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The binding of Bcl-2 to Beclin-1 reduces Beclin-1's capacity to induce autophagy. Here, we have tested whether the interaction is reciprocated by loss of Bcl-2's anti-apoptotic function. We targeted Bcl-2 to mitochondria or endoplasmic reticulum (ER) and induced apoptosis using several apoptotic stimuli that initiate ER and/or mitochondrial signaling pathways (UV radiation, TNF and cycloheximide, staurosporine, thapsigargin and tunicamycin). When Beclin-1 and Bcl-2 were expressed together in HeLa cells, Beclin-1 (but not Beclin-1 lacking the Bcl-2-binding domain) followed Bcl-2 to the appropriate organelle with complete or near-complete overlap (comprising 60 and 30% of cells, respectively). The interaction between Beclin-1 and Bcl-2 was verified by immunoprecipitation, and a membrane-proximate localization of Beclin-1 was shown by immunoelectron microscopy. Apoptosis was followed by measuring changes in nuclear morphology, caspase-3 activity, poly-ADP-ribose polymerase cleavage or punctation of mRFP-Bax on mitochondria. Binding of Beclin-1 to Bcl-2 did not modify apoptosis irrespective of Bcl-2 concentration, location or apoptotic stimulus. A similar result was obtained in Atg5-/- cells that are autophagy-deficient, arguing against compensation for the loss of protection by Bcl-2 by autophagy-mediated survival induced by Beclin-1. Hence, although Beclin-1 contains a BH3-only motif typical of pro-apoptotic proteins, it is a negligible modulator of Bcl-2's anti-apoptotic function.

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinised. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

We reported that administration of anti-tumor necrosis factor alpha (anti-TNF-alpha) antibodies exacerbates the course of a Salmonella infection in both susceptible and resistant mice by preventing the suppression of bacterial growth in the reticuloendothelial system. In the present study, we evaluated the effect of in vivo neutralization of TNF-alpha on the histopathology of primary Salmonella infections. We show that in primary infections, the suppression of bacterial growth in the reticuloendothelial system coincides with granuloma formation in the spleen and liver. Administration of anti-TNF-alpha globulins on day -1 of salmonellosis affected neither the histological picture nor the course of the infection in the early stages of the disease (days 1 to 3), with splenic and hepatic lesions consisting mainly of polymorphonuclear leukocytes (PMNs); conversely, later in infection (days 3 to 7), the treatment inhibited the formation of granulomas. When the anti-TNF-alpha treatment was started well after the suppression of bacterial growth in the reticuloendothelial system and the formation of granulomatous lesions in the spleen and liver, a prompt relapse of the infection and regression of already established granulomas were seen. In anti-TNF-alpha-treated mice, salmonellae were found inside macrophages and PMNs and extracellularly in the necrotic tissue of the spleen, while in the liver the organisms were seen mainly in inflammatory mononuclear cells, resident Kupffer cells, and hepatocytes and occasionally in the extracellular compartment within necrotic lesions. The bacteria appeared most often in clusters, being morphologically intact when in the extracellular space or within hepatocytes, while undergoing various degrees of degeneration when inside phagocytes. The results suggest that TNF-alpha is required for granuloma formation in salmonellosis and that its neutralization does not completely abrogate the bactericidal activity of macrophages and PMNs. Salmonellae were observed to grow within both hepatocytes and phagocytes but were killed only in the latter.

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.

The ultrastructure of cardiac ventricular myocytes isolated from normal and chronically K-depleted rabbits was analysed using morphometric techniques. Myocytes from normal animals showed a quantitatively similar distribution of organelle volumes to those previously reported for isolated myocytes from rat, and for whole myocardium in a variety of species. Myocytes from animals that had been fed a K-deficient diet for 25 days had a significantly increased surface density of junctional sarcoplasmic reticulum with the T-tubules and a significant increase in the surface density of the T-system itself. There were no other differences. The increase in junctional SR and T-system may be related to the defect in cardiac contractility that has been reported for rabbit following K depletion, and may represent an adaptive mechanism acting to protect the heart against Ca overload.

The present investigation was undertaken in rats to determine whether sensory nerves exist in apposition to the bronchial microvessels which may function as rapidly adapting receptors (RAR). The primary and secondary bronchi on both sides were removed and processed for light and electron microscopy. Nerves were frequently found in relation to venules external to the muscle coat of bronchi. They comprised myelinated axons which ended individually as non-myelinated convoluted terminals enclosed within a loose capsule of attenuated cells. Serial sections showed that these terminals were not related to ganglion cells. Cervical vagal section and injection of HRP-WGA into the nodose ganglion provided corroborative evidence of the sensory nature of these terminals. Vagal section caused degenerative changes in the encapsulated nerve terminals in the bronchial walls and horseradish peroxidase labelling was demonstrable in such terminals. Moreover, immunocytochemical studies demonstrated the presence of calcitonin gene regulated peptide and substance P in these structures. It is suggested that they comprise the RAR. Encapsulated nerve terminals were not found in the epithelial layer, in the submucous coat or in the muscularis of bronchi.

The differentiation of the myocardial rudiment was examined in mouse embryos, isolated between the afternoons of the eighth and ninth days of gestation, by means of transmission electron microscopy of ultrathin sections and of freeze-fracture replicas; some of the material used for sectioning was labelled with ruthenium red. Formation of the presumptive pericardial cavity commences during the late presomite stage (afternoon of the eighth day) and the myocardial rudiment originates in situ as a thickening of the splanchnic pericardial lining. Initially, the myocardium comprises an epithelium or plate directly exposed to the pericardial lumen and overlying a separate layer of endocardial elements. As the heart tube bulges into the pericardial coelom, it becomes surrounded by a sleeve of myocardium which thickens and stratifies during the ninth day and subsequently (on the tenth day) acquires an epicardial covering of flattened cells. The myocardium commences pulsations at or about the 3-4 somite stage (morning of the ninth day) by which time the myoblasts already contain striated myofibrillae and specialised cell junctions. From its earliest appearance, the myocardial plate contains tight junctions and desmosomes between the lateral borders of the apical parts of the myoblasts. Gap junctions soon appear in the same regions and they increase in number and extent, as the myoblasts elongate and divide, thus establishing contact in various planes; they are supplemented by the formation of fasciae adherentes and of more desmosomes. On the other hand, tight junctions decline in extent and are eventually confined to the epicardium. Other features such as caveolae and T-tubules are not established by the end of the ninth day and coupling arrangements of the sarcoplasmic reticulum are only in the rudimentary stages of formation.

Atrial and ventricular myocardium from young golden hamsters, 3-6 months of age, was examined under the transmission electron microscope, representative samples from each animal having been stained histochemically for acid phosphatase (AP) by a lead precipitation technique. Our observations indicated that AP positive primary lysosomes are produced at the sites of Golgi-endoplasmic reticulum-lysosome (GERL) complexes and the main pathway for lipofuscin production is by fusion of primary lysosomes with mitochondria in both atrial and ventricular myocytes. The mitochondria are then progressively degraded, their matrix becomes denser and there is an accompanying increase of AP activity. After reaching a peak level, AP activity declines until an AP negative terminal pigment body with a smooth outline is produced. Another pathway, but much less frequent at this age leads to the formation of larger bodies, irregular in outline and containing small globules. Their genesis entails the participation of autophagic vacuoles which engulf lipid droplets, mitochondria or amorphous debris. Like the main variety of lipofuscin body, they contain AP while digestion progresses but eventually become AP negative. A few lipofuscin granules are eliminated by being pinched off within fine cell protrusions and are subsequently engulfed by scavenger cells.

The ontogenesis of specific heart granules and of the related natriuretic peptide activity in heart muscle was studied in fetal and neonatal rats and golden hamsters by ultrastructural analysis including immunogold labelling for ANP-28 and by radioimmunoassay. In both species, immunoreactive granules first appear in the myocardial sleeve of the embryonic heart tube during the looping stages which precede chamber formation and the peptide becomes detectable by radioimmunoassay two or three days later by which time the chambers are identifiable. Granule density and ANP concentration in the rat are higher than in the hamster at all stages of development. Almost all atrial myocytes express ANP in fetal hearts whereas, in the ventricular wall, cells containing immunoreactive granules are scattered. The density of granules in atrial myocytes increases during further stages of fetal and neonatal development, while it decreases markedly even in those ventricular myocytes which are immunoreactive. Changes in the ultrastructural appearance of ventricular SHG suggest that the mode of production of ANP changes in ventricular myocytes after birth but does not change in atrial cells. There is no correlation between the distribution of immunoreactive ventricular myocytes and that of the conducting system. In both species, the concentration of ANP in the atrial well is higher than ventricular levels from the outset and the disparity becomes exaggerated with development till, in six months old adult animals, the atrial to ventricular concentration ratio is about 3 x 10(3):1 in the rat and 1.5 x 10(3): 1 in the hamster. In the hamster, a distinct gradient of ANP concentration between the right and left atria is already established in the early fetal period and it becomes enhanced in the neonatal period. In the rat, however, a slight difference becomes discernible only after birth.