Bovine herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo.

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency that enables episome tethering to mitotic chromosomes and modulates cellular pathways implicated in growth and survival of latently infected cells. Comparison of pORF73 orthologues encoded by rhadinoviruses reveals important variations in amino acid sequence length and composition. Bovine herpesvirus 4 (BoHV-4) encodes by far the shortest ORF73 orthologue, with a size equivalent to only 22 % of that of the largest orthologues. The present study focused on determining whether BoHV-4 ORF73 is a bona fide gene and investigating whether it is essential for latency, as established for larger ORF73 orthologues. Our results demonstrate that BoHV-4 ORF73 is transcribed as immediate-early polycistronic mRNA together with ORF71. Using a BoHV-4 bacterial artificial chromosome clone, we produced a strain deleted for ORF73 and a revertant strain. Deletion of BoHV-4 ORF73 did not affect the capacity of the virus to replicate in vitro, but it prevented latent infection in vivo using a rabbit model. Interestingly, the strain deleted for ORF73 induced an anti-BoHV-4 humoral immune response comparable to that elicited by the wild type and revertant recombinants. Together, these results demonstrate that, despite its relatively small size, BoHV-4 ORF73 is a functional homologue of larger rhadinovirus ORF73 orthologues, and highlight the potential of ORF73 deletion for the development of BoHV-4 as a vector in vaccinology.

Immunoregulation in experimental autoimmune myasthenia gravis--about T cells, antibodies, and endplates.

Experimental autoimmune myasthenia gravis (EAMG) can be induced in a large number of animal species by active immunization (AI) AChR, by passive transfer (PT) of anti-AChR antibodies, by autologous bone marrow transplantation and cyclosporin (BMT-Cy), or spontaneously. Depending on the model used, different immunological mechanisms are operational. In the AI model, the T cell is pivotal in directing the anti-AChR antibody production towards pathogenic, that is, cross-linking and complement-fixing antibodies. Injection of anti-AChR antibodies alone suffices to induce EAMG, excluding the role of specific cell-mediated immune responses in the effector phase of the disease. Aged animals are resistant to the induction of AI and PT EAMG. This resistance is localized at the postsynaptic membrane containing more AChR-anchoring proteins, including S-laminin and rapsyn in aged animals. In BMT-CyA EAMG, a dysregulation of the immune system in the absence of immunization is capable of inducing myasthenia. The role of these animal models in relation to pathogenesis and immunotherapy is discussed.

New protocol for DNA extraction of stool.

Present methods for DNA isolation of stool have various limitations such as the amount of stool used, the requirement of lavage fluids or the use of fresh stool. In this paper, a new method is described for the isolation of human nucleic acids from stool, which is independent from the moment of collection. Fecal samples as dry as possible were collected from 75 patients; two grams of stool were mixed with a lysis buffer containing phenol. DNA yields of crude stool were variable and ranged from 9-1686 micrograms/g of feces. With dot blots in 9 of the 75 cases, the human DNA was identified and ranged from 0.06%-46%. In the remaining 66 cases, human genomic DNA was detected by nested PCR, using human K-ras gene amplification as an example. Amplification products were confirmed for human K-ras with the exonuclease-amplification coupled capture technique (EXACCT). In conclusion, the developed DNA isolation method can be used for the study of large numbers of stool samples, is independent of the age or method of stool collection and is suitable for large-scale screening studies.

Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins.

The behavior of chimeric proteins consisting of A-type lamins and green fluorescent protein (GFP) was studied to investigate the localization and dynamics of nuclear lamins in living cells. Cell line CHO-K1 was transfected with cDNA constructs encoding fusion proteins of lamin A-GFP, lamin Adelta10-GFP, or lamin C-GFP. In the interphase nucleus lamin-GFP fluorescence showed a perinuclear localization and incorporation into the lamina for all three constructs. Our findings show for the first time that the newly discovered lamin A 10 protein is localized to the nuclear membrane. The GFP-tagged lamins were processed and behaved similarly to the endogenous lamin molecules, at least in cells that expressed physiological levels of the GFP-lamins. In addition to the typical perinuclear localization, in the majority of transfected cells each individual A-type lamin-GFP revealed an extensive collection of branching intra- and trans-nuclear tubular structures, which showed a clear preference for a vertical orientation. Time-lapse studies of 3-D reconstructed interphase cells showed a remarkable stability in both number and location of these structures over time, while the lamina showed considerable dynamic movements, consisting of folding and indentation of large parts of the lamina. Fluorescence recovery after bleaching studies revealed a low protein turnover of both tubular and lamina-associated lamins. Repetitive bleaching of intranuclear areas revealed the presence of an insoluble intranuclear fraction of A-type lamins. Time-lapse studies of mitotic cells showed that reformation of the lamina and the tubular structures consisting of A-type lamins did not occur until after cytokinesis was completed.

Comparison of A and B-type lamin expression in reactive lymph nodes and nodular sclerosing Hodgkin's disease.

AIMS: In order to clarify the differentiation and proliferation status of the Reed-Sternberg and Hodgkin cells we studied A and B-type lamin expression with specific monoclonal antibodies in nodular sclerosing Hodgkin's disease. Its normal counterpart, the reactive lymph node, was also examined for lamin subtype expression. METHODS AND RESULTS: The CD20 positive centrocytes and centroblasts of the follicle centre in the reactive lymph nodes expressed lamin B1, but were not or only very weakly positive for lamin B2 or A-type lamin antibodies. Mantle zone lymphocytes displayed lamins B1 and B2, but were negative for A-type lamins. Furthermore, CD3- and CD20-positive lymphocytes in the medulla and paracortex lacked A-type lamins, but were positive for both B-type lamins. Finally, the proliferation marker Ki67 was mainly detected in the centroblasts, but also in a fraction of the A-type lamin negative cells in the paracortex and medulla. In Hodgkin's disease, all cells expressed lamins B1 and B2, whereas A-type lamins were primarily observed in CD30-positive Reed-Sternberg and Hodgkin cells. About 20% of the Reed-Sternberg and Hodgkin cells expressed Ki67, with co-expression of lamin A in most of these cells. CONCLUSIONS: Ki67 and A-type lamin staining were in general mutually exclusive in lymph nodes, indicating that A-type lamin positive cells are not proliferative. This suggests also that the A-type lamin expression in Reed-Sternberg and Hodgkin cells is correlated with a relatively mature phenotype of these malignant cells. However, some of these differentiated malignant cells still have a capacity to proliferate as indicated by Ki67 positivity. Our observation that lamin B2 expression in the follicle centre cells of the reactive lymph node is low or absent indicates that this lamin subtype is not always expressed in nucleated cells, which is in clear contrast to the results obtained in previous studies in other diseases and in normal tissues. Absence of lamin B2 expression may be associated with the follicle centre stage of B-cells.

Detailed analysis of cell cycle kinetics upon proteasome inhibition.

We have studied specific effects of proteasome inhibition on cell cycle progression. To this end, the protease inhibitors MG115, calpain inhibitor I, and calpain inhibitor II, which display differential inhibitory effects on proteasomes, were used. Cell kinetic studies using bromodeoxyuridine pulse labeling revealed a complete block of G1/S and metaphase transitions and a delayed progression through S phase in cell cultures treated with 54 microM of MG115. Calpain inhibitor I in similar concentrations displayed a fivefold lower effect on cell cycle kinetics. Calpain inhibitor II and MG2M, which is a structural analogue of MG115, had no effect on the cell cycle. The inhibitory effect of MG115 treatment was reversible, because the cell cycle was immediately resumed when the MG115-containing culture medium was replaced by fresh culture medium. Because ubiquitinated proteins accumulated after MG115 treatment, it was confirmed that ubiquitin-dependent protein degradation, and thus proteasomal activity were blocked. By comparison of biochemical and in vitro proteasome inhibition experiments, it was hypothesized that chymotrypsin-like activity of proteasomes may play an important role in cell cycle kinetics.

A- and B-type lamins are differentially expressed in normal human tissues.

A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies.

Nuclear lamin expression in normal testis and testicular germ cell tumours of adolescents and adults.

Nuclear A- and B-type lamins are differentially expressed in tissues, depending on the degree of cellular differentiation and proliferative status. By studying lamin expression in testis parenchyma and testicular germ cell tumours, further insight may be gained into the degree of cellular differentiation in normal testis and into the whole spectrum of differentiation lineages found in testicular germ cell tumours. Frozen tissue sections of normal testis and the different types of testicular germ cell tumours were immunostained with monoclonal antibodies to distinct lamin subtypes. Lamin reactivity was evaluated in relation to the lineage and degree of cellular differentiation and the reactivity patterns were compared with each other and with those in normal testis. In normal testis, both A- and B-type lamins were expressed in Sertoli, Leydig, and peritubular cells, while in spermatogonia only B-type lamins were found and spermatocytes showed weak reactivity with the A-type lamin antibodies. Carcinoma in situ was most often positive for both of the B-type lamins and negative for the A-type lamins (lamins A and C). In testicular germ cell tumours, B-type lamins were always expressed, while A-type lamins were differentially expressed. Differentiated non-seminomas were positive for both of the A-type lamins, whereas embryonal carcinomas were positive for lamin C and negative for lamin A. Seminomas were negative for both of the A-type lamins, with the exception of seminomas containing a Ras mutation. Spermatogonia and seminoma cells, which follow a differentiation pathway along the spermatogenic lineage and show characteristics of germ cells, do not express A-type lamins. Non-seminomas, showing embryonal or extraembryonal differentiation, express A-type lamins to varying degrees, distinguishing embryonal carcinoma cells from other non-seminomatous components. This may aid in the evaluation of the percentage of embryonal carcinoma in non-seminomatous testicular germ cell tumours as a prognostic parameter.

Subcellular localization of proteasomes in apoptotic lung tumor cells and persistence as compared to intermediate filaments.

We have studied the subcellular localization and expression levels of proteasomes during apoptosis in a lung cancer cell line. Apoptosis was induced by exposing the cells to 200 microM olomoucine, a specific cyclin-dependent kinase inhibitor. The morphological changes characteristic for apoptotic cells were visible: the cells reduced in size, the chromatin condensed and the membranes became convoluted. As the process continued, the nuclei became fragmented, and the cells broke up into cytoplasmic vesicles and apoptotic bodies. Immunocytochemically, apoptotic cells were detected by the ability to bind annexin V at their surface. During the initial stages of apoptosis, proteasomes were present in the nucleus as well as in the cytoplasm. Upon increased chromatin condensation, nuclear proteasomes were found predominantly surrounding the chromatin, while the chromatin itself remained devoid of staining. That the proteasomes persisted relatively long in the apoptotic cells was shown by immunoblotting of non-denaturing gels, which indicated that both 20S and 26S proteasomes were present in apoptotic cells. In immunofluoresence microscopy the proteasome fluorescence intensity of apoptotic cells seemed higher than that of non-apoptotic cells. These differences in intensity were even more pronounced after Triton X-100 extraction. Flow cytometry revealed that the absolute levels of proteasome staining in cells were decreased after Triton X-100 extraction. However, no differences in staining levels were detected between apoptotic and non-apoptotic cells. A relative increase of proteasome concentration through cell shrinkage or a concentration in certain cell compartments may be the origin of the apparently increased signal that was seen in immunofluorescence microscopy. Furthermore, proteasomes were clearly detectable in the apoptotic bodies and cytoplasmic vesicles at the time immunocytochemical reactivity for cytokeratins and lamins had diminished to a large extent. Immunoblotting of denaturing polyacrylamide gels confirmed the results obtained by flow cytometry. The proteasome content was retained only partially in the cells after Triton X-100 extraction, while the intermediate filaments were not detectable anymore in the apoptotic cells.

An alternative splicing product of the lamin A/C gene lacks exon 10.

Expression of the A-type lamins was studied in the lung adenocarcinoma cell line GLC-A1. A-type lamins, consisting of lamin A and C, are two products arising from the same gene by alternative splicing. Northern blotting showed in GLC-A1 a relatively low expression level of lamin C and an even lower expression level of lamin A as compared to other adenocarcinoma cell lines. Immunofluorescence studies revealed highly irregular nuclear inclusions of lamin A, suggesting protein or gene expression abnormalities. Reverse transcriptase-polymerase chain reaction-based cDNA analysis followed by sequencing indicated the presence of an as yet unidentified alternative splicing product of the lamin A/C gene. This product differs from lamin A by the absence of the 5' part of exon 10 (90 nucleotides). Therefore we propose to designate this product lamin Adelta10. Deletion of the 30 amino acids encoded by exon 10 was predicted to result in a shift in pI of the protein from 7.4 to approximately 8.6, which was confirmed by two-dimensional immunoblotting. mRNA analysis in a variety of cell lines, normal colon tissue as well as carcinomas demonstrated the presence of lamin Adelta 10 in all samples examined, suggesting its presence in a variety of cell types.

Abnormal A-type lamin organization in a human lung carcinoma cell line.

We have studied the expression of lamins A and C (A-type lamins) in a lung carcinoma cell line using type-specific monoclonal antibodies. Using immunofluorescence and immunoblotting studies it was noted that several irregularities in lamin expression exist in the cell line GLC-A1, derived from an adenocarcinoma. First, the expression of the A-type lamins was lower than in other adenocarcinoma cell lines of the lung. Also the ratio between lamins A and C proteins was 1:8 instead of the 1:1 ratio seen in the other cell lines. Northern blotting confirmed the altered level of A-type lamin expression. Secondly, an abnormal localization of lamin A was observed. Intensely fluorescing lamin A aggregates were observed in the nucleus, rather than the typical perinuclear staining pattern. Confocal scanning laser microscopy revealed that the lamin A aggregates were indeed present throughout the internal nucleus. When these cells were extracted with Triton X-100 the nucleoplasmic aggregates disappeared, which indicates that the A-type lamins are not properly incorporated into the lamina. The A-type lamins in other cell lines derived from adenocarcinomas remained present in the nuclear periphery after extraction with the non-ionic detergent. Immunoblotting studies of the Triton X-100 soluble and insoluble fractions showed that lamin A and an apparently truncated product, which was detected with the lamin A antibody, were present in the insoluble fraction of GLC-A1. This truncated product is partly Triton X-100 soluble since it was also detected in the detergent soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in immunocytochemical detectability of proteasome epitopes depending on cell growth and fixation conditions of lung cancer cell lines.

The localization of proteasome epitopes in the lung cancer cell lines NCI-H82, derived from a small cell lung cancer, and MR65, derived from a squamous cell lung carcinoma, was studied in relation to cell growth conditions. For this purpose the proteasome monoclonal antibodies MCP34 and MCP20 were applied to the cells growing under different nutritional conditions, resulting in different proliferative states. Using indirect immunofluorescence microscopy with brief fixation in methanol (5 sec, -20 degrees C) followed by three dips in acetone (5 sec at room temperature), it became obvious that the intracellular detectability of the proteasomes changes depending on the nutritional and proliferative status of the tumor cells. Two types of experiments were carried out: (1) cells were grown for two days at different cell densities, with an excess of culture medium, and (2) cells were seeded in a low cell density and monitored for 6 days without change of medium. In cells grown at low density, the proteasomes can be detected mainly in the nuclei, while the nucleoli are almost devoid of staining, and the cytoplasm is only slightly stained. In cells grown at high density, the staining pattern changes with a much less pronounced nuclear staining than in the cells at low density, while the cytoplasm remains slightly stained. In the nutrient depletion experiment similar changes were seen. In cells growing under favorable conditions (1 or 2 days in fresh medium) proteasomes are detected mainly in the nuclei, whereas when the medium becomes depleted of nutrients (4 or 5-day-old medium) the staining pattern changes to one with a much less pronounced nuclear staining. However, in immunofluorescence studies on cells grown under similar conditions but fixed in ethanol (-20 degrees C) for 15 min, the changes in proteasome localization pattern were not detected during medium depletion. Using this fixation protocol the proteasomes are detected mainly in the nuclei at all stages of the medium exhaustion experiment. These apparently contrasting results suggest that upon nutrient depletion the proteasome epitopes become less accessible to the antibodies used. Apparently, the epitopes can regain accessibility if an extended ethanol fixation is used. This hypothesis was confirmed by flow cytometry and immunoblotting experiments. In flow cytometry of ethanol-fixed cells the fluorescence intensity of only a minor part of the cell population decreases to some extent with medium depletion, but in the majority of the cells fluorescence remains at its initial level. The immunoblotting experiments show no quantitative changes in proteasome content of the tumor cells at the different growth conditions.

RFLP markers linked to the root knot nematode resistance gene Mi in tomato.

The Mi gene originating from the wild tomato species Lycopersicon peruvianum confers resistance to all major root knot nematodes (Meloidogyne spp.). This single dominant gene is located on chromosome 6 and is very closely linked to the acid phosphatase-1 (Aps-1) locus. Resistance to nematodes has been introgressed into various cultivars of the cultivated tomato (L. esculentum), in many cultivars along with the linked L. peruvianum Aps-1 (1) allele. By using a pair of nearly isogenic lines differing in a small chromosomal region containing the Mi and Aps-1 loci, we have identified two RFLP markers, GP79 and H6A2c2, which are located in the introgressed L. peruvianum region. Analysis of a test panel of 51 L. esculentum genotypes of various origins indicated that GP79 is very tightly linked to the Mi gene and allows both homozygous and heterozygous nematode-resistant genotypes to be distinguished from susceptible genotypes, irrespective of their Aps-1 alleles. Marker H6A2c2 is linked to the Aps-1 locus and is capable of discriminating between the L. peruvianum Aps-1 (1) allele and the L. esculentum Aps-1 (3) and Aps-1 (+) alleles. In combination, these RFLP markers may provide a powerful tool in breeding tomatoes for nematode resistance.