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.

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).

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.

Improved detection of p53 point mutations by dideoxyfingerprinting (ddF).

Two screening techniques for identifying point mutations (single-strand conformational polymorphism (SSCP) and dideoxyfingerprinting (ddF)) were compared to sequencing to determine their efficiency in detecting mutations in exons 5-8 of the p53 tumor suppressor gene. Twelve human glioblastoma cell lines were studied by each of the three methods. Ten mutations were identified by sequencing; of these, 10/10 were detected by ddF, while SSCP detected 6/10 true mutations and falsely identified two presumed mutations not confirmed by sequencing. We examined the impact of parameters which influence DNA conformation (gel temperature, gel composition, and PCR product size) on the ability of SSCP and ddF to detect mutations. The sensitivity of SSCP varied with both gel temperature and the size of the PCR product; in contrast, ddF was not influenced by either gel temperature or product length (up to 460 nucleotides). We conclude that the increased sensitivity of ddF, together with its greater ease of application due to the lack of need for optimization, provides significant advantages over SSCP in screening DNA sequences for the presence of point mutations. Our results also suggest that the incidence of p53 mutations may be underestimated in studies of human cancers which utilize SSCP as the method of mutational screening.

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.

Bioaccumulation of metals by bivalves from the Limski Kanal (North Adriatic Sea). IV. Zinc distribution between Mytilus galloprovincialis, Ostrea edulis and ambient water.

From June 1979 to June 1980 the accumulation of zinc by the mussel Mytilus galloprovincialis and the oyster Ostrea edulis, from the Limski Kanal on the west coast of Istria, SR Croatia, Yugoslavia, was determined. The distribution of zinc within tissues and organs of mussels and oysters of the same length was related to the zinc concentration in the ambient water. Three physico-chemical forms of zinc can be distinguished in seawater: "ionic" dissolved (Zn electrochemically determined at the natural pH of seawater); "total" dissolved (Zn determined after sample acidification to pH 2); and particulate Zn (bound to suspended particles with a diameter 0.45 micron). The zinc content of different tissues and organs of both species increased significantly with an increase in their condition factors. Zinc concentration decreased significantly in all body parts of the oyster, but, in the mussel, only the foot was affected. Zinc concentrations in organs that are directly involved in the reproductive cycle showed a seasonal variation. The zinc concentration in the mantle of mussels was significantly and positively correlated with the "ionic" and "total" dissolved zinc content of water. Elevated amounts of particulate material suspended in the ambient water increase the filtration rate, which resulted in an accelerated accumulation of zinc by mussels, but not by oysters. Differences in zinc concentration between samples suggest that the dynamics of accumulation and loss may differ during the year; first, mainly as a consequence of the reproductive cycle in bivalves and the concentration of stimulants for filtration in the surrounding water, and second, by coastal input of Zn and by the amount of metal remobilized from the sediment.

Bioaccumulation of metals by bivalves from the Limski Kanal (North Adriatic Sea). III. Copper distribution between Mytilus galloprovincialis (Lmk.) and ambient water.

Copper accumulation was studied in native mussels, Mytilus galloprovincialis, Lmk., from the Limski Kanal, North West Yugoslavia (Peninsula Istria), from June 1979 to June 1980. The distribution of copper between different body parts of the mussels is correlated with the concentrations of the different physico-chemical forms of copper in the ambient seawater. Free and labile complexes of dissolved Cu were electrochemically determined in a sample at a natural pH of approximately 8, "total" dissolved Cu was determined in acidified samples (pH 2), and Cu after acid decomposition of the suspended particulate matter was collected on 0.45 micron Millipore filters. The copper content correlates with the weight of the organs and with enhanced gametogenetic activity in the mussels. The copper concentrations in the total soft part and in the various organs are highly correlated with the dissolved "ionic" copper content of the ambient water. The particulate matter in the water column influences copper accumulation and its distribution within organs of the mussel. This conclusion arises because the concentration of copper in the mussels is highly correlated with the quantity of particulate material. The copper concentration varies very significantly with condition factors of the foot. Therefore, as the foot can be easily dissected, we propose this organ as a "sentinel" part of the mussel's body for "mussel watch", in the global monitoring program for copper surveillance.

Uptake of mercury species by transplanted mussels Mytilus galloprovincialis under estuarine conditions (Krka river estuary).

Biometric features and physico-chemical conditions are responsible for many of the variables for metal concentrations in indigenous populations of mussels. In order to reduce variations and promote the utility of mussels as bioindicator organisms for environmental mercury concentration levels, individuals of approximately uniform biometric characteristics, selected from a culture of 1-year-old mussels (Mytilus galloprovincialis, Lmk., were transplanted to four locations in the Krka river estuary and nearby coastal area (Eastern Adriatic coast). Biometric parameters of mussels and their total and methylmercury content were analysed four times over a period of 270 days in 1988/89. It was found that total and methylmercury concentrations were significantly correlated with shell weight, wet weight and dry weight of mussels. The estimate of minimal methylmercury concentration in the water of the Krka River Estuary (approximately 20 pg/l), derived from the mussels data, is in a good agreement with recently determined concentration levels of methylmercury (between 50 and 100 pg/l) in waters of the same area. The accumulation efficiency for methylmercury is about 20-50 times higher than for total mercury. Only 1% of the total mercury content, and 20-50% of the methylmercury content in water filtered by the mussels, is accumulated in the shellfish tissues.

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.

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

Serotonin is an aromatic monoamine found in parafollicular cells of rats, bats and in a human medullary thyroid carcinoma cell line. We have examined the hypothesis that serotonin stimulates follicular cells. The effect of serotonin in presence and absence of thyrotropin was studied in a cell line of rat follicular cells FRTL-5 (Fischer Rat Thyroid cells in Low serum) by measuring [3H]thymidine incorporation into cell DNA (growth assay) and by transmission electron microscopy. The cell line was used to avoid the contamination with serotonin secreted from parafollicular cells. Results show that serotonin at 3 microM, 10 microM, 30 microM, and 100 microM concentrations increases [3H]thymidine incorporation into cell DNA. Serotonin at a 1000 microM concentration reduces sharply the [3H]thymidine incorporation into cell DNA. All cytoplasmic organelles completely disappear, only a thin fibrous membrane remains. The toxic effect of serotonin is observed in the nuclei, too. As expected, thyrotropin stimulates follicular cells. Serotonin does not have any influence on that stimulative effect of thyrotropin. Rough endoplasmic reticulum consists of round vesicles, several mitochondria are present in the cytoplasm. From the surface few pseudopodia extend into the culture medium.

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.

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.

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.

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.

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.

Histology of laryngeal mucosa.

The larynx is a complex tubular segment of the respiratory system formed by irregularly shaped plates of hyaline and elastic cartilage. The mucosa form two pairs of folds, false and true vocal cords, which extend into the lumen of the larynx. The laryngeal epithelium corresponding to the mechanically exposed areas consists of stratified squamous nonkeratinized epithelium. Suprabasally in this epithelium, dendritic antigen-presenting Langerhans cells (LCs) can be found. In the rest of the larynx, the epithelium is ciliated columnar pseudostratified with a rich population of goblet cells. Except in the true vocal cords, lamina propria consists of rather loose connective tissue and contains groups of small, branched tubuloalveolar glands.

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.

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.

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.

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.