Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins?

Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane-bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein-framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes.

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor.

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

In vitro nuclear transport of ribosomal ribonucleoprotein: temperature affects quantity but not quality of exported particles.

The in vitro export of ribosomal ribonucleoprotein (rRNP) from Tetrahymena nuclei was investigated at the optimal growth temperature of 28 degrees C and at the nonlethal temperature of 8 degrees C. At both temperatures, nuclei exported ribosomal precursor particles that revealed the same physical qualities of size, appearance in negative-staining electron microscopy, sedimentation coefficient, buoyant density, and rRNA pattern. Surprisingly, fewer rRNP particles were exported at 8 than at 28 degrees C, as was revealed by a lower saturation plateau in the export kinetics from nuclei prelabeled with [3H]uridine. Upon a temperature increase from 8 to 28 degrees C, additional rRNP particles were exported. We conclude that nuclei export only a defined portion of rRNP particles at a given temperature, although enough potentially transportable rRNP particles are present in nuclei. Obviously, the reactivity of at least one of the reactants involved directly or indirectly in rRNP export changes with temperature.

Thermal diminution and augmentation of the retention of transportable rRNA in nuclear envelope-free nuclei.

We have examined the effect of temperature on the rRNA transport from nuclei isolated from Tetrahymena after removal of both nuclear membranes and pore complexes by 1% Triton X-100. These nuclei export rRNA as precursor ribosomal ribonucleoprotein particles at both 28 degrees C and 8 degrees C which are qualitatively the same in terms of rRNA pattern, sedimentation coefficients and buoyant densities. At 8 degrees C, however, significantly fewer ribosomal ribonucleoprotein particles can be maximally exported than at 28 degrees C, though nuclei contain enough potentially transportable particles. These are increasingly released with increasing temperatures. Under conditions non-permissive for export, temperature elevation decreases the number of the potentially transportable ribosomal ribonucleoprotein particles in nuclei. Our data show: transportable ribosomal ribonucleoprotein particles inside nuclei are not 'free', but rather are subject to a complex temperature-sensitive retention: this retention is gradually diminished under export conditions and augmented under non-permissive export conditions with increasing temperatures. These retention mechanisms operate at an intranuclear level preceding the ribosomal ribonucleoprotein passage through the nuclear envelope pore complexes, i.e., the nuclear envelope regulates neither the number of potentially transportable ribosomal ribonucleoprotein particles in nuclei nor the number of those particles which can be maximally exported from nuclei at a given temperature. We suggest that these retention mechanisms involve temperature-sensitive domains of the nuclear matrix.

Similar effects of electroporational stress and treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate on vimentin expression in mouse plasmacytoma cells.

In mouse plasmacytoma cells (MPC-11), an activation of the normally repressed vimentin gene was observed as a response to transfectional stress. Effects of electroporation on vimentin gene expression were compared at the cellular and chromatin level to those caused by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). At the cellular level, similar changes in vimentin gene activity and cell-cycle distribution were observed by flow cytometry, whereas at the chromatin level similar changes in patterns of hypersensitive regions were detected by DNase I mapping. Additionally, a region located 700 bp upstream of the transcriptional start became hypersensitive to DNase I digestion upon electroporation and TPA treatment. This region overlaps two adjacent AP-1-like binding elements and generates specific DNA/AP-1 complexes in bandshift experiments. Therefore, the transcription factor AP-1 seems to play a central role in the activation of vimentin gene expression induced by these 2 different forms of stress.

Induction of vimentin synthesis in a murine myeloma cell line by TPA is strongly dependent on the composition of the cell culture medium.

MPC-11 mouse plasmacytoma cells virtually lacking intermediate filament (IF) proteins can be induced to synthesize and accumulate the IF protein vimentin by treatment with the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Like MPC-11 cells, X63-Ag8.6.5.3 mouse myeloma cells (Ag8) proved to be vimentin-negative, as assayed by immunoblotting of whole cellular protein using goat antiserum to vimentin and [125I]protein A. Vimentin synthesis could be elicited by a TPA concentration as low as 10(-9) M in cells grown in HB-102 serum-free medium. Transfer of these cells to medium containing 15% fetal calf cerum (FCS) greatly reduced the ability of these cells to synthesize vimentin upon TPA treatment. After 50 generations of culture in the presence of FCS, induction of vimentin synthesis was barely detectable even at a TPA concentration of 10(-6) M. Addition of FCS to cells grown in serum-free medium partially suppressed vimentin induction by TPA. This suppression seems to be due, at least in part, to nondialyzable, heat-sensitive components of FCS, since the dialyzable fraction even enhanced vimentin induction by TPA. When cells grown in the presence of FCS were transferred back to serum-free medium, their ability to synthesize vimentin in response to TPA treatment was readily restored. The individual components of serum-free medium which proved to support vimentin induction by TPA were insulin and the unsaturated fatty acids oleic acid and linoleic acid. An even stronger TPA response could be elicited by a combination of these components.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of vimentin synthesis in mouse myeloma cells MPC-11 by 12-0-tetradecanoylphorbol-13-acetate.

Mouse myeloma cells (MPC-11 cell line) known to lack intermediate filaments were treated with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). Asynchronous cell cultures were screened for vimentin by indirect immunofluorescence microscopy, whole cell lysates derived from such cultures by immunoblotting using goat antiserum to vimentin. The minimum TPA concentration sufficient for the induction of vimentin synthesis was found to be 3 X 10(-9) M; substantially larger amounts of vimentin could be detected after treatment of cells with TPA at a concentration of 3 X 10(-8) M. At each effective TPA concentration tested, the maximum level of vimentin was reached after 18 to 24 h; it was dependent on the TPA concentration. In addition, vimentin synthesis was demonstrated employing two-dimensional polyacrylamide gel electrophoresis in combination with either fluorography or immunoblotting and autoradiography. Vimentin purified from TPA-treated MPC-11 cells as well as a protein species in whole lysates from cells labelled with [35S]methionine after TPA treatment for at least 2 h comigrated with vimentin isolated from Ehrlich ascites tumor cells. The fact that only poly(A) +RNA from TPA-treated MPC-11 cells was able to direct vimentin synthesis in vitro suggests that in MPC-11 cells vimentin production is regulated at the transcriptional level.

Expression of vimentin and nuclear lamins during the in vitro differentiation of human promyelocytic leukemia cells HL-60.

We have reported previously that the human promyelocytic leukemia cell line HL-60, in its undifferentiated state, is devoid of cytoplasmic intermediate filament proteins and nuclear lamins A and C, but does express lamin B. Using immunofluorescence and immunoblotting techniques, we have further investigated the expression of vimentin and lamins A and C during differentiation of these tumor cells along the macrophage or granulocytic pathway in response to the inducing effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide. Our results show that, while the expression of lamin B remains largely unchanged, the synthesis of vimentin and lamins A and C is dramatically enhanced during the maturation of HL-60 cells along both hemopoietic pathways. Northern blot analysis of cellular RNAs isolated from untreated and TPA-treated HL-60 cell populations as well as from control HeLa cells was performed using two oligonucleotides, one complementary to the 5' region common to human lamin A/C mRNAs and the other to the 5' region of hamster vimentin mRNA. Very low but still detectable amounts of vimentin and lamin A/C mRNAs were found in untreated HL-60 cell population, in accordance with the detection of small quantities of vimentin and lamins A and C in these populations. This is probably due to the presence of a small number of spontaneously differentiating cells. On the other hand, strong signals comparable to those obtained with RNA from control HeLa cells were detected for the three mRNA species from TPA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis.

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.

Differentiation and retrodifferentiation of U937 cells: reversible induction and suppression of intermediate filament protein synthesis.

Significant morphological and functional changes were observed when human monoblastoid U937 tumor cells growing in suspension were induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h to differentiate along the monocyte/macrophage pathway. These include adherence of the cells to each other and to the substratum, alterations in cell-surface antigen expression and cessation of autonomous proliferation. In this study, we show by both, hybridization analysis of RNA and immunoblotting that an enhanced expression of the intermediate filament (IF) subunit proteins vimentin, lamin A and lamin C accompanied the TPA-induced differentiation process. After long-term culture of differentiated U937 cells in the absence of TPA (more than 28 days), however, the adherent cells retracted their pseudopodia, detached and started again to proliferate. This "retrodifferentiation" process, not previously described was paralleled by a rapid down-regulation of both, IF mRNA and protein synthesis back to the level of undifferentiated U937 control cells. These data suggest a functional relationship between the expression of vimentin and lamins A and C and the differentiation process taking place in these cells.

Estradiol binding to cell surface raises cytosolic free calcium in T cells.

The Fura-2 method is used to examine a possible action of 17beta-estradiol (E2) on [Ca2+]i of splenic T cells isolated from female C57BL/10 mice. E2 concentrations between 10 fM and 10 nM induce a rapid and dose-dependent increase in [Ca2+]i due to Ca2+ influx and release of Ca2+ from intracellular stores. Ca2+ influx is mediated by Ca2+ channels which are completely blockable by Ni2+ and partly by nifedipine. The antiestrogen tamoxifen does not inhibit the E2-induced rise in [Ca2+]i. Ca2+ influx and Ca2+ release from intracellular stores is also inducible by plasma membrane impermeable E2 conjugated to BSA. E2-BSA-FITC binds to the surface of T cells of both the CD4+ and CD8+ subset. Our data suggest a novel E2-signalling pathway in T cells which is not mediated through the classical nuclear estrogen receptor response but rather through putative plasma membrane receptors for E2.

Non-immunologically-mediated cytotoxicity of Lactobacillus casei and its derivative peptidoglycan against tumor cell lines.

Lactobacillus casei, which shows antitumoral activity mediated by the stimulation of cellular defence mechanisms, and its peptidoglycan were tested for their ability to inhibit in vitro the viability of various murine (Yac-1, P815, Ehrlich ascites tumor, mammary carcinoma) and human (K562, KB) tumor cell lines through primary cytotoxic activity. Treatment of these tumor line with L. casei or its peptidoglycan at different doses and for different times demonstrated a decrease in viability by 25-30%. This cytotoxic activity was revealed by 51Cr release, succinate dehydrogenase (SDH) activity, ATP assays and morphological alterations in the treated tumor cells. Immunoenzymatic assays (ELISA) showed a precise ratio of binding between Ehrlich ascites or YAC-1 cell membranes and peptidoglycan. This binding is discussed with regard to the structure of the peptidoglycan molecule. The results suggest that L. casei and its derivative peptidoglycan have both a stimulating activity in normal cells and an inhibiting activity in tumor cells, as has been found for other immunomodulatory complexes.

Polymerase chain reaction-assisted papillomavirus detection in cervicovaginal smears: stratification by clinical risk and cytology reports.

Seven hundred and twelve patients from cancer screening, pregnancy care, outpatient clinics for patients at risk for cervical dysplasia and human immunodeficiency virus (HIV) infection were tested simultaneously for cytological aberrations and human papillomavirus (HPV). Classification of these cases, and of all cytology records throughout 1991 and 1992 was performed according to the "Münchner Nomenklatur" and the Bethesda classification. HPV-directed polymerase chain reaction analysis was carried out with general primers, patients at risk for cervical dysplasia were tested by subsequent hybridization with HPV 16 and 18 probes. Patients from cancer screening and pregnancy care showed similar HPV prevalences ranging between 19.4%-24.6%. In contrast, patients from dysplasia and HIV units were infected in 56.2%-62.3% and 75.0%-76.9% respectively in centre of disease control stage III-IV, HPV detection rates in patients from dysplasia and HIV units increased gradually from 40.1%-52.9% in non-suspicious smears to 80.8%-100% in atypical smears. High risk HPV 16 and 18 infections were detected in 64% of smears with cytological evidence of HPV infection (koilocytosis) to 84.2% in severe dysplasia. Following the Bethesda guidelines, 2.9%-14.7% of all smears initially reported as Pap 2 K (suggestive of HPV infection) would be qualified as risk lesions (low grade squamous intraepithelial lesions), although they tested HPV negative in more than a third of cases. Thus, when using the Bethesda system, HPV analysis is needed to prevent overclassification and overtreatment. The "Münchner Nomenklatur" avoids this dilemma by not mixing morphological statements on infection, atypia and cancer risk.

Schistosomiasis in women: manifestations in the upper reproductive tract.

Female genital schistosomiasis (FGS) is a neglected disease entity which may give rise to considerable suffering among women of child-bearing age in areas where schistosomiasis (especially due to Schistosoma haematobium) is prevalent. The close relation between the vessels in genital organs and the urinary bladder enables the parasite to easily change location to virtually any organs in the female pelvic area. Symptoms concur with the anatomical location of worm pairs and their ova. Lesions of the lower female genital tract can easily be investigated by cytology, histology or direct demonstration of eggs in scrapings or biopsies whereas schistosomiasis of the upper genital tract is clinically indecipherable and less accessible for examination. In the literature there are references to FGS as a cause of infertility, complications of pregnancy, menstrual disorders, problems related to sexual intercourse, diagnostic similarities to STDs and cancer, unspecified complaints related to blood loss, chronic abdominal pain, social segregation and related psychological problems. The diagnosis of female upper genital schistosomiasis is difficult and the authors point out possible diagnostic procedures which might be helpful for further understanding of this complex entity.

Female genital schistosomiasis due to Schistosoma haematobium. Clinical and parasitological findings in women in rural Malawi.

A total of 51 women with urinary schistosomiasis haematobium were examined in order to identify diagnostic indicators for female genital schistosomiasis (FGS). Patients were selected at random from the outpatient department of the Mangochi District Hospital, Malawi. The medical histories were recorded according to a pre-designed questionnaire and the women were subjected to a thorough gynaecological examination including colposcopy and photographic documentation of lesions. Microscopy of genital biopsies revealed that 33 of the 51 women had S. haematobium ova in cervix, vagina and/or vulva in addition to the presence of ova in urine. The most sensitive diagnostic procedure was beside microscopic examination of a wet cervix biopsy crushed between two glass slides, which revealed 25 of the 33 genital infections. There was a significant correlation between the size of genital lesions and the number of ova counted per mm2 of crushed tissue. Women with FGS had significantly more tumours in the vulva than women with schistosomiasis limited to the urinary tract. Most of the observed genital pathology could easily be identified by the naked eye, but colposcopic examination yielded valuable additional information like the demonstration of neovascularisation around cervical sandy patches. Few of the symptoms previously regarded as indicators for FGS could be linked to the presence of schistosome ova in genital tissue. Husbands of infertile women with FGS had children with other women significantly more often than husbands of women who only had urinary schistosomiasis. This, together with the finding that the majority of the divorced women had FGS, indicates that the manifestation of this disease may have implications for the marital and sexual life of the affected women.

Urine reagent strips for diagnosis of schistosomiasis haematobium in women of fertile age.

Hematuria, proteinuria and leukocyturia were semiquantitatively assessed by reagent strips in single morning urine of women of fertile age visiting the outpatient department of the Mangochi district hospital, Malawi. This was part of a diagnostic approach to female genital schistosomiasis (FGS). In 51 women ova of Schistosoma haematobium were detected in urine by a filtration technique. In 33 of these women ova were also present in genital tissue as demonstrated by microscopic examination of biopsies. In 209 women no ova were found in the single urine filtered. There were significantly higher scores for hematuria, proteinuria and leukocyturia as well as of the combined reagent strip index (RSI) in egg-excreting than in egg-negative women. The sensitivity of a single hematuria, proteinuria and leukocyturia reading was 98, 84 and 73%, respectively. However, the respective specificity was only 24, 22 and 23%. The best prediction of urinary schistosomiasis was achieved by a +2 score for hematuria, of which the sensitivity was 94% and the specificity was 61%. The high false-positive rates can probably be explained by contamination of urine by vaginal secretion. Moreover, cases of schistosomiasis have probably been overlooked because only a single morning urine sample was examined. The total absence of hematuria, proteinuria and leukocyturia, however, may be used to rule out heavy infections in community surveys. There was no difference in reagent strip scores between women with genital and urinary schistosomiasis as compared with those with urinary tract lesions alone. Thus urine analysis reagent strip readings do not help to discriminate between S. haematobium infected women with and without FGS.

Diagnosis of female genital schistosomiasis by indirect disease markers: determination of eosinophil cationic protein, neopterin and IgA in vaginal fluid and swab eluates.

Based on assumptions about the pathophysiology of egg-related lesions in the lower reproductive tract, putative indirect disease markers were investigated in vaginal fluids from 54 Malawi adolescent girls and women infected with S. haematobium. These women received a careful gynecological examination during which biopsies were taken from the cervix, and, if present, also from suspicious lesions in the vagina and the vulva. If the biopsies, either in wet crushed preparations or in histological sections, contained eggs the patients were considered to have female genital schistosomiasis (FGS; n = 33). The remainder (n = 21) were classified as having urinary schistosomiasis only. Eosinophil cationic protein (ECP), a cytotoxic granule protein of eosinophils, neopterin, a second messenger molecule generated during the activation of macrophages, and IgA as an indicator of local B-cell activation were quantitatively determined in vaginal fluid. To clarify the origin of ECP, this protein was also looked for in histological sections by an immunohistochemical method. In order to explore whether such disease markers can be detected after absorption to a tampon-like material, ECP and IgA were also assessed after elution from a non-porous, polypropylene fibre web impregnated with vaginal fluid. The concentration of ECP in vaginal fluid and the degree of immunohistochemical staining in histological sections were significantly higher in patients with FGS than in women with urinary schistosomiasis only. The amount of ECP detected in histological sections correlated to the number of eggs/mm2 of compressed genital tissue (rho = 0.36, P = 0.02), and the concentration of ECP in vaginal fluid correlated to the concentration of neopterin as well as to that of IgA (rho = 0.52, P = 0.004 and rho = 0.37, P = 0.02, respectively). Median neopterin concentration in vaginal fluid was also higher in the FGS group, but the difference was not statistically significant. ECP could also be detected in eluates from impregnated fibre webs, but the concentration was approximately one power of 10 less than in the original vaginal fluid. These results demonstrate that indicators of immunological mechanisms related to the egg-granuloma might be useful as indirect disease markers for women with FGS if assessed in vaginal washings or swab eluates.

Female genital schistosomiasis (FGS): relationship between gynecological and histopathological findings.

Schistosomiasis of the lower female reproductive tract manifests itself in a broad spectrum of clinical features. However, clinical and histopathological findings have never been studied in a synoptic manner. Based on the assumption that any type of pathology present in the female reproductive tract is the expression of a complex pathophysiological reaction towards eggs sequestered in the genital tissues, we decided to analyze colposcopic and histopathological findings in a comprehensive manner. Thirty-three women in Malawi with urinary and genital schistosomiasis were examined parasitologically and gynecologically. A thorough colposcopic examination with photodocumentation was performed and biopsies were taken from the cervix, the vagina and/or the vulva for histological sectioning and immunohistochemistry. The predominant colposcopic findings were sandy patches on the cervical surface similar to those seen in the bladder and polypous/papillomatous tumors with irregular surface on the vaginal wall and in the vulvar area. The histopathological sections of sandy-patch-like lesions demonstrated only a small cellular reaction around S. haematobium eggs in various stages of disintegration. In contrast, in the case of polyps the histology revealed a more pronounced immunological reaction characterized by a heavy cellular infiltrate. One case of invasive squamous cell carcinoma of the cervix was diagnosed. We conclude that colposcopy is a useful tool in the detection of FGS related pathology in the lower female reproductive tract and that the synoptic assessment of surface and of corresponding histological sections helped to understand the pathophysiology of S. haematobium associated disease in genital tissue.