Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen. We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments. Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells [PBMCs]) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected. In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity. Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87%. Augmentation of cellular calcium flux with ionomycin had no restorative effect. Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80-87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion. Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in Ig cultures. Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.     

Premature centromere division dominantly inherited in a subfertile family

An increased frequency of mitoses showing premature centromere division (PCD) in every chromosome was found in lymphocyte cultures from four members of a subfertile family. These cells were observed in both the presence and absence of colchicine. Cultured fibroblasts from the proband showed only normal diploid metaphases. PCD cells seemed to have a shorter cell cycle. The anomaly was transmitted in a way compatible with autosomal dominant inheritance in this family.     

Transgenic expression of the viral FLIP MC159 causes lpr/gld-like lymphoproliferation and autoimmunity

Death receptor-induced programmed cell death (PCD) is crucial for the maintenance of immune homeostasis. However, interference of downstream death receptor signaling by genetic ablation or transgenic (Tg) expression of different apoptosis inhibitors often impairs lymphocyte activation. The viral FLICE (caspase-8)-like inhibitor proteins (v-FLIPs) are potent inhibitors of death receptor-induced apoptosis and programmed necrosis. We generated Tg mice expressing the v-FLIP MC159 from Molluscum contagiosum virus under the control of the H2Kb class I MHC promoter to examine the role of death receptor-induced PCD in the control of immune functions and homeostasis. We found that expression of MC159 led to lymphoproliferation and autoimmunity as exemplified by T and B lymphocyte expansion, accumulation of TCRalphabeta+ CD3+ B220+ CD4- CD8- lymphocytes in secondary lymphoid organs, elevated serum Ig levels, and increased anti-dsDNA Ab titers. These phenotypes were caused by defective death receptor-induced apoptosis, but not by defective passive cell death in the absence of mitogenic stimulation. Lymphocyte activation was normal, as demonstrated by normal thymidine incorporation and CSFE dilution of T cells stimulated with anti-CD3 and anti-CD28 Abs. In addition, effector CD8+ T cell responses to acute and memory lymphocytic choriomeningitis virus infections were unaffected in the Tg mice. These phenotypes are reminiscent of the lpr and gld mice, and show that the v-FLIP MC159 is a bona fide PCD inhibitor that does not interfere with other essential lymphocyte functions. Thus, the MC159-Tg mice provide a model to study the effects of PCD in immune responses without hampering other important lymphocyte functions.     

Modeled microgravity causes changes in the cytoskeleton and focal adhesions, and decreases in migration in malignant human MCF-7 cells

Because cells are sensitive to mechanical forces, microgravity might act on stress-dependent cell changes. Regulation of focal adhesions (FAs) and cytoskeletal activity plays a role in cell maintenance, cell movement, and migration. Human MCF-7 cells were exposed to modeled microgravity (MMG) to test the hypothesis that migration responsiveness to microgravity is associated with cytoskeleton and FA anomalies. MMG acts on MCF-7 cells by disorganizing cytoskeleton filaments (microfilaments and microtubules). Microfilaments in MMG did not display their typical radial array. Likewise, microtubules were disrupted in MCF-7 cells within 4 h of initiation of MMG and were partly reestablished by 48 h. FAs generated in microgravity were less mature than those established in controls, shown by reduced FAs number and clustering. In parallel, MMG decreased kinases activity (such as FAK, PYK2, and ILK) of FAs in MCF-7 cells. The expression of both integrinβ1 and integrinβ4 were downregulated by MMG. We conclude that cytoskeletal alterations and FAs changes in MMG are concomitant with cell invasion and migration retardation. We suggest that reduced migration response in MCF-7 cells following MMG is linked to changes of cytoskeleton and FAs.     

Force and surface mechanomyogram frequency responses in cat gastrocnemius

Muscle surface displacement is a mechanical event taking place simultaneously with the tension generation at the tendon. The two phenomena can be studied by the surface mechanomyogram signal (MMG) (produced by a laser distance sensor) and the force signal (from a load cell). The aim of this paper was to provide data on the reliability of the laser detected MMG in muscle mechanics research. To this purpose it was verified if the laser detected MMG was suitable to estimate a frequency response in the cat medial gastrocnemius and its frequency response was compared with the one retrieved by the force signal at the tendon level. The force and MMG from the exposed medial gastrocnemius of four cats were analysed. The frequency response was investigated by sinusoidally changing the number of orderly recruited motor units, in different trials, in the 0.4-6 Hz range. It resulted that it was possible to model the force and MMG frequency response by a critically damped second-order system with two real double poles and a pure time delay. On the average, the poles were at 1.83 Hz (with 22.6 ms delay) and at 2.75 Hz (with 38 ms delay) for force and MMG, respectively. It can be concluded that MMG appears to be a reliable tool to investigate the muscle frequency response during stimulated isometric contraction. Even though not statistically significant. the differences in the second-order system parameters suggest that different components of the muscle mechanical model may specifically affect the force or MMG.     

Chromosome abnormalities in tuberous sclerosis

Summary In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.     

Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells

Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.     

Exposure to modeled microgravity induces metabolic idleness in malignant human MCF-7 and normal murine VSMC cells

We investigated the effect of modeled microgravity (MMG) on normal vascular smooth muscle cells (VSMC) and neoplastic human breast cancer cells (MCF-7). In both cell types, MMG induced partial arrest in G2M and increased p14-3-3, HSP70, HSP60 and p21 expression. Cells synchronized by 24h starvation reentered the normal cycle within 24h if released in complete medium and exposed to normal gravity, but not if exposed to MMG. Similarly, MMG prevented VSMC and MCF-7 cells from overcoming growth arrest and re-synthesizing DNA. This study shows that cells adjust their metabolic rate in response to MMG.     

C-anaphases in lymphocyte cultures versus premature centromere division syndromes

Summary Different cases in which chromosomes obtained from short-term lymphocyte cultures appear with separated centromeres are reviewed. These include: (1) C-anaphases in the colchicine-resistance mutation, (2) C-anaphases as a common variant, (3) premature centromere division (PCD) in elderly women, (4) PCD in Alzheimer's disease, (5) early splitting and puffing of centromeres in Roberts' syndrome.     

植物生长发育中程序性细胞死亡

本文简要介绍了植物细胞凋亡的一些特点以及植物在营养生长和生殖生长过程中发生的细胞凋亡现象。指出细胞凋亡是植物生长发育过程中正常的生理现象。     

The breakdown of the cytokine network subsequent to human immunodeficiency virus infection

The acquired immunodeflciency syndrome (AIDS) is a clinically multifaceted disease induced by infection with the human immunodeficiency virus (HIV). HIV infection results in a complex pattern of immunologic alterations that leads to the development of AIDS in the majority of HIV seropositive (HIV+) individuals. The reduction in CD4 T lymphocyte counts is the hallmark of HIV infection; nevertheless, long before the reduction in CD4 counts reaches critical levels, a series of profound and complex defects that impair the function of CD4 T lymphocytes can be detected. Thus, HIV infection is characterized by quantitative and qualitative defects affecting CD4 T lymphocytes. It was suggested recently that programmed cell death (PCD) is an important mechanism leading to CD4 depletion in HIV infection, and that susceptibility of peripheral lymphocytes to PCD is differentially regulated by diverse cytokines. Thus, type 1 cytokines would protect CD4 lymphocytes against PCD, whereas type 2 cytokines would not protect against, and could augment, PCD. We suggest that the qualitative alterations of the immune response provoke the CD4 depletion characteristic of HIV disease via type 2 cytokinemediated augmentation of PCD, and are therefore ultimately responsible for the progression of HIV infection. Finally, we summarize recent data showing that three correlates of disease progression: emergence of HIV strains with syncitium-inducing ability (SI), type 1-to-type 2 cytokine shift, and CD4 depletion, are significantly associated, suggesting a complex interconnected virologic-immunologic pathogenesis of HIV infection.     

Redox regulation in plant programmed cell death

Programmed cell death (PCD) is a genetically controlled process described both in eukaryotic and prokaryotic organisms. Even if it is clear that PCD occurs in plants, in response to various developmental and environmental stimuli, the signalling pathways involved in the triggering of this cell suicide remain to be characterized. In this review, the main similarities and differences in the players involved in plant and animal PCD are outlined. Particular attention is paid to the role of reactive oxygen species (ROS) as key inducers of PCD in plants. The involvement of different kinds of ROS, different sites of ROS production, as well as their interaction with other molecules, is crucial in activating PCD in response to specific stimuli. Moreover, the importance is stressed on the balance between ROS production and scavenging, in various cell compartments, for the activation of specific steps in the signalling pathways triggering this cell suicide process. The review focuses on the complexity of the interplay between ROS and antioxidant molecules and enzymes in determining the most suitable redox environment required for the occurrence of different forms of PCD.     

Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS- induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.     

Basic characteristics between mechanomyogram and muscle force during twitch and tetanic contractions in rat skeletal muscles

The mechanomyogram (MMG) is a signal measured by various vibration sensors for slight vibrations induced by muscle contraction, and it reflects the muscle force during electrically induced-contraction or until 60%–70% maximum voluntary contraction, so the MMG is considered an alternative and novel measurement tool for muscle strength. We simultaneously measured the MMG and muscle force in the gastrocnemius (GC), vastus intermedius (VI), and soleus (SOL) muscles of rats. The muscle force was measured by attaching a hook to the tendon using a load cell, and the MMG was measured using a charged-coupled device-type displacement sensor at the middle of the target muscle. The MMG-twitch waveform was very similar to that of the muscle force; however, the half relaxation time and relaxation time (10%), which are relaxation parameters, were prolonged compared to those of the muscle force. The MMG amplitude correlated with the muscle force. Since stimulation frequencies that are necessary to evoke tetanic progression have a significant correlation with the twitch parameter, there is a close relationship between twitch and tetanus in the MMG signal. Therefore, we suggest that the MMG, which is electrically induced and detected by a laser displacement sensor, may be an alternative tool for measuring muscle strength.     

Epidermal growth factor triggers an original, caspase-independent pituitary cell death with heterogeneous phenotype

Programmed cell death (PCD) is physiologically involved in the regulation of cell division and differentiation. It encompasses caspase-dependent mitochondrial and nonmitochondrial pathways. Additional caspase-independent pathways have been characterized in mitochondrial PCDs but remain hypothetical in nonmitochondrial PCDs. Epidermal growth factor (EGF) has been shown to inhibit division of pituitary somato-lactotrope cells occurring in parallel with EGF-mediated differentiation of these precursors into lactotrope cells. We show here that in somato-lactotrope pituitary cell line GH4C1, EGF triggers a PCD characterized by an apoptosis-like DNA fragmentation, insensitivity to broad-range caspase inhibitors, and absence of either cytochrome c or apoptosis-inducing factor release from mitochondria. Dying cells display loose chromatin clustering and numerous cytoplasmic vacuoles, a fraction of which are autophagic, thus conferring a heterogeneous phenotype to this PCD. Moreover, overexpression of cell death inhibitor Bcl-2 prevented not only the EGF-induced PCD but also its prodifferentiation effects, thus pointing to a mechanistic relationship existing between these two phenomena. Overall, the characterized differentiation-linked cell death represents an original form of caspase-independent PCD. The mechanisms underlying this PCD involve combinatorial engagement of discrete death effectors leading to a heterogeneous death phenotype that might be evolutionary related to PCD seen during the differentiation of some unicellular organisms.     

Time and frequency domain responses of the mechanomyogram and electromyogram during isometric ramp contractions: a comparison of the short-time Fourier and continuous wavelet transforms.

The purposes of this study were to examine the mechanomyographic (MMG) and electromyographic (EMG) time and frequency domain responses of the vastus lateralis (VL) and rectus femoris (RF) muscles during isometric ramp contractions and compare the time-frequency of the MMG and EMG signals generated by the short-time Fourier transform (STFT) and continuous wavelet transform (CWT). Nineteen healthy subjects (mean+/-SD age=24+/-4 years) performed two isometric maximal voluntary contractions (MVCs) before and after completing 2-3, 6-s isometric ramp contractions from 5% to 100% MVC with the right leg extensors. MMG and surface EMG signals were recorded from the VL and RF muscles. Time domains were represented as root mean squared amplitude values, and time-frequency representations were generated using the STFT and CWT. Polynomial regression analyses indicated cubic increases in MMG amplitude, MMG frequency, and EMG frequency, whereas EMG amplitude increased quadratically. From 5% to 24-28% MVC, MMG amplitude remained stable while MMG frequency increased. From 24-28% to 76-78% MVC, MMG amplitude increased rapidly while MMG frequency plateaued. From 76-78% to 100% MVC, MMG amplitude plateaued (VL) or decreased (RF) while MMG frequency increased. EMG amplitude increased while EMG frequency changed only marginally across the force spectrum with no clear deflection points. Overall, these findings suggested that MMG may offer more unique information regarding the interactions between motor unit recruitment and firing rate that control muscle force production during ramp contractions than traditional surface EMG. In addition, although the STFT frequency patterns were more pronounced than the CWT, both algorithms produced similar time-frequency representations for tracking changes in MMG or EMG frequency.     

Programmed cell death in plants: distinguishing between different modes

Programmed cell death (PCD) in plants is a crucial componentof development and defence mechanisms. In animals, differenttypes of cell death (apoptosis, autophagy, and necrosis) havebeen distinguished morphologically and discussed in these morphologicalterms. PCD is largely used to describe the processes of apoptosisand autophagy (although some use PCD and apoptosis interchangeably)while necrosis is generally described as a chaotic and uncontrolledmode of death. In plants, the term PCD is widely used to describemost instances of death observed. At present, there is a vastarray of plant cell culture models and developmental systemsbeing studied by different research groups and it is clear fromwhat is described in this mass of literature that, as with animals,there does not appear to be just one type of PCD in plants.It is fundamentally important to be able to distinguish betweendifferent types of cell death for several reasons. For example,it is clear that, in cell culture systems, the window of timein which ‘PCD’ is studied by different groups varieshugely and this can have profound effects on the interpretationof data and complicates attempts to compare different researcher'sdata. In addition, different types of PCD will probably havedifferent regulators and modes of death. For this reason, inplant cell cultures an apoptotic-like PCD (AL-PCD) has beenidentified that is fairly rapid and results in a distinct corpsemorphology which is visible 4–6 h after release of cytochromec and other apoptogenic proteins. This type of morphology, distinctfrom autophagy and from necrosis, has also been observed inexamples of plant development. In this review, our model systemand how it is used to distinguish specifically between AL-PCDand necrosis will be discussed. The different types of PCD observedin plants will also be discussed and the importance of distinguishingbetween different forms of cell death will be highlighted. Key words: Apoptosis, apoptosis-like programmed cell death (AL-PCD), Arabidopsis, autophagy, mitochondria, necrosis, programmed cell death (PCD) Received 5 June 2007; Revised 13 September 2007 Accepted 20 September 2007     

镉胁迫引起烟草悬浮细胞程序性死亡

镉胁迫会造成烟草悬浮细胞大规模死亡。通过TUNEL技术和琼脂糖凝胶电泳技术的检测发现,这种细胞死亡伴随有典型的DNA“梯形带”出现,表明这种由Cd胁迫引起的细胞死亡是一种程序性死亡。受胁迫细胞氧化性增强及细胞中丙二醛(MDA)水平升高,说明Cd胁迫时会在细胞中造成大量活性氧(ROS),暗示烟草细胞的程序性死亡可能与ROS有关。     

Mean power frequency and amplitude of the mechanomyographic and electromyographic signals during incremental cycle ergometry.

The purpose of this investigation was to determine the relationships for mechanomyographic (MMG) amplitude, MMG mean power frequency (MPF), electromyographic (EMG) amplitude, and EMG MPF versus power output during incremental cycle ergometry. Seventeen adults volunteered to perform an incremental test to exhaustion on a cycle ergometer. The test began at 50 W and the power output was increased by 30 W every 2 min until the subject could no longer maintain 70 rev min(-1). The MMG and EMG signals were recorded simultaneously from the vastus lateralis during the final 10 s of each power output and analyzed. MMG amplitude, MMG MPF, EMG amplitude, EMG MPF, and power output were normalized as a percentage of the maximal value from the cycle ergometer test. Polynomial regression analyses indicated that MMG amplitude increased (P<0.05) linearly across power output, but there was no change (P>0.05) in MMG MPF. EMG amplitude and MPF were fit best (P<0.05) with quadratic models. These results demonstrated dissociations among the time and frequency domains of MMG and EMG signals, which may provide information about motor control strategies during incremental cycle ergometry. The patterns for amplitude and frequency of the MMG signal may be useful for examining the relationship between motor-unit recruitment and firing rate during dynamic tasks.