Polymorphism L26V in the cathepsin B gene may be associated with a risk of prostate cancer and differentiation.

Cathepsin B is a lysosomal enzyme thought to be involved in tumour cell invasion and metastasis. This study was designed to investigate the presence of a known leucine to valine mutation at position 26 (L26V) single nucleotide polymorphism (SNP) in the cathepsin B (CTSB) gene in a Slovenian Caucasian population, and to evaluate the association with risk of prostate adenocarcinoma (PCa). A total of 168 PCa patients were compared with 168 controls. There was a significant difference between the frequency of alleles in control subjects and PCa patients: the VV genotype was found in 35.7% of the controls versus 48.8% of the PCa patients. The relative risk for the VV genotype in PCa patients was 1.71. When evaluating the frequency of alleles of the CTSB gene according to tumour grade, increased frequency of the VV genotype was associated with less differentiated tumours. The VV genotype of the CTSB L26V SNP may indicate an increased risk for PCa and less differentiated cancer (higher Gleason score).

Morphometrical evaluation of germ cell apoptosis in infertile men.

Apoptosis associated with programmed cell death plays an essential role in the control of germ cell number in the testes. Although male germ cell apoptosis has been well characterized in different animal models, only a few studies of apoptosis in human testes are presently available. In 43 infertile men with azoospermia of varying aetiology, testicular tissue was obtained by testicular biopsy. Apoptosis of testicular germ cells was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method in situ. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive cells were found in the testicular tissue of all patients with azoospermia, except in Sertoli cell-only syndrome. The apoptotic index was higher in germ cell hypoplasia and in normal spermatogenesis in comparison with germ cell arrest. This study was performed to confirm the presence and determine the frequency of apoptosis in infertile men.

p53, Bcl-2 and AgNOR tissue markers: model approach in predicting prostate cancer characteristics.

The balance between proliferation and apoptosis is represented by changes in the expression of the tissue markers, Bcl-2 and p53, and the presence of silver-stained nucleolar organizing regions (AgNOR) on DNA in prostate adenocarcinomas. Identifying a mathematical model that would take into account the opposing nature of both processes and relate this to cancer stage and grade would be a useful adjunct for studying disease behaviour. This retrospective study investigated tissue marker expression in prostate adenocarcinoma biopsy samples from 17 patients. Staining for p53 was inversely correlated with patient age. Staining for Bcl-2 correlated with the presence of advanced metastatic cancer and American Joint Committee on Cancer (AJCC) disease stage. A mathematical model was developed which combined coded staining intensity data for Bcl-2 and AgNOR, as markers of proliferation, and for p53, as a marker of apoptotis. The mathematical model significantly correlated with Gleason score, AJCC stage and serum prostate specific antigen level, whereas each tissue marker alone did not correlate with all these measures.

Germ cell apoptosis in the human testis.

Apoptosis is a widespread phenomena during development. It represents a form of cell death and has a crucial role in tissue homeostasis. Apoptosis is also involved in a number of pathological conditions. Spermatogenesis is a dynamic process of germ cell proliferation and differentiation. During regular spermatogenesis, the number of testicular germ cells degenerate by an apoptotic process. The significance of regulating cell population by apoptosis is more apparent when sperm production is halted. The presence and frequency of apoptosis in germ cells of human testis biopsy specimens were tested. The results confirm the presence of germ cell apoptosis but not the apoptosis of Sertoli cells. The increased apoptotic index was observed in patients with azoospermia in comparison with normal but obstructed spermatogenesis.

Apoptosis in co-cultures of human muscle and rat spinal cord during the formation of the neuromuscular junction.

The motor neurons and skeletal muscle fibers they innervate are strongly dependent on each other. Important communication between both tissues is mediated through the neuromuscular junction. However, release and reception of various factors at other parts of both tissues must also be considered as means of mutual influences. In vitro innervated human muscle is the only experimental model to investigate nerve - muscle interactions during synaptogenesis of human neuromuscular junction. Here we studied the occurrence of programmed cell death in this model. In order to find out if mutual interactions between neurons and myotubes control the extent of apoptosis in both tissues, we compared the number of apoptotic cells in spinal cord explants and myotubes in co-cultures and mono-cultures of these tissues. Apoptotic cells were detected by TUNEL at selected time intervals (10, 21 and 30 days after co-culture) coresponding to various stages of synaptogenesis of neuromuscular junction. The number of apoptotic cells in spinal cord explants was higher at earlier (day 10) in comparison to later stages (days 21 and 30). Co-cultures and mono-cultures did not differ in this respect. TUNEL positive cells were not found in mono-cultured human myotubes under our experimental conditions. These results suggest that the process of apoptosis seems to be rather independent on neuron - muscle interactions at least at the time points examine.

Restoration of the rat urothelium after cyclophosphamide treatment.

Processes leading to the recovery of a normal three-layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive loss of cells from urothelium, but the remaining cells started to express epidermal growth factor receptor (EGFR) in their plasma membranes. On day 2 after CP injection, proliferating cell nuclear antigen (PCNA) immunohistochemistry showed a rapid increase in positively stained nuclei, from which a hyperplastic urothelium developed, composed of undifferentiated cells expressing EGFR over the entire plasma membrane. Subsequently, EGFR gradually disappeared from the apical plasma membrane but remained in the basolateral membranes. After day 6, PCNA-positive nuclei in all cell layers decreased, except in basal cells. Apoptotic cells were detectable by the TUNEL assay at day 2, and increased in number in all layers of the hyperplastic urothelium until day 10, returning to the control levels by day 14. Electron microscopic evidence showed that apoptotic cells were either pinched off into the bladder lumen or phagocytosed by the neighbouring urothelial cells. Thus, the urothelium responds to the damage by intense proliferation for a week, resulting in an undifferentiated hyperplastic state. Differentiation of superficial cells then begins and damaged cells are gradually removed by apoptosis until the three-layered urothelium is fully restored by two weeks following CP treatment.

Postnatal restoration of the mouse urinary bladder urothelium.

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.

Defective binding of factor XI-N248 to activated human platelets.

Variants of factor XI containing Gln226 to Arg (Q226 to R) and Ser248 to Asn (S248 to N) substitutions were first identified in an African American family with a history of excessive bleeding. The substitutions have recently been identified in unrelated individuals, suggesting they are relatively common. Both amino acids are located in the third apple domain of factor XI, an area implicated in binding interactions with factor IX and activated platelets. Recombinant factor XI-R226 and factor XI-N248 were compared with wild-type factor XI in assays for factor IX activation or platelet binding. Factor XI-R226 activates factor IX with a Michaelis-Menten constant (K(m)) about 5-fold greater than wild-type protein. The catalytic efficiency of factor IX activation is similar to wild-type protein, however, due to an increase in the turnover number (k(cat)) for the reaction. Iodinated factor XI-N248 binds to activated platelets with a dissociation constant (K(d)) more than 5-fold higher than wild-type protein (55 nM and 10 nM, respectively). Activation of factor XI-N248 by thrombin in the presence of activated platelets is slower and does not progress to the same extent as activation of the wild-type protein under similar conditions. Factor XI-N248 activates factor IX normally in a purified protein system and has relatively normal activity in activated partial thromboplastin time (aPTT) assays. Factor XI-N248 is the first factor XI variant described with a clear functional difference compared with wild-type protein. Importantly, the defect in platelet binding would not be detected by routine clinical evaluation with an aPTT assay.

Conserved worldwide linkage disequilibrium in the human factor XI gene.

We have identified, in four diverse human populations, five common single-nucleotide polymorphisms (SNPs) in the coding region of the gene for the blood coagulation protease factor XI. Each SNP has an allele frequency >5% in at least one population. Three of the SNPs (C472T, A844G, and T1234C), spread out over approximately 10 kb of genomic DNA, are in marked linkage disequilibrium (LD) with one another (P < 10(-4)). Interestingly, haplotypes associated with the linked SNPs are conserved across all populations studied, despite significantly different allele frequencies between populations. The presence of such common, widely dispersed haplotypes could complicate the interpretation of LD studies and emphasizes the need for a better understanding of general patterns of LD to facilitate identification of genes for common disorders.

Familial thrombophilia associated with fibrinogen paris V: Dusart syndrome.

We report on a family with a history of venous thromboembolism associated with fibrinogen Paris V (fibrinogen Aalpha-Arg554-->Cys). Ten members experienced thrombotic events, including 4 with fatal pulmonary emboli. Pulmonary embolism was the presenting feature in 4. Those with the mutation and a history of thrombosis had somewhat higher fibrinogen concentrations than those with the mutation and no thrombosis (294 +/- 70 mg/dL vs 217 +/- 37 mg/dL, respectively). The Paris V mutation consistently caused a prolongation of the reptilase time, and fibrin clots containing the abnormal fibrinogen were more translucent than normal clots. Given the early onset of symptoms and the initial presentation with pulmonary embolism in some family members, it was justifiable to offer prophylactic anticoagulation with warfarin to carriers of the mutation. Fibrinogen Paris V has now been reported in 4 apparently unrelated families, indicating that it is a relatively common cause of dysfibrinogenemia-associated thrombosis.

Morphometry of the FRTL-5 cells after irradiation.

The morphological changes of in vitro irradiated FRTL-5 cells and their ability to grow in semi-solid medium were studied morphometrically. FRTL-5 cells were grown in medium with 4 different concentrations of TSH (0, 0.1, 1, 10 mU/ml). After irradiation with 0 Gy, 2 Gy and 4 Gy, the cells were seeded on glass cover-slips and in methocel. Fourteen days after irradiation, the morphometric analysis of FRTL-5 cells and their nuclei was performed. The results showed that irradiation and different doses of TSH have influence on FRTL-5 cell size, more on their nuclei than on the cells as a whole. Growing of FRTL-5 cells in the methocel indicates the possible transformation of these cells after long-culturing in the TSH medium and after irradiation.

Morphology of FRTL-5 cell colonies in a semi-solid medium.

For the first time, 3, 7 and 10 days growth of normal thyroid follicular FRTL-5 cell colonies in a semi-solid medium of 0.6% methocel over 1% agar base was morphometrically analyzed. It was found that the areas of FRTL-5 colonies as well as their perimeters and maximum diameters increased, while according to their form factors the FRTL-5 colonies were regular in shape. After 10 days in a semi-solid medium, 83% of the FRTL-5 colonies had maximum diameters greater than 50 microm. It is obvious that after long culturing of FRTL-5 cells under the influence of the pituitary thyroid-stimulating hormone (TSH) these cells are not uniform anymore and that they grow in a semi-solid medium.

Factor XI messenger RNA in human platelets.

The bleeding diathesis associated with congenital deficiency of factor XI (FXI) is variable and correlates poorly with standard coagulation assays. Platelets are reported to contain FXI activity that may substitute for the plasma protein. The presence of this platelet activity in some patients deficient in plasma FXI could partly explain the variable bleeding associated with the deficiency state. Polyclonal antibodies to plasma FXI recognize a 220 kD platelet membrane protein distinct in structure from plasma FXI. The messenger RNA (mRNA) coding for this protein has been postulated to be an alternatively spliced FXI message lacking the fifth exon found in the liver (wild type) message. We analyzed RNA from platelets, leukocytes, and bone marrow for FXI mRNA by reverse transcription polymerase chain reaction (RT-PCR) technology. Single FXI mRNA species were identified by RT-PCR in platelet and bone marrow RNA, but not leukocyte RNA, that are the same size as the message from liver RNA. Sequencing of PCR products confirmed that the FXI mRNA species in platelets is identical to the one in liver. Wild-type FXI mRNA was also identified in three leukemia cell lines with megakaryocyte features (MEG-01, HEL 92.1.7, and CHRF-288-11). The data show that platelets contain wild-type FXI mRNA. FXI protein, therefore, may be present in platelets and may be released during platelet activation. The data do not support the premise that the 220 kD platelet protein that cross-reacts with FXI antibodies is a product of an alternatively spliced mRNA from the FXI gene.

Detection of mutations by automated fluorescence/RNA-based dideoxy fingerprinting (ARddF)

Dideoxy fingerprinting (ddF) is a hybrid technique which combines aspects of single strand conformational polymorphism (SSCP) and dideoxy sequencing to detect the presence of single base changes in a defined fragment of nucleic acid. ddF is no more technically demanding than SSCP, yet it is more sensitive in detecting point mutations. We describe here the adaptation of conventional ddF to an automated sequencing system using fluorescent Cy5 labeled primers. We show that automated RNA-based ddF (ARddF) has several advantages over conventional radioisotope-based ddF, including: (1) analysis of larger nucleic acid fragments (up to 10(3) bp), due to the ability to continuously analyse and compile sequencing information; (2) greater reliability for distinguishing mutant sequences from wild type sequences (particularly when the mutation leads to gain or loss of a dideoxy termination segment); (3) the use of fluorescent labeled primers, making ARddF less hazardous than methods requiring radionucleotides. The use of ARddF in conjunction with new methods for isolating RNA from a [corrected] small number of cells facilitates mutational analysis of small tissue biopsies and other limited samples, and will allow more widespread application of mutational screening in the setting of clinical diagnostic laboratories.

Activation and motivation of medical students for learning histoembrylogy.

The paper described the present learning/teaching activities for the basic subject in the medical curriculum called histoembryology. Various forms of teaching are presented, but a special emphasis is put on computer assisted testing. The leading idea in the teaching activities is to improve the activation and motivation of the students. This goal has been only partly achieved presumably because of insufficient coordination and integration in the curriculum. The plans for further improvements in histoembryology teaching are presented, including the improvements in computer assisted testing.

Identification of mutations and polymorphisms in the factor XI genes of an African American family by dideoxyfingerprinting.

Congenital deficiency of factor XI is a rare condition associated with a mild to moderate bleeding diathesis that is most commonly found in persons of Jewish ancestry. The disorder has been reported sporadically in a number of other ethnic groups, but rarely in the black population. We report on the genetic analysis of the factor XI genes of two African American patients: a 9-year-old boy (the propositus) with mild factor XI deficiency and his mother. Both individuals have lifelong histories of excessive bleeding. Dideoxyfingerprinting, a technique combining components of single-strand conformational polymorphism analysis and dideoxy-chain termination sequencing, was used in the analysis. Both patients were found to be heterozygous for a mutation changing serine 248 to asparagine [corrected], whereas the propositus was heterozygous for an additional mutation on the paternal allele changing glutamine 226 to arginine. Both mutations reside in the third apple domain of the factor XI heavy chain, an area that has been shown to contain binding sites for factor IX, platelets, and glycosaminoglycans. Previously reported mutations in the factor XI gene seem to cause deficiency primarily by reducing protein expression. Because both alleles in the propositus contain amino acid substitutions, the significant amount of circulating factor XI in his plasma must be comprised entirely of abnormal molecules. Factor XI circulates as a homodimer, and the presence of mutations in both alleles of the factor XI gene suggests that his bleeding disorder is caused in part by the effect of the two abnormal gene products forming dimers in different combinations. Three neutral (not associated with amino acid changes) DNA polymorphisms were also identified in the two subjects: a C to T change at nucleotide 472 in exon 5, A to G at nucleotide 844 in exon 8, and T to C at nucleotide 1234 in exon 11. Analysis of a random sample of normal volunteers showed that these polymorphisms are relatively common, with allele frequencies of 7.4%, 19%, and 18%, respectively. This suggests that there is considerable genetic heterogeneity in the factor XI gene.

The influence of calcium on thyroid follicular cells FRTL-5 in vitro.

The present work is based on the results of in vivo experiments on rats, which had shown that hypercalcemia had led to morphological and biochemical hyperfunction of thyroid follicular cells. The regulation of the activity of follicular cells should directly, or indirectly via paracrine action of serotonin secreted from parafollicular cells, depend on the presence of calcium ions. The effect of calcium was studied on a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) using three methods: measuring the quantity of produced cAMP (cyclic adenosine 3',5'-monophosphate), measuring [3H]thymidine incorporation into cell DNA and transmission electron microscopy. Results show that calcium has no effect on cAMP production. Calcium at 1.3 mM, 3 mM, 10 mM, 20 mM and 30 mM concentrations increases [3H]thymidine incorporation into cell DNA when compared with controls without calcium. Calcium at the concentration of 30 mM has no effect on FRTL-5 cell morphology. TSH (thyrotropin) stimulates follicular cells; at higher extracellular concentrations (3 mM, 10 mM, 20 mM, 30 mM), calcium diminishes its effect, presumably by activation of a cAMP phosphodiesterase which disintegrates cAMP and/or by inhibition of adenyl cyclase.

The effect of irradiation on FRTL-5 cell morphology.

The morphological changes and proliferative activity of FRTL-5 cells irradiated in vitro with one dose of 0, 8, 12, 16, 24 or 32 Gy were studied morphometrically 7, 14 and 21 days after irradiation. The number of cells irradiated with 24 Gy and 32 Gy decreased during the 3 weeks of the experiment. Morphometrical analysis confirmed that the cells repaired their injuries up to 16 Gy of irradiation, while doses of 24 and 32 Gy damaged them irreversibly. Thyroglobulin was detected in cells after all doses of irradiation.

Folate deficiency delays the onset but increases the incidence of leukemia in Friend virus-infected mice.

Clinical studies have indicated that folate deficiency may enhance the development of various malignancies. In animal studies that examined the effect of folate deficiency on malignancies, conflicting results have been reported. In some studies, folate deficiency increased the development and growth of malignant tumors; in others, it decreased the development and growth of malignancies. We examined the effect of transient folate deficiency on the development of leukemia in mice infected with the anemia-inducing strain of Friend leukemia virus. Friend virus disease can be considered as a model for human acute leukemias that are preceded by a preleukemic period. These include leukemias that develop in patients who received previous chemotherapy and/or radiation therapy, as well as patients with chronic granulocytic leukemia or myelodysplasia. Folate deficiency around the time of Friend virus-infection delayed the onset but increased the incidence of leukemia. The rates of rearrangement of the Spi-1 (PU.1 ) oncogene by provirus integration and alteration of the p53 tumor-suppressor gene were the same in leukemia cell lines derived from folate-deficient mice as they were in cell lines from control mice. These results indicate that folate deficiency did not exert its enhancement of leukemogenesis through changes in either Spi-1 or p53, even though these two genes have been found to be the most frequently altered ones in Friend virus-induced leukemias. Our results suggest that folate deficiency may enhance the development of acute leukemia in patients who are at high risk for this disease.

Transforming growth factor beta as a potential tumor progression factor among hyperdiploid glioblastoma cultures: evidence for the role of platelet-derived growth factor.

Among early-passage, near-diploid gliomas in vitro, transforming growth factor type beta (TGF beta) has been previously shown to be an autocrine growth inhibitor. In contrast, hyperdiploid (> or = 57 chromosomes/metaphase) glioblastoma multiforme (HD-GM) cultures were autocrinely stimulated by the TGF beta. The mechanism of this 'conversion' from autocrine inhibitor to mitogen is not understood; previous studies have suggested that platelet-derived growth factor (PDGF) might be modulated by TGF beta. The similar expression of TGF beta types 1-3, PDGF-AA; -BB, as well as the PDGF receptor alpha and beta subunits (a/beta PDGFR) between biopsies of the HD-GM and near-diploid, TGF beta-inhibited glioblastomas (GM) by immunohistochemistry did not explain the discrepancy in their regulatory responses. Flow cytometry demonstrated that TGF beta's mitogenic effect was selective for the aneuploid subpopulations of two of three selected HD-GM cultures, while the diploid cells were inhibited. Among the HD-GM, TGF beta 1 induced the RNA of PDGF-A, c-sis and TGF beta 1. The amount of PDGF-AA secreted following TGF beta treatment was sufficient to stimulate the proliferation of a HD-GM culture. Antibodies against PDGF-AA, -BB, -AB, alpha PDGFR and/or beta PDGFR subunits effectively neutralized TGF beta's induction of DNA synthesis among the HD-GM cell lines, indicating that PDGF served as the principal mediator of TGF beta's growth stimulatory effect. By comparison, TGF beta induced only the RNA of PDGF-A and TGF beta 1 among the near-diploid GM, c-sis was not expressed at all. However, the amount of PDGF-A which was secreted in response to TGF beta 1 was insufficient to prevent TGF beta's arrest of the near-diploid cultures in G1 phase. Thus, the emergence of hyperdiploidy was associated with qualitative and quantitative differences in TGF beta's modulation of PDGF-A and c-sis, which provided a mechanism by which the aneuploid glioma cells might achieve 'clonal dominance'. We hypothesize that TGF beta may serve as an autocrine promoter of GM progression by providing a selective advantage to the hyperdiploid subpopulation through the loss of a tumor suppressor gene which mediates TGF beta's inhibitory effect.