Birth of the life sciences in The Netherlands and Belgium.

Small countries can seldom compete in science with larger, more powerful nations. The Netherlands and Belgium, however, are excellent examples of centres that fertilized scientific activity in neighbouring countries for over 400 years.

Walther Flemming: pioneer of mitosis research.

The German anatomist Walther Flemming began his pioneering studies of mitosis almost 150 years ago. What were his achievements, and where have his discoveries led?

From Galen to Golgi: birth of the life sciences in Italy.

Between the sixteenth and twentieth centuries, the study of biology was intimately intertwined with progress in medicine. So how, when and where did research into the life sciences begin?

Lovastatin induces mitotic abnormalities in various cell lines.

We examined the effects of lovastatin, a common anti-atherosclerotic drug and a blocker of the cell cycle, on the process of mitosis. It is known that lovastatin induces an arrest or a retardation of the cell cycle in many cell types not only at the G(1)phase, but also at the G(2)/M transition. After 24-48 h incubation of epithelial PtK(2), T24, HeLa cells and fibroblastic L929 cells in the presence of 1. 0-60.0 microm lovastatin, diverse mitotic perturbations have been observed. The most noteworthy phenomena recorded were prometaphase retardation and chromosome lagging during metaphase and anaphase. After the recovery in lovastatin-free media, the cells continued mitosis without any disturbances. Mevalonic acid prevented the effects of lovastatin. We conclude that the effects were specific for lovastatin-induced inhibition of mevalonic acid synthesis. Immunofluorescence studies with anticentromeric antibodies suggested that one of the possible causes of the lovastatin-induced mitotic disorder could be an interference with the development and function of the centromeres.

The influence of pneumoperitoneum used in laparoscopic surgery on an intraabdominal tumor growth.

BACKGROUND: Numerous clinical reports have raised the possibility that laparoscopic cancer surgery has an inherently detrimental effect on tumor growth. The aim of the current study was to examine the influence of a pneumoperitoneum on the morphology of the peritoneum and the intraperitoneal tumor cell implantation and growth in the case of intraabdominal spread of tumor cells. METHODS: Black mice were stratified into 2 groups of 36 animals each. A CO(2) pneumoperitoneum was induced for 30 minutes in Group 1 and 200,000 cells of a malignant melanoma were injected intraperitoneally. In Group 2 only tumor cells were injected. After a defined period of hours, 4 animals were killed and the peritoneum was examined by scanning electron microscopy. RESULTS: In Group 1, starting 2 hours after release of the pneumoperitoneum, strong alterations of the peritoneum became visible and parts of the underlying basal lamina were laid bare. Tumor cells attached to the free basal lamina and formed predominantly diffuse metastases throughout the peritoneum within the next 96 hours. No diffuse changes of the peritoneal surface and no diffuse metastases were observed in the second group. Singular metastases followed gravity and involved the anterior abdominal wall. CONCLUSIONS: Because these findings explain the clinical findings of intraabdominal metastases after laparoscopy the authors conclude that the pneumoperitoneum provokes particular damage to the peritoneum that induces a specific intraperitoneal tumor growth. [See also editorial counterpoint on pages 747-8 and reply to counterpoint on pages 749-50, this issue.

[A scanning microscopy study of the peritoneum in mice after application of a CO2-pneumoperitoneum]. / Rasterelektronenmikroskopische Untersuchungen am Peritoneum der Maus nach Anlage eines CO2-Pneumoperitoneums.

OBJECTIVE: The application of a CO2-pneumoperitoneum in operative laparoscopy presumably leads to basic alterations of the intraperitoneal homeostasis. In order to better understand the pathophysiology of this phenomenon, the morphologic alterations of the mesothelium after CO2-application will be examined. MATERIAL AND METHODS: In 36 mice (C 57, black mice) a CO2-pneumoperitoneum with an intraperitoneal pressure of 6 mm Hg was applied for 30 minutes. After 1, 2, 6, 12, 24, 48, 72 and 96 hours each of 4 animals were killed and the entire peritoneum was examined by scanning electron microscopy. RESULTS: Already after 1 hour mesothelial cells became cuboidal, were detached and showed condensation. After 2 hours this initial reaction reached its peak; immature cells then attached to the free basal membrane. After 96 hours the entire mesothelium was regenerated. CONCLUSIONS: The morphologic integrity of the mesothelium is temporarily disturbed by a CO2-pneumoperitoneum. Reasons for this phenomenon may be either the abdominal pressure or a CO2-induced surface acidosis. In further studies, the influence of the described phenomena on intraperitoneal formation of metastases will be examined.

Characteristic alterations of the peritoneum after carbon dioxide pneumoperitoneum.

OBJECTIVE: Any route of entry into the abdomen contributes to alterations of the intraperitoneal organs with different clinical consequences. Characteristic alterations of the peritoneum after CO2 pneumoperitoneum used in laparoscopic surgery is examined. METHODS: A CO2 pneumoperitoneum with an intraperitoneal pressure of 6 mmHg was applied for 30 min in 32 nude mice. In the course of 4 days, the animals were killed and the peritoneal surface of the abdominal wall was studied by means of scanning electron microscopy. RESULTS: Already 2 h after release of the pneumoperitoneum, mesothelial cells were bulging up. The intercellular clefts thereby increased in size, and the underlying basal lamina became visible. This reaction peaked after 12 h. Subsequently, peritoneal macrophages and lymphocytes filled all gaps, thereby recovering the basal lamina. CONCLUSION: The morphologic integrity of the peritoneum is temporarily disturbed by a CO2 pneumoperitoneum.

Use of PCR to screen for promoter elements in genomic DNA library clones.

We report a modified PCR strategy to screen for promoter elements of genes of interest that is based upon consecutive rounds of PCR and appropriate subcloning. Following preliminary identification and sequencing of intron 1 by standardized PCR, the application of a suited cDNA/intron primer combination renders a succeeding PCR-mediated screening of cosmid or P1-derived artificial chromosome (PAC) libraries possible, thus identifying genomic clones comprising the searched promoter elements. We tested our approach in comparison with a commercially available promoter finder kit by searching the promoter elements of the CENP-C gene from the human and mouse genomes. Applying the kit system, we amplified the anticipated promoter from mouse, but failed in isolating human promoter elements. Our approach made use of a 5'-UTR/intron 1 primer combination in the second round of PCR, enabling the identification of positive clones from genomic DNA within a human PAC library possible. Subcloning and final PCR amplification revealed the successful isolation of the human promoter. Therefore, we conclude that our approach might represent a helpful alternative to identify promoter elements, especially when prior art genome walking, STS-based strategies or anchored PCR failed.

Proteasome activator (PA28) subunits, alpha, beta and gamma (Ki antigen) in NT2 neuronal precursor cells and HeLa S3 cells.

The catalytic activity of the 20S proteasome can be modulated by endogenous proteins. A proteasome activator protein termed PA28 or 11S regulator, composed of two homologous subunits (alpha and beta) and a separate but related protein termed Ki antigen or PA28gamma have been characterized. To explore the functional relationship of these proteins, NT2 clone D1 human neuronal precursor cells, as well as HeLa S3 cells were labeled by immunofluorescence and immunoelectron microscopy with three different antisera directed against peptides derived from their sequences. It was found that both PA28alpha and PA28beta antisera label the cytoplasm and the nucleoli. In contrast, the PA28gamma antiserum labels the nucleus but not the nucleoli while in the cytoplasm it labels two different classes of structures identified as microtubular-like extensions and inclusion bodies that are most likely autophagosomes. The latter do not contain proteasome delta subunit antigen. The microtubular-like structures colocalize with beta-tubulin, are dispersed by nocodazole and are not affected by brefeldin A treatment. PA28alpha and PA28beta are co-localized in the cell whereas PA28gamma has a different distribution. PA28gamma complexed with the proteasome may serve a function other than or in addition to activation and may also have a proteasome-independent function.

Cdc2-independent induction of premature mitosis by okadaic acid in HeLa cells.

The induction of premature mitosis by okadaic acid (OA) in HeLa cells in S-phase or in G2-phase has been studied using light microscopy, immunofluorescence, and immunochemical techniques. The observations indicate an involvement of a cdc2-independent pathway in these cells. It has been claimed that inhibition of an OA-sensitive phosphatase, possibly of PP1, induces activation of a kinase which is sensitive to staurosporine and Zn2+. This kinase brings about mitosis-specific cytoskeletal rearrangements, chromosome condensation, and nuclear envelope breakdown, inducing a mitosis-like state. However, other mitotic events do not follow. The possibility that this kinase may be a NIMA-like Nek2 kinase is discussed.

Detection of intranucleolar chromatin using an ultrastructural immunolabelling technique.

The intranucleolar chromatin has been detected by feeding HeLa cells with bromodeoxyuridine and then using an immunocytochemical technique for its detection at the ultrastructural level. Labelling was mainly observed on the dense fibrillar component (DFC), which surrounds the fibrillar centres (FC). Labelling could also be seen on the network within the granular component (GC) of the nucleoli. Ring shaped nucleoli with large FCs showed labelling, in addition, on the FCs. Nucleoli from spread preparations of cells treated with hypotonic salt solution for different periods revealed a nucleolonemal network, consisting of units comparable to rDNA transcriptional units in length and associated with tufts of fibrils and granules. Thread-like axes could be detected running through the units and in between two units. Chromatin could also be identified within these units by the immunolabelling technique. The observations strongly indicate that these units are identical with the rDNA transcriptional units. A dynamic nature of the nucleolar chromatin and nucleolar organization has been discussed.

Induction of premature mitosis in cells blocked in S-phase: possibilities of its use in cancer therapy.

Oncogenesis leads to weak regulations of many cell cycle checkpoints. This could be useful for cancer therapy. To test this assumption, five human malignant cell lines were used to induce premature mitosis by overriding the S-phase control, which ultimately leads to cell death. Two cell lines showed a significantly high frequency of premature mitotic cells induced by okadaic acid; whereas the others did not. One cell line, although having a high cyclin B level in cells blocked in S-phase, failed to show premature mitosis. The cyclin B level in cells blocked in S-phase alone could not explain the differential behavior of the cell lines. It was observed that in addition, the activation of cdc25 in these cells could also play an important role in the induction of premature mitosis in such cells.

Effects of low concentrations of okadaic acid in HeLa cells.

HeLa cells treated for prolonged period with okadaic acid (OA; 5-10nM) inhibiting protein phosphatase 2A (PP2A) and also protein phosphatase 1 (PP1) partially showed prolonged effects on mitotic progression. In the presence of OA cells progressed normally in mitosis almost upto 4 hr, then a progressive accumulation of mitotic cells could be noticed. Most of the mitotic cells seemed to be arrested at the metaphase-anaphase transition point. In arrested mitotic cells the chromosomes remained arranged at the equiatorial plate, but with prolonged treatment the chromosomes got either scattered or clumped. However, a slow release into anaphase could also be observed after 15 hr treatment. Immunofluorescence studies for microtubules and electron microscope investigations indicated the dearrangement of spindle fibres, and a prolonged treatment led to the formation of multipolarity. This was also confirmed by spread preparations of chromosomes and the formation of multinucleate cells in preparations released from the mitotic block. Chromosomes became highly condensed showing mostly nondisjunction, but separation of sister chromatids could be observed in many cells. Immunoblot assays indicated a degradation of cyclin A, but the cyclin B1 level was significantly higher in the arrested mitotic cells after 12 hr treatment. After 24 hr of treatment the cyclin B1 level was slightly lower in arrested cells. Possible roles of protein phosphatase 2A inhibition and a prolonged partial inhibition of PP1 on the mitotic progression and the cyclin degradation at the metaphase-anaphase transition have been discussed.

Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome.

HeLa cells growing in vitro were treated with the peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the proteasome N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). Immunofluorescence studies of treated cells revealed the formation of massive perinuclear aggregates rich in ubiquitin and proteasomal antigens, which on the ultrastructural level appeared as perinuclear aggregates of electron-dense material, usually in the vicinity of Golgi cisternae. Histochemical studies disclosed that these cells contained protein-rich perinuclear aggregates detected by amido black staining, while unusual accumulations of lipids, carbohydrates, or nucleic acids were not present. Inhibition of protein synthesis by cycloheximide prevented the formation of aggregates, whereas microtubule disruption by nocodazole induced a dispersion of the aggregates. We hypothesize that aggregates induced by PSI treatment correspond to accumulations of proteasome-substrate complexes in a well-defined region, where the proteolytic processes of the ubiquitin-proteasome pathway seem to be somehow centered. We propose to call this region the proteolysis center.

The centromeres of the Indian muntjac: evidence for the existence of multiple centromeres?

Unlike the centromeres of other species, the "compound' centromeres of the Indian muntjac span over exceptionally extended regions (Brinkley et al., 1984). We extend this concept and show that some of these centromeres are divisible into several chromomeres in which the light staining regions alternate with the dark staining C-band positive segments. Unlike the centromeres of other species where the centromere replicates as one unit, the replication of the sub-units constituting the centromere of the X-chromosome in the muntjac occurs at different times as at least three independent segments. The CREST staining of the centromere regions of even the smallest (Y2) chromosome is interrupted by non-staining segments. Electron microscopy shows similar interruptions in the continuity of the trilamellar kinetochore. Sister chromatid exchanges occur in the region of the centromeres and chromatid breaks within the centromere region occur in the non-fluorescent segments. We interpret these data to suggest that the centromere regions of the Indian muntjac are made up of independent multiple centromeres interrupted by non-centromeric chromatin. Relevance of these parameters in mutagenesis is briefly discussed.

Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control.

Are proteasomes involved in the formation of the kinetochore?

Proteasomes catalyse the degradation of proteins responsible for the regulation of mitosis enabling the cell to complete cell division. We have studied the effect of an inhibitor of the chymotrypsin-like activity of the proteasome on the trilaminar structure of the kinetochore in HeLa cells. Whereas a role for the proteasome in the degeneration of the kinetochore was predicted, we found instead that the inhibitor strongly regarded kinetochore development. We observed different 'developmental' stages of the kinetochore from the fibrous ball of a 'prekinetochore' to the 'mature' kinetochore in one cell. The data presented here support the proposition that proteasomes are involved in kinetochore formation.

Okadaic acid overrides the S-phase check point and accelerates progression of G2-phase to induce premature mitosis in HeLa cells.

Okadaic acid has been shown to induce premature mitosis in HeLa cells belonging to both S-phase and G2-phase. The response is dependent on concentration and perhaps also on the differential susceptibility of cells to okadaic acid (OA). OA resembles caffeine in its ability to override the DNA-replication check point but differs in being capable of inducing premature mitosis in cells in G2-phase, thereby accelerating mitosis. Chromosomes from mitotic cells induced in G2-phase show a typical banding pattern similar to G-banding. From immunoblot experiments and survey of the relevant literature it has been argued that OA may modulate both a tyrosine kinase and a tyrosine phosphatase activity to override the S-phase checkpoint and accelerate the activation of MPF in cells in G2-phase, respectively.

Nucleolar organization as revealed in cell spreads.

The nucleolar organization has been studied in spreads of cells either untreated or treated with hypotonic salt solution for different periods. A network corresponding to the nucleolonema becomes evident with progressive hypotonic treatment. The network reveals units comparable to the rDNA transcriptional units in length and is associated with tufts of fibrils and granules. Spread preparations from cycloheximide treated cells reveal a thread-like axis and often 'Christmas tree'-like configurations within these units. Spacers joining the units can also be detected. It is supposed that the transcriptional units move outwards with their transcriptional products where the processing takes place. In loose nucleoli, this network forms the nucleolonema, which remains associated with the granules, the processed transcriptional products. In compact nucleoli the network is obliterated by the granules and they form the major component of the nucleoli. Such organization represents all the events in the transcription and processing of ribosomal RNA.