Nuclear Margination Research Articles

Cytokinin-induced apoptotic nuclear changes in cotyledons of Solanum aviculare and Lycopersicon esculentum

Cytokinin-induced nuclear changes have been observed in epidermal and sub-epidermal layers of cells in cotyledons of Lycopersicon esculentum and Solanum aviculare which compare closely to those observed during apoptosis in mammalian, insect and nematode species. The nuclei, which show invagination of the nuclear membrane, margination and eventual breakdown of the chromatin, are positive with the TUNEL reaction. Such changes are not induced by treatment of the cotyledons with 2,4-dichlorophenoxyacetic acid. In contrast, mesophyll cells induced to form vessels (programmed cell death) by treatment with 2,4-dichlorophenoxyacetic acid together with either 6-benzylamino-purine or zeatin, retain their diploid nuclei until the end of differentiation.

Types of Programmed Cell Death: Two Variants Expressed by Neonatal Murine Hepatocytes

Neonatal livers examined with the terminal transferase-mediated dUTP nick end labeling (TUNEL) method contained numerous positive cells. Although the majority of dying cells are either hematopoietic cells including erythroids and granulocytes or macrophages, a few hepatocytes were also positive. As for the ultrastructural features of these dying hepatocytes, two different types, type I and II, could be identified. The early features of type I appeared in the cytoplasm, which was characterized by dilated rough endoplasmic reticulum, and the cell fragments displayed a round, foamy appearance. Type II was characterized by nuclear compaction and margination of heterochromatin resulting in the formation of sharply circumscribed masses, followed by the condensation of the cytoplasm. The cell death of type I, characterized by the formation of massive vacuolization of the endoplasmic reticulum, corresponds to cytoplasmic type degeneration or nonapoptotic death, while that of type II corresponds to nuclear type cell death or classical apoptotic death. In the two types of programmed cell death, the incidence of nonapoptotic cell death was much higher than that of classical apoptosis in neonatal murine hepatocytes.

Thalamic retrograde degeneration in the congenitally hydrocephalic rat is attributable to apoptotic cell death.

Congenitally hydrocephalic HTX rats develop ventricular dilatation with extensive damage of the cerebral white matter. Recently, we have reported that neuronal cell death also occurs in the thalamus of HTX rats. To investigate the mechanism underlying this thalamic degeneration in these animals, we carried out a histopathological study of the brain at different phases of postnatal development. Eosinophilic neurons with condensed chromatin or fragmented nuclei were observed in the thalamus from postnatal day 17 onward. The incidence of cell death in the thalamus increased with the progression of hydrocephalus. Ultrastructurally, thalamic neurons occasionally had apoptotic features including nuclear chromatin condensation and marginalization. Immunohistochemically, single-stranded DNA-positive neuronal nuclei were found in the thalamus. They were also positively stained with the TUNEL method. Marked loss of myelin and axons with many TUNEL-positive oligodendrocytes were found in the cerebral white matter. These findings suggest that the neuronal cell death observed in the thalamus in hydrocephalic HTX rats is retrograde degeneration due to extensive damage of axons in the cerebral white matter and that the thalamic retrograde degeneration is attributable to apoptotic cell death.

Effects of exogenous nitric oxide on oral squamous cell carcinoma: An in vitro study

Purpose: Nitric oxide (NO) is a newly found unstable free radical gas, serving as an important mediator, messenger, and signal transduction molecule and involved in a variety of pathophysiologic processes. Recently, NO has been reported to have cytotoxic effects on several tumor cells as an effector molecule of activated macrophage. The objective of this study was to investigate the effects of exogenous NO on oral squamous cell carcinoma cell line and to try to clarify the possible mechanisms by which it kills tumor cells. Methods: TSCCa cell line, established from a patient with oral squamous cell carcinoma of the tongue, was exposed to various concentrations of exogenous NO that were released from an NO donor, sodium nitroprusside (SNP), for 48 hours. Nitrite/nitrate levels in the culture supernatant were determined with a commercial available NO kit. Both morphologic and ultrastructural changes were evaluated by reverse phase contrast microscopy or transmission electron microscopy. The DNA was harvested from SNP-treated or untreated TSCCa cells and assessed by agarose gel electrophoresis. Results: SNP released NO into medium in a dose-dependent manner. NO had a concentration-dependent cytotoxicity against TSCCa cells. NO induced tumor cell death through apoptosis, which was characterized by incompleteness of nuclear membrane, disappearance of nucleole and nuclear condensation, chromatin margination, or chromatin homogenization. Agarose gel electrophoresis showed a typical internucleosomal DNA cleavage pattern (DNA ladder), a reliable indicator of apoptosis. Conclusions: Our results suggested that NO had a tumoricidal potential against oral cancer cells. NO might exert its cyotoxicity as an effector molecule of activated microphage through at least apoptosis. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:905-910, 2002

Executioner Caspase-3, -6, and -7 Perform Distinct, Non-redundant Roles during the Demolition Phase of Apoptosis

Apoptosis is orchestrated by a family of cysteine proteases known as the caspases. Fourteen mammalian caspases have been identified, three of which (caspase-3, -6, and -7) are thought to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the relative contributions that the "executioner" caspases make to the demolition of the cell remains speculative. Here we have used cell-free extracts immuno-depleted of either caspase-3, -6, or -7 to examine the caspase requirements for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We show that caspase-3 is the primary executioner caspase in this system, necessary for cytochrome c/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleoprotein, DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-linked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addition, caspase-3 was also essential for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse in this system. Surprisingly, although caspase-6 and -7 are considered to be important downstream effector caspases, depletion of either caspase had minimal impact on any of the parameters investigated, calling into question their precise role during the execution phase of apoptosis.

DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV).

Fifty black tiger shrimp Penaeus monodon from commercial cultivation ponds in Malaysia were examined by Tdt-mediated dUTP nick-end labeling (TUNEL) fluorescence assay and agarose gel electrophoresis of DNA extracts for evidence of DNA fragmentation as an indicator of apoptosis. From these specimens, 30 were grossly normal and 20 showed gross signs of white spot syndrome virus (WSSV) infection. Of the 30 grossly normal shrimp, 5 specimens were found to be positive for WSSV infection by normal histology and by nested polymerase chain reaction (PCR) analysis. All of the specimens showing gross signs of WSSV infection were positive for WSSV by normal histology, while 5 were positive by nested PCR only (indicating light infections) and 15 were positive by 1-step PCR (indicating heavy infections). Typical histological signs of WSSV infection included nuclear hypertrophy, chromatin condensation and margination. None of the 25 grossly normal shrimp negative for WSSV by 1-step PCR showed any signs of DNA fragmentation by TUNEL assay or agarose gel electrophoresis of DNA extracts. The 10 specimens that gave PCR-positive results for WSSV by nested PCR only (i.e., 5 grossly normal shrimp and 5 grossly positive for WSSV) gave mean counts of 16 +/- 8% TUNEL-positive cells, while the 25 specimens PCR positive by 1-step PCR gave mean counts of 40 +/- 7% TUNEL-positive cells. Thus, the number of TUNEL positive cells present in tissues increased with increasing severity of infection, as determined by gross signs of white spots on the cuticle, the number of intranuclear inclusions in histological sections, and results from single and nested PCR assays. DNA extracts of PCR-positive specimens tested by agarose gel electrophoresis showed indications of DNA fragmentation either as smears or as 200 bp ladders. Given that DNA fragmentation is generally considered to be a hallmark of apoptosis, the results suggested that apoptosis might be implicated in shrimp death caused by WSSV.

Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of exoS.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Using a tissue culture system, invasive strains of P. aeruginosa were discovered to induce apoptosis at high frequency in HeLa and other epithelial and fibroblast cell lines. This apoptotic phenotype in the infected cells was determined by several criteria including (i) visual changes in cell morphology, (ii) induction of chromatin condensation and nuclear marginalization, (iii) the presence of a high percentage of cells with subG1 DNA content, and (iv) activation of caspase-3 activity. Induction of the type III secretion machinery, but not invasion of P. aeruginosa is required for induction of apoptosis. The apoptosis phenotype is independent of the cytoskeletal rearrangements that occur in the host cell early after infection. Mutants in P. aeruginosa exoS fail to induce apoptosis and complementation with wild-type exoS restored the apoptosis-inducing capacity, demonstrating that ExoS is the effector molecule. Analysis of exoS activity mutants shows that the ADP-ribosylating capacity of ExoS is essential for inducing the apoptotic pathway.

Pathologic alterations in adult rainbow trout , Oncorhynchus mykiss, exposed to dietary 2,3,7,8-tetrachlorodibenzo- p-dioxin

Adult female rainbow trout ( Oncorhynchus mykiss) fed [ 3H]2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) impregnated diet at 0, 1.8, 18 or 90 ng/kg food for up to 320 days were evaluated by clinical pathology, gross pathology and histopathology procedures. Gross pathological changes were limited to a slight increase in the incidence of lesions of the caudal fins in the treated groups. Mixed mononuclear inflammatory infiltrates were present in multiple organs of fish from control and treatment groups, but did not appear to be treatment related. Lesions associated with exposure to TCDD were observed histologically in liver and spleen after 100 and 250+ days of exposure. The livers of TCDD-treated fish contained less hepatocellular glycogen, more mitotic figures, greater anisokaryosis, anisocytosis, nuclear chromatin clumping and margination. Prominent nucleoli were directly proportional to TCDD dose. Hepatocellular changes in fish exposed to TCDD also included single cell necrosis and clear cytoplasmic vacuoles consistent with lipid. Some fish from all TCD exposed groups had lower lymphoid density compared to controls at all time intervals. Fish exposed to the highest TCDD dose had decreased peripheral leukocyte counts after 50 and 100 days. The lowest observable adverse effect level (LOAEL) for these effects was 5.69 ngTCDD/kg in diet and 0.90 ng TCDD/kg liver.

Intrahippocampal injection of paraquat produces apoptotic cell death which is prevented by the lazaroid U74389G, in rats

Injection of paraquat, a redox-cycling compound, into the rat hippocampus produces limbic seizures and hippocampal damage. Here we report that a proportion of the neuronal cell death caused by the herbicide occurs via an apoptotic mechanism which appears to be mediated by oxygen free radicals. Adult male Wistar rats (n = 12) received a single dose of paraquat (25 nmol 0.5 μl ; 0.5 μl min rate) and were sacrificed 24 h later. Paraquat caused DNA fragmentation, nuclear chromatin marginalization and compaction in all hippocampal subsectors, 24 h after its injection, as revealed by both the TUNEL procedure and hematoxylin eosin staining of coronal brain tissue sections. Pre-treatment with the free radical scavenger lazaroid U74389G (30 mg kg given ip 30 min beforehand) significantly reduced paraquat-induced apoptosis, but did not protect against non apoptotic neuronal cell loss caused by the herbicide.

Heat Shock Proteins in the Regulation of the Apoptotic Response

Cell growth, differentiation and death are highly regulated events during the normal development of cells. Following the pioneering work of Kerr [1], it has become apparent that dying eukaryotic cells can undergo two fundamentally different types of death, necrosis or accidental cell death, and apoptosis or programmed cell death. Apoptosis, unlike necrosis, occurs through the activation of a cell-intrinsic death cascade which can be modulated by many exogenous signals. The dying cell undergoes a relatively ordered form of cell death characterised morphologically by cytoplasmic shrinkage, cellular crenation, nuclear margination and condensation, internucleosomal DNA fragmentation and ~nally formation of apoptotic bodies [2–5]. A critical part of apoptosis is the ef~cient recognition and removal of these cells by tissue macrophages without an in_ammatory response [6]. This involves the rearrangement and biochemical alteration of the plasma membrane in the dying cell aiding recognition of the apoptotic cell by macrophages [7]. This mechanism prevents the release of the cytotoxic contents of apoptotic proin_ammatory cells and limits the possibility of neighbouring host cell injury [8]. The apoptotic process is fundamentally distinct from cell death by necrosis or lysis where cells release their contents into the surrounding tissues and perpetuate the local in_ammatory response. Apoptosis plays an essential part in normal tissue homeostasis with important roles in embryological remodelling, in_ammation, and immune tolerance [2,9,10]. Dysregulated apoptosis is associated with a variety of human diseases, including cancer, autoimmunity, viral infections, allergic disorders and neurodegenerative diseases [5,11,12]. Heat shock proteins (HSPs) are stress response proteins found in all species which are thought to play a protective role in cells under stress [13,14]. Recent evidence supports a role for HSPs in modulating a cell’s response to an apoptotic stimulus. In general, HSPs have been shown to protect cells from an apoptotic inducing signal [15]. Cells exposed to ever increasing increments of a noxious stressor respond generally in a graded manner [16]. Low stress levels appear to induce HSPs altering cellular biochemistry to promote survival at levels of environmental stress which otherwise may be lethal to the cell. This graded resistance is kinetically linked to the synthesis of HSPs [17]. When the inducing stress is removed these cells continue to function normally and HSP levels return to normal. As stress levels increase cellular damage exceeds the protective ability of HSPs and programmed cell death or apoptosis is triggered. At extremes of environmental stress the cell is no longer capable of controlling its fate, and necrosis or degenerative cell death becomes the predominant form of cell death. This view of the universal cellular protective effect of HSPs has, however, recently come under challenge with the revelation that HSPs can, depending on the cell type and stimulus evoked, induce increased apoptosis [18,19,20]. A better understanding of the heat shock response, with speci~c regard to the apoptotic effect of HSPs, may provide important insight into the regulation of apoptosis and may suggest paradigms for therapeutic interventions into diseases where dysregulated apoptosis occurs.

N-Methyl- d-aspartate (NMDA) and non-NMDA glutamate receptor antagonists protect from apoptosis induced in the lateral geniculate nucleus of rabbits exposed to the dark

Mono or binocular deprivation, during early postnatal development, produces dramatic effects on the anatomy and physiology of the visual system. Here we report that dark exposure induces apoptotic cell death in the lateral geniculate nucleus (LGN) of adult rabbit and this may be related to activation of N-methyl- d-aspartate (NMDA) and non-NMDA subtypes of glutamate receptors. In situ DNA fragmentation (TUNEL) was observed in the LGN of rabbits exposed to dark for 48 h. Morphological changes were confirmed on hematoxylin-eosin stained brain tissue coronal sections. Systemic treatment with CGP 040116 or MK 801, two NMDA receptor antagonists, and with GYKI 52466, a non-NMDA receptor antagonist, prevented in situ DNA fragmentation and nuclear chromatin marginalization and condensation. In no instance was apoptosis seen in rabbits kept under a normal light–dark cycle. Our findings indicate that glutamate, acting on NMDA and non-NMDA receptors, may be involved in the mechanisms of apoptotic cell death induced in the LGN of adult rabbits exposed to darkness.

T-2 Toxin-induced Apoptosis in Peyer's Patches of Mice.

Lymphocytic apoptosis in Peyer's patches in the small intestine of mice orally inoculated with T-2 toxin (10mg/kg b.w.) were examined for up to 48 hours after inoculation (HAI). The number of apoptotic lymphocytes observed by in situ detection method of fragmented DNA increased significantly from 6HAI and reached a plateau at 24HAI. Ultrastructurally, lymphocytes characterized by shrinkage of the cell body and condensation of nuclear chromatin and/or margination of condensed chromatin along the nuclear membrane were observed from 6HAI. Some cell nuclei were fragmented into small pieces, and these small pieces were sometimes ingested by macrophages.

Kainate-induced apoptotic cell death in hippocampal neurons

We have examined the role apoptosis plays in epileptic brain damage using intra-amygdaloid injection of kainate. With the silver staining technique of Gallyas, argyrophylic (dying) neurons were observed, a few hours after the injection, in the amygdala and in the vulnerable pyramidal neurons of the hippocampal CA3 region. In both areas, cell death has apoptotic features, including: (i) nuclear chromatin condensation and marginalization with light and electron microscopy; (ii) DNA fragmentation with a typical ladder pattern on agarose gel electrophoresis; (iii) positive nuclear labelling with a selective in situ DNA fragmentation staining method. Combined in situ DNA labelling and silver staining showed that the DNA fragmentation occurred in dying neurons. CA1 or granule cells which do not degenerate following intra-amygdaloid injection of kainate were not stained with the in situ DNA labelling or the argyrophylic technique. Administration of diazepam blocked the kainate-induced seizures and prevented DNA fragmentation in CA3 but not in the amygdala. Therefore, apoptosis contributes to the local and distant damage induced by kainate.

Ultrastructural changes of tracheal epithelial cells of chicks experimentally infected withCryptosporidiumsp.

The ultrastructural changes in the tracheal epithelial cells of chicks with experimental cryptosporidiosis were examined. There was deciliation and formation of compound cilia whereas the microvilli showed Y-shaped formation, atrophy or blunting, fusion and branching. The basal bodies and striated rootlets were irregular and sometimes wanting while there was an increase in centrioles beneath the cilia. Nuclear atypia was common whereas the nucleoli were hypertrophic with reticular nucleolonema. Double nucleoli were mostly reticular and rarely ring-shaped or of thin-shelled type. There was an increase in well-developed rough endoplasmic reticulum and Golgi complex, ribosomes, coated vesicles, smooth-membraned vacuoles, mitochondria and mitochondrial-RER associations. The smooth and rough endoplasmic reticula had dilated cisternae. Nuclear bodies, triple nucleoli, nucleolar margination and segregation, ring-shaped and thin-shelled nucleoli, cytoplasmic imagination, autolysosomes, multivesicular bodies, electron-dense fibres and vesicular rough endoplasmic reticulum were rare. Cryptosporidium sp. attached even to the goblet cells, which confirmed that the microvilli played an important role in the attachment process of the parasite. The surface changes of the cilia and microvilli may denote either an adaptation or compensatory response to cryptosporidiosis. The major changes, however, indicated that the tracheal epithelial cells have the same capacity to increase their protein synthesis as the chicken bursae, in response to the parasitism.

Transformation of anterior midgut and hepatopancreas cells by monodon baculovirus (MBV) in Penaeus monodon postlarvae

The transformation of anterior midgut and hepatopancreas cells of the shrimp Penaeus monodon by monodon baculovirus (MBV) was investigated by means of light and electron microscopy with particular emphasis on the cytoplasmic organelles. The three types of MBV-infected hepatopancreas cells and the anterior midgut cells exhibit similarities in nuclear alterations such as nuclear hypertrophy, chromatin margination, virion assembly and formation of occlusion bodies. Similarities are also found in some changes of the cell organelles, e.g. reduction of rough endoplasmic reticulum, enhancement of ribosomes or occurrence of microfilament patches. In contrast, further alterations are largely cell specific and depend on the organelle ultrastructure prior to the virus infection. Specific changes are, for instance, proliferation of smooth endoplasmic reticulum around lipid droplets, and elongation and proliferation of dictyosomc cisternae in R-cells, or formation of huge dictyosomes with heavily dilated cisternae and membrane labyrinths at the nuclear envelope in F-cells. The cytoplasmic compartments of the B-cells and the anterior midgut cells change only moderately compared to R- and F-cells. The observed cytological alterations are discussed with respect to the function of the cell type.

A pathologic study of photoreceptor cell death in retinal photic injury

In the studies of retinal photic injury in the rat model, about 14-47% of the photoreceptor cell loss occurs in the first 24 hours. To understand the mechanism of this massive cell death and subsequent dissolution, we studied the early morphologic changes and examined the effect of cycloheximide, a protein synthesis inhibitor, on photic injury in rats. Groups of 2 dark-adapted albino Lewis rats were sacrificed immediately after 8, 16 or 24 hr of continuous exposure to green fluorescent light (intensity, 160-180 foot-candles; wavelength, 490-560 nm). An additional 2 rats were sacrificed 8 hr after a 24 hr light exposure, and 2 animals served as unexposed controls. The morphologic findings of the degenerating photoreceptor cells were assessed by light and electron microscopy. The integrity of the nuclear chromatin was investigated using a monoclonal anti-DNA antibody. Most of the photoreceptor cell loss was observed between 16 and 24 hr of exposure. No inflammatory or macrophagic cells were seen. Different stages of nuclear condensation and chromatin margination could be defined. The chromatin showed a progressive decrease in DNA labelling density. Scattered photoreceptor cells showed early cytoplasmic densification. To study the effect of cycloheximide, 4 rats were treated with 5 mg/kg subcutaneously at the start of a 24 hr exposure period and were sacrificed 6 hr after the exposure. Four untreated animals served as exposed controls for morphometric comparison of the outer nuclear layer (ONL). The control rats showed a 24% decrease in the thickness of the ONL when compared to cycloheximide-treated rats (p less than 0.001). The observations of mitochondrial changes and early DNA digestion were consistent with necrosis as the mechanism of cell death. However, in scattered photoreceptor cells, cytoplasmic densification, margination of nuclear chromatin, the lack of associated inflammation and the protective effect of cycloheximide were suggestive of apoptosis as another mechanism of cell death.

Nuclear changes in ovine luteal cells in response to PGF 2α

Percentages of normal and apoptotic parenchymal cells, fibroblasts and endothelial cells in ovine corpora lutea at 12, 24 and 36 hr following administration of a luteolytic dose of PGF 2α were determined and compared to percentages for identical cell types in corpora lutea removed from control ewes on days 10 (n = 5) and 12 (n = 6) postestrus. In corpora lutea obtained from control ewes ≥ 95% of nuclei examined were scored normal for each of the respective cell types with no difference (P > .05) observed between luteal tissue obtained on days 10 and 12 postestrus. Following treatment with PGF 2α there were significant (P < .05) reductions in the percentages of nuclei scored normal. Compared to controls the percentage of endothelial cell nuclei scored normal was reduced at 12 hr following PGF 2α-treatment; however significant reductions in percentages of parenchymal and fibroblast nuclei scored normal were not evident until 24 and 36 hr, respectively. Consistent with the concept of apoptosis, nuclear condensation and/or margination indicative of apoptosis did not occur synchronously within a given cell type: i.e, irrespective of the time point examined some cells appeared normal, whereas others had undergone nuclear condensation and/or margination. A sequence of events to explain structural and functional changes that occur during luteolysis following the interaction of PGF 2α with specific receptors in large steroidogenic luteal cells is discussed.

Apoptosis in Wool Follicles During Mouse Epidermal Growth Factor (mEGF)-Induced Catagen Regression

Depilatory infusions of mouse epidermal growth factor (mEGF) induced regressive involution (catagen) in the wool follicles of Merino sheep. The follicles were examined by transmission electron microscopy prior to infusion and at intervals during catagen regression in order to determine the mechanism(s) involved in follicle involution. Cell deletion by apoptosis occurred in all cell types in the proximal region of catagen follicles between 12 h and 6 days after the beginning of infusion. Apoptosis also occurred in the basal layer of involuting sebaceous glands at 2 and 3 days, following earlier mEGF-induced proliferation. This process involved nuclear chromatin condensation and margination in single, scattered cells which subsequently fragmented and were ultimately phagocytosed and degraded by adjacent unaffected cells. It was concluded that during mEGF-induced catagen, wool follicle involution was accomplished largely through cell death and deletion by apoptosis.

Intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that ICP 4 is associated with progeny virus DNA.

The localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diamidino-2-phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication.

Ultrastructural Postmortem Changes in Chicken Kidneys at 27 C

Ultrastructural postmortem changes of glomeruli, proximal tubules (PT), distal tubules (DT), and cortical collecting ducts (CD) were studied in adult chickens held at 27 C for 0, 1, 5, 10, 30, 60, or 360 minutes. There were marked ultrastructural alterations in tissues considered normal by light microscopy. The earliest changes, evident in immediately fixed samples, consisted of apical cell swelling of PT and DT epithelium and formation of smaller projections from glomerular capillary endothelium. Swelling increased with time, and gaps in cell membranes of tubular cytoplasmic blebs were evident by 10 minutes postmortem. The urinary space also contained cellular debris in immediately fixed samples and was filled by swollen epithelial cells by 10 minutes postmortem. Organelle changes were pronounced by 5 minutes in PT epithelium and by 10 minutes in DT epithelium. These initial changes consisted of dilation of mitochondrial and endoplasmic reticulum. Flocculent mitochondrial matrix densities were present by 5 minutes in PT epithelium and enlarged and appeared in other tubular segments as the time postmortem increased. Nuclear chromatin margination occurred first in 10-minute samples of PT and DT, and it was present by 30 minutes in glomerular epithelial cells. Chromatin clumping was present in PT and DT epithelium at 30 minutes and was followed by pyknosis and karyorrhexis at 360 minutes. These latter nuclear changes were not found in glomerular cells during the study. Nuclei of CD showed little change during the periods studied. The glomerular basement membrane appeared as a single hyaline membrane by 30 minutes postmortem, but this membrane and podocyte foot processes showed little further change by 360 minutes postmortem.