Measurements of the elasticity of monolayers consisting of lipids from nerve membranes.

For measurement of viscoelastic properties of monolayer-covered interfaces a longitudinal wave is generated in the plane of the interface, using a horizontal oscillating barrier. The wave propagation depends on the values of the viscoelastic parameters of the monolayer. The technique is applied here to study the surface elasticity of layers consisting of lipids extracted from nerve membranes. It is concluded that mechanical disturbances are propagated as longitudinal waves. The possibility that longitudinal waves occur in nerve membranes and the role they might play in the transmission of information in biological membranes is discussed.

The role of the tumor suppressor p53 in spermatogenesis.

The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in spermatogonia, suggesting that, in contrast to other reports, p53 does have a role in spermatogonia. To determine the possible role of p53 in spermatogonia, histological analysis was performed in testes of both p53 knock out C57BL/6 and FvB mice. The results indicate that p53 is an important factor in normal spermatogonial cell production as well as in the regulation of apoptosis after DNA damage. First, p53 knock out mouse testes contained about 50% higher numbers of A1 spermatogonia, indicating that the production of differentiating type spermatogonia by the undifferentiated spermatogonia is enhanced in these mice. Second, 10 days after a dose of 5 Gy of X-rays, in the p53 knock out testes, increased numbers of giant sized spermatogonial stem cells were found, indicating disturbance of the apoptotic process in these cells. Third, in the p53 knock out testis, the differentiating A2-B spermatogonia are more radioresistant compared to their wild-type controls, indicating that p53 is partly indispensable in the removal of lethally irradiated differentiating type spermatogonia. In accordance with our immunohistochemical data, Western analysis showed that levels of p53 are increased in total adult testis lysates after irradiation. These data show that p53 is important in the regulation of cell production during normal spermatogenesis either by regulation of cell proliferation or, more likely, by regulating the apoptotic process in spermatogonia. Furthermore, after irradiation, p53 is important in the removal of lethally damaged spermatogonia.

Dynamic and differential Oct-1 expression during early Xenopus embryogenesis: persistence of Oct-1 protein following down-regulation of the RNA.

As a first step towards the elucidation of the role of the transcription factor Oct-1 in development, we prepared a monoclonal antibody to study the spatio-temporal distribution of Oct-1 protein in vivo. Here we report differential expression of the Oct-1 gene in the Xenopus embryo both at the RNA and the protein level. Transcripts and protein are detected in ectodermal and mesodermal cell lineages, in which the expression exhibits a pattern of progressive spatial restriction in the course of development. The Oct-1 expression as reported here is not correlated with cell density or cell proliferation in the embryo. Our results suggest a role of Oct-1 in the specification and differentiation of neuronal and neural crest cells. In many other cells, the developmental decision to down regulate Oct-1 is delayed, probably due to a high stability of the protein.

Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression.

Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx-1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx-2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain-binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.

Regulatory role of p27kip1 in the mouse and human testis.

p27kip1 is a cyclin-dependent kinase inhibitor that regulates the G1/S transition of the cell cycle. Immunohistochemical analysis showed that during mouse testicular development p27kip1 is induced when the fetal germ cells, gonocytes, become quiescent on day 16 postcoitum, suggesting that p27kip1 is an important factor for the G1/G0 arrest in gonocytes. In the adult mouse and human testis, in general, spermatogonia are proliferating actively, except for undifferentiated spermatogonia that also go through a long G1/G0 arrest. However, none of the different types of germ cells immunohistochemically stained for p27kip1. During development, Sertoli cells are proliferating actively and only occasionally were lightly p27kip1 stained Sertoli cells observed. In contrast, in the adult testis the terminally differentiated Sertoli cells heavily stain for p27kip1. Twenty to 30% of both fetal and adult type Leydig cells lightly stained for p27kip1, possibly indicating the proportion of terminally differentiated cells in the Leydig cell population. In p27kip1 knockout mice, aberrations in the spermatogenic process were observed. First, an increase in the numbers ofA spermatogonia was found, and second, abnormal (pre)leptotene spermatocytes were observed, some of which seemingly tried to enter a mitotic division instead of entering the meiotic prophase. These observations indicate that p27kip1 has a role in the regulation of spermatogonial proliferation, or apoptosis, and the onset of the meiotic prophase in preleptotene spermatocytes. However, as p27kip1 is only expressed in Sertoli cells, the role of p27kip1 in both spermatogonia and preleptotene spermatocytes must be indirect. Hence, part of the supportive and/or regulatory role of Sertoli cells in the spermatogenic process depends on the expression of p27kip1 in these cells. Finally, we show that the expression of p27kip1 transiently increases by a factor of 3 after x-irradiation in whole testicular lysates. Hence, p27kip1 seems to be involved in the cellular response after DNA damage.

Microplate washing: process description and improvements.

Heterogeneous immunoassays require wash steps in order to separate bound from free constituents. In this paper we demonstrate that in microplate assays the washing process includes two separate physical processes: (1) a rapid and wash volume-dependent direct dilution of the droplet-shaped residual volume, and (2) a diffusion-limited and strongly time-dependent dilution of a residual layer of liquid, which necessitates the use of time-consuming soak times in the immunoassay. We have shown that optimizing the motion of the wash fluid effectively reduces the residual layer thickness that results in extended soak times. This results not only in improved washing efficiency and reduced background variance in the immunoassay, it also yields a significantly improved immunoassay sensitivity.

Role of cell cycle control in radiosensitization of mouse spermatogonial stem cells.

PURPOSE: To investigate the possible role of cell cycle arrest in the radiosensitization of mouse spermatogonial stem cells due to small conditioning X-ray exposures. MATERIALS AND METHODS: A 24 h fractionation interval between conditioning (1 Gy) and challenging (8 or 9 Gy) exposures was used. Two approaches were followed: the first in the Swiss random-bred wild-type mouse of the radiation-induced cell cycle arrest-evading agents 3-aminobenzamide (3-AB) and caffeine; and, second, using the C57BL/6 mouse of different p53 status. As biological parameter stem cell survival was analysed by the repopulation index (RI) method and chromosomal translocations were recorded using spermatocyte analysis at appropriate posttreatment periods. RESULTS: In the Swiss wild-type mouse, the application of 3-AB or caffeine significantly suppressed the sensitization of stem cells towards killing or translocation induction. In the C57BL/6 mouse, somewhat more variability in response was observed but no significant differences in sensitization between the p53 +/+, +/- or -/- mouse were recorded, suggesting no involvement of p53 in this process. CONCLUSIONS: The results indicate that p53-independent cell cycle regulation plays an important role in the radiosensitization of mouse spermatogonial stem cells.

An easy-to-build-timer for kinetic measurements in flow cytometry.

Measuring kinetics of any time-dependent intracellular process on a cell-to-cell basis is a powerful extension of flow cytometry (FCM) possibilities. A digital timer is described that is easy to build and that fits with all commercial or custom made multiparameter equipment. Of all possible applications of this timer (phagocytosis kinetics, staining of cell components, enzyme reactions, etc.), one example is given: enzymatic intracellular precipitation owing to myeloperoxidase in human peripheral blood cells.

Calculation of S-phase numbers of four major cell categories in human bone marrow from DNA-flow cytometry and counterflow centrifugation data.

Fractionation of heterogeneous cell populations into a number of fractions differing in cell size and composition, followed by measurement of DNA profiles and differential morphology of the samples allows the calculation, using a mathematical deconvolution procedure, of the cell proliferation of the individual categories. The method was applied to human bone marrow, fractionated by counterflow centrifugation into 10 to 15 fractions. Calculated percentage of S-phase cells in four major categories, including myeloid, erythroid, monocytic, and lymphoid cells, were in good agreement with data obtained by tritiated thymidine autoradiography.

A single-parameter analog level discriminator: a cheap and powerful extension of FCM data acquisition.

Modern flow cytometers offer the possibility to measure more parameters of individual cells than the normally available data acquisition equipment can store. To enlarge the number of parameters that is taken into account while recording data, an analog level discriminator is described that is easy to build and easy to implement on any existing FCM system. An example of gated data acquisition in light scattering measurements on nucleated human bone marrow cells is given. The level discriminator may give a cheap and yet valuable extension of expensive data acquisition equipment.

Continuous measurement and analysis of staining kinetics by flow cytometry.

The measurement of color development with time in cells following the start of a staining reaction is of interest in a number of biological systems. These include the subsets of peripheral white blood cells after acridine orange staining, the uptake by cells and nuclei of fluorescent agents, especially antitumor drugs, and measurement of intracellular enzyme kinetics using fluorogenic or absorbing substrates. The present work describes a simple computer program for analyzing flow cytometric (FCM) data versus time, including both the population kinetics of color development and the variability of staining speed within one population of cells. A single-channel absorption measurement in flow (Technicon Hemalog D) was used to record peroxidase kinetics in peripheral blood cells. Every 5 s, a 64-channel absorption histogram was recorded, up to a maximum of 64 histograms. The data were then analyzed by a computer program which searched for the peak channel of each histogram. A least-squares fit was computed for these maxima. The asymmetries of the 64 absorption histograms were compared to see if there was more than one population present with different time constants. Although developed for enzyme kinetic measurements, this program may have wider usefulness in any measurements of time-dependent phenomena by FCM.

Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse.

Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D(1) is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D(1) in spermatogonial proliferation, in particular during the G(1)/S phase transition. Cyclin D(2) is first expressed at the start of spermatogenesis when gonocytes produce A(1) spermatogonia. In the adult testis, cyclin D(2) is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A(al) spermatogonia differentiate into A(1) spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D(2) during spermatogenesis, cyclin D(2) expression was studied in vitamin A-deficient testis. Cyclin D(2) was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A(1) spermatogonia by administration of retinoic acid. Overall, cyclin D(2) seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A(1) spermatogonia. Cyclin D(3) is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D(3) expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D(3) may control G(1)/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D(1) and D(3) seem to be involved in cell cycle regulation, whereas cyclin D(2) likely has a role in spermatogonial differentiation.

P21(Cip1/WAF1) expression in the mouse testis before and after X irradiation.

During spermatogenesis, the radiosensitivity of testicular cells changes considerably. To investigate the molecular mechanisms underlying these radiosensitivity differences, p21(Cip1/WAF1) expression was studied before and after irradiation in the adult mouse testis. P21(Cip1/WAF1) is a cyclin-dependent kinase inhibitor (CDI) and has a role in the G1/S checkpoint and differentiation. P21(Cip1/WAF1) expression was observed in the normal testis, using Western blotting analysis. After a dose of 4 Gy, but not after 0.3 Gy, an increase in p21(Cip1/WAF1) expression could be determined in whole testis lysates. To investigate which germ cells are involved in p21(Cip1/WAF1) protein expression, immunohistochemical analysis was performed on irradiated testis. In the normal testis a weak staining for p21(Cip1/WAF1) was found in pachytene spermatocytes in epithelial stage V up to step 5 spermatids. A dose of 4 Gy of X-irradiation resulted in a transient increase of p21(Cip1/WAF1) staining in these cells with a maximum at 6 h post irradiation, despite the fact that the irradiation did not induce an increase in the number of apoptotic spermatocytes. When a dose of 0.3 Gy was given, no increase in p21(Cip1/WAF1) staining was observed. Using the TUNEL technique, a 10-fold increase in apoptotic spermatogonia was found after a dose of 4 Gy. However, no staining for p21(Cip1/WAF1) was observed in spermatogonia, suggesting that these cells do not undergo a p21(Cip1/WAF1)-induced G1 arrest prior to DNA repair or apoptosis. These data imply that p21(Cip1/WAF1) is a factor which could be important during the meiotic prophase in spermatocytes and repair mechanisms in these cells, but not in spermatogonial cell cycle delay or apoptosis induction.

Effects of gramicidin and tryptophan-N-formylated gramicidin on the sodium and potassium content of human erythrocytes.

In order to get a better understanding in the mechanism by which tryptophan-N-formylated gramicidin (NFG) and gramicidin kill the malaria parasite Plasmodium falciparum in vitro, we studied the capacity of these peptides to change the potassium, as well as the sodium, composition of normal human erythrocytes, and their ability to cause cell lysis. It is shown that both peptides are able to induce potassium leakage from, and sodium flux into, erythrocytes in such a manner that it is most likely that they are able to form cation channels in the membrane of these cells. For both peptides, potassium efflux proceeds at a faster rate than sodium influx, but this difference is greater for NFG than for gramicidin. This explains the observation that gramicidin is more lytic than NFG is, even when comparing concentrations that show the same antimalarial activity. The finding that gramicidin is approximately 10 times more active than NFG in causing potassium efflux from normal erythrocytes, as well as in killing the malaria parasite, supports the hypothesis that peptide-induced parasite death is related to their capacity to induce potassium leakage from infected erythrocytes. Finally, the observation that erythrocytes are able to restore their normal ion contents after losing more than 50% of their potassium content by incubation with NFG or gramicidin, suggests that, in vivo, and upon treatment with drug concentrations that cause full inhibition of parasite growth, these cells would not be irreversibly damaged by action of the drugs.

Apoptosis regulation in the testis: involvement of Bcl-2 family members.

Using immunohistochemical techniques and Western blot analysis, the possible role of Bcl-2 family members Bax, Bcl-2, Bcl-x(s), and Bcl-x(l) in male germ cell density-related apoptosis and DNA damage induced apoptosis was studied. The apoptosis inducer Bax was localized in all mouse and human testicular cell types, but despite the fact that irradiation induces its transcriptional activator, p53 in the human, Bax expression did not change after irradiation. The apoptosis inhibitor Bcl-2 appeared to be present in late spermatocytes and spermatids and was up-regulated in these cells after a dose of 4 Gy of X-rays. Finally, Bcl-x was expressed in both the mouse and human testis. The apoptosis inhibiting long transcripts of Bcl-x, Bcl-x(l), were expressed in spermatogonia and spermatocytes and were up-regulated after X-irradiation. The apoptosis inducing shorter form of Bcl-x, Bcl-x(s), was found to be expressed only in somatic cells, like peritubular and Leydig cells. While Bax is important in germ cell density regulation, Bax expression did not change after DNA damage inflicted by X-radiation. Hence, spermatogonial apoptosis after X-irradiation may not be induced via the apoptosis inducer Bax. Furthermore, as Bcl-x(l), but not Bcl-2, is present in spermatogonia and spermatocytes, Bcl-x(l) may regulate germ cell density, possibly in cooperation with Bax. As Bcl-x(l) expression is enhanced after irradiation, this protein may also have a role in the response of spermatogonia and spermatocytes to irradiation.

Bone tissue-specific transcription of the osteocalcin gene: role of an activator osteoblast-specific complex and suppressor hox proteins that bind the OC box.

Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.

Effective generation of very low density lipoprotein receptor transgenic mice by overlapping genomic DNA fragments: high testis expression and disturbed spermatogenesis.

The generation of functional transgenes via microinjection of overlapping DNA fragments has previously been reported to be successful, but it is still not a widely applied approach. Here we show that the method is very reliable, and should be considered, in case a single large insert clone of the desired gene is not available. In the present study, two large DNA fragments consisting of overlapping cosmids, together constituting the human very low density lipoprotein receptor (VLDLR) gene (35 kb), were used to generate VLDLR transgenic (VLDLR-Tg) mice. Three transgenic founders were born, of which two (strain #2 and #3) generated transgenic offspring. Using Fiber-FISH analysis, the integration site was shown to contain at least 44 and 64 DNA fragments in mouse strains #2 and #3, respectively. This copy number resulted in integration sites of 1.5 and 2.5 megabase in size. Notably, over 90% of the fragments in both mouse strains #2 and #3 were flanked by their complementary fragment. In line with this observation, Southern blot analysis demonstrated that the correct recombination between fragments predominated in the transgenic insertion. Human VLDLR expression was detected in testis, kidney and brain of both mouse strains. Since this pattern did not parallel the endogenous VLDLR expression, some crucial regulatory elements were probably not present in the cosmid clones. Human VLDLR expression in testis was detected in germ cells up to the meiotic stage by in situ mRNA analysis. Remarkably, in the F1 generation of both VLDLR-Tg mouse strains the testis was atrophic and giant cells were detected in the semineferous tubuli. Furthermore, male VLDLR-Tg mice transmitted the transgene to their progeny with low frequencies. This could imply that VLDLR overexpression in the germ cells disturbed spermatogenesis.