Cytoplasmic Damage Research Articles

Effects of Varying Salinity on Leaf Ultrastructure of Zannichellia Palustris

Zanichellia palustris is a submerged halophyte which occurs in waters of low salinity (5%o to 10%o). It is not capable of surviving at high salinity. Reasons for its inability to tolerate hypersaline conditions is important to our understanding of its biology. in the present study, leaf ultrastructure of plants growing at normal salinity was compared with plants growing at high salinity so as to assess the effects of hypersalinity. Attention was focused on ultrstructural changes occurring in leaf blade epidermal cells.Plants were grown in seawater at two salinities : 10%o (control) and 20%o (high salinity). Pieces of mature leaf blades from both treatments were harvested and prepared for Transmission Electron Microscopy (TEM) following standard procedures. in addition, the overall distribution and concentration of chlorine (Cl) in the leaves was ascertained since it is the most abundant anion in seawater and is important in considerations of salt tolerance in submerged halophytes. Cl was determined by means of a nuclear microprobe. Pieces of sectioned leaves were rapidly frozen, freeze dried and carbon coated. Elemental distribution maps of Cl were obtained using the true elemental imaging system (Dynamic Analysis). Two complementary techniques, Proton Induced X-Ray Emission (PLXE) and Proton Back Scattering (BS) were performed simultaneously using the GeoPIXE suite of programmes. Sample thickness and composition of major elements were obtained from the BS spectra.The ultrastructural morphology of leaf blade epidermal cells, which possess transfer cell characteristics differed at each salinity. While the cytoplasm was well preserved in epidermal cells of plants at the control salinity : 10%o (Fig 1), considerable cytoplasmic damage occurred in epidermal cells of plants at high salinity, in the form of breakdown of membranes such as the plasmamembrane and tonoplast, and senescence of various organelles including chloroplasts and mitochondria (Fig. 2).

Immature germ cell conception-in vitro germ cell manipulation.

Experimental studies in laboratory animals have shown that successful conception can be achieved by fertilizing oocytes with immature male germ cells. This gave rise to the concept that immature germ cells recovered from the testes of azoospermic men with maturation arrest may be used for assisted reproduction. However, in contrast to using germ cells recovered from healthy animals, clinical application to the treatment of male sterility is burdened by inherent defects in germ cells attributable to underlying testicular pathology. The recent introduction of in vitro germ cell culture/manipulation techniques makes it possible, in some cases, to overcome the in vivo maturation arrest by allowing an additional meiotic and post-meiotic differentiation and the selective harvesting of cells devoid of apoptosis-related nuclear and cytoplasmic damage. These techniques enabled the first births of normal infants fathered by azoospermic men with maturation arrest at the primary spermatocyte stage and improved the efficacy of assisted reproduction in men with maturation arrest at the round spermatid stage.

Correlation of endothelium-dependent and -independent vasodilatation with liver function tests during prolonged perfusion of the rat liver

Twelve male Wistar rats were anaesthetized with pentobarbitone (3 mg 100g −1 i.p.), the livers were excised and perfused in vitro through the hepatic artery and portal vein at constant flow rates of 0.32 ± 0.01 (mean ± S.E.) and 0.98 ± 0.03 ml min −1 g liver −1, respectively. The tone of the preparation was raised by methoxamine (7.5×10 −6 M). Responses to mid-range doses of acetylcholine (−11 log mol) and sodium nitroprusside (−9 log mol) produced submaximal degrees of vasodilatation (−log mol ED 50 = 12.18 ± 0.08) and (−log mol ED 50 = 9.95 ± 0.23), respectively, which did not subside until 5.5 h of perfusion. These did not coincide with the increase in activities of lactic acid dehydrogenase (LDH) and aspartate serine transaminase (AST) activity at 2.5 h, which were indicative of hepatocellular mitochondrial and cytoplasmic damage, respectively. Vascular responses suggested that there was little deterioration in endothelial or smooth muscle function in the hepatic artery up to 5 h perfusion. This model can be reliably used to investigate endothelium-dependent and -independent vasodilators in vascular pharmacological studies of the rat liver although some minimal increases may occur in AST and LDH activity before hemodynamic changes appear at 5.5 h.

The Effects of Delta Rays on the Number of Particle-Track Traversals per Cell in Laboratory and Space Exposures

It is a common practice to estimate the number of particle-track traversals per cell or cell nucleus as the product of the ion's linear energy transfer (LET) and cell area. This practice ignores the effects of track width due to the lateral extension of delta rays. We make estimates of the number of particle-track traversals per cell, which includes the effects of delta rays using radial cutoffs in the ionization density about an ion's track of 1 mGy and 1 cGy. Calculations for laboratory and space radiation exposures are discussed, and show that the LET approximation provides a large underestimate of the actual number of particle-track traversals per cell from high-charge and energy (HZE) ions. In light of the current interest in the mechanisms of radiation action, including signal transduction and cytoplasmic damage, these results should be of interest for radiobiology studies with HZE ions.

Immunohistochemical analysis of gel-transferred cells in cytologic preparations following smear division

Fine-needle aspirations on solid tumors are used increasingly as a means of obtaining a primary diagnosis. In many cases, a panel of immunostains performed on these aspirates is necessary to further characterize the cytologic interpretation. The amount of material obtained through aspiration, however, is often quite limited and is present on few glass slides. Previous studies have demonstrated the success of dividing cytologic smear preparations into smaller parts that could then be used for a panel of immunohistochemical stains. These results, however, did not compare the immunoreactivities of various antibodies before and after tissue transfer on cytologic preparations. In the present study, 41 immunohistochemical stains that employed 16 antibodies on 15 tumor preparations were performed following smear partition using the tissue-transfer technique. The percentage of cells that stained positive after transfer was determined and was correlated quantitatively to the untransferred controls. Specific immunoreactivity was demonstrated in 30 of 38 cases (79%) but was significantly decreased or lost in 8 of 38 cases (21%), which included antibodies for S-100, estrogen and progesterone receptors, chromogranin, neuron-specific enolase, and cytokeratin. Morphology was well preserved following tissue transfer, although limited cytoplasmic damage was seen in up to 25% of tumor cells. Immunopositive samples were found to be easily interpretable. Because sporadic cases fail to show immunohistochemical staining reactions following cytologic smear division and transfer, negative immunohistochemical stains in such preparations should be approached with caution.

Alterations of Bcl-2 family proteins precede cytoskeletal proteolysis in the penumbra, but not in infarct centres following focal cerebral ischemia in mice

Apoptosis has drawn attention in ischemic neuronal death recently. However, studies of apoptosis in cerebral ischemia have concentrated largely in DNA fragmentation, a late phase in apoptotic nuclei, at the expense of possible primary ischemic targets at the subcellular level and of upstream apoptotic signalling. To assess those issues, we used an intraluminal middle cerebral artery occlusion model in mice with or without reperfusion, and examined sequential changes of Bcl-2 family proteins modulating apoptotic signalling immunohistochemically and studied nuclear DNA fragmentation, to compare their chronology in relation to the development of infarct as detected by loss of microtubule-associated protein-2, an early marker of cytoplasmic damage. In the centre of the lesion, Bax protein increased and Bcl-2 and Bcl-x proteins decreased after loss of microtubule-associated protein-2 antigenicity occurred, but at the border of the lesion, the former changes preceded loss of microtubule-associated protein-2 antigenicity. Additionally, close morphologic analysis of DNA fragmentation in situ indicated that transient ischemia predominantly induced apoptotic cells but permanent ischemia produced necrosis of cells in the centre of the lesion.The contrasting cell death mechanisms, apoptosis and necrosis, are selectively involved in the pathology of cerebral ischemia, depending on its severity.

Evidence for an active type of cell death with ultrastructural features distinct from apoptosis: The effects of 3-acetylpyridine neurotoxicity

3-Acetylpyridine is a niacinamide antagonist with potent neurotoxic properties in vitro and in vivo. 3-Acetylpyridine neurotoxicity was associated with positive DNA end-labelling and displayed features of active cell death without the ultrastructural changes of apoptotic cell death. After systemic administration in rats (70 mg/kg), we detected labelled nuclei in the inferior olive using in situ DNA end-labelling. However, the conventional chromatin stain did not show chromatin condensation or fragmentation and electron microscopy studies failed to reveal features of apoptosis. Although areas of condensed chromatin were present in some nuclei, cytoplasmic damage with extensive organelle swelling was the most prominent finding. In vitro, 3-acetylpyridine (0.1–1 mM) induced degeneration of cerebellar granule neurons in a concentration- and time-dependent manner. The protein synthesis inhibitor cycloheximide (10 μg/ml) and the transcriptional inhibitor actinomycin D (10 μM) protected against 3-acetylpyridine toxicity. In contrast, neither the free radical scavenger α-phenyl-N-tertbutylnitron (100 μM), nor glutathione ethyl ester (10–100 μM), N-acetyl-cysteine (10–200 μM) or 3-aminobenzamide (0.1–4 mM), an inhibitor of poly(ADP-ribose) synthesis, were effective. 3-Acetylpyridine-induced neuronal death in vitro was associated with positive in situ DNA labelling. However, DNA fragmentation could not be demonstrated prior to neuronal cell loss and no DNA “laddering” was detected by DNA gel electrophoresis. Correspondingly, no apoptotic nuclei were revealed upon electron microscopy but organelle swelling and extensive vacuolization, changes similar to autophagocytosis. In conclusion, 3-acetylpyridine induces an active form of cell death that required de novo protein synthesis but is distinct from apoptosis. A loss of glutathione accompanies, but does not precede, cell death.

Observations on the surface coat of Blastocystis hominis.

The surface coat of Blastocystis hominis was studied in the electron microscope. In some cells the surface coat was seen in two layers; the external layer was more electron-dense and fragmented than the internal layer. It appears that the surface coat is being continuously formed by the parasite and shed in the environment. The fibrillar material of the surface coat attaches to the bacteria, in some cases, completely surrounding them, possibly causing cytoplasmic damage to the bacterial cell as indicated by loss of electron density.

Destruction of Lacis Cells of the Juxtaglomerular Apparatus in the Hypercholesterolemic Rabbit

During a study of experimentally induced atheromatosis in hypercholesterolemic rabbits we found a heavy infiltration of the lacis cell field (G field) by lipids in the kidney. A systematic study was done in 12 rabbits receiving a dietary cholesterol supplement of 0.3% for 26 weeks and in 8 animals receiving a supplement of 2% for 8 weeks. Standard histology, immunohistochemistry for a-smooth muscle cell actin and with the macrophage specific antibody RAM 11, and transmission electron microscopy were performed. In the low cholesterol, long duration group, 90% of the G fields were involved. The lipid accumulation was mainly extracellular. The lacis cells contained moderate numbers of medium sized lipid vacuoles and showed signs of cytoplasmic damage leading to disintegration. The mesangial cells contained few dipid droplets and the matrix was normal. The arterioles were not involved. The lesion suggests an impairment of the clearance of lipids, leading to interstitial accumulation and cellular damage.

Morphological changes in cultured human periodontal ligament cells exposed to dental materials

The cytotoxicity of a range of dental restorative cements was assessed by continuous observation of cultures with inverted microscopy and by light microscopic study of fixed preparations, using an in vitro model with cultured test cells derived from human periodontal ligament. The sequential morphological changes observed over a seven day period showed different degrees of cell loss and patterns of injury in response to different restorative materials, reflecting primarily either nuclear or cytoplasmic damage. Attempts at recovery were frequently identified as the culture period was extended and were characterized by recolonization of denuded areas of the culture well. It was concluded that differing dental cements damage cells through a variety of mechanisms and that the test cells exhibit differing degrees of susceptibility to injury. Assays based on short-term cultures may overestimate cytotoxicity by not allowing for cell recovery from reversible injury or repopulation of monolayers by proliferation of resistant cells.

Responses of maize ( Zea mays L.) plants to copper stress—I. Growth, mineral content and ultrastructure of roots

Changes in the morphology, physiology and ultrastructure of root systems of Zea mays cv. Aris under various Cu treatments were investigated. A progressive decrease of root length and biomass with increasing Cu in nutrient solution was observed. Mineral content and distribution were markedly affected by Cu. The roots accumulated significantly higher amounts of Cu than the above ground parts. Significant reduction of root calcium and iron contents as well as extensive damage to root epidermal cells occurred at the higher Cu concentration. In the remainder of the root, the effects of Cu (80 μM) varied within the same tissues. Thus, there were cortical or stelar cells with disintegrated cytoplasm, next to cells with well preserved plasmalemma, tonoplast and cytoplasm including cell organelles. Multi-lamellar bodies inside the vacuoles indicated an increased activity in digestion of the cytoplasmic components under Cu stress. Deposits of a less dense and less compact material were found behind the plasmalemma associated with the cell wall as well as an accumulation of dense material attached to the cell walls; cells with such deposits in their walls revealed extensive cytoplasmic damage. However, root ultrastructure was affected less than the morphology and physiology. The occurrence of well preserved cells indicates that Zea mays root cells do not respond uniformly to stressful conditions and suggests the development of a resistance strategy of maize roots to Cu-toxicity.

The behaviour of nuclear domains in the course of apoptosis

Programmed cell death is activated, by different stimuli and in many cell types, to regulate cell population balance during tissue proliferation and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca(2+)-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characterized by deep nuclear changes, consisting of typical cap-shaped chromatin marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late stage of the process and the greatest part of the phenomenon appears to take place despite good preservation of the plasma membrane and organellar component. In the present study we analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were processed for morphological examination in order to investigate the behaviour of the different nuclear domains. To follow DNA and RNA localization, we utilized osmium ammine and DNase-colloidal gold cytochemical reactions. The concentration of most DNA in the cap-shaped structures was demonstrated by these reactions. Confocal microscopy of cells processed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.

Soluble and membrane-bound TNF-alpha are involved in the cytotoxic activity of B cells from tumor-bearing mice against tumor targets.

Splenic B cells from BALB/c mice bearing mammary adenocarcinomas are capable of performing Ab-dependent cellular cytotoxicity. Effector-target conjugation after 18 h results in minimal cytoplasmic damage, whereas extensive nuclear disintegration is observed. To determine whether splenic B cells from tumor-bearing mice can effect direct cytotoxicity against tumor cells, L929 and WEHI 164 cells were used as targets. B lymphocytes from tumor-bearing mice, but not from normal animals, were capable of lysing these two types of tumor cells. However, only a low level of cytotoxicity could be detected when the nontumorigenic 3T3 cells were used as targets. To elucidate the mechanism of cytotoxicity of these killer B cells, RNase protection assays were performed using perforin, granzyme A, TNF-alpha, and lymphotoxin probes. No perforin, granzyme A, or lymphotoxin RNA could be detected in purified preparations of B cells from normal and tumor-bearing mice. B cells from normal mice did not have TNF-alpha RNA. In contrast, B cells from tumor bearers expressed TNF-alpha RNA. TNF-alpha could be detected in supernatants from both unstimulated and stimulated tumor bearers' splenic B cells, as measured by ELISA, and its lytic activity was neutralized by anti-TNF-alpha Ab. Western blots revealed the presence of TNF-alpha on the surface of the killer B cells. Paraformaldehyde-fixed B cells from tumor-bearing mice but not from normal animals were able to lyse TNF-alpha-sensitive tumor targets. This cytotoxicity was neutralized by anti-TNF-alpha Ab. These results suggest that TNF-alpha in soluble and membrane-bound forms may be involved in the mechanism of cytotoxicity exerted by B cells from tumor-bearing mice.

Ultrastructural evidence of the mechanisms responsible for interleukin‐4‐activated rejection of a spontaneous murine adenocarcinoma

The ultrastructural pattern of the anti-tumor response elicited by interleukin-4 (IL-4) was investigated by using a spontaneous mammary adenocarcinoma (TS/A) unable to elicit protective immunity in syngeneic BALB/c mice as suggested by a variety of preimmunization-challenge experiments. A subcutaneous lethal challenge of TS/A tumor cells was inhibited in a significant number of BALB/c mice receiving recombinant murine IL-4 injected daily for 10 days around the tumor-draining lymph node. Tumor rejection was mainly the result of direct membrane and cytoplasmic damage to tumor cells by eosinophils, neutrophils and macrophages that deeply penetrated the proliferating tumor mass. Lymphocytes and fibroblasts participated in the reaction by interacting with tumor cells, granulocytes and each other. The most frequent cell interactions in the peri- and intra-tumoral areas and in the tumor-draining lymph nodes are illustrated. The efficiency with which the IL-4-activated reaction leads to tumor inhibition and induction of a T-lymphocyte-dependent tumor-specific immune memory appears to depend on interactions between distinct leukocytes.

Seasonal variations of semen quality in male goats: Study of sperm abnormalities

The type, frequency and seasonal variation of sperm abnormalities in male goats inhabiting in Mediterranean region were evaluated. Semen was collected twice a week with an artificial vagina from 9 10-month-old Murciano-Granadina male goats weighing 41 to 48 kg. Distal cytoplasmic droplets and acrosomal damage were the predominant abnormalities. Increase in age (10 to 27 months) did not reveal itself as a main effect on the occurrence of sperm abnormalities. All types of sperm abnormalities studied showed significant (P<0.01) seasonal variation. The poorest quality semen was collected during winter and spring. In spite of a seasonal variation in sperm quality, semen of Murciano-Granadina goats in the Mediterranean area is good all year long, since the proportion of abnormal spermatozoa was well within the accepted range for normal fertility.

Histopathological Lesions in Heteropneustes fossilis Subjected to Acute Thiodan Toxicity

AbstractThe common Indian freshwater catfish, Heteropneustes fossilis, was exposed to the 96 h‐LC50 (0.007‐0.013 ppm) of the organochloride thiodan (endosulfan) for 4 days. The stressed fishes showed various histopathological changes. In the liver, extensive degeneration of cytoplasm, pycnosis of nuclei, and loss of glycogen was observed most commonly. Stasis of circulation and constriction of bile duct lumen was also seen frequently. In the kidney, shrinkage of glomeruli, cytoplasmic damage and epithelial desquamation in tubules, and extensive degeneration of hemopoietic stroma was commonly observed. In the intestine, the damage was comparatively negligible, the most common effect was an increased mucus cell activity leading to accumulation of mucoid exudates in the lumen.

A test system for the rapid detection of nuclear and cytoplasmic damage in Chinese hamster ovary cells

The effect of various agents which cause cell inactivation on the growth curves and RNA synthesis rates of chinese hamster ovary (CHO) calls has been studied. Agents which are known to act on DNA cause a reduced cell growth and RNA synthesis rate. Agents with a cytoplasmic target in eucaryotic cells cause a lengthened lag-phase, followed by growth and RNA-synthesis comparable to that of the control cells. From experiments with acrylonitrile, trichloroethylene, p-tetrachlorohydroquinone, cadmium chloride, and mercury chloride it is concluded that such studies can be used as a rapid test system for preliminary information as to whether a particuliar agent primarily damages the DNA of the cell nucleus or cytoplasmic structures in CHO cells.

Effects of cyclosporin A on cultured human epidermal keratinocytes. Growth and 5-bromo-2'-deoxyuridine incorporation.

We have studied the in vitro effect of CsA at various doses (0.5, 1, 5, 10 micrograms/ml) on the growth and 5-bromo-2'-deoxyuridine (BrdU) incorporation by cultured normal human epidermal keratinocytes (EK). CsA was found to reduce the growth of EK at all doses used after 24, 48 and 72 h in culture, but the difference with appropriate controls became statistically significant for the dose of 10 micrograms/ml after a 48- and 72-h culture. On the other hand, CsA-treated EK comprised a reduced fraction of BrdU + (S-phase) cells, the difference being statistically significant for the dose of 10 micrograms/ml at 24, 48 and 72 h. Ultrastructural examination of CsA-treated EK, despite the presence of cytoplasmic vacuoles also observed in control EK did not show signs of severe cytoplasmic or nuclear damage. These results confirm the antiproliferative effect of CsA on cultured EK and suggest that at the concentration used, CsA acts through a cytostatic rather than a cytotoxic mechanism, most likely by blocking EK in an early phase (G0/G1) of the cell cycle.

Endotheliitis in Hepatic Allografts

Endotheliitis (EN) is a feature of hepatic allograft rejection, characterized by the adherence of immunocytes to the endothelium of veins, often leading to endothelial damage, endophlebitis, and, sometimes, panphlebitis. We found EN at least once in 28 of 41 allografts (68%) that had survived 6 months or longer. In approximately half the affected cases, the condition recurred. The EN was mild in most instances; moderate or severe manifestations were found in only 13% of the cases. The histologic changes were present for about 1 week in approximately half the cases; a duration of more than 2 weeks was noted in 17%, and then EN usually persisted. After retransplantation, recurrence of EN was observed in all of nine cases. We were unable to establish specific clinicopathologic and laboratory correlations for EN. The immunocytes in EN consisted mainly of helper and suppressor/cytotoxic T cells as well as natural killer cells. Sometimes, the immunocytes assumed a blastlike appearance; in these instances, the condition was severe. Such blastlike changes may be specific for EN. The immunocytes were attached to the endothelium by pseudopodia, broad bases, or both; some also were interconnected by cytoplasmic bridges. Underlying endothelial cells often showed evidence of cytoplasmic damage. The pathogenesis of EN is not completely understood; the immunocytes probably attach themselves to antigenic epitopes. Their nature, however, has not been clearly identified; HLA-A, B, and C and HLA-DR were displayed in areas of EN, but the antigens also were found in vessels without EN.

Cytoplasmic damage gradients in dendrites after transection lesions.

Ultrastructural changes in the cytoplasm of transected dendrites have been studied with serial section electron microscopy 10 to 12 min after the lesion. Dendrites from large multipolar neurons in mouse spinal monolayer cultures were selected 4 weeks after seeding and cut with a pulsed UV laser microbeam. The data reveal cytoplasmic damage gradients which decrease in severity with distance from the lesion. The damage is expressed primarily in cytoskeletal disruption, the swelling and vacuolation of mitochondria (MC), and the swelling and vesiculation of the endoplasmic reticulum (ER). For a 3-4 microns diameter dendrite lesioned 150 microns from the soma and fixed 10 min after transection, the following results can be reported. The number of microtubules (MT) decreases almost linearly with a slope of 40 MT/10 microns until MT completely disappear 20 microns from the lesion. Neurofilaments (NF) decrease at approximately 50 NF/10 microns, with 20% of these structures still intact at or near the lesion. MC show both gradual and explosive swelling in the damaged process. The onset of these two phases of MC swelling are correlated with the initial and total loss of MT respectively. MT and NF densities in cut dendrites are drastically different from those in control processes and suggest a possible retrograde redistribution of these organelles. Damage from external shockwaves that cause process distortion but not transection results in a less severe but nevertheless similar loss of MT and NF.