints were clinical herniation pressure and hydroxyproline concentration (HP) as well as number of fibroblasts and collagen fibers at 7, 14, and 90 days after implantation. RESULTS: In both experimental groups (REL and FG) higher herniation pressures, hydroxyproline contents, and number of fibroblasts/collagen fibers were found at all times of measurement compared to the CG. The PGA mesh alone showed a significant lack of stability after 14 days which can be compensated for by the investigated components. Significant differences (P < 0.05) were observed regarding the herniation pressure (REL vs CG at 7 and 14 days; FG vs CG at 14 days) and the number of collagen fibers (REL vs CG at 14 days). CONCLUSIONS: These results suggest that the quality of a PGA mesh repair can be improved by application of fibrin glue or platelet releasates in the described experimental setting.

Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p.

Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Delta mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles.

Pex19p, a farnesylated protein essential for peroxisome biogenesis.

We report the identification and molecular characterization of Pex19p, an oleic acid-inducible, farnesylated protein of 39.7 kDa that is essential for peroxisome biogenesis in Saccharomyces cerevisiae. Cells lacking Pex19p are characterized by the absence of morphologically detectable peroxisomes and mislocalization of peroxisomal matrix proteins to the cytosol. The human HK33 gene product was identified as the putative human ortholog of Pex19p. Evidence is provided that farnesylation of Pex19p takes place at the cysteine of the C-terminal CKQQ amino acid sequence. Farnesylation of Pex19p was shown to be essential for the proper function of the protein in peroxisome biogenesis. Pex19p was shown to interact with Pex3p in vivo, and this interaction required farnesylation of Pex19p.

Maturation of peroxisomes in differentiating human hepatoblastoma cells (HepG2): possible involvement of the peroxisome proliferator-activated receptor alpha (PPAR alpha).

We have studied the alterations of peroxisomes in the human hepatoblastoma cell line HepG2, induced to differentiate by long-term cultivation (20 days without passaging) using morphological and biochemical techniques as well as mRNA analysis. Ultrastructural studies revealed alterations in shape and size of peroxisomes, with significant increases in mean diameter and formation of small clusters exhibiting heterogeneous staining for catalase after 20 days in culture. These alterations of peroxisomes correspond to the changes described during the maturation process from prenatal to adult human hepatocytes. As revealed by Northern and Western blotting there was marked elevation of the mRNA (190%) and protein (180%) of the peroxisomal branched-chain acyl-CoA oxidase. This protein is the key regulatory enzyme for the side chain oxidation of cholesterol for bile acid synthesis, a pathway associated with mature hepatocytes. Concomitantly a marked increase of bile canaliculi was noted by light and electron microscopy. This differentiation process was confirmed also by the increase of albumin synthesis (mRNA: 160%; protein: 190%) which is generally used as a differentiation marker of hepatocytes in culture. Interestingly, the mRNA for peroxisome proliferator-activated receptor alpha (PPAR alpha) increased drastically by almost 390% and its corresponding protein by 150%, suggesting its involvement in maturation of the peroxisomal compartment in differentiating HepG2 cells. In contrast to the wellknown increases during the drug-induced peroxisome proliferation of cytochrome P450 4A, multifunctional enzyme 1, palmitoyl-CoA oxidase and the 70-kDa peroxisomal membrane protein, those proteins were either not altered or only slightly elevated during the differentiation process, suggesting that peroxisome proliferation and maturation are two distinct and differentially regulated processes.

Detection of mRNAs encoding peroxisomal proteins by non-radioactive in situ hybridization with digoxigenin-labelled cRNAs.

We have used a non-radioactive in situ hybridization (ISH) protocol for the detection of mRNAs encoding proteins localized in peroxisomes. In this presentation the literature on detection of "peroxisomal mRNAs" is reviewed and the results obtained by application of the non-radioactive method are compared with those obtained by hybridization with radioactive probes. Moreover, the special processing conditions and the application of the method for the specific visualization of mRNAs coding for several peroxisomal proteins with different abundance levels and distinct tissue distributions are presented. The combination of the following technical details in the ISH procedure were found to be essential for obtaining optimal sensitivity and good histological quality of the preparations: (a) perfusion-fixation with a fixative containing 4% depolymerized paraformaldehyde/0.05% glutaraldehyde, (b) the use of paraffin embedding instead of frozen sections, (c) specific proteinase K-digestion time for each tissue, and (d) the use of digoxigenin-labelled cRNA probes (hydrolyzed to a length of about 200 bases) for detection. By using this technique, we were able to localize several peroxisome-specific mRNAs with different degrees of abundance: (1) high-level (catalase and urate oxidase) and (2) low-level (all beta-oxidation enzymes and the 70-kDa peroxisomal membrane protein) in rat liver and kidney. The specificity of the method was confirmed by the negative results obtained with corresponding sense controls and the distinct positive staining patterns obtained for albumin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. All transcripts for mRNAs encoding peroxisomal proteins were localized to the cytoplasm of hepatocytes, with all nuclei as well as epithelial cells of bile ducts and sinusoidal cells remaining negative. In rat kidney, the catalase transcripts were confined to proximal tubular epithelial cells, which is consistent with the high abundance of peroxisomes in this part of the nephron. In contrast, no transcripts for urate oxidase were present in the kidney, corresponding to the absence of that protein in this organ. The transcripts for GAPDH on the other hand were localized in proximal and distal tubular epithelial cells as well as in collecting ducts. The application of this technique to the rat adrenal gland and testis in recent unpublished studies have revealed exclusive localization of catalase transcripts to the adrenal cortex and to interstitial cells of Leydig, which are known to be rich in microperoxisomes. These observations demonstrate the suitability of this technique for accurate localization of mRNAs encoding peroxisomal proteins and for the analysis of alterations in the expression of the corresponding genes under different experimental conditions.

Application of in situ hybridization, cytochemical and immunocytochemical techniques for the investigation of peroxisomes. A review including novel data. Robert Feulgen Prize Lecture 1997.

In situ hybridization, cytochemical and immunocytochemical techniques have contributed significantly to the understanding of the biology of peroxisomes, since they permit in situ demonstration of the sites of synthesis and distribution of peroxisomal proteins without the necessity of homogenization and subcellular fractionation of tissues or cultured cells. This article reviews the results of research on mammalian peroxisomal metabolism, biogenesis and proliferation in which morphological techniques have played a significant role in the elucidation of the biological problem. Some new data on peroxisomal heterogeneity and morphogenesis are included. The morphological methods applied have made it possible to characterize the differences in distribution of mRNAs encoding peroxisomal proteins in different tissues, as well as to monitor the marked heterogeneity in the protein composition and in the activity of specific enzymes in the peroxisomal population of single cells, or in tissues with complex organization (e.g. liver and kidney). In addition, the dynamic alterations and high plasticity of the peroxisomal compartment--partly dependent on contact of the peroxisomes to the microtubular network-are presented.

A mouse model for Zellweger syndrome.

The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.