Membrane-bound Bodies Research Articles

ICTV Virus Taxonomy Profile: Fimoviridae 2024.

Members of the family Fimoviridae are plant viruses with a multipartite negative-sense enveloped RNA genome (-ssRNA), composed of 4-10 segments comprising 12.3-18.5 kb in total, within quasi-spherical virions. Fimoviruses are transmitted to plants by eriophyid mites and induce characteristic cytopathologies in their host plants, including double membrane-bound bodies in the cytoplasm of virus-infected cells. Most fimoviruses infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the ICTV Report on the family Fimoviridae, which is available at ictv.global/report/fimoviridae.

Dynamic correlations in lipid bilayer membranes over finite time intervals.

Recent single-molecule measurements [Schoch et al., Proc. Natl. Acad. Sci. U. S. A. 118, e2113202118 (2021)] have observed dynamic lipid-lipid correlations in membranes with submicrometer spatial resolution and submillisecond temporal resolution. While short from an instrumentation standpoint, these length and time scales remain long compared to microscopic molecular motions. Theoretical expressions are derived to infer experimentally measurable correlations from the two-body diffusion matrix appropriate for membrane-bound bodies coupled by hydrodynamic interactions. The temporal (and associated spatial) averaging resulting from finite acquisition times has the effect of washing out correlations as compared to naive predictions (i.e., the bare elements of the diffusion matrix), which would be expected to hold for instantaneous measurements. The theoretical predictions are shown to be in excellent agreement with Brownian dynamics simulations of experimental measurements. Numerical results suggest that the experimental measurement of membrane protein diffusion, in complement to lipid diffusion measurements, might help to resolve the experimental ambiguities encountered for certain black lipid membranes.

The morphology and differentiation of stromal cells in the cortex of follicles in the bursa of Fabricius of the chicken.

Mesenchymal reticular cells (MRCs) form a supporting system in the cortex of the bursal follicle. The stellate-shaped MRCs exhibit a low electron density, which is helpful for their identification. A remarkable feature of MRC is the formation of multiple blebs in the nuclear envelope. The large, irregularly shaped blebs-which are perinuclear spaces-may be detached from the nuclear membrane, creating a sac-like granular endoplasmic reticulum (GER). Inside the bleb, membrane-bound bodies originate from cytoplasmic impressions. The cytoplasm contains a few round mitochondria, in which the internal membranes form either ovoid vesicles or the entire internal structure is indistinct. These mitochondria may be associated with the blebs. The classical Golgi complex with cis and trans faces cannot be recognized, but the accumulation of very small vesicles occurs around two or three stacked flat cisterns. The MRC forms a continuous layer along the corticomedullary basal lamina (CMBL), and during cell migration between the cortex and medulla, it may contribute to the temporary closure of the gap in the CMBL. At the outer surface of the cortex, transitory cells between the MRC and fibrocytes of the interfollicular connective tissue are present, and both cells can produce GER by blebbing. This finding suggests that MRCs and fibrocytes may have a common origin. The other stromal cell is the macrophage (Ma), which may fuse together to form multinucleated giant cells. The definition of histological classification of the third type of stromal cell is questionable, but certain morphological features may be referred to as progenitors of MRCs.

To die or not to die: Programmed cell death responses and their interactions with Coxiella burnetii infection.

Coxiella burnetii is a Gram-negative, obligate intracellular, macrophage-tropic bacterium, and the causative agent of the zoonotic disease Q fever. The epidemiology of Q fever is associated with the presence of infected animals; sheep, goats, cattle, and humans primarily become infected by inhalation of contaminated aerosols. In humans, the acute phase of the disease is characterized primarily by influenza-like symptoms, and approximately 3%-5% of the infected individuals develop chronic infection. C. burnetii infection activates many types of immune responses, and the bacteria's genome encodes for numerous effector proteins that interact with host immune signaling mechanisms. Here, we will discuss two forms of programmed cell death, apoptosis, and pyroptosis. Apoptosis is a form of non-inflammatory cell death that leads to phagocytosis of small membrane-bound bodies. Conversely, pyroptosis results in lytic cell death accompanied by the release of proinflammatory cytokines. Both apoptosis and pyroptosis have been implicated in the clearance of intracellular bacterial pathogens, including C. burnetii. Finally, we will discuss the role of autophagy, the degradation of unwanted cellular components, during C. burnetii infection. Together, the review of these forms of programmed cell death will open new research questions aimed at combating this highly infectious pathogen for which treatment options are limited.

Sesquiterpene biosynthesis in a leafy liverwort Radula lindenbergiana Gottsche ex C. Hartm

Liverworts (Marchantiophyta) are among the earliest diverging lineages of extant land plants. Among their unique features, most liverworts contain membrane-bound oil bodies, organelles that accumulate diverse secondary metabolites, especially terpenoids. In contrast to the rich information on liverwort terpenoid chemistry, little is known about their biosynthesis. Recently, terpenoid biosynthesis was studied in a model thalloid species Marchantiapolymorpha, in which sesquiterpenes and monoterpenes are biosynthesized by a new type of terpene synthases termed microbial terpene synthase-like (MTPSL) proteins. Here we study terpenoid biosynthesis in a leafy liverwort Radula lindenbergiana. Vegetative plants of R.lindenbergiana were found to contain a mixture of sesquiterpenes, with (E,E)-α-farnesene/β-curcumene and (Z)-β-bisabolene being the most abundant constituents. From the analysis of the R. lindenbergiana transcriptome, five full-length MTPSL genes were identified. They were designated RlMTPSL1-5, respectively. Recombinant RlMTPSL proteins were produced in Escherichia coli and tested for sesquiterpene synthase activities using farnesyl diphosphate (FPP) as substrate. All except RlMTPSL5 were demonstrated to catalyze the formation of different sesquiterpenes. RlMTPSL1 produced multiple sesquiterpenes with eremophilene and an unidentified sesquiterpene as major products. The major products of RlMTPSL2 and RlMTPSL3 were β-elemene and an unidentified sesquiterpene, respectively. RlMTPSL4 was also a multi-product sesquiterpene synthase with an unidentified sesquiterpene being the major product. Homology-based structural modeling was performed to understand the structural basis underlying different product profiles of the RlMTPSLs proteins. Most of the sesquiterpene products of the four active RlMTPSLs were also detected in R. lindenbergiana plants. Expression levels of the four RlMTPSL genes encoding active enzymes in vegetative plants were compared. In phylogenetic analysis, RlMTPSL genes were found to cluster together, indicating lineage-specific expansion of MTPSL genes in lineages leading to R.lindenbergiana and M. polymorpha. This study strengthens evidence for the contribution of MTPSL genes to terpenoid biosynthesis in liverworts.

Visual system pathology in a canine model of CLN5 neuronal ceroid lipofuscinosis

CLN5 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disease characterized by progressive neurological decline, vision loss and seizures. Visual impairment in children with CLN5 disease is attributed to a progressive decline in retinal function accompanied by retinal degeneration as well as impaired central nervous system function associated with global brain atrophy. We studied visual system pathology in five Golden Retriever littermates homozygous for the CLN5 disease allele previously identified in the breed. The dogs exhibited signs of pronounced visual impairment by 21–22 months of age. Electroretinogram recordings showed a progressive decline in retinal function primarily affecting cone neural pathways. Altered visual evoked potential recordings indicated that disease progression affected visual signal processing in the brain. Aside from several small retinal detachment lesions, no gross retinal abnormalities were observed with in vivo ocular imaging and histologically the retinas did not exhibit apparent abnormalities by 23 months of age. However, there was extensive accumulation of autofluorescent membrane-bound lysosomal storage bodies in almost all retinal layers, as well as in the occipital cortex, by 20 months of age. In the retina, storage was particularly pronounced in retinal ganglion cells, the retinal pigment epithelium and in photoreceptor cells just interior to the outer limiting membrane. The visual system pathology of CLN5-affected Golden Retrievers is similar to that seen early in the human disease. It was not possible to follow the dogs to an advanced stage of disease progression due to the severity of behavioral and motor disease signs by 23 months of age. The findings reported here indicate that canine CLN5 disease will be a useful model of visual system disease in CLN5 neuronal ceroid lipofuscinosis. The baseline data obtained in this investigation will be useful in future therapeutic intervention studies. The findings indicate that there is a fairly broad time frame after disease onset within which treatments could be effective in preserving vision.

New Insights on the Mobility of Viral and Host Non-Coding RNAs Reveal Extracellular Vesicles as Intriguing Candidate Antiviral Targets

Intercellular communication occurring by cell-to-cell contacts and via secreted messengers trafficked through extracellular vehicles is critical for regulating biological functions of multicellular organisms. Recent research has revealed that non-coding RNAs can be found in extracellular vesicles consistent with a functional importance of these molecular vehicles in virus propagation and suggesting that these essential membrane-bound bodies can be highjacked by viruses to promote disease pathogenesis. Newly emerging evidence that coronaviruses generate non-coding RNAs and use extracellular vesicles to facilitate viral pathogenicity may have important implications for the development of effective strategies to combat COVID-19, a disease caused by infection with the novel coronavirus, SARS-CoV-2. This article provides a short overview of our current understanding of the interactions between non-coding RNAs and extracellular vesicles and highlights recent research which supports these interactions as potential therapeutic targets in the development of novel antiviral therapies.

Are unusual ultrastructural features occurring in the pollen endomembrane system of Cyperaceae and other angiosperms?

Cyperaceae representatives present peculiar microsporogenesis and microgametogenesis, which raises the question of how regular the sedge pollen is. In order to answer this question, preanthesis pollen grains of Cyperaceae individuals were analyzed under different tools such as light and transmission electron microscopy, which included cytochemistry and immunogold procedures. The results showed that maturing pollen in Cyperaceae presents some unusual endomembrane behaviors. Endoplasmic reticulum and dictyosomes are concerned in classic secretion pathways in vegetative cells, and possibly the late breakdown of degenerative microspores. However, cortical and concentric endoplasmic reticulum are also present and are possibly related to other functions aside the biosynthetic pathway. Unconventional secretion of large membrane-bound bodies containing cell wall precursors was also observed and confirmed by immunogold. However, since these bodies most likely receive material from dictyosomes, as observed in silver nitrate reaction, the "unconventional" status of this secretion is debatable. Reports of the literature show that these unusual endomembrane formations are not exclusive of the sedge pollen, but little attention have been given to them so far. This could represent an opportunity to re-examine our understanding on the endomembrane system in pollen cells in general.

Cellular preservation of excysting developmental stages of new eukaryotes from the early Ediacaran Weng’an Biota

The Ediacaran Weng’an Biota provides a unique window on marine diversity during the interval in which the fundamental animal body plans were being established. Here we describe a previously unreported component of the assemblage, millimeter-scale encysted spheres that exhibit a characteristic but simple slit-shaped excystment mechanism (Sporosphaera guizhouensis n. gen. n. sp.), reminiscent of acritarchs. The cysts contain a large inner body or numerous small discrete membrane-bounded bodies. It is possible that the inner bodies represent disaggregated cells of a multicellular body, like an embryo, but there is no evidence to support this interpretation and the occurrence of the excystment structure is not readily compatible with an embryo interpretation. Rather, we interpret the encysted organisms as multicellular stages within the lifecycle of otherwise probably unicellular eukaryotes. The developmental mode exhibited by Sporosphaera, incorporating a resting stage, implies an adaptation to adverse environmental conditions. This parallels the appearance of Large Ornamented Ediacaran Microfossils (LOEMs) which have been interpreted as diapause stages in the embryology of early animals. Sporosphaera is distinct from LOEMs by ornamentation instead of size, which may implicate that not all LOEMs are animal embryos, if any.

ICTV Virus Taxonomy Profile: Fimoviridae.

Members of the family Fimoviridae, order Bunyavirales are plant viruses with segmented, linear, single-stranded, negative-sense RNA genomes. They are distantly related to orthotospoviruses and orthobunyaviruses of the families Tospoviridae and Peribunyaviridae, respectively. The family Fimoviridae includes the genus Emaravirus, which comprises several species with European mountain ash ringspot-associated emaravirus as the type species. Fimoviruses are transmitted to plants by eriophyid mite vectors and induce similar characteristic cytopathologies in their host plants, including the presence of double membrane-bound bodies in the cytoplasm of the virus-infected cells. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Fimoviridae, which is available at www.ictv.global/report/fimoviridae.

Identification of the Key Sequence in the FliK C-Terminal Domain for Substrate Specificity Switching in the Flagellar Protein Secretion.

The flagellar hook is a short tubular structure located between the external filament and the membrane-bound basal body. The average hook length is 55 nm and is determined by the soluble protein FliK and the integral membrane protein FlhB. Hook elongation is terminated by FliK-mediated cessation of hook protein secretion, followed by the secretion of filamentous proteins. This process is referred to as the substrate specificity switch. Switching of the secretion modes results from a direct interaction between the FliK C-terminal domain (FliKC) and the secretion gate in FlhB. FliKC consists of two α-helices and four β-strands. Loop 2 connects the first two β-sheets and contains a conserved sequence of 9 residues. Genetic and physiological analyses of various fliK partial deletion mutants pointed to loop 2 as essential for induction of a conformational change in the FlhB gate. We constructed single-amino-acid substitutions in the conserved region of loop 2 of FliK and discovered that the loop sequence LRL is essential for the timely switching of secretion modes. Flagellar protein secretion is controlled by the soluble protein FliK. We discovered that the loop 2 sequence LRL in the FliK C terminus was essential for timely switching of secretion modes. This mechanism is applicable to type three secretions systems that secrete virulence factors in bacterial pathogens.

Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.

Endosomal sorting complex required for transport (ESCRT)-III-mediated membrane invagination and scission are a critical step in multivesicular body (MVB) sorting of ubiquitinated membrane receptors, and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein Alix is critically involved in multiple ESCRT-III-mediated, membrane-remodelling processes in mammalian cells. However, Alix knockdown does not inhibit degradation of the activated epidermal growth factor receptor (EGFR) in mammalian cell lines, leading to a widely held notion that Alix is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In the present study, we demonstrate that, despite its non-essential role in degradation of the activated EGFR, Alix plays a critical role in its MVB sorting and silencing Epidermal growth factor (EGF) stimulation of mammalian cell lines induces Alix's interaction with the ubiquitinated EGFR via the Alix V domain, and increases Alix's association with membrane-bound charged multivesicular body protein 4 (CHMP4) via the Alix Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of Alix's interaction with membrane-bound CHMP4, inhibition of Alix dimerization through the V domain or Alix knockdown dramatically inhibits MVB sorting of the activated EGFR and promotes sustained activation of extracellular-signal regulated kinase (ERK)1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of the activated EGFR. These findings indicate that Alix is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.

Disruption of fas-fas ligand signaling, apoptosis, and innate immunity by bacterial pathogens.

Fas ligand (FasL, CD95L) is a type-II membrane protein withinthe tumor necrosis factor (TNF) superfamily of death receptors [1].FasL shares 25%–30% sequence homology with related familymember proteins such as tumor necrosis factor alpha (TNFa) andTNF-related apoptosis-inducing ligand (TRAIL), with the mostsimilarity present in the C-terminal homology ectodomain thatextends into the extracellular space for receptor binding [2]. FasLengages and trimerizes the death receptor Fas (CD95) on cellsurfaces to initiate the extrinsic apoptosis pathway [3]. The Fas-FasL interaction recruits the Fas-associated death domain adapterprotein (FADD) via death domain binding, which interacts withdimerized procaspase-8 to form the death-inducing signalingcomplex (DISC) [4]. Caspase-8 catalyzes its autoactivation,followed by the proteolytic conversion of downstream effectorcaspases such as caspase-3 and -7 into their mature forms [5].Effector caspases direct cell death by apoptosis, which results innuclear and cytoplasmic condensation followed by cellularfragmentation into membrane-bound apoptotic bodies [6].Caspase-activated DNase (CAD) cleaves genomic DNA betweennucleosomes to generate short fragments prior to cellularcondensation and membrane blebbing [7]. Membrane fragmentsare usually taken up by other cells and degraded in phagosomesvia a process known as efferocytosis. Efferocytosis of apoptotic cellscontributes to the resolution of inflammation by rapidly clearingcytotoxic cellular debris, and defects in this process can lead toinflammatory diseases such as acute lung injury [8].Although FasL is expressed by many cell types, it is primarilyrecognized as associated with activated T lymphocytes and naturalkiller (NK) cells. FasL-dependent apoptosis plays important rolesin tissue remodeling and the deletion of potentially autoreactivethymocytes to maintain immune tolerance during development,and it also contributes to tumor cell clearance by effector NK cells[9–11]. Though apoptosis has traditionally been defined asnoninflammatory during these processes, FasL-induced cell deathhas been shown to be highly proinflammatory in the context ofmicrobial infections [12].

Nonredundant Function of Zeins and Their Correct Stoichiometric Ratio Drive Protein Body Formation in Maize Endosperm

Zeins, the maize (Zea mays) prolamin storage proteins, accumulate at very high levels in developing endosperm in endoplasmic reticulum membrane-bound protein bodies. Products of the multigene α-zein families and the single-gene γ-zein family are arranged in the central hydrophobic core and the cross-linked protein body periphery, respectively, but little is known of the specific roles of family members in protein body formation. Here, we used RNA interference suppression of different zein subclasses to abolish vitreous endosperm formation through a variety of effects on protein body density, size, and morphology. We showed that the 27-kilodalton (kD) γ-zein controls protein body initiation but is not involved in protein body filling. Conversely, other γ-zein family members function more in protein body expansion and not in protein body initiation. Reduction in both 19- and 22-kD α-zein subfamilies severely restricted protein body expansion but did not induce morphological abnormalities, which result from reduction of only the 22-kD α-zein class. Concomitant reduction of all zein classes resulted in severe reduction in protein body number but normal protein body size and morphology.

Cellular Effects After Laser In Situ Keratomileusis Flap Formation With Femtosecond Lasers: A Review

To provide an overview of the cellular effects of femtosecond laser in laser in situ keratomileusis flap formation. Literature review. The IntraLase is the only femtosecond laser with sufficient histopathological and confocal studies to allow review of the cellular effects of laser application. Histopathological analyses have demonstrated that the energy per pulse and total energy delivered play important roles in the inflammatory reaction to the surgery. The IntraLase laser triggers cellular necrosis (death accompanied by the release of lysosomal enzymes and other components from membrane-bound intracellular compartments) in the corneal stroma surrounding the lamellar cut rather than apoptosis (gentler form of cell death in which most intracellular components remain confined to membrane-bound apoptotic bodies) that is predominant with the microkeratome. Necrosis is a more inflammatory form of cell death that attracts more inflammatory cells. This is likely why earlier femtosecond lasers, such as the 15-kHz IntraLase laser, which requires higher total energy delivery to cut a flap, are associated with more corneal inflammation and diffuse lamellar keratitis. The design of the 60-kHz IntraLase model allows for much lower energy delivery to cut the flap and, therefore, a substantial reduction in keratocyte necrosis to the point that the overall inflammatory response is not significantly different from the microkeratome. Histopathological analysis performed with the Femtec femtosecond laser noted little change in the corneal stromal structure. Confocal microcopy studies performed with the IntraLase laser showed keratocyte "activation" in the stroma and greater fibrotic scarring at the interface than that induced by a mechanical microkeratome. The morphologic alterations in the corneal stroma produced by currently available models of the IntraLase laser are comparable to those produced by mechanical microkeratomes. Advances that have resulted in a reduction in the total amount of energy delivered by the laser when it cuts the flap have resulted in a decrease in the inflammatory response associated with femtosecond flap formation to the point that it is indistinguishable from the microkeratome at the cellular level. Further study of each of the femtosecond laser models, including the 150-kHz IntraLase laser, is needed to fully characterize the corneal response to these lasers.

Apoptosis of Epithelial Cells and Macrophages due to NonpigmentedSerratia marcescensStrains

Serratia marcescens strains are opportunistic pathogens that are increasingly recognized as a cause of severe nosocomial infections. In this study we observed interactions between nonpigmented strains with human epithelial and macrophage-like cells. The strains revealed hemolytic activity only after the contact of the cells with erythrocytes. The contact of the bacteria with the host cells was also essential to their cytotoxicity. Moreover, all strains revealed adherence ability and were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation of the HEp-2 and J774 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin and formation of membrane-bound apoptotic bodies. The lowest apoptotic index in HEp-2 cells did not exceed 25%, whereas the highest reached 59% at 24 h and 72% at 48 h after infection. Most of the strains (60%) induced fragmentation of nuclear DNA. The process depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. This study provided new insights into the mechanisms of nonpigmented S. marcescens pathogenesis. The results revealed that the strains produce cell-contact toxins that facilitate bacterial invasion, induce hemolysis, cytotoxicity, and apoptosis of host cells.

The historical evolution of the concept of apoptosis in rheumatic diseases

Even if the idea of apoptosis may probably be detected for the first time in a Hippocratic treatise, it was Rudolph Virchow, the founder of cellular pathology in the XIX century, who gave the first structured description of the processes underlying the term. In the course of the XX century a body of systematic observations on cell death was elaborated in an organic way. In 1908 Mechnikov won the Nobel Prize for his studies on phagocytosis, while in the '30s and '40s many studies on metamorphosis were carried out, and Fell and Canti identified cell death in chondrocytes in culture. At the end of the '40s Saunders began to observe cell death in chick limbs, and Hamburger and Levi Montalcini began their exploration of nerve growth factors. In mid '50s research on lysosomes began, and in the '60s Kerr defined shrinkage necrosis. The term apoptosis was definitely introduced by Kerr in 1972 to indicate a particular form of death in which cells commit suicide by dissolving themselves into membrane-bounded apoptotic bodies. In the '80s ced-3 was described and at the beginning of the '90s apoptosis genes were identified. In more recent times new instruments have allowed a wide range of cellular deaths to be described, including apoptosis as currently defined. Extremely interesting are its role and function in many different diseases; in particular in rheumatic ones recent evidence indicates that, far from being simple, brief and monomorphic, apoptosis is a complex, long-lasting, and pleiomorphic process.

Granulovacuolar degeneration bodies of Alzheimer's disease resemble late‐stage autophagic organelles

Granulovacuolar degeneration involves the accumulation of large, double membrane‐bound bodies within certain neurons during the course of Alzheimer's disease. Because of the two‐layer membrane morphology, it has been proposed that the bodies are related to autophagic organelles. To test this hypothesis, spatial colocalization of autophagic and endocytic markers with casein kinase 1 delta, a marker for granulovacuolar degeneration bodies (GVBs), was investigated in hippocampal sections prepared from post mortem Alzheimer's disease cases using double‐label confocal fluorescence microscopy. Results revealed that GVBs colocalized most strongly with LAMP1 (lysosome‐associated membrane protein 1) and CHMP2B (charged multivesicular body protein 2B), but weakly with lysosomal marker cathepsin D or early‐stage autophagy markers LC3 and p62. The resultant immunohistochemical signature suggests that GVBs do contain late‐stage autophagic markers, and accumulate at the nexus of autophagic and endocytic pathways. The data further suggest that failure to complete autolysosome formation may be an important correlate of GVB accumulation. This work was supported in part by the National Institute on Aging (AG14452, JK).

Granulovacuolar degeneration (GVD) bodies of Alzheimer's disease (AD) resemble late-stage autophagic organelles

Granulovacuolar degeneration involves the accumulation of large, double membrane-bound bodies within certain neurones during the course of Alzheimer's disease (AD) and other adult-onset dementias. Because of the two-layer membrane morphology, it has been proposed that the bodies are related to autophagic organelles. The aim of this study was to test this hypothesis, and determine the approximate stage at which the pathway stalls in AD. Spatial colocalization of autophagic and endocytic markers with casein kinase 1 delta, a marker for granulovacuolar degeneration (GVD) bodies, was evaluated in hippocampal sections prepared from post mortem Braak stage IV and V AD cases using double-label confocal fluorescence microscopy. GVD bodies colocalized weakly with early-stage autophagy markers LC3 and p62, but strongly with late-stage marker lysosome-associated membrane protein 1 (LAMP1), which decorated their surrounding membranes. GVD bodies also colocalized strongly with charged multivesicular body protein 2B (CHMP2B), which colocalized with the core granule, but less strongly with lysosomal marker cathepsin D. The resultant immunohistochemical signature suggests that granulovacuolar degeneration bodies (GVBs) do contain late-stage autophagic markers, and accumulate at the nexus of autophagic and endocytic pathways. The data further suggest that failure to complete autolysosome formation may be an important correlate of GVB accumulation.

Cytotoxic Activities of Physalis minima L. Chloroform Extract on Human Lung Adenocarcinoma NCI‐H23 Cell Lines by Induction of Apoptosis

Physalis minima L. is reputed for having anticancer property. In this study, the chloroform extract of this plant exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (after 24, 48 and 72 h of incubation). Analysis of cell-death mechanism demonstrated that the extract exerted apoptotic programed cell death in NCI-H23 cells with typical DNA fragmentation, which is a biochemical hallmark of apoptosis. Morphological observation using transmission electron microscope (TEM) also displayed apoptotic characteristics in the treated cells, including clumping and margination of chromatins, followed by convolution of the nuclear and budding of the cells to produce membrane-bound apoptotic bodies. Different stages of apoptotic programed cell death as well as phosphatidylserine externalization were confirmed using annexin V and propidium iodide staining. Furthermore, acute exposure to the extract produced a significant regulation of c-myc, caspase-3 and p53 mRNA expression in this cell line. Due to its apoptotic effect on NCI-H23 cells, it is strongly suggested that the extract could be further developed as an anticancer drug.