DNA response to bleomycin in mammalian cells with variable degrees of chromatin condensation

BLM induces DNA degradation in living cells. We used CHO cells with maximal chromatin compactness (cells synchronized in metaphase), cells with chromatin decondensed by Na butyrate treatments, and control cells with normal chromatin condensation in order to analyze the correlation between chromatin compactness, DNA sensitivity to BLM, efficiency of repair of BLM-induced DNA lesions, and cell viability. We found that the DNA sensitivity to BLM and the efficiency of DNA repair is inversely correlated with the degree of chromatin coiling. Cells with decondensed chromatin are those showing higher DNA sensitivity to BLM but also those having the best efficiency to mend the damage. Accordingly, these cells show an amount of residual DNA lesions and a curve of growth similar to that of control cells. The situation is just the opposite for metaphase cells. The DNA of these cells is more resistant to BLM, but the damage is poorly repaired. The final result is that BLM induces a higher concentration of residual DNA lesions and a lower viability in metaphase than in control cells. Our results suggest that chromatin structure influences the quantity and repairability of the BLM-induced lesions, producing a higher incidence of double strand break in the DNA of cells with marked chromatin condensation. © 1993 Wiley-Liss, Inc.     

Adenosine receptor ligands protect against a combination of apoptotic and necrotic cell death in cerebellar granule neurons

Agonists at A₁ receptors and antagonists at A_2A receptors are known to be neuroprotective against excitotoxicity. We set out to clarify the mechanisms involved by studying interactions between adenosine receptor ligands and endogenous glutamate in cultures of rat cerebellar granule neurons (CGNs). Glutamate and the selective agonist N-methyl-d-aspartate (NMDA), applied to CGNs at 9 div (days in vitro), both induced cell death in a concentration-dependent manner, which was attenuated by treatment with the NMDA receptor antagonists dizocilpine, d-2-amino-5-phosphono-pentanoic acid (d-AP5) or kynurenic acid (KYA), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glutamate toxicity was reduced in the presence of all of the following: cyclosporin A (CsA), a blocker of the membrane permeability transition pore, the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-fmk), the poly (ADP-ribose) polymerase (PARP-1) inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), and nicotinamide. This is indicative of involvement of both apoptotic and necrotic processes. The A₁ receptor agonist, N ⁶-cyclopentyladenosine (CPA), and the A_2A receptor antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) afforded significant protection, while the A₁ receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and the A_2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS21680) had no effect. These results confirm that glutamate-induced neurotoxicity in CGNs is mainly via the NMDA receptor, but show that a form of cell death which exhibits aspects of both apoptosis and necrosis is involved. The protective activity of A₁ receptor activation or A_2A receptor blockade occurs against this mixed profile of cell death, and appears not to involve the selective inhibition of classical apoptotic or necrotic cascades.     

Glutamate and aspartate immunoreactivity in cortico-cortical neurons of the sensorimotor cortex of rats

Summary Retrograde transport of tracers and immunocytochemistry have been used to determine if association and callosal neurons in the primary motor and somatosensory cortex of rats contain high levels of glutamate or aspartate and may, thus, use these amino acids as neurotransmitter. After tracer injections in these areas, about 65% of the retrogradely labeled neurons in layer V in the ipsilateral or contralateral hemisphere are immunopositive for glutamate. Lower percentages of double-labeled neurons are found in layers III, VI, and II. Similar results are obtained when sections are processed for aspartate immunoreactivity. About 90% of retrogradely labeled neurons are immunopositive in sections incubated with a mixture of both glutamateand aspartate antisera. These results suggest that a large fraction of cortico-cortical neurons are immunoreactive for either one amino acid but not for both. It is proposed that neurons with high levels of one amino acid use this as neurotransmitter; high levels of glutamate and aspartate are likely to be present in a fraction of neurons which may release both amino acids or a substance closely related to these.     

Effects of intracellular calcium on GABAA receptors in mouse cortical neurons

 Using the patch-clamp technique, we studied the effect of intracellular Ca²⁺ on Cl^– current gated by type A γ-aminobutyric acid receptors (GABA_A) in mouse cortical neurons. When the rapid Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) was in the pipette solution, the GABA-activated Cl^– current amplitude decreased over time to 49 ± 7% of control. In contrast, equimolar replacement of BAPTA with ethylenebis(oxonitrilo)tetraacetate (EGTA) caused a 60 ± 10% increase in GABA current. An increased intracellular Ca²⁺ concentration caused a transient augmentation of the GABA current. This effect of Ca²⁺ was concentration dependent (10 nM to 34 μM). Ca²⁺ increased the amplitude of the current by enhancing the maximal response to GABA rather than by changing the affinity of the receptor to GABA (EC₅₀ = 5 ± 0.4 μM vs. 7 ± 0.3 μM). Both calmodulin (CaM) and a CaM kinase II inhibitor (200 μM) blocked the potentiating effect of Ca²⁺ suggesting that it was mediated by activation of CaM kinase II. We found that regulation of GABA_A receptors by intracellular Ca²⁺ in cortical neurons has important physiological implications since the potentiating effect of increasing the intracellular Ca²⁺ on responses to GABA was mimicked by activating excitatory receptors with 100 μM N-methyl-D-aspartate (NMDA). These findings suggest that modulation of GABA_A receptor activity by glutamate may be brought about via changes in intracellular Ca²⁺. Received: 20 May 1997 / Received after revision: 12 August 1997 / Accepted: 1 September 1997     

CREB-shRNA对氧糖剥夺/复氧皮层神经元线粒体形态和凋亡的影响

目的:探讨沉默CREB基因对氧糖剥夺/复氧神经元线粒体形态及细胞凋亡的影响。方法:3条靶向CREB基因的shRNA片段插入慢病毒载体pLenti Lox3.7(PLL),与包装质粒psPAX2和pMD2.G共转染293T细胞包装病毒颗粒后感染原代皮层神经元,Western blot检测CREB蛋白的沉默情况;分别用Mito Tracker red、TUNEL和Western blot实验检测氧糖剥夺/复氧诱导CREB基因沉默后神经元的线粒体形态、细胞凋亡和Bcl-2、Bax的表达情况。结果:pLL-CREB-shRNA1为最有效的shRNA,其抑制皮层神经元CREB基因表达率高达80%。CREB基因沉默促进氧糖剥夺/复氧诱导的皮层神经元线粒体向点状、片段化形态改变,同时降低Bcl-2表达、促进Bax的表达并加重细胞凋亡(P0.05)。结论:CREB-shRNA重组慢病毒载体可特异性抑制神经元CREB基因的表达,CREB基因沉默加重氧糖剥夺/复氧诱导的皮层神经元线粒体形态改变,促进细胞凋亡。     

骨髓造血细胞DNA氧化损伤与骨细胞凋亡在早期激素性股骨头坏死中的表现

背景：激素导致股骨头缺血坏死是一个复杂的生物学过程,其发病机制仍不清楚,探明其病因学机制,仍然是该领域研究的重点。目的：观察骨髓造血细胞DNA氧化损伤与骨细胞凋亡在早期激素性股骨头缺血坏死中的病理改变。方法：4月龄日本大耳白兔40只,随机数字表法分为4组,每组10只。其中3组兔分别给予激素联合内毒素、单纯激素、单纯内毒素处理,另1组做空白对照。分别于最后一次给药后第2,4周处死实验动物,每次每组5只。光镜下计数各组兔股骨头空缺骨陷窝、电镜观察骨细胞形态变化、TUNEL法检测骨细胞凋亡、免疫组织化学法检测骨髓造血细胞DNA氧化损伤。结果与结论：与其他3组比较,激素联合内毒素组第2,4周空缺骨陷窝率、骨髓造血细胞DNA氧化损伤率及第4周骨细胞凋亡率均显著增高(P < 0.01);其他3组之间空缺骨陷窝率、骨髓造血细胞DNA氧化损伤率及骨细胞凋亡率各指标比较差异无显著性意义。激素联合内毒素组空缺骨陷窝率与细胞凋亡率呈正相关(r=0.793)。结果提示在激素性股骨头缺血坏死早期,骨髓造血细胞DNA氧化损伤与骨细胞凋亡参与了激素性股骨头缺血坏死的病理变化过程,前者先于后者发生,骨细胞的死亡方式为凋亡。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

3,4-dihydroxybenzoic acid from Smilacis chinae rhizome protects amyloid beta protein (25-35)-induced neurotoxicity in cultured rat cortical neurons

The neuroprotective effect of 3,4-dihydroxybenzoic acid (3,4-DHBA) isolated from Smilacis chinae rhizome against Abeta (25-35)-induced neurotoxicity on cultured rat cortical neurons was found in this study. The protective effect of 3,4-DHBA against Abeta (25-35)-induced neuronal cell death was investigated by measuring cell viability via a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. 3,4-DHBA (1 and 10 microM) concentration-dependently inhibited 10 microM Abeta (25-35)-induced neuronal apoptotic death. 3,4-DHBA (1 and 10 microM) inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)), which was measured by a fluorescent dye, Fluo-4 AM. 3,4-DHBA also inhibited glutamate release into medium, reactive oxygen species (ROS) generation, and caspase-3 activation, which were induced by 10 microM Abeta (25-35). These results suggest that 3,4-DHBA prevents Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.     

Flunarizine blocks voltage-gated Na(+) and Ca(2+) currents in cultured rat cortical neurons: A possible locus of action in the prevention of migraine

Although flunarizine (FLN) has been widely used for migraine prophylaxis with clear success, the mechanisms of its actions in migraine prophylaxis are not completely understood. It has been hypothesized that migraine is a channelopathy, and abnormal activities of voltage-gated Na(+) and Ca(2+) channels might represent a potential mechanism of cortical hyperexcitability predisposing to migraine. The aim of the present study was to investigate the effects of FLN on Na(+) and Ca(2+) channels of cultured rat cortical neurons. Sodium currents (I(Na)) and calcium currents (I(Ca)) in cultured rat cortical neurons were monitored using whole-cell patch-clamp recordings. Both I(Na) and I(Ca) were blocked by FLN in a concentration-dependent manner with IC(50) values of 0.94μM and 1.77μM, respectively. The blockade of I(Na) was more powerful at more depolarizing holding potentials. The steady-state inactivation curve of I(Na) was shifted towards more hyperpolarizing potentials by FLN. FLN significantly delayed the recovery from fast inactivation of I(Na). Furthermore, the action of FLN in blocking I(Na) was enhanced at higher rates of channel activation. Blockades of these currents might help explain the mechanism underlying the preventive effect of FLN on migraine attacks.     

Age-related changes in the structure and function of chromatin: a review

Due to rapid advancement in biochemical and biophysical techniques during the last decade, extensive studies have been undertaken to understand the structure and function of chromatin. Several interesting results have been reported regarding the changes in basic organization and function of chromatin during the life time of a eukaryotic cell. The data accumulated so far have been obtained with different organs and organisms and widely differing methods, and the conclusions drawn from them are sometimes contradictory. In this paper, therefore, the available data on the age-associated alterations in the composition, structure and function of chromatin have been discussed, and an attempt has been made to correlate the structural changes in chromatin with alteration in gene expression during aging.     

Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response

Protein misfolding neurodegenerative diseases arisethrough neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer's disease, Huntington's disease, Parkinson's disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding--induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion--like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion--induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease.     

PGC-1α过表达逆转OGD/R诱导的神经元线粒体功能降低和凋亡

目的:探讨过氧化物酶体增殖物激活受体γ辅助活化因子1α(PGC-1α)基因过表达对氧糖剥夺/复氧(OGD/R)诱导的神经元线粒体功能及细胞凋亡的影响。方法:采用RT-PCR的方法从C57BL/6乳鼠大脑皮层获取PGC-1α的全基因序列,并克隆到真核表达载体p EGFP-N1上,经PCR初步鉴定后转染原代皮层神经元,Western blot鉴定PGC-1α的表达情况,成功构建PGC-1α真核表达载体p EGFP-N1-PGC-1α。分别将转染p EGFP-N1和p EGFP-N1-PGC-1α载体的皮层神经元进行OGD/R处理,分别采用Mito Tracker Red染色、流式细胞术、ATP代谢检测试剂盒和TUNEL细胞凋亡检测试剂盒检测线粒体质量分数、活性氧簇(ROS)和ATP生成、细胞凋亡以及caspase-3激活的变化。结果:PGC-1α过表达可抑制OGD/R诱导的神经元线粒体生成能力的降低和ROS的生成(P0.05),增强ATP的合成能力(P0.01),抑制神经元的凋亡(P0.01)并降低caspase-3的激活(P0.05)。结论:PGC-1α过表达可通过促进线粒体生成、抑制ROS的产生和维护线粒体功能而抑制OGD/R诱导的神经元凋亡。PGC-1α可以作为开发脑缺血再灌注损伤药物的靶标之一。     

Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis

In this study the relative levels of ADP and ATP have been measured in cells undergoing apoptosis. Using HL60, CEM7, Jurkat and U937 cell lines and cytotoxic agents known to induce apoptosis, there was a significant correlation (P<0.01 for all models) between the ADP:ATP ratio and the degree of apoptosis measured by TUNEL and estimation of the sub G₀ fraction by propidium iodide staining and flow cytometry. The ratio measured in viable proliferating cells was found to be less than 0.11 compared with ratios between 0.11 and 1.0 seen in cells undergoing apoptosis. The higher the percentage of hypodiploidy the greater the ratio. Necrosis induced by heat shock resulted in ADP:ATP ratios in excess of 15.0. When primary cultures of AML blast cells were used, there was again a significant correlation between the ADP:ATP ratio and the degree of hypodiploidy. Recent evidence suggests that apoptosis is accompanied by opening of the mitochondrial permeability pores, leading to disruption of the mitochondrial transmembrane potential (ΔΨ_m). This results in caspase activation due to the release of cytochrome c and apoptogenic factors into the cytosol. In five experiments using CEM7 and dexamethasone the mitochondrial transmembrane potential was assessed using the fluorescent cyanine dye JC-1 and flow cytometry. Functioning mitochondria concentrate the JC-1 to produce red fluorescence. Loss of mitochondrial transmembrane potential results in green fluorescence only. The percentage of cells exhibiting red fluorescence correlated positively with the ATP values and negatively with the ADP:ATP ratio.     

Induction of DNA damage by urethane in mouse testes: DNA binding and unscheduled DNA synthesis

The extent and persistence of DMA damage and repair were investigated in mouse spermatogenic cells exposed in vivo to urethane (ethyl carbamate, EC). Adult male mice exposed to [³H]EC at 10–1,000 mg/kg were sacrificed 12 hr later. EC/metabolite binding to liver and testicular DNA and to sperm heads from the vasa deferentia was measured. Other male mice were exposed to EC at 50–750 mg/kg, and unscheduled DNA synthesis (UDS) induction was investigated in early spermatid stages. Similar experiments were conducted with vinyl carbamate (VC; putative EC metabolite) at 10–75 mg/kg. [³H]EC bound to liver and testicular DNA and to whole sperm heads. Testicular DNA binding increased linearly with dose, although binding was at least 2 orders of magnitude lower than with liver DNA. Sperm head binding also increased linearly with dose. Dose response studies with the UDS assay showed that EC and VC induced a small but significant increase of the UDS response in early spermatid stages. However, the induced UDS responses were quite variable and did not consistently increase with the administered dose. To determine the time kinetics of UDS induction, [³H]dThd was injected at various times after treatment with 500 mg/kg of EC or 60 mg/kg of VC. A slight but significant UDS increase was observed 4 hr after treatment with EC but not with VC. Overall, these results suggest that EC metabolites bind to testis DNA and cause low-level DNA damage in mouse sper-matogenic cells. This type of DNA damage apparently does not have significant genetic consequences. © 1994 Wiley-Liss, Inc.     

The relationship between callosal axons and cortical neurons in the planum temporale: alterations in schizophrenia

The relationship between “connectivity” measures such as DTI and the cellular alterations in the cortex that give rise to those connections remains unclear. Cytoarchitectural changes in the planum temporale (PT) suggest impaired layer III feedforward projection neurons in schizophrenia. Altered hemispheric asymmetry of the PT has been reported in patients, along with altered white matter density in the corpus callosum, and there is anomalous activation of the PT during auditory hallucinations. We measured layer III cell density and pyramidal neuron size in PT of both hemispheres of post-mortem brains from patients with schizophrenia (n = 16) and control subjects (n = 16). We found reduced cell density and the loss of a correlation between magnopyramidal neuron density and axon number in the isthmus of the corpus callosum in schizophrenia. The normal asymmetry indicated that magnopyramidal neurons tend towards being larger and denser in the left PT but this asymmetry is significantly reduced in schizophrenia. The findings offer cytoarchitectural insight into the relationship between PT cortex and callosal white matter abnormalities in schizophrenia.     

Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation

Background: Programmed cell death is an essential event during mammalian morphogenesis which eliminates unnecessary cells to accomplish histogenesis and organogenesis. Cell death in interdigital spaces of the developing limb is a classical example of morphogenetic cell death. We investigated whether classical programmed cell death in the interdigital tissue of the developing limb in mice is apoptosis with fragmentation of nuclear DNA and also examined sequentially the occurrence of programmed cell death and cell proliferation in the developing limb of mouse fetuses to analyze their interrelation. Methods: We examined the occurrence of apoptotic cell death in the developing limbs of mouse fetuses by using Nile blue sulphate staining, agarose gel electrophoresis for detecting DNA laddering, and a cytochemical labeling of DNA fragmentation. We also labeled proliferating cells using BrdU/anti-BrdU immunohistochemistry and examined the interrelation between apoptotic programmed cell death and cell proliferation. Results: DNA ladders, a biochemical evidence of apoptosis, were detected in DNA extracts from the interdigital tissue of day 13 mouse fetuses by agarose gel electrophoresis. Programmed cell death and DNA fragmentation were detected by Nile blue staining and cytochemical labeling of DNA fragmentation, respectively, in the interdigital mesoderm and in the regions of presumptive joints of the digit. BrdU/anti-BrdU immunohistochemistry for identifying proliferating S-phase cells revealed that interdigital mesenchymal cells cease DNA synthesis before programmed cell death and DNA fragmentation begin. Conclusions: We confirmed that both cytological apoptotic alterations and fragmentation of nuclear DNA occur in the interdigital tissue and presumptive joint areas of fetal mouse limbs, and they appear to play a significant role in the separation of digits as well as the formation of joint cavities. © 1995 Wiley-Liss, Inc.     

Branching cortical neurons in cat which project to the colliculi and to the pons: a retrograde fluorescent double-labeling study

Summary The fluorescent double-labeling technique has been used to determine whether the corticopontine and the corticotectal fibers in the cat are derived from two different sets of neurons or whether they are derived from branching neurons which distribute collaterals to the pontine grey and the colliculi. After unilateral DY.2HCl injections in the pontine grey and FB injections in the ipsilateral colliculi, large numbers of FB-DY.2HCl double-labeled neurons were present in the cortex of the ipsilateral hemisphere. However, the labeled neurons in its rostral part may have represented pyramidal tract neurons which were labeled retrogradely because their fibers descended through the DY.2HCl injection area. Therefore, also DY.2HCl injections were made in the pyramid (i.e. caudal to the pons) and the cortical pyramidal tract area, containing the retrograde DY.2HCl-labeled neurons, was delineated. In the rest of the experiments only the DY.2HCl-labeled neurons in the caudal two thirds of the hemisphere (outside the pyramidal tract area) were taken into account because only these neurons could, with confidence, be regarded as corticopontine neurons. In some anterograde HRP transport experiments the trajectories of the corticotectal and the corticopontine fibers were visualized. On the basis of the findings the DY.2HCl injections in the pontine grey were placed such that they could not involve any of the corticotectal fibers passing from the cerebral peduncle to the colliculi. Thus artifactual doublelabeling of cortical neurons was avoided. However, also under these circumstances many double-labeled neurons were present in the caudal two thirds of the hemisphere. This led to the conclusion that in the cat a large proportion of the corticopontine neurons in the caudal two thirds of the hemisphere represent branching neurons which also distribute collaterals to the colliculi. The parietal (anterior part of the lateral gyrus, middle and posterior suprasylvian gyri) and the cingulate areas together contained three quarters of all labeled corticopontine neurons outside the pyramidal tract area. In the parietal areas roughly 25% of them were double-labeled and in the cingulate area 14%. However, in the visual areas 18 and 19 a much larger percentage (30–60%) was doublelabeled. In a recent study from our laboratory it was found that in the cat the pyramidal tract fibers distribute an abundance of collaterals to the pontine grey. Therefore, a large proportion of all corticopontine connections in this species appear to be established by branching neurons which also distribute fibers to other cell groups in the brain stem and the spinal cord.Abbreviations A.E. anterior ectosylvian sulcus - a.e.s. anterior ectosylvian sulcus - BC brachium conjunctivum - BCI brachium colliculus inferior - BP brachium pontis - cor. sulc. coronal sulcus - CP cerebral peduncle - CR. cruciate sulcus - CUN cuneiform nucleus - DBC decussation brachium conjunctivum - DLP dorsolateral pontine nucleus - IC inferior colliculus - inf. coll. inferior colliculus - INS. insula cortex - IO inferior olive - IP interpeduncular nucleus - LAT. lateral sulcus - l.s. lateral sulcus - MG medial geniculate body - LL lateral lemniscus - ML medial lemniscus - MLF medial longitudinal fascicle - NdG dorsal nucleus of Gudden - NLL nucleus lateral lemniscus - NRTP reticular tegmental pontine nucleus - ORB. orbital sulcus - P pyramid - PAG periaqueductal grey - P.E. posterior ectosylvian sulcus - RF reticular formation - PG pontine grey - RB restiform body - RN red nucleus - S. sylvian sulcus - SC superior colliculus - SN substantia nigra - SO superior olive - SPV spinal trigeminal complex - S.S. suprasylvian sulcus - s.syl.s. suprasylvian sulcus - S.SPL. suprasplenial sulcus - SPL. splenial sulcus - spl.s. splenial sulcus - sup. coll. superior colliculus - syl.s. sylvian sulcus - TB trapezoid body - VC vestibular complex - Vm trigeminal motor nucleus - Vs trigeminal principle nucleus - III oculomotor nucleus - IV trochlear nucleus - VI abducens nucleus - VII facial nerve - VIII vestibulo-trochlear nerve Supported in part by grant 13-46-91 of FUNGO/ZWO (Dutch Organization for Fundamental Research in Medicine)     

Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation

We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression.