A mechanistic view of mitochondrial death decision pores.

Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

Adventitial layer enlargement correlates with the percentage of medial thickness in peripheral pulmonary arteries from patients with congenital heart defects.

Arterial walls undergo modifications during the course of pulmonary hypertension, particularly in the medial and intimal layers, leading to progressive occlusion of the lumen. Adventitial layer enlargement has been described as being present in the experimental hypoxic model and in the persistent pulmonary hypertension of the newborn. It was suggested that this enlargement may be related to stimulating factors derived from the medial smooth muscle cells. This study was designed to verify if different degrees of medial hypertrophy are correlated to the volume density of the adventitial layer in pulmonary hypertension secondary to congenital heart defects. Reviewing 21 lung biopsies from patients with congenital heart defects, we concluded that there is a statistically significant positive linear correlation between the mean percentage of medial arterial thickness and the volume density of the adventitial layer in the biopsies showing isolated medial hypertrophy. On the other hand, in biopsies showing frequent intimal proliferative lesions and irregular medial layer hypertrophy the correlation coefficient was lower. These findings suggest that the adventitial layer participates in the arterial remodeling process in secondary pulmonary hypertension, and that its enlargement depends on the qualitative degree of pulmonary vaso-occlusive disease.

Evidence of type II pneumocyte apoptosis in the pathogenesis of idiopathic pulmonary fibrosis (IFP)/usual interstitial pneumonia (UIP).

BACKGROUND/AIMS: The pathogenesis of idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP), a chronic and incurable human respiratory disease, is not well established. This study was designed to investigate whether the apoptosis of type II pneumocytes could be the precipitating factor in the pathogenesis of IPF. METHODS: Nineteen specimens obtained by retrospective review of the medical and pathological records of 55 patients with IPF, four normal subjects, and 10 disease control lungs were analysed. The selected specimens had normal alveoli with intervening patchy scarring of the lung parenchyma, fulfilling the pathological criteria for UIP. To identify individual cells undergoing apoptosis in the normal alveoli, electron microscopy and in situ end labelling of fragmented DNA were performed on paraffin was embedded sections using digoxigenin-11-dUTP and the enzyme terminal deoxynucleotidyl transferase. RESULTS: Apoptosis was detected in the normal alveoli of 17 of the 19 patients with IPF/UIP and was absent in the controls. Electron microscopy demonstrated apoptotic changes in type II pneumocytes. These results indicate that apoptotic type II pneumocyte death occurs in normal alveoli of IPF/UIP and could be the principal cause of several events that account for the histological, clinical, and functional alterations seen in IPF/UIP. CONCLUSIONS: In conclusion, numerous type II pneumocytes from the normal alveoli of most patients with IPF/UIP actively undergo programmed cell death. This finding may shed new light on the pathogenesis of this disease, with implications mainly for the treatment of affected patients.

Large unilamellar vesicles as trehalose-stabilised vehicles for vaccines: storage time and in vivo studies.

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein from Mycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with large unilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehalose avoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrapped protein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on the liposomal membrane integrity.

Oxidation of LDL enhances the cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester transfer rate to HDL, bringing on a diminished net transfer of cholesteryl ester from HDL to oxidized LDL.

Cholesteryl ester transfer protein (CETP) plays a controversial role in atherogenesis by contributing to the net transfer of high density lipoprotein (HDL) cholesteryl ester (CE) to the liver via apolipoprotein-B-containing lipoproteins (apoB-LP). We evaluated in vitro the CETP-mediated bidirectional transfer of CE from HDL to the chemically modified pro-atherogenic low density lipoprotein (LDL) particles. Acetylated or oxidized (ox) LDL, either unlabeled or [3H]-CE labeled, were incubated with [14C]-CE-HDL in the presence of the lipoprotein-deficient plasma fraction (d>1.21 g/ml) as the source of CETP. The amount of radioactive CE transferred was determined after dextran sulfate/MgCl(2) precipitation of LDL. The results showed a 1.4-2.8-fold lower HDL-CE transfer to acetylated LDL while no effect was observed on the CE transfer to oxidized LDL. However, the reverse transfer rate of [3H]CE-LDL to HDL was 1.4-3.6 times greater when LDL was oxidized than when it was intact. Overall, HDL(2) was better than HDL(3) as donor of CE to native LDL, probably reflecting the relatively greater CE content of HDL(2). Oxidation of LDL enhanced the CETP-mediated cholesteryl ester transfer rate to HDL, bringing on a reduced net transfer rate of cholesteryl ester from HDL to ox LDL. This may diminish the oxLDL particle's atherogenic effect.

Morphometric studies on venom secretory cells from Bothrops jararacussu (Jararacuçu) before and after venom extraction.

A comparative morphometrical analysis was carried out on secretory cells from Bothrops jararacussu venom glands, before manual extraction of the venom (milking) and 4 and 8 days after milking. At the 8th day after milking, the cytoplasmic volume increased by 160%. The rough endoplasmic reticulum (RER) volume density increase, up to the 8th day after milking, is mainly due to widening of the intra-scisternal space. The total volume and membrane surface of the RER. Golgi apparatus and subcomponents, secretory vesicles and mitochondria, increased during the experimental period while the volume and surface densities of these organelles, with the exception of the RER, did not vary. The numerical density of Golgi-associated microvesicles per Golgi volume unit also increased. The greatest relative increments in these parameters occurred within the first 4 days. These results are compatible with an increased rate of membrane synthesis and transport in the milked glands and suggest that the membrane biogenesis, degradation and circulation that takes place in the first week after milking is achieved through coordinated cellular mechanisms that maintain the rate between total membrane surface and total cytoplasmic volume unaltered.

A kinetic and structural study of two-step aggregation and fusion of neutral phospholipid vesicles promoted by serum albumin at low pH.

The addition of bovine serum albumin (BSA) to 25 +/- 5 nm diameter single bilayer phosphatidylcholine (PC) vesicles (SBV) (pH 3.5) gives rise to readily visible transient turbidity. Studies of this system, employing a series of techniques, including time-dependent turbidity changes, membrane filtration, centrifugation, Sepharose chromatography and freeze fracture electron microscopy demonstrated that the process involves aggregation and fusion of the vesicles. At least three distinct time-dependent steps have been characterized: (1) the rapid initial formation (in approx. 5 min) of large aggregates (responsible for the visible turbidity) composed of SBV interconnected by BSA in its F form. The formation of these aggregates may be reversed by raising the pH or adding excess BSA to the system at this stage; (2) spontaneous collapse of these large aggregates, in an irreversible step, to form a heterogeneous population of vesicles; (3) fusion produces as the final product of the process, a relatively homogeneous population of larger (50 +/- 10 nm diamter) vesicles. This system serves as a convenient and simple model system for the detailed study of protein-mediated aggregation and fusion of membranes at the molecular level.

Long-term stability of lipid emulsions with parenteral nutrition solutions.

The stability of the mixture of peripheral vein parenteral nutrition (PN) solution with 10% lipid emulsions (Intralipid or Lipofundin MCT) was tested during a prolonged period of refrigerated storage. The analysis included gross visual examination of the bottle, pH determination, and examination by electron microscope. The mixtures of fat emulsions with PN solution demonstrated no physical instability or pH alteration. Examination under electron microscope revealed no alterations after 4 wk, but the surface layer of fat globules was disrupted after 10 and 18 wk. This study demonstrates that complete nutritive mixtures can be prepared and stored in refrigeration for at least 4 wk before clinical use.

Morphometric study of normal and adenomatous pituitary somatotrophs in humans.

Somatotrophs from ten pituitary adenomas were evaluated morphometrically by light and electron microscopy using the following parameters: a) nuclear, cytoplasmic and cell volumes; b) volume density, total volume, surface density, total surface and surface/volume ratio of secretory granules, mitochondria, rough endoplasmic reticulum and Golgi apparatus, and c) the number of secretory granules and mitochondria per micron3 of cytoplasm and per cell. The results were compared (p less than 0.05 and p less than 0.10) with those obtained from somatotrophs identified in five normal pituitaries. The data obtained indicate that: a) in the adenomas, the number of secretory granules per cell cannot be accurately evaluated from their apparent number in sectioned cell profiles; b) there are two basic sub-types of adenomatous somatotrophs defined according to the mean secretory granule diameter; cells in which granule diameter is inferior to 180 nm exhibit distinct morphological features such as nuclear pleomorphism, the presence of gross bundles of intermediate sized filaments or fibrous bodies in the cytoplasm and a variable number of secretory granules. Adenomas constituted mainly by these cells were found in younger patients, suggesting the more aggressive nature of these tumours, thus warranting close clinical follow-up of such patients; and c) in both types of adenomatous cells, the organelles directly involved in the secretory process, i.e., the rough endoplasmic reticulum and Golgi apparatus, are larger than in the control cells; however, the ratio between the surfaces of these two compartments does not differ among the three groups studied.

Cell population growth in the rat parotid gland during postnatal development.

The growth kinetics of different cell populations in the rat parotid was studied. The evolution of the frequency and absolute number of each cell type was determined morphometrically by a particle-counting method and the evolution of the [(3)H]thymidine labeling indices of the same cell types was determined by autoradiography. The data obtained for the evolution of cell number in each gland compartment, i.e. acini, intercalated ducts, striated ducts and stroma, were adjusted by exponential equations, permitting estimation of the effective cell accumulation rate in the compartment for each population, i.e. the mean population duplication time (T(D)). In addition, the cell production rate in each gland compartment was determined using the mean labeling index for the period studied and a mathematical estimation of the mean cell generation time (T(G)), assuming an exponential growth pattern for the acinar, intercalated duct and striated duct populations during the period from 5 to 20 days of postnatal development. Analysis of the relation between effective cell accumulation (T(D)) and presumed cell production (labeling index and T(G)) for each intralobular parenchymal compartment of the rat parotid during this period suggests that the proliferative activity of the acinar cell population was sufficient to guarantee marked growth of its compartment and provided cells that presumably dedifferentiated into intercalated duct cells, whereas cells produced in the intercalated duct compartment migrated to, and differentiated into, cells of the striated duct compartment.

Cytochemical analysis of acid phosphatase activity in the venom secretory cells of Bothrops jararaca.

A study of the histochemical reaction for acid phosphatase (AcPase) in venom gland secretory cells from Bothrops jararaca was done to investigate the distribution of lysosomes and related structures in stages of high- and low-protein synthesis. From this analysis, it was expected to gain insight into the cellular pathway by which AcPase is secreted into the venom. Two subtypes of AcPase reactivities were detected in the venom gland secretory cells: one was found in lysosomes and related structures and in some trans-Golgi network (TGN) elements and reacts with beta-glycerophosphate (betaGP) as substrate; the other was found in secretory vesicles, apical plasmalemma, lysosomes and related structures, and in some TGN elements, and reacts with cytidine monophosphate (CMP). The results are compatible with the possibility that there is a secretory via for AcPase in the venom gland of B. jararaca and that the elements composing this pathway are noted only when CMP is used as substrate. Large autophagosomes reactive to both betaGP and to CMP were commonly observed in the basal region of the secretory cells, and they were more abundant in the glands during the stage of low activity of protein synthesis.

Structural elements common to mitosis and apoptosis.

Both mitotic and apoptotic cells display hypercondensation of the chromatin and loss of the nuclear envelope (Lazebnik et al., 1993). Herein, we describe a third similarity between the two processes. We have observed, initially in apoptotic cells of the PC-12 lineage clusters of 40-60 (approximately 50) nm vesicles adjoined by a minor contingent of tubule vesicular elements of 100-200 nm which are indistinguishable from their vesicular counterparts in mitotic PC-12 cells. The clusters of approximately 50 nm vesicles were subsequently observed in all studied rat tissue cells in apoptosis (plasma cells and macrophages, secretory epithelial cells from pancreatic acini, ventral lobe of prostate and mammary gland). Clusters of approximately 50 nm vesicles comparable to those of the PC-12 cells were found in HeLa cells treated with human alfa TNF, in WEHI-3 cells exposed to VM 26 (a teneposide) (Sesso et al., 1997) and in HL-60 cells treated with thapsigargin. PC-12 and HeLa cells affixed to coverslips were double labelled and examined with the fluorescence microscope to reveal simultaneously the disposition of the chromatin with Hoechst stain and the distribution of the fluorescence of Golgi or of Golgi-associated proteins. A common pattern of fluorescence was observed in a minor proportion of apoptotic cells using three different antibodies used. The label frequently appeared as finely dispersed granules in the cytoplasm. In some apoptotic cells, relatively coarse granules were observed. This pattern of label distribution is compatible with the disposition of vesicular clusters we have encountered in apoptotic PC-12 cells sectioned serially or semi serially. In such sections of both mitotic and apoptotic PC-12 cells, we noticed that the conglomerates of 50 nm vesicles were frequently associated with cisternae of the rough ER. Vesicles of similar size were also noted pinching off from the extremities of Golgi cisternae reduced in size. These cisternae diminish in length and width when they are in the process of disassembling at the very beginning of mitosis and in apoptosis.

The use of subcutaneously implanted araldite disks for microstructural studies of inflammatory macrophages.

Subcutaneously implanted epoxy resin disks can be used instead of glass coverslips in mice to recruit inflammatory macrophages. Implanted araldite disks exhibited large numbers of macrophages on both surfaces which could be observed conveniently by light and phase contrast microscopy. Most importantly, the disks can be embedded directly in an araldite mixture for the preparation of thin sections for electron microscope examination.

Morphometric evaluation of acinar cell number and volume in rat pancreas treated with dl-ethionine.

1. The morphological changes of the pancreas induced by administration of dl-ethionine were determined for treated rats and for a group of pair-fed untreated controls in order to separate the direct effect of dl-ethionine from the effect of accompanying reduction of food intake. 2. Adult male Wistar rats were studied in 3 groups of 10 animals each: 1) fed ad libitum and treated daily with dl-ethionine (35 mg/100 g body weight, ip) for 10 days; 2) treated daily with vehicle (saline) as group 1 and pair-fed to group 1; 3) treated daily with vehicle (saline) and fed ad libitum. Two rats from each group were killed on days 2, 4, 6, 8 and 10 to characterize the pancreas in terms of morphological properties using morphometric analysis. 3. Exposure to dl-ethionine induced a progressive and significant decrease in both pancreas weight and acinar cell number and volume. Pair feeding induced less pronounced decreases in pancreas weight and acinar cell volume. Pancreas weight was 125 mg/100 g body weight for dl-ethionine-treated vs 205 mg/100 g body weight for pair-fed controls and 320 mg/100 g body weight for ad libitum-fed controls after 10 days. Acinar cell number (x +/- sigma/square root of n): 175 x 10(6) +/- 28.4 per micron3 for dl-ethionine-treated vs 221 x 10(6) +/- 17.3 per micron3 for pair-fed controls and 271 x 10(6) +/- 23.9 per micron3 for ad libitum-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Thin-section and freeze-fracture study of post-mortem changes in dog myocardium.

1. Fragments of dog hearts submitted to 1, 6, 10, 24 and 48 h of autolysis at 20 degrees C were studied with freeze-fracture and thin-section techniques under the transmission electron microscope. 2. The freeze-fracture replicas revealed maximal reduction in the mean number and clustering of intramembrane particles at 6 h post mortem, indicating irreversible cellular damage. However, signs of lethal damage (intramitochondrial amorphous dense bodies) were not observed in thin sections of the same material. 3. The present study indicates that signs of irreversible damage similar to that occurring in in vivo ischemic alterations can be detected earlier by the freeze-fracture technique than by the thin-section technique.

Nephrotoxicity of human Bence Jones protein in rats: proteinuria and enzymuria profile.

The effect of intravenous administration of 80 mg purified human Bence Jones protein twice weekly for 5 weeks was investigated in male Wistar rats (N = 7; 2 months old). A state of immunological tolerance was demonstrated by the absence of a B-cell response (plaque-forming cells and hemagglutination titers) and by the absence of detectable antigen or antibody deposition in glomeruli, as indicated by light and electron microscopy. No rise in blood urea level was detected (33.9 +/- 4.3 vs 32.8 +/- 1.3 mg%). There was an increase in proteinuria (5.3 +/- 0.9 vs 32.8 +/- 4.0 mg/day), mainly due to Bence Jones protein excretion (0 vs 29.2 +/- 5.2 mg/day), with a slight but significant increase in albuminuria (0.2 +/- 0.1 vs 1.0 +/- 0.2 mg/day). There was a significant increase of lysosomal N-acetyl-beta-D-glucosaminidase in the urine (6.1 +/- 1.3 vs 72.7 +/- 18.8 mU/mg in creatinine). Lysosomal accumulation of Bence Jones protein in proximal tubular cells was evidenced by immunoelectronmicroscopy with protein A-gold. These results clearly showed proximal tubular dysfunction induced by chronic Bence Jones protein administration, without interference of autologous immune response as demonstrated by immunological state of tolerance.

Must fat emulsions and parenteral nutrition solutions be given through separate circuits?

To test the stability of fat emulsions with parenteral nutrition (PN) solutions, 10% soybean fat emulsion (FE) was suspended in PN solutions containing amino acids, electrolytes, vitamins, and different glucose concentrations. Six different mixtures were prepared and analyzed after 15 minutes or 24 hours, under transmission electron microscope: 10% fat emulsion (FE); FE + PN solution with 5% glucose (15 minutes); FE + PN solution with 5% glucose (24 hours); FE + PN with 12.5% glucose (24 hours); FE + PN with 25% glucose (24 hours); and FE + pure glucose 50% (24 hours). The addition of FE to PN solutions containing 5% to 25% glucose did not cause coalescence, aggregation, or confluence of the liposomes or damage to their surface in the first 24 hours after admixture. However, the suspension of fat emulsion in a 50% glucose solution coalesced liposomes and the droplets were nearly all aggregated. We conclude that mixtures of FE with PN solutions with glucose are stable for at least 24 hours and are suitable for clinical use.

One fate of bloodstream trypomastigote forms of Trypanosoma cruzi after immune clearance: an ultrastructural study.

The fate of bloodstream forms of Trypanosoma cruzi in tissues of mice was studied after immune elimination from circulation. Observations using transmission electron microscopy showed platelet thrombi occluding small vessels in the lung, liver, and spleen, and phagocytosed parasites in different stages of destruction within macrophages, neutrophils, and eosinophils. It is suggested that no particular cell population is a potential effector, but that different cells act in concert to destroy the parasites. The mechanism of this destruction might be related to intra- and extracellular mechanisms with trypanolytic activity.

Acid secretory response in the late follow-up of proximal gastric vagotomy for duodenal ulcer without Helicobacter pylori eradication.

BACKGROUND/AIMS: The profile of acid secretory responses was studied in 20 patients who had had proximal gastric vagotomy (PGV) surgery performed 11-22 years previously in order to treat duodenal ulcers (DU). The presence of Helicobacter pylori was detected in all of the patients. METHODOLOGY: The recurrence of DU was diagnosed in 10 patients and the other 10 remained without recurrence during the follow-up period. The control groups included 10 DU patients with refractory responses to H2 receptor antagonists and 10 "normal" subjects. Both control groups had untreated Helicobacter pylori infection. Measures of 1) basal acid output, 2) acid output for 30 min under continuous i.v. infusion of 0.2 ug/kg/h of pentagastrin acid, and 3) the response for 30 and 60 min after starting a sham feeding, modified by the "chew and spit" technique under simultaneous i.v. infusion of 0.2 ug/kg/h of pentagastrin were performed. Serum gastrin was measured during fasting and at sham feeding. The densities of the gastrin cells of antrum and duodenum were estimated by morphometric counting. RESULTS: Both basal output and acid response to sham feeding plus pentagastrin infusion were higher in the DU controls and DU recurrence patients. The response to pentagastrin infusion did not show any discriminant value. Fasting serum gastrin values increased after PGV, either with or without DU recurrence. Gastrin cell hyperplasia was not demonstrated in any of these groups. CONCLUSIONS: The secretory profile of patients with both late DU recurrence after PGV and Helicobacter pylori infection lies between DU patients refractory to the H2 receptor antagonist approach and those free of DU recurrence after PGV--both of them with current Helicobacter pylori infection. The characteristic pattern of late DU recurrence after PGV and untreated Helicobacter infection is that of increased basal acid output and higher acid secretion responsiveness to sham feeding plus pentagastrin in the presence of higher serum levels of gastrin.

Occurrence of retrovirus-like particles in various cellular and intercellular compartments of the venom glands from Bothrops jararacussu.

Retrovirus-like particles were detected in venom glands from Bothrops jararacussu during electron microscopy. Type C-like particles were found inter- and intracellularly in gland and vessel lumina and scattered in the connective tissue. They were about 100 nm in diameter, with an electron dense core and bilaminar external membrane. Shapes suggestive of a budding process from the plasma membrane were also observed. Less frequently, type A-like particles, about 80 nm diameter with an electronlucent core, appeared in association with the membranes of the endoplasmic reticulum of the secretory cells.