Nucleolar segregation during apoptosis of haemopoietic stem cell line FDCP-Mix.

The programmed elimination of cells during apoptosis is distinct from necrosis both morphologically and biochemically. Currently, the morphological description of apoptosis discriminates between the segregation of the nucleolus and the so called 'chromatin condensation'. The latter originates from observations of electron dense material adjacent to the nuclear envelope of apoptotic nuclei. Although there is ample evidence for an involvement of DNA in electron dense marginations, their true nature is still unknown. By studying apoptosis in FDCP-Mix, a pluripotent murine haemopoietic stem cell line, we found morphological and histochemical evidence that electron dense material at the nuclear envelope (NE) has emerged as a result of the segregation of nucleoli in association with the nuclear membrane. The remaining electron dense and homogenous bulk of the nucleolus labels for RNAse-gold, but even more intensely for DNAse-gold, and therefore could possibly be mistaken as 'condensed chromatin' in the light microscope. The labelling of the electron dense material for DNase-gold in FDCP-Mix could be explained by a migration of DNA into the bulk of the nucleoli at an early stage of cell death.

Macromolecular substructure in nuclear pore complexes by in-lens field-emission scanning electron microscopy.

Scanning electron microscopy (SEM) has produced a wealth of novel images that have significantly complemented our perception of biological structure and function, derived initially from transmission electron microscopy (TEM) information. SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by reduced resolution in comparison with TEM. Recently, SEM resolution has been considerably improved by the advent of high-brightness sources used in field-emission instruments (FEISEM) which have produced resolution of around 1 nm, virtually equivalent to TEM "working resolution." Here we review our findings in the use of FEISEM in the imaging of nuclear envelopes and their associated structures, such as nuclear pore complexes, and the relationships of structure and function. FEISEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding its functional significance.

Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.

Accessing nuclear structure for field emission, in lens, scanning electron microscopy (FEISEM).

Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding, its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces.

The isolation and culture of adult mouse colonic epithelium.

A technique is described for the reproducible primary culture of colonic epithelium from adult mice. A collagenase-dispase digestion technique (adapted form Evans et al. 1992) is used to release the epithelium, followed by differential sedimentation to produce a high purity crypt preparation with maintained structural integrity and minimal mesenchymal contamination. The crypt units attach to collagen coated plastic within 24 h and the epithelial cells quickly begin to migrate outwards producing a monolayer surrounding the attached crypts. Electron microscopy revealed that the migrating epithelial cells possessed both desmosomes and microvilli. Proliferation in the colony supports the outward migration of cells until the migratory cells of adjacent colonies connect and a confluent monolayer begins to form. Proliferation is routinely maintained for 10 days (although cultures have now been maintained without subculturing for 35 days) and is demonstrated by increased cell numbers in spite of continuous cell loss into the culture media. Culture growth is enhanced by increasing concentrations of fetal calf and mouse serum and EGF but does not appear to respond significantly to added transferrin. Growth is also stimulated by a murine small intestinal extract thought to contain a potentially novel growth factor or cocktail of factors. This culture model has considerable potential for studies on growth factor control of this carcinoma susceptible tissue and its differentiated function as well as studies into the mechanisms of carinogenesis.

ATP dependent histone phosphorylation and nucleosome assembly in a human cell free extract.

Physiologically spaced nucleosome formation in HeLa cell extracts is ATP dependent. ATP hydrolysis is required for chromatin assembly on both linear and covalently closed circular DNA. The link between the phosphorylation state of histones and nucleosome formation has been examined and we demonstrate that in the absence of histone phosphorylation no stable and regularly spaced nucleosomes are formed. Phosphorylated H3 stabilizes the nucleosome core; while phosphorylation of histone H2a is necessary to increase the linker length between nucleosomes from 0 to approximately 45 bp. Histone H1 alone, whether phosphorylated or unphosphorylated, does not increase the nucleosome repeat length in the absence of core histone phosphorylation. Phosphorylations of H1 and H3 correlate with condensation of chromatin. Maximum ATP hydrolysis which is necessary to increase the periodicity of nucleosomes from approximately 150 to approximately 185 bp, not only inhibits H1 and H3 phosphorylation but facilitates their dephosphorylation.

The effects of decerebration on the reflex response to pulmonary vein distension.

1. A method is described for the decerebration of dogs using high frequency coagulation. Animals made decerebrate by this method showed a slowing of the heart rate and a decrease in arterial pressure.2. Distension of the pulmonary vein-left atrial junctions by the inflation of small balloons caused an increase in heart rate in intact and decerebrate dogs. The magnitude of the response was not significantly different in the two states.3. The increase in heart rate caused by pulmonary vein distension was shown to be a reflex. It was significantly reduced by injection of propranolol both before and after decerebration. Cervical vagotomy always prevented any response.4. The similarity of the responses before and after decerebration suggests that structures rostral to the superior colliculus are not required for the appearance of the full reflex effect.5. The magnitude of the response remaining after administration of propranolol raised the question of the efficacy of propranolol as a beta-blocking agent in this experimental situation, or alternatively suggests the possibility of the existence of an efferent vagal component to the reflex response.