Biomedical subjects

L Konrad

Publications and source records attributed to L Konrad.

At least 19 recordsLinked to original sources

PubMed2006

Effects of TGF-betas and a specific antagonist on apoptosis of immature rat male germ cells in vitro.

Massive apoptosis of pubertal male germ cells is important for the development of functional spermatogenesis in the adult testis. Although the trigger(s) for male germ cell loss at puberty remain undefined, we have hypothesized that transforming growth factor-betas (TGF-betas) play an active role. Here we demonstrate that the three mammalian TGF-beta isoforms, TGF-beta1, TGF-beta2 and TGF-beta3, induce distinct apoptosis of pubertal spermatogonia and spermatocytes in a dose-dependent manner. Induction of male germ cell death by activation of caspase-3 was most pronounced with TGF-beta2 compared to TGF-beta1 and TGF-beta3. Furthermore, we found colocalization of activated caspase-3 with apoptotic protease-activating factor-1 (Apaf-1) in apoptotic germ cells, thus indicating the importance of the intrinsic mitochondrial pathway in TGF-beta-induced apoptosis. The specificity of the TGF-beta effects was proven by addition of recombinant latency-associated peptide against TGF-beta1 (rLAP-TGF-beta1) which completely abolished TGF-beta1-induced and TGF-beta3-induced germ cell apoptosis. Although TGF-beta2-triggered germ cell death also was significantly reduced by rLAP-TGF-beta1, inhibition was not maximal. Our results suggest that the three TGF-beta isoforms induce apoptosis of pubertal male germ cells via the mitochondrial pathway in vitro and are thus likely candidates involved in the excessive first wave of apoptosis of male germ cells during puberty.

Animals↗

PubMed2005

The influence of bone allograft processing on osteoblast attachment and function.

In order to assess the influence of eight different sterilisation and disinfection methods for bone allografts on adhesion, proliferation, and differentiation of human bone marrow stromal cells (BMSC), cells were grown in culture and then plated onto pieces of human bone allografts. Following processing methods were tested: autoclavation (AUT), low-temperature-plasma sterilisation of demineralised allografts (D-LTP), ethylene oxide sterilisation (EtO), fresh frozen bone (FFB), 80 degrees C-thermodisinfection (80 degrees C), gamma-irradiation (Gamma), chemical solvent disinfection (CSD), and Barrycidal-disinfection (BAR). The seeding efficiency was determined after one hour to detect the number of attached cells before mitosis started. The cell viability was determined after 3, 7, and 21 days. Tests to confirm the osteoblastic differentiation included histochemical alkaline phosphatase staining and RT-PCR for osteocalcin. Human BMSC showed greatest attachment affinities for D-LTP-, 80 degrees C-, and CSD-allografts, whereas less cells were found attached to AUT-, EtO-, FFB-, Gamma-, and BAR-probes. Cell viability assays at day 3 revealed highest proliferation rates within the FFB- and 80 degrees C-groups, whereas after 21 days most viable cells were found in D-LTP-, 80 degrees C-, CSD-, and Gamma-groups. BAR-treatment showed a considerably toxic effect and therefore was excluded from all further experiments. Highest AP-activity and gene expression of osteocalcin were detected in the D-LTP-group in comparison with all other groups. In summary, our results demonstrate that cell adhesion, final population, and function of BMSC are influenced by different disinfection and sterilisation methods. Therefore, processing-related alterations of BMSC-function may be important for the success of bone grafting. The experimental setup used in the present work may be useful for further optimisation of bone allograft processing.

Alkaline Phosphatase↗

PubMed2004

Localization and synthesis of zona pellucida proteins in the marmoset monkey (Callithrix jacchus) ovary.

In most species, the zona pellucida (ZP), an extracellular matrix surrounding the mammalian oocyte, is composed of three glycoproteins: ZPA, ZPB and ZPC. Based mainly on results with mice, the site of zona pellucida biosynthesis has been suggested to be exclusively in the oocyte cytoplasm. However, evidence is accumulating that among various species cumulus/granulosa cells may be involved. Because knowledge of ZP biosynthesis in primates is lacking, we used the common marmoset (Callithrix jacchus) to acquire information about the localization and the site of synthesis of ZP proteins in this species. Using antibodies against synthetic ZPA and ZPC peptides, immunoreactivity was found in the marmoset ZP and in surrounding cumulus cells. Interestingly, the amounts of ZPA and ZPC proteins expressed appeared to differ depending on the stage of folliculogenesis. RT-PCR analysis of mRNA from marmoset oocytes and from oocyte-free follicle cells revealed expression of ZPA, ZPB and ZPC in oocytes and in follicle cells of different stages of marmoset monkey folliculogenesis. Our data suggest that the biosynthesis of marmoset ZPA, ZPB and ZPC proteins takes place both in oocytes and in follicle cells of different follicle stages, although the abundance of ZP glycoproteins may differ depending on the individual ZP protein.

Animals↗

PubMed2004

Dihydroorotate dehydrogenase mRNA and protein expression analysis in normal and drug-resistant cells.

To follow the expression of the fourth enzyme of pyrimidine de novo synthesis dihydroorotate dehydrogenase (DHODH) in cells and tissues, we studied the DHODH mRNA expression by means of RT-PCR in rat tissues. Rabbit polyclonal anti-DHODH immunoglobulins were applied for immunochemical quantification of the enzyme protein by Western blotting. In mouse B-lymphocytes, which were adapted to tolerate up to a 50-fold concentration of the DHODH inhibitor leflunomide, a 20 fold protein overexpression was measured. Southern blotting indicated DHODH gene amplification.

Animals↗

PubMed2003

Bioengineered human bone tissue using autogenous osteoblasts cultured on different biomatrices.

Surgical treatment of critical-size posttraumatic bone defects is still a challenging problem, even in modern bone and joint surgery. Progress in cellular and molecular biology during the last decade now permits novel approaches in bone engineering. Recent conceptual and technical advances have enabled the use of mitotically expanded, bone-derived cells as a therapeutic approach for tissue repair. Using three different tissue carrier systems, we successfully cultivated human osteoblasts in a newly developed perfusion chamber. We studied cell proliferation and the expression of osteocalcin, osteopontin, bone morphogenetic protein-2A, alkaline phosphatase, and vascular endothelial growth factor as parameters for osteoblast function and viability. Adherence of highly enriched human osteoblasts had already started after 1 h and resulted in completely overgrown human bone pieces after 10 days. Expression analysis of bone-specific alkaline phosphatase indicated differentiating osteoblasts, whereas the high mRNA expression of osteocalcin and osteopontin revealed terminally differentiated osteoblasts and the process of mineralization. Additionally, gene expression was significantly higher when demineralized bone was used as biomatrix, compared to autoclaved bone and hydroxyapatite ceramics. We conclude that with our newly developed perfusion culture system, vital autogenous bone implants of clinically applicable size can be generated within 17 days in order to manage critical-size bone defects.

Animals↗

PubMed2002

Effects of dexamethasone on proliferation of and fibronectin synthesis by human primary prostatic stromal cells in vitro.

Glucocorticoids are anti inflammatory stress hormones and have been suggested to be involved in a large number of pathological processes. To test the effects of glucocorticoids on stromal prostatic cell growth and proliferation in vitro, the influence of a synthetic glucocorticoid (dexamethasone, dex) on recently established human primary cells from prostatic stroma (hPCPs) was analysed. The localization and distribution of the glucocorticoid receptor (GR) was investigated by immunohistochemistry. In addition, expression of the active isoform of the receptor (alpha-GR) was examined by reverse transcription PCR, and the effect of different doses of dex on proliferation of the stromal cells evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and amido black assays. alpha-GR mRNA was expressed by the hPCPs, and the GR protein was detected in the cytoplasm and nucleus of these cells. Incubating the cells with dex resulted in an enhanced cell proliferation that was mainly restricted to the fibroblasts. Moreover, fibronectin (FN) gene expression and secretion of the protein was increased by high doses of dex (> or = 10(-8) M), whereas low doses of dex (10(-10)M) showed no effect. Human prostatic stromal cells show sensitivity to dex in vitro, resulting in an increase in cell proliferation and FN synthesis. The authors assume that locally accumulating glucocorticoids can also influence the regulation of cell growth and extracellular matrix synthesis in the human prostate in vivo and may play a role in the pathologically altered prostate.

Cell Division↗

PubMed2002

Post-natal development of the rat testis: steroid hormone receptor distribution and extracellular matrix deposition.

The control of testicular development and differentiation depends on hormones and a variety of cell-cell interactions mediated mainly by paracrine factors. In the second and third weeks of post-natal development important changes take place in the rat testis, e.g. the tubular lumen starts to open on post-natal day 10, the blood-testis barrier starts to form on day 15, and Sertoli cell proliferation ceases on day 15. In the present study the expression in different testicular compartments of the androgen receptor (AR), progesterone receptor (PR), and extracellular matrix proteins such as laminin, entactin-1 (nidogen-1) and fibronectin, during post-natal development was examined using immunohistochemistry and semiquantitative image analysis. An intratubular AR peak on days 14-17, an increase in intratubular PR expression on days 14-16, and an increase in peritubular entactin-1 expression during the second and third weeks post-partum are demonstrated. These results suggest that a variety of changes occur at the cellular level during this period when certain milestones of testicular development occur, substantiating the hypothesis of a particular role for paracrine interactions during the development of the rat testis.

Aging↗

PubMed2001

VDAC2 (porin-2) expression pattern and localization in the bovine testis.

In this study, sequencing of voltage-dependent anion channel 2 (VDAC2, porin-2) cDNA from bovine testis is reported. High identity to the murine, rabbit, and human subtypes at both the nucleotide and amino acid levels is demonstrated. mRNA analysis revealed expression of VDAC2 in bovine testis, whereas high levels of VDAC2 proteins were found in late spermatocytes, spermatids, and spermatozoa. In contrast, VDAC1 (porin-1) is exclusively localized in Sertoli cells. The possible role of testicular VDAC2 in providing energy metabolites and in germ cell apoptosis is discussed.

Amino Acid Sequence↗

PubMed2000

Cell lineage characteristics of human prostatic stromal cells cultured in vitro.

BACKGROUND: An in vitro model of prostatic stromal cells suitable for experimental studies of the pathogenesis of BPH is still lacking. We therefore standardized the isolation, cultivation, and characterization of human prostatic stromal cell lineages. METHODS: Stromal cells were isolated from a surgical specimen of BPH. Using antibodies specific for either epithelial or stromal cells of the human prostate, the isolated cells were morphologically and immunohistochemically characterized. Viability and functional activity were assessed by proliferation assays and stimulation experiments. Gene expression was monitored by RT-PCR. RESULTS: In early passages (P8), cells showed a high purity (>/=98%) for stromal markers; about 60% displayed the characteristics of fibroblasts, and the remaining 40% were classified as smooth muscle cells. In late passages (P20), the proportion of muscle cells declined to 10%. Stimulation experiments including basic fibroblast growth factor (bFGF) resulted in enhanced proliferation, whereas dihydrotestosterone (DHT), estrogen, and flutamide did not influence proliferation. Gene expression studies demonstrated a positive signal for androgen receptor and keratinocyte growth factor (KGF). CONCLUSIONS: Prostatic stromal cells can be propagated several times and show karyotypic stability for up to 18 subculture experiments. The ratio of myoid and fibroblastic cells can be used for standardization of cell cultures with stable characteristics.

Cell Division↗

PubMed2000

The distribution of aminoacylase I among mammalian species and localization of the enzyme in porcine kidney.

Aminoacylase I (Acy-1, EC 3.5.1.14) is found in many mammalian tissues, with highest activities occurring in kidney. The enzyme hydrolyzes a variety of N-acylated amino acids; however, the physiological role and the exact cellular localization of Acy-1 are still a matter of debate. The comparison of Acy-1 activities in kidney and liver homogenates of 11 mammalian species showed that the enzyme is most abundant in true herbivores such as sheep and cattle as well as in omnivores, while activities were very low in both rodents and the cat. Acy-1 activity was not detected in livers of dogs of five different breeds. Using in situ hybridization of porcine kidney sections with DIG-labeled RNA probes, Acy-1 mRNA was shown to be evenly distributed throughout the tubular system, while glomeruli and the interstitium were free of stain. During subcellular fractionation, porcine Acy-1 behaved like a typical cytosolic enzyme. Commonly, Acy-1 is thought to catalyze hydrolytic reactions, i.e., the formation of free amino acids from acylated derivatives. Based on the present results and literature data, we propose a novel hypothesis, i.e., that Acy-1 catalyzes the synthesis (rather than the hydrolysis) of hippurate that is formed as a detoxification product of aromatic compounds.

Amidohydrolases↗

PubMed2000

Antiproliferative effect on human prostate cancer cells by a stinging nettle root (Urtica dioica) extract.

In the present study the activity of a 20% methanolic extract of stinging nettle roots (Urtica dioica L., Urticaceae) on the proliferative activity of human prostatic epithelial (LNCaP) and stromal (hPCPs) cells was evaluated using a colorimetric assay. A concentration-dependent and significant (p < 0.05) antiproliferative effect of the extract was observed only on LNCaP cells during 7 days, whereas stromal cell growth remained unaltered. The inhibition was time-dependent with the maximum of growth reduction (30%) at a concentration of 1.0E-6 mg/ml on day 5 compared to the untreated control. On day 4 and 6, the reduction in proliferation of LNCaP cells showed the minimal effective dose at 1.0E-9 mg/ml. No cytotoxic effect of ME-20 on cell proliferation was observed. The antiproliferative effect of ME-20 of stinging nettle roots observed both in an in vivo model and in an in vitro system clearly indicates a biologically relevant effect of compounds present in the extract.

Antineoplastic Agents, Phytogenic↗

PubMed2000

Mesenchymal entactin-1 (nidogen-1) is required for adhesion of peritubular cells of the rat testis in vitro.

Epithelial-like Sertoli cells isolated from immature rat testis aggregate to form tubule-like structures when cultured on a monolayer of mesenchyme-derived peritubular cells. At the end of this morphogenetic process both cell types are separated by a basement membrane. In this study the gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells was examined using DD-RT-PCR. One of the isolated cDNA clones showed high homology to the cDNA encoding the basement membrane component entactin-1 (nidogen-1). Even though the entactin-1 (nidogen-1) gene is transcribed in peritubular cells, Sertoli cells, and in direct cocultures, the mRNA is translated only by the peritubular cells. No entactin-1 (nidogen-1) was detected in the Sertoli cells by Western blotting. Moreover, peritubular cell monocultures and cocultures showed the presence of one single band at 152 kDa in the supernatant, whereas in cell lysates two bands were detectable at 152 kDa and 150 kDa. Perturbation experiments using monoclonal antibodies directed against entactin-1 (nidogen-1) were performed with peritubular cells and Sertoli cells, respectively, and demonstrated loss of cell adhesion of the peritubular cells, while the Sertoli cells remained adherent. From these data we conclude that entactin-1 is exclusively produced and secreted by mesenchymal peritubular cells, and affects adhesion of peritubular cells in an autocrine manner.

Animals↗

PubMed2000

Transforming growth factor-beta2 mediates mesenchymal-epithelial interactions of testicular somatic cells.

Transforming growth factor-beta2 (TGFbeta2) is an important mediator of growth and differentiation. We here describe for the first time the complete sequence of the TGFbeta2 complementary DNA derived from peritubular myoid cells of the rat testis. The size of the rat TGFbeta2 complementary DNA was 1245 bp, and the deduced protein sequence contained 414 amino acids. Sequence comparison with the human and mouse amino acid sequences demonstrated 96.4% and 97.9% sequence identities, respectively. To elucidate the functional role of TGFbeta2 in testicular somatic cells, we studied its secretion in vitro in monocultures and cocultures of mesenchymal peritubular and epithelial Sertoli cells. The highest amounts of TGFbeta2 protein were secreted in the cocultures and by peritubular cells, whereas Sertoli cells secreted only minor amounts. Stimulation experiments with FSH revealed a reduced secretion of TGFbeta2 in cocultures, probably mediated by a paracrine interaction of the FSH-responsive Sertoli cells. In contrast, TGFbeta2 secretion by peritubular cells was increased after stimulation with glucocorticoids and after addition of recombinant TGFbeta2, indicating an autoregulation of TGFbeta2. Furthermore, application of recombinant TGFbeta2 to cocultures resulted in an enhanced aggregation and cell clustering of Sertoli cells, pointing to an important role of TGFbeta2 in the paracrine interaction of peritubular and Sertoli cells of the developing rat testis.

Amino Acid Sequence↗

PubMed2000

Glucocorticoid receptor distribution in rat testis during postnatal development and effects of dexamethasone on immature peritubular cells in vitro.

In this study, the occurrence of the glucocorticoid receptor in the rat testis during early stages of postnatal development and its potential functional significance were investigated. Quantitative analyses of immunohistochemically labelled paraffin sections revealed that the receptor was present during all stages of postnatal development in the nuclei of interstitial cells such as Leydig cells, macrophages and fibroblasts, and endothelial cells of blood vessels. The labelling index increased initially, with maximum levels reached within the second week of postnatal development, and decreased thereafter. Within the seminiferous tubules, the glucocorticoid receptor could be detected in the nuclei of germ cells as well as Sertoli cells, reaching the highest levels in 3-week-old rats, mainly due to immature germ cell staining. In contrast, approximately 50% of the peritubular cell nuclei were stained throughout postnatal development. In vitro experiments on immature and immortalized peritubular cells demonstrated a dose-dependent and significant decrease in proliferation and fibronectin secretion after administration of dexamethasone. The data of this study suggest that glucocorticoids have a consistently repressive effect on peritubular cells throughout postnatal development. In summary, labelling of germ cells, especially in immature rats, might indicate an inhibition of spermatogenesis by corticosteroids.

Animals↗

PubMed1999

Calcyclin is differentially expressed in rat testicular cells.

Immature rat Sertoli cells aggregate and form tubule-like structures when cultured on a monolayer of peritubular myoid cells. In this study, differential gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells were examined. One of the cDNA clones isolated showed high homology to calcyclin and a microvascular differentiation gene, CEC5, which was reported to be highly homologous to CASK, a membrane-associated guanylate kinase homolog. Sequencing and mRNA analysis of rat calcyclin demonstrated that the gene was differentially expressed and was found only in peritubular cells and cocultures with increased levels. In contrast, CASK was expressed by Sertoli cells, peritubular cells, and cocultures, whereas CEC5 was never found in the testicular somatic cells. Our findings point to a paracrine regulation of calcyclin expression in testicular peritubular fibroblasts which seems to be related to tubular growth.

Amino Acid Sequence↗

PubMed1999

Prostate specific membrane antigen (PSM) is expressed in various human tissues: implication for the use of PSM reverse transcription polymerase chain reaction to detect hematogenous prostate cancer spread.

Detection of prostate-specific membrane antigen (PSM)-mRNA expression in blood samples using reverse transcription polymerase chain reaction (RT-PCR) is discussed as a new diagnostic marker of circulating micrometastases in prostate cancer patients. We applied the RT-PCR technique to different human tissues and obtained positive signals for PSM transcripts in human genital and multiple extra-genital tissue sites. The cDNAs were prepared from different human tissues and prostatic cell lines. RT-PCR and nested RT-PCR for PSM was performed with primers derived from the published PSM cDNA. The RT-PCR fragments obtained were cloned and showed 100% sequence homology to PSM. Southern blot hybridization with labeled probes was used to confirm the specificity of the amplicons. In addition to the known PSM expression in the human brain, PSM-mRNA was detected in cDNA isolated from human testis, epididymis and seminal vesicles and in the PC-3 prostatic cancer cell line. Furthermore, we found PSM-mRNA in heart, liver, lung, kidney, spleen, and thyroid gland. The results indicate that PSM expression is not restricted to the prostate gland, but represents a more general component of genital and extra-genital human tissues. This must be considered when RT-PCR and nested RT-PCR screening for PSM expression is performed as a diagnostic measure in blood from prostate cancer patients.

Antigens, Surface↗

PubMed1999

Fibronectin in human prostatic cells in vivo and in vitro: expression, distribution, and pathological significance.

In the present study we examined the expression and release of the extracellular matrix glycoprotein fibronectin (FN) in a prostate cancer cell line (LNCaP) and in primary prostatic stromal cells using the reverse transcription-polymerase chain reaction (RT-PCR) and by an enzyme-linked immunosorbent assay. Perturbation experiments in vitro using antibodies directed against FN and the FN receptor were also performed. Immunohistochemistry was used to show the in vivo distribution of FN and the FN receptor in tissue sections of normal human prostate, benign prostatic hyperplasia, and prostate carcinoma. The expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue was investigated by RT-PCR. The FN mRNA was expressed by LNCaP and primary prostatic stromal cells, respectively. Both cell types released FN into the medium in a time-dependent manner, whereby FN secretion was about 2.5-fold higher in cultures of stromal cells relative to LNCaP cells. Blocking FN with anti-FN antibodies resulted in a significant decrease in cell adhesion for LNCaP cells and a change in morphology for the primary stromal cells. FN was located mainly in the stromal compartment of the prostate, showing a distinct distribution pattern in prostate carcinoma, whereas the FN receptor was detectable only in the prostate epithelia. RT-PCR experiments showed the expression of the oncofetal FN ED-B segment in benign prostatic hyperplasia and prostate carcinoma tissue, with a 3.5-fold higher expression in the prostate carcinoma probes. Our data point to an important role for FN in cell adhesion of prostatic cells and show that an alternatively spliced FN mRNA is upregulated in the pathologically altered human prostate.

Animals↗

PubMed1999

Neurogenic origin of human prostate endocrine cells.

OBJECTIVES: To determine the histogenetic origin of prostate neuroendocrine cells in human embryos. METHODS: Prostatic tissue in human fetuses, ranging in gestational age from early week 10 to term, and infantile and pubertal glands were studied immunohistochemically. The distribution of neuroendocrine cells within the developing gland was semiquantitatively determined. Antibodies against the neuroendocrine markers chromogranin A (CgA) and protein gene product 9.5 (PGP), along with markers of prostatic secretion (prostate-specific antigen [PSA], prostatic acid phosphatase [PAP]), were used. They were applied either individually or in double-labeling experiments, as well as in experiments combining CgA immunohistochemical analysis with in situ hybridization or in situ end-labeling. RESULTS: In embryos of less than 65-mm crown-rump length (CRL) (ie, younger than 12 weeks of gestation), the epithelium of the urogenital sinus was free of endocrine cells. On either side of the future prostatic mesenchyme, paraganglia containing CgA-immunoreactive cells are present, which start to penetrate the urogenital mesenchyme. In the late 10th week, these CgA-immunoreactive cells are found dispersed in the urogenital mesenchyme. In embryos of 65-mm CRL, when prostatic anlagen start to sprout from the urogenital epithelium, very few (but typically shaped) neuroendocrine cells appear in the urogenital sinus epithelium. Later, after the 12th week, when solid prostatic ducts have started forming, CgA-immunoreactive neuroendocrine cells are also present in these buds. The number of neuroendocrine cells in the urethral epithelium is considerably increased, and with the continuous sprouting and lumen formation of prostatic anlagen, neuroendocrine cells are transported into the future gland. Neuroendocrine cells observed in stroma of prenatal and postnatal prostates may also contribute to the neuroendocrine cell population of the gland. CONCLUSIONS: Our results provide the first evidence that human prostate neuroendocrine cells represent a cell lineage of their own, being of neurogenic origin and therefore distinct from the urogenital sinus-derived prostate secretory and basal cells.

Adolescent↗