Accumulation Of Dense Bodies Research Articles

Antifungal Activity of Alpha-Mangostin against Colletotrichum gloeosporioides In Vitro and In Vivo.

We investigated alpha-mangostin (α-mangostin, α-MG), a xanthone natural product extracted from the pericarp of mangosteen (Garcinia mangostana), for its antifungal activities and possible mechanism against Colletotrichum gloeosporioides, which causes mango anthracnose. The results demonstrated that α-MG had a relatively high in vitro inhibitory activity against C. gloeosporioides among 20 plant pathogenic fungi. The median effective concentration (EC50) values of α-MG against mycelial growth were nearly 10 times higher than those of spore germination inhibition for both strains of C. gloeosporioides, the carbendazim-sensitive (CBD-s) and carbendazim-resistant (CBD-r). The results suggested that α-MG exhibited a better inhibitory effect on spore germination than on the mycelial growth of C. gloeosporioides. Further investigation indicated that the protective effect could be superior to the therapeutic effect for mango leaves for scab development. The morphological observations of mycelium showed that α-MG caused the accumulation of dense bodies. Ultrastructural observation further revealed that α-MG caused a decrease in the quantity and shape of the swelling of mitochondria in the mycelium cells of C. gloeosporioides. In addition, bioassays disclosed that the inhibitory activity of α-MG on spore germination was reduced by adding exogenous adenosine triphosphate (ATP). These results suggested that the mode of action of α-MG could be involved in the destruction of mitochondrial energy metabolism. The current study supports α-MG as a natural antifungal agent in crop protection.

Synthesis and fungicidal activity of novel 2,5-disubstituted-1,3,4- thiadiazole derivatives containing 5-phenyl-2-furan.

A series of 2,5-disubstituted-1,3,4-thiadiazoles were synthesized using Lawesson’s reagent by an efficient approach under microwave irradiation in good yields. Their structures were characterized by MS, IR, 1H NMR, 13C NMR, and elemental analysis. Their in vitro and in vivo fungicidal activities revealed that the title compounds exhibited considerable activity against five selected fungi, especially to Phytophthora infestans. In order to illustrate the mechanism of title compounds against P. infestans, scanning electron micrographs (SEM) and transmission electron micrographs (TEM) were applied. The morphological and ultrastructural studies demonstrated that compound I18 led to swelling of hyphae, thickening and proliferating multilayer cell walls, excessive septation and accumulation of dense bodies. The bioassay results indicated compound I18 might act on cell wall biosynthesis, and blocked the nutrition transportation and led to cells senescence and death. Meanwhile, compound I18 had broad fungicidal activity against other twenty different kinds of fungi. These results suggested that title compounds were eligible to be development candidates and compound I18 as a promising lead compound was worthy to be further discovery, especially against P. infestans.

Study of Inhibitory Effects and Action Mechanism of the Novel Fungicide Pyrimorph against Phytophthora capsici

The antifungal activity of the novel fungicide pyrimorph, (E)-3-[(2-chloropyridine-4-y1)-3-(4-tert-butylpheny1)acryloyl]morpholin, against Phytophthora capsici was investigated in vitro. Pyrimorph inhibited different stages in the life cycle of P. capsici including mycelial growth, sporangium production, zoospore release, and cystospore germination with EC(50) values of 1.84, 0.17, 4.92, and 0.09 microg mL(-1), respectively. The effect of pyrimorph on mycelial growth was reduced by the addition of different concentrations of ATP, which suggested that the action mechanism of pyrimorph was connected with impairment of the energy generation system. Meanwhile, pyrimorph exhibited certain inhibition on metabolic approaches of Embden-Meyerhof-Parnas (EMP), tricarboxylic acid cycle (TCA), and hexosemonophosphate (HMP) by measuring the oxygen consumption of pyrimorph combining with three representative inhibitors to the metabolic approaches. The results indicated that pyrimorph could inhibit the approach of HMP significantly. Morphological and ultrastructural studies showed that pyrimorph caused excessive septation and swelling of hyphae, distortion and disruption of most vacuoles, thickening and development a multilayer cell wall, and accumulation of dense bodies. These results suggested pyrimorph exhibited multiple modes of action including impairment of the energy generation system and effect on cell wall biosynthesis directly or indirectly.

The immunosuppressant FK506 elicits a neuronal heat shock response and protects against acrylamide neuropathy

Acrylamide (AC) is a known industrial neurotoxic chemical that has been recently found in carbohydrate-rich foods cooked at high temperatures. Repeated AC administration produces a pronounced neuropathy characterized by flaccid paralysis and ataxia and represents a well-established animal model of progressive axonal loss. AC also elicits prominent morphologic alterations (e.g., eccentrically placed nuclei, infolding of the nuclear membrane, accumulations of dense bodies, and clusters of smooth endoplasmic reticulum (SER) associated with numerous microtubules) in cerebellar Purkinje cells that may contribute to the pronounced ataxia in these animals. Here, we examined the neuroprotective action of FK506 (tacrolimus) in male and female rats given daily intraperitoneal injections of AC (30 mg/kg) for 4 weeks. Daily subcutaneous injections of FK506 (2 mg/kg/day) dramatically reduced the behavioral signs of neuropathy (i.e., paralysis and ataxia), markedly protected against axonal loss (by 82% and 73% in the tibial nerves of male and female rats, respectively), and reduced the pathologic changes in Purkinje cells. In a separate study, subcutaneous injections of FK506 (2 or 10 mg/kg) for 2 weeks markedly increased heat shock protein-70 (Hsp-70) immunostaining in sensory neurons, motor neurons, Purkinje cells, and other regions of the brain (in particular, the amygdala) from nonintoxicated and AC-intoxicated rats compared to controls. In contrast, AC-intoxicated animals not given FK506 demonstrated reduced Hsp-70 staining. Thus, the ability of FK506 to increase Hsp-70 expression may underlie its neuroprotective action. We suggest that compounds capable of eliciting a heat shock response may be useful for the treatment of human neuropathies.

Quantifying dense bodies and lipofuscin during aging: a morphologist's perspective

Secondary lysosomes, residual or dense bodies containing lipofuscin or age pigment accumulate in post-mitotic and inter-mitotic cells during aging. The consensus is that the accumulation of this auto-fluorescent material is an index of cellular senescence. Biochemical and morphological studies have independently demonstrated marked age-related increases in the cell and tissue contents of lipofuscin. Most morphological studies on aging have been qualitative, have included only two or three age groups and have not yielded data that are easily correlated with biochemical analyses. One of the best documented age-related changes in hepatocytes and cardiac myocytes is the accumulation of dense bodies and lipofuscin inclusions. Independent stereologic studies reported two- to eightfold age-related increases in the dense body volume fraction of rat hepatocytes. Furthermore, we reported a fourfold increase in the dense body volume fraction of cardiac myocytes in rats between 6 and 30 months of age. These and other studies confirm the use of quantitative morphology to estimate the increases in dense body and lipofuscin inclusions as indices of age. Whether or not the accumulated lipofuscin compromises cell functions in senescent animals has not been adequately addressed. On the one hand, there is little evidence that several-fold increases in this subcellular compartment impair the functional capacities of either hepatocytes or cardiac myocytes. On the other hand, the age-related accumulation of immunoprecipitable, but catalytically inactive, lysosomal enzymes in both liver and heart muscle may be a reflection of increased lipofuscin deposits in the dense bodies.

A novel nonnucleoside inhibitor specifically targets cytomegalovirus DNA maturation via the UL89 and UL56 gene products.

3-Hydroxy-2,2-dimethyl-N-[4([[5-(dimethylamino)-1-naphthyl]sulfonyl]amino)-phenyl]propanamide (BAY 38-4766) is a novel selective nonnucleoside inhibitor of cytomegalovirus (CMV) replication with an excellent safety profile. This compound and structural analogues inhibit neither viral DNA synthesis nor viral transcription and translation. Accumulation of dense bodies and noninfectious enveloped particles coincides with inhibition of both concatemer processing and functional cleavage at intergenomic transitions, pointing to interference with viral DNA maturation and packaging of monomeric genome lengths. Resistant virus populations, including a murine CMV (MCMV) isolate with 566-fold-decreased drug sensitivity, were selected in vitro. Sequencing of the six open reading frames (ORFs) known to be essentially involved in viral DNA cleavage and packaging identified mutations in ORFs UL56, UL89, and UL104. Construction of MCMV recombinants expressing different combinations of murine homologues of mutant UL56, UL89, and UL104 and analysis of drug susceptibilities clearly demonstrated that mutant ORFs UL89 exon II (M360I) and M56 (P202A I208N) individually confer resistance to BAY 38-4766. A combination of both mutant proteins exhibited a strong synergistic effect on resistance, reconstituting the high-resistance phenotype of the in vitro mutant. These findings are consistent with genetic mapping of resistance to TCRB (2,5,6-trichloro-1-beta-D-ribofuranosyl benzimidazole) (P. M. Krosky et al., J. Virol. 72:4721-4728, 1998) and provide further indirect evidence that proteins encoded by UL89 and UL56 function as two subunits of the CMV terminase. While these studies also suggest that the molecular mechanism of BAY 38-4766 is distinct from that of benzimidazole ribonucleosides, they also offer an explanation for the excellent specificity and tolerability of BAY 38-4766, since mammalian DNA does not undergo comparable maturation steps.

Structure and ultrastructure of the ventral prostate of isogenic mice (C57B1/6J) submitted to chronic alcohol ingestion

Morphological and functional alterations caused by chronic alcohol ingestion in testes and accessory sex organs have been studied both in man and in laboratory animals. The aim of the present study was to examine the possible occurrence of deleterious effects of chronic alcohol ingestion on the secretory epithelium of the ventral prostate of mice. Twenty-four adult male C57BL/6J mice were divided into three groups. The alcohol-treated group was allowed to drink only 6% (v/v) ethanol, the isocaloric group received a diet of water/sucrose with a calorie content equivalent to a 6% alcohol solution and the control group received water. Both groups were fed ad libitum with solid Purina rat chow. After 120 days, animals from each group were anesthetized with ethyl ether, weighed and processed for light and transmission electron microscopy. The results demonstrated reduction in the glandular epithelium cell height and disorganization of the Golgi complex. Moreover, abundant membrane-bound structures, most likely representing cytoplasmic material, were observed, as well as accumulation of dense bodies. Statistical analysis showed that bodyweight gain was similar for both groups. In conclusion, chronic alcohol ingestion has harmful effects on the secretory epithelium cells of the ventral lobe of the prostate of mice after 120 days of treatment.

Liver function and phase I drug metabolism in the elderly: a paradox.

Aging is accompanied by marked changes in the physiology of many organs, as well as in their constituent cells. These nonpathological alterations in structure and/or function may affect normal physiological processes in the elderly (individuals > 65 years), for example drug disposition. The liver plays a major role in drug clearance and aging has been reported to diminish this hepatic capacity, particularly the clearance of drugs that undergo mandatory oxidation by the microsomal cytochrome P450-dependent mono-oxygenase systems. Liver volume and blood flow decline with age in humans and, no doubt, this contributes to the diminished clearance of drugs that exhibit first-pass kinetic profiles. Changes in liver morphology with aging that have been described in rodents are limited to the hepatocytes, for example accumulation of dense bodies and loss of smooth surfaced endoplasmic reticulum. There is no evidence that the increase in intracellular lipofuscin adversely affects hepatocyte functions. A number of studies have documented significant age-related declines in the amounts, specific activities and rates of induction of liver microsomal mono-oxygenases in inbred male rats. On the basis of a variety of clinical tests, most liver functions in humans appear to be well preserved. The most remarkable characteristic of liver function in the elderly is the increase in interindividual variability, a feature that may obscure age-related differences. Most in vitro studies using nonhuman primate or human liver tissue did not detect age-related deficiencies in cytochrome P450-dependent microsomal mono-oxygenases. On the other hand, there have been recent reports of age-related, but not gender-related, declines in the in vitro activities of several human liver mono-oxygenases, for example the cytochrome P450 isoform CYP3A. Nevertheless, reduced liver volume and blood flow in the elderly permit the reconciliation of: the in vivo clinical pharmacokinetic data indicative of reduced hepatic drug clearance; and the absence of significant age-related declines in the amounts or in vitro activities of liver microsomal mono-oxygenases.

Ultrastructural features of degeneration of the gallbladder during lamprey biliary atresia

Thin sections and freeze-fracture replicas were used to examine the fine structural features of degeneration of the gallbladder during lamprey biliary atresia. The cells of the epithelium undergo a progressive accumulation of dense bodies and vacuoles, loss of glycogen, condensation of the filamentous ectoplasm, fragmentation of microvilli, and dilation of cisternae of rough endoplasmic reticulum but eventually disappear by stage 4 of metamorphosis. Zonulae occludentes in the epithelium show a progressive increase in apical-basal depth as the junctional strands fragment. The possibility of an influence of transformed, subepithelial cells on degeneration of epithelial cells is suggested by close contact of the former with the thickened, highly pleated, epithelial basal lamina. The smooth muscle cells of the larval gallbladder are believed to transform during lamprey metamorphosis into these subepithelial cells which shed their external lamina, become intimately associated with collagen and other microfibrils, and which may be capable of phagocytosis. The events of gallbladder degeneration during lamprey metamorphosis show features of apoptosis.

Morphology of the pineal gland in pregnant and nursing sows.

The pineal glands of pregnant and nursing sows were studied by light and electron microscopy. Similar structural changes in the pineal gland during pregnancy and lactation were noted as follows: a proliferation of glial cells concurrent with a decrease in the number of pinealocytes, a relative increase in the number of pinealocytes with cytoplasmic accumulation of dense bodies, and an increase in number of presumptive microglia. It appears that pregnancy and lactation exert a complex influence on pineal function, with a predominantly stimulating effect.

Ultrastructural pathology of dendritic spines in epitumorous human cerebral cortex.

Ultrastructural changes in the human epitumorous cerebral cortex were examined. A swelling of nerve cell perikarya, dendrites and axon terminals, and an occasional hyperplasia and disarray of microtubules and neurofilaments were observed. Accumulations of lysosomes, tubuloreticular structures and intracytoplasmic or intramitochondrial crystalloid inclusions were also found. Some myelinated axons were degenerated. Astrocytes and their processes were focally swollen. A mild swelling was found also in microgliocytes. Oligodendrocytes occasionally contained accumulations of dense bodies. Special attention was paid to dendritic spines. The spine surface morphology changed distinctly on swollen dendritic segments. The necks of most spines were short and wide, and numerous sessile forms were present in this location. The spine apparatus was often hypertrophied and disorganized. Smoothing out of spines on swollen dendrites is described and a possible functional significance of the observed changes in the symptomatology of brain tumors is hypothesized.

Morphologic effects of ammonia on primary astrocyte cultures. I. Light microscopic studies.

To evaluate the astrocytic alterations commonly seen in hepatic encephalopathy and other hyperammonemic states, primary astrocyte cultures derived from neonatal rats were exposed to varying concentrations of ammonia for one to ten days. Ammonia-treated cultures initially showed an increase in basophilia, prominent cytoplasmic processes and increased cytoplasmic granularity and vacuolization. Nucleoli were increased in size and there was an increase in nucleolar/nuclear ratio. Later, fragmentation and loss of cytoplasmic processes, formation of dense bodies and frank cellular disintegration were noted. The changes were proportional to the concentration and duration of ammonia treatment. Our studies show that ammonia is capable of directly causing morphologic alterations in astrocytes. We believe that the use of primary cultures provides a means of exploring the precise role of astrocytes in hyperammonemic states.

Morphologic effects of ammonia on primary astrocyte cultures. II. Electron microscopic studies.

Light microscopic studies of primary astrocyte cultures following exposure to ammonia have shown several alterations. To determine the nature and significance of these changes, electron microscopic studies were performed. Ultrastructural changes consisted of proliferation, pleomorphism and swelling of mitochondria, condensation of the mitochondrial matrix, cytoplasmic lucency and vacuolization, disaggregation of polyribosomal clusters, an initial increase followed by degranulation of rough endoplasmic reticulum, proliferation of smooth endoplasmic reticulum, an accumulation of dense bodies and a loss of intermediate glial filaments. The early alterations appeared reactive and perhaps reflected ammonia detoxification. Some changes were degenerative and support the view that ammonia exerts a direct toxic effect on astrocytes. It is postulated that these changes may interfere with critical astroglial functions and thereby play a key role in the neurologic dysfunction seen in hyperammonemia.

Evolution of the intracellular changes in neurons caused by trimethyltin.

Rats have been given a single dose of trimethyltin (10 mg/kg) and the intracellular events have been followed particularly in hippocampus, cerebral cortex, cerebellum and spinal ganglion cells. The earliest change visible occurs 12 h after this dose and is found to be dense membrane-bound bodies, probably derived from branching tubulo-vesicular smooth endoplasmic reticulum formations. These occur in close connection with rought endoplasmic reticulum and polyribosomes and appear also to have some association with the Golgi complex. At 24 h there is a general vacuolation of Golgi cisterns and SER membranes, and the membrane-bound dense body formation is greatly increased. SER abnormalities are particularly conspicuous in Purkinje cells. In spinal ganglion cells, while vacuolation of Golgi cisterns is intense, dense bodies are inconspicuous and are replaced by increased autophagosomes, often of great complexity. By 48 h vacuolation of Golgi cisterns has waned, but accumulation of dense bodies and secondary lysosomes has steadily increased. In spinal ganglion cells autophagosomes only are increased as the Golgi vacuolation declines. At later times steady increases of lysosomal dense bodies is seen generally accompanied in hippocampal pyramidal cells and dentate fascia cells by abundant cell death. The suggestion is put forward that the Golgi complex may be the seat of the critical metabolic lesion and disturbances to protein transfer and protein synthesis follow. No explanation for the selective loss of hippocampal h1-5 (CA1-CA4 except Sommer's sector) pyramidal cells and of small dentate fascia neurons can be derived from these conclusions.

An Ultrastructural Study of the Corpora Amylacea of the Optic Nerve Head and Retina

We studied corpora amylacea in the optic nerve heads and retinas of four human eyes with increased intraocular pressure by light and electron microscopy. Ultrastructurally they consisted of a mass of filamentous tangle discretely bordered by normal or abnormal axoplasm within an axonal swelling. We found neurotubules, mitochondria, and the accumulation of dense bodies in the axoplasm surrounding the filamentous tangle. We noted ultrastructural similarities between corpora amylacea and cytoid bodies, swollen axons with transport block, and experimentally induced neurofibrillary tangles. We concluded that corpora amylacea are intracellular organelles found in axonal swellings and are not glial in origin, as previously believed. There is a possible relationship between corpora amylacea and distrubance of axoplasmic transport.

Structural aspects of [formula omitted] aging rabbit blood platelets

The structural and density changes that occur in aging blood platelets may help to evaluate the age distribution of platelets in a blood sample. Separation of normal rabbit platelets on human serum albumin discontinuous density gradient (HSAG), provided four bands of cells, usually in the 18,19,22 and 23% solutions of HSA. Large and small platelets were found in each of the four bands. No significant differences were found in the mean size of the cells taken from the light fractions (18% and 19%) when compared to cells taken from the heavier fractions (22% and 24%). However cells of the light fractions usually contained less Dense Bodies (DB) than cells from the heavier fractions. Newly formed platelets obtained from rabbits recovering from induced thrombocytopenia after injection of goat antirabbit platelet serum or neuraminidase, are generally large and found in the solution of 21% HSA (S.G. 1.0606). These platelets contain glycogen particles and practically no DB. After several days of maturation and regeneration, smaller platelets containing higher number of DB, are found in the circulation. On the 10th day, after the beginning of regeneration, platelets are distributed above and below the solution of 21% HSA, similarly to the normal platelet population. The in vivo aging of platelets, seems to be associated with accumulation of DB within these cells.

Abnormal ultrastructural appearances in axons of feline pericruciate cortex after lateral funiculotomy.

Following left lateral funiculotomy, axons of cat pericruciate cortex exhibited neurofilamentous hyperplasia and complex, adaxonal, oligodendrocytic invaginations into electron-lucent or (commonly) electron-dense, degenerating axoplasm. These changes were absent form sham-operated and unoperated animals. Neurofilamentous hyperplasia was exclusively right-sided and appeared in myelinated axons 5--49 days postoperatively and in nonmyelinated axons 14--153 days after surgery. Oligoglial invaginations were present 1--49 days after surgery and were predominantly right-sided. Intramyelinic, axo-dendritic synapses appeared in operated cats 5--10 days postoperatively. Intra-axonal accumulations of ribosomes were found also. These changes also occurred exclusively or predominantly contralateral to spinal surgery. Other ultrastructural abnormalities, e.g., amorphous transformation of axoplasm and accumulations of dense bodies in intra-myelinic, dark cytoplasm, had a less certain relationship to lateral funiculotomy. The axonal alterations that were limited to operated cats possibly represent a true retrograde axonal degeneration occurring at a distance from the site of axonic interruption and unaccompanied by evidence of nerve cell death.