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2.
J Chem Phys ; 141(3): 034306, 2014 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-25053319

RESUMO

Measurements of the grand total and total positronium formation cross sections for positron scattering from uracil have been performed for energies between 1 and 180 eV, using a trap-based beam apparatus. Angular, quasi-elastic differential cross section measurements at 1, 3, 5, 10, and 20 eV are also presented and discussed. These measurements are compared to existing experimental results and theoretical calculations, including our own calculations using a variant of the independent atom approach.


Assuntos
Elétrons , Uracila/química , Elasticidade , Modelos Moleculares , Teoria Quântica
3.
J Virol ; 77(23): 12852-64, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14610206

RESUMO

The induction of apoptosis or programmed cell death in virus-infected cells is an important antiviral defense mechanism of the host, and some herpesviruses have evolved strategies to modulate apoptosis in order to enhance their survival and spread. In this study, we examined the ability of varicella-zoster virus (VZV) to induce apoptosis in primary human dorsal root ganglion neurons and primary human foreskin fibroblasts (HFFs). Three independent methods (annexin V, TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] staining, and electron microscopy) were used to assess apoptosis in these cells on days 1, 2, and 4 postinoculation. By all three methods, apoptosis was readily detected in VZV-infected HFFs. In stark contrast, apoptosis was not detected during productive VZV infection of neurons. The low-passage clinical isolate Schenke and the tissue culture-adapted ROka strain both induced apoptosis in HFFs but not in neurons, suggesting that this cell-type-specific apoptotic phenotype was not VZV strain specific. These data show that the regulation of apoptosis differs markedly between HFFs and neurons during productive VZV infection. Inhibition of apoptosis during infection of neurons may play a significant role in the establishment, maintenance, and reactivation of latent infection by promoting survival of these postmitotic cells.


Assuntos
Apoptose/fisiologia , Herpesvirus Humano 3/fisiologia , Neurônios Aferentes/virologia , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/virologia , Imunofluorescência , Gânglios Espinais/virologia , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Confocal , Microscopia Eletrônica
4.
J Virol ; 77(20): 11139-49, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14512561

RESUMO

Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h postinfection, wild-type (wt) HSV-2 (186) abortively infected murine bone marrow-derived DC and induced early cell death compared to UV-inactivated HSV-2 or mock-infected DC. HSV-2-induced loss of DC viability was more rapid than that induced by HSV-1 and was due, in part, to apoptosis, as shown by TEM, caspase-3 activation, and terminal deoxynucleotidyl transferase-mediated dCTP biotin nick end labeling. HSV induced type-specific changes in the murine DC immunophenotype. At 12 h postinfection, wt HSV-2 upregulated DC major histocompatibility complex (MHC) class II expression, and in contrast to UV-inactivated HSV-2, downregulated expression of MHC class I, but it had no effect on surface CD40, CD80, or CD86. Wt HSV-1 (MC-1) induced only CD40 upregulation. More-profound effects on the DC immunophenotype were observed in HSV-2-infected neonatal DC. Wt HSV of either serotype impaired murine DC-induced T-cell alloproliferation and lipopolysaccharide-induced DC interleukin-12 secretion. Thus, there are marked differences in the levels of HSV-induced cytolysis in DC according to the HSV serotype, although HSV-2 displays immunomodulatory effects on the DC immunophenotype and function similar to those of HSV-1.


Assuntos
Apoptose , Células Dendríticas/fisiologia , Herpesvirus Humano 2/patogenicidade , Animais , Antígenos de Histocompatibilidade Classe I/análise , Imunofenotipagem , Interleucina-12/biossíntese , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Necrose , Proteínas Virais/análise
5.
J Virol ; 74(4): 1827-39, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10644356

RESUMO

The mechanism of anterograde transport of herpes simplex virus was studied in cultured dissociated human and rat dorsal root ganglion neurons. The neurons were infected with HSV-1 to examine the distribution of capsid (VP5), tegument (VP16), and glycoproteins (gC and gB) at 2, 6, 10, 13, 17, and 24 h postinfection (p.i.) with or without nocodazole (a microtubule depolymerizer) or brefeldin A (a Golgi inhibitor). Retrogradely transported VP5 was detected in the cytoplasm of the cell body up to the nuclear membrane at 2 h p.i. It was first detected de novo in the nucleus and cytoplasm at 10 h p.i., the axon hillock at 13 h p.i., and the axon at 15 to 17 h p.i. gC and gB were first detected de novo in the cytoplasm and the axon hillock at 10 h p.i. and then in the axon at 13 h p.i., which was always earlier than the detection of VP5. De novo-synthesized VP16 was first detected in the cytoplasm at 10 to 13 h p.i. and in the axon at 16 to 17 h p.i. Nocodazole inhibited the transport of all antigens, VP5, VP16, and gC or gB. The kinetics of inhibition of VP5 and gC could be dissociated. Brefeldin A inhibited the transport of gC or gB and VP16 but not VP5 into axons. Transmission immunoelectron microscopy confirmed that there were unenveloped nucleocapsids in the axon with or without brefeldin A. These findings demonstrate that glycoproteins and capsids, associated with tegument proteins, are transported by different pathways with slightly differing kinetics from the nucleus to the axon. Furthermore, axonal anterograde transport of the nucleocapsid can proceed despite the loss of most VP16.


Assuntos
Transporte Axonal , Capsídeo/metabolismo , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Neurônios/virologia , Proteínas do Envelope Viral/metabolismo , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Brefeldina A/farmacologia , Proteínas do Capsídeo , Linhagem Celular , Células Cultivadas , Gânglios Espinais/citologia , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/ultraestrutura , Humanos , Cinética , Microscopia Confocal , Microscopia Eletrônica , Microscopia Imunoeletrônica , Neurônios/citologia , Neurônios/metabolismo , Nocodazol/farmacologia , Nucleocapsídeo/metabolismo , Ratos , Ratos Wistar , Células Tumorais Cultivadas
6.
J Virol ; 73(10): 8503-11, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10482603

RESUMO

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.


Assuntos
Gânglios Espinais/virologia , Herpes Simples/metabolismo , Neurônios/virologia , Simplexvirus/fisiologia , Proteínas Virais/metabolismo , Axônios/ultraestrutura , Axônios/virologia , Transporte Biológico , Células Cultivadas , Feminino , Gânglios Espinais/patologia , Herpes Simples/patologia , Humanos , Microscopia Eletrônica , Neurônios/ultraestrutura , Gravidez
7.
J Microsc ; 192(Pt 1): 69-72, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9848271

RESUMO

Retaining the ultrastructural arrangement of a mixed-cell culture on a solid support while processing for immunocytochemical study is a technical challenge. We developed a technique to study the axonal transport of the Herpes simplex virus from dorsal root ganglia sensory neurones to epidermal cells. Autologous explants of human foetal dorsal root ganglia and skin were cultured on plastic cover slips. Axon fascicles grew from the ganglia to the epidermal cells and the ganglia were inoculated selectively with virus. The whole preparation, retained on the cover slip, was fixed with formaldehyde 4% (freshly prepared from paraformaldehyde)/glutaraldehyde 0.1%, processed by freeze substitution, and embedded in Lowicryl HM20 resin. The edges of the cover slip in the block were trimmed, allowing clean and complete separation from the resin block, which retained the tissue. The resin block was placed in fresh HM20 and repolymerized. The polymerizing resin bonded strongly to the existing block. After trimming, serial sections were easily obtained and successfully immunolabelled for viral proteins. This is a convenient technique for immunolabelling tissue grown on cover slips in which the preservation of the ultrastructural interactions between different cells is important. It should be adaptable to a number of cell-culture applications and has a number of advantages over other techniques.


Assuntos
Axônios/virologia , Gânglios Espinais/virologia , Queratinócitos/virologia , Simplexvirus/metabolismo , Pele/virologia , Axônios/ultraestrutura , Capsídeo/análise , Proteínas do Capsídeo , Técnicas de Cultura , Gânglios Espinais/embriologia , Humanos , Queratinócitos/ultraestrutura , Microscopia Imunoeletrônica , Microtomia/métodos , Pele/embriologia , Inclusão do Tecido/métodos
8.
J Am Soc Nephrol ; 9(1): 77-84, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9440090

RESUMO

Proteinuria has been invoked as a cause of tubulointerstitial injury in chronic renal disease, and in vivo studies have suggested indirectly the particular nephrotoxicity of one urinary protein holotransferrin (Tf-Fe). However, to date there has been no direct evidence for the nephrotoxicity of Tf-Fe. To examine the potential cytotoxicity of Tf-Fe and the mechanism involved, and to compare this to another urinary protein albumin, rat proximal tubule cells were studied in primary culture. Tf-Fe at pH 6.0 caused functional and ultrastructural injury, but no cytotoxicity was seen with cells exposed to albumin, apotransferrin (transferrin), or Tf-Fe at pH 7.4. The influence of pH on Tf-Fe-induced cytotoxicity was not due to pH per se, but could be explained by an effect on Tf-Fe uptake. At pH 6.0, uptake of 125I-Tf-Fe (3.55 +/- 0.05 versus 1.25 +/- 0.10 fmol/dish, P < 0.01) and intracellular iron concentration (1.14 +/- 0.25 versus 0.46 +/- 0.23 nmol/dish, P < 0.01) were increased compared with values at pH 7.4. In contrast, pH 6.0 did not increase iron uptake from FeCl3. Lysine (100 mM) inhibited Tf-Fe uptake, decreased intracellular iron concentration, and attenuated Tf-Fe-induced cytotoxicity. The iron chelator des-ferrioxamine (200 microM) and hydroxyl radical scavenger dimethylpyrroline N-oxide (32 mM) abolished lactate dehydrogenase leakage induced by Tf-Fe at pH 6.0. Lipid peroxidation, as assessed by production of malondialdehyde, preceded lactate dehydrogenase leakage. In summary, holotransferrin, but not albumin, is toxic to rat proximal tubule cells, a pH-dependent effect involving its uptake into tubule cells, its iron moiety, and its lipid peroxidation.


Assuntos
Túbulos Renais Proximais/efeitos dos fármacos , Albumina Sérica/farmacologia , Transferrina/intoxicação , Animais , Células Cultivadas , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Ferro/metabolismo , Túbulos Renais Proximais/citologia , L-Lactato Desidrogenase/metabolismo , Lisina/farmacologia , Masculino , Malondialdeído/metabolismo , Ratos , Ratos Wistar , Transferrina/farmacocinética
9.
Kidney Int ; 45(4): 1006-13, 1994 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8007569

RESUMO

Reactive oxygen species (ROS) have been implicated in progression of disease in the rat remnant kidney (RK) model of chronic renal failure. Substantial amounts of iron accumulate in proximal tubular lysosomes of RK and could damage tubules by ROS generation. The effect of dietary protein intake on ROS, tubular damage and iron accumulation assessed by energy dispersive analysis was determined in RK (5/6 nephrectomy, N = 12) and sham-operated kidneys (SO, N = 10). In RK, mean lysosomal iron concentration, urinary iron and protein excretion and morphological damage were increased and GFR decreased. Dietary protein loading (40% vs. 12%) increased the number of iron-containing lysosomes (P < 0.05) and the mean lysosomal iron (P < 0.02) in proximal tubular cells after four weeks. In RK, high protein diet increased renal weight (P < 0.01), numerical density of iron-containing lysosomes and tubular damage (both P < 0.05). ROS generation, assessed by tissue and plasma malondialdehyde (MDA), was also increased (both P < 0.05). Plasma MDA correlated with tubular iron accumulation (r = 0.75). In RK fed a high protein diet (N = 18) treatment with the iron-chelator desferrioxamine reduced serum iron, urinary volume, and tubular iron accumulation and damage compared to controls (P < 0.01). In summary, in RK dietary protein manipulation altered urinary iron and protein excretion, proximal tubular iron accumulation, renal cortical ROS generation and ultrastructural damage. Desferrioxamine treatment reduced tubular lysosomal iron and ultrastructural damage. These results suggest a role for tubular iron as a determinant of tubular injury associated with dietary protein loading in rats with partial nephrectomy.


Assuntos
Proteínas Alimentares/farmacologia , Ferro/metabolismo , Túbulos Renais/metabolismo , Rim/fisiopatologia , Animais , Desferroxamina/farmacologia , Taxa de Filtração Glomerular , Glutationa/análise , Rim/patologia , Rim/cirurgia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Masculino , Malondialdeído/análise , Nefrectomia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/análise , Transferrina/metabolismo
10.
J Am Soc Nephrol ; 4(8): 1598-607, 1994 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8025233

RESUMO

Iron has been implicated in the pathophysiology of several models of acute and chronic renal disease. In this study, energy-dispersive x-ray spectrometry was used to quantify and localize iron in rat remnant kidneys (RK) and normal kidneys (NK) and to determine its pathophysiologic significance. Substantial iron accumulation occurred in proximal tubular cell secondary lysosomes of RK (P < 0.001 versus NK) and reached a plateau at 8 wk after partial nephrectomy. In NK, minor increases of iron also occurred with aging (P < 0.02). Proximal tubular iron accumulation correlated independently with protein excretion (r = 0.90) and impairment of GFR (r = 0.70) and was associated with tubular damage and phosphate accumulation (both P < 0.001). Iron nitrilotriacetate (1 mg/kg ip) increased tubular lysosomal iron accumulation and tubular damage (P < 0.001 versus nitrilotriacetate) in NK, comparable to levels seen in untreated RK, and increased cortical cytosolic malondialdehyde, consistent with reactive oxygen species generation. The iron chelator deferoxamine (30 mg/kg per day ip) significantly reduced iron accumulation and tubular damage in RK at 4 wk, compared with deferoxamine chelated to iron and untreated RK. These results suggest that filtered iron enters the remnant tubular lysosomes across the brush border membrane by endocytosis and may produce tubular damage in chronic renal disease by the generation of reactive oxygen species.


Assuntos
Ferro/metabolismo , Falência Renal Crônica/metabolismo , Rim/química , Animais , Creatinina/metabolismo , Desferroxamina/uso terapêutico , Modelos Animais de Doenças , Microanálise por Sonda Eletrônica , Compostos Férricos/toxicidade , Infarto/metabolismo , Ferro/toxicidade , Rim/irrigação sanguínea , Falência Renal Crônica/patologia , Túbulos Renais Proximais/química , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Masculino , Microscopia Eletrônica , Nefrectomia/efeitos adversos , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/toxicidade , Proteinúria/etiologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio , Transferrina/urina
11.
Ren Fail ; 16(3): 367-81, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8059020

RESUMO

Iron accumulates within proximal tubular lysosomes in several models of renal disease and may play a role in the progression of proteinuric chronic renal disease by the generation of reactive oxygen species. In this study, tubular iron was examined at an ultrastructural level by energy dispersive x-ray spectrometry in streptozotocin (STZ) and BB diabetic rats, and in humans with diabetic nephropathy, and compared to their respective nondiabetic controls. Substantial amounts of iron accumulated in the secondary lysosomes of proximal tubules in STZ diabetic rats (4.16 +/- 0.47 iron-containing lysosomes/microns 2 x 10(-3) tubular area vs. 0.90 +/- 0.29 in controls, p < 0.001). Proximal tubular iron was related independently with urinary protein and transferrin excretion, suggesting increased cellular uptake of iron from the tubular fluid. Lysosomal iron accumulation was also associated with tubular damage (r = 0.55, p < 0.001). Minimal amounts of tubular iron were observed in BB diabetic and nondiabetic littermates. In humans with diabetic nephropathy, increased proximal tubular lysosomal iron concentration (35.6 +/- 13.0 mg% Fe vs. 9.5 +/- 2.7, p < 0.05) and numbers of iron-containing lysosomes were observed compared to nondiabetic controls, and the latter correlated with elevation of serum creatinine (r = 0.94, p = 0.016). These results suggest that filtered iron enters proximal tubular lysosomes across the brush-border membrane and are consistent with a role for iron in causing the tubular damage of diabetic nephropathy.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Ferro/metabolismo , Túbulos Renais Proximais/metabolismo , Lisossomos/metabolismo , Animais , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Microanálise por Sonda Eletrônica , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos BB , Ratos Wistar
12.
Diagn Cytopathol ; 9(5): 541-6, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8287763

RESUMO

An intraoperative cytological diagnosis of a rare solid and papillary epithelial tumor of the pancreas was made at laparotomy in a 15-year-old Indian female. The characteristic cytological features were the presence of numerous large branching papillary clusters with central vascular stalks lined by multiple layers of uniform bland cells. Perivascular metachromatic material was prominent on Romanovsky stain. Ultrastructural identification of endocrine and exocrine features supports a multipotential cell origin in small pancreatic ductules. DNA analysis, not previously reported for this tumour, demonstrated a diploid population of tumour cells with a low S-phase fraction (0.4%). This would explain the bland nuclear morphology and favourable prognosis with a high rate of surgical cure for this neoplasm.


Assuntos
Carcinoma/patologia , Neoplasias Pancreáticas/patologia , Adolescente , Carcinoma/genética , Carcinoma/ultraestrutura , Feminino , Citometria de Fluxo , Humanos , Cariotipagem , Microscopia Eletrônica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/ultraestrutura
13.
Am J Kidney Dis ; 20(6): 580-4, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1462986

RESUMO

Iron, which has been shown to accumulate within proximal tubule lysosomes in proteinuric models of renal disease, may play a role in the progression of chronic renal disease by the generation of reactive oxygen species. Therefore, renal biopsies from humans with proteinuria and/or chronic renal failure were examined at an ultrastructural level for iron by energy dispersive analysis and compared with normal biopsies. Iron accumulated in proximal tubular lysosomes in renal disease (P < 0.05 v normals), accompanied in some cases by phosphorus and silicon. Both the number of iron-containing lysosomes per tubular cross-section (1.86 +/- 0.41 v 0.66 +/- 0.22, P < 0.05) and the mean concentration of lysosomal iron (254.5 +/- 73.4 mg/dL v 81.2 +/- 23.8, P < 0.001) was greater in patients with nephrotic syndrome (n = 12) than in those without (n = 8). Iron accumulation (number of iron-containing lysosomes/tubule) correlated with protein excretion (r = 0.68, P = 0.003, n = 20), but not with glomerular filtration rate. Damaged tubules contained greater amounts of iron than tubules with less damage (288.5 +/- 68.5 mg/dL v 80.4 +/- 13.9, P < 0.01). Further studies are needed to define the possible role of iron in causing tubular damage and progression of renal disease.


Assuntos
Ferro/metabolismo , Nefropatias/metabolismo , Túbulos Renais Proximais/metabolismo , Doença Crônica , Microanálise por Sonda Eletrônica , Glomerulonefrite/metabolismo , Humanos , Nefropatias/patologia , Nefropatias/fisiopatologia , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiopatologia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Proteinúria/metabolismo
14.
Pathology ; 17(1): 67-70, 1985 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-4000714

RESUMO

A case is presented of a patient in whom over 90% of spermatozoa had distinctive round heads as seen by light microscopy. Other features of his semen were only slightly abnormal. Electron microscopy revealed spermatozoa with no acrosome confirming the sterility of the patient. With such men other means of achieving a family must be considered.


Assuntos
Infertilidade Masculina/patologia , Cabeça do Espermatozoide/ultraestrutura , Espermatozoides/ultraestrutura , Adulto , Humanos , Masculino , Microscopia Eletrônica
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