Alterations in the Proteome of the Euprymna scolopes Light Organ in Response to Symbiotic Vibrio fischeri

During the onset of the cooperative association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, the anatomy and morphology of the host's symbiotic organ undergo dramatic changes that require interaction with the bacteria. This morphogenetic process involves an array of tissues, including those in direct contact with, as well as those remote from, the symbiotic bacteria. The bacteria induce the developmental program soon after colonization of the organ, although complete morphogenesis requires 96 h. In this study, to determine critical time points, we examined the biochemistry underlying bacterium-induced host development using two-dimensional polyacrylamide gel electrophoresis. Specifically, V. fischeri-induced changes in the soluble proteome of the symbiotic organ during the first 96 h of symbiosis were identified by comparing the protein profiles of symbiont-colonized and uncolonized organs. Both symbiosis-related changes and age-related changes were analyzed to determine what proportion of the differences in the proteomes was the result of specific responses to interaction with bacteria. Although no differences were detected over the first 24 h, numerous symbiosis-related changes became apparent at 48 and 96 h and were more abundant than age-related changes. In addition, many age-related protein changes occurred 48 h sooner in symbiotic animals, suggesting that the interaction of squid tissue with V. fischeri cells accelerates certain developmental processes of the symbiotic organ. These data suggest that V. fischeri-induced modifications in host tissues that occur in the first 24 h of the symbiosis are independent of marked alterations in the patterns of abundant proteins but that the full 4-day morphogenetic program requires significant alteration of the host soluble proteome.     

The effect of Rho kinase inhibitor Y-27632 on endotoxemia-induced intestinal apoptosis in infant rats

The aim of this study was to evaluate the effect of Rho kinase inhibitor, Y-27632 on the intestinal apoptosis in endotoxemic infant rats. Wistar albino 15–17-day-old rat pups (n = 21) were randomized to three experimental groups: (1) controls; (2) endotoxemia (LPS); and (3) endotoxemia treated with Y-27632 (LPS + Y-27632). Endotoxemia was induced in rats by intraperitoneal (i.p) injection of lipopolysaccharide (Escherichia coli serotype 0111:B4; 10 mg/kg). Y-27632 was administered 5 mg/kg i.p at three times, just, 8 and 16 h after LPS injection. Twenty-four hours after LPS injection, intestinal apoptosis was assessed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and immunohistochemistry for active caspase-3. Endotoxemia induced extensive apoptotic injury in the intestinal tissues. The administration of Y-27632 to endotoxemic infant rats caused a marked decrease in the number of apoptotic cells in both intestinal epithelium and lamina propria. In conclusion, the inhibition of Rho kinase with Y-27632 diminished the intestinal apoptotic damage induced by endotoxemia in infant rats.     

Roles of Vibrio fischeri and Nonsymbiotic Bacteria in the Dynamics of Mucus Secretion during Symbiont Colonization of the Euprymna scolopes Light Organ

During light organ colonization of the squid Euprymna scolopes by Vibrio fischeri, host-derived mucus provides a surface upon which environmental V. fischeri forms a biofilm and aggregates prior to colonization. In this study we defined the temporal and spatial characteristics of this process. Although permanent colonization is specific to certain strains of V. fischeri, confocal microscopy analyses revealed that light organ crypt spaces took up nonspecific bacteria and particles that were less than 2 μm in diameter during the first hour after hatching. However, within 2 h after inoculation, these cells or particles were not detectable, and further entry by nonspecific bacteria or particles appeared to be blocked. Exposure to environmental gram-negative or -positive bacteria or bacterial peptidoglycan caused the cells of the organ's superficial ciliated epithelium to release dense mucin stores at 1 to 2 h after hatching that were used to form the substrate upon which V. fischeri formed a biofilm and aggregated. Whereas the uncolonized organ surface continued to shed mucus, within 48 h of symbiont colonization mucus shedding ceased and the formation of bacterial aggregations was no longer observed. Eliminating the symbiont from the crypts with antibiotics restored the ability of the ciliated fields to secrete mucus and aggregate bacteria. While colonization by V. fischeri inhibited mucus secretion by the surface epithelium, secretion of host-derived mucus was induced in the crypt spaces. Together, these data indicate that although initiation of mucus secretion from the superficial epithelium is nonspecific, the inhibition of mucus secretion in these cells and the concomitant induction of secretion in the crypt cells are specific to natural colonization by V. fischeri.     

<Emphasis Type="Italic">Cotesia plutellae</Emphasis> bracovirus suppresses expression of an antimicrobial peptide,cecropin, in the diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis>, challenged by bacteria

An endoparasitoid wasp, Cotesia plutellae, induces significant immunosuppression of host insect, Plutella xylostella. This study was focused on suppression in humoral immune response of P. xylostella parasitized by C. plutellae. An EST database of P. xylostella provided a putative cecropin gene (PxCec) which is 627 bp long and encodes 66 amino acids. A signal peptide (22 amino acids) is predicted and two putative O-glycosylation sites in threonine are located at positions 58 and 64. Without bacterial infection, PxCec was expressed in pupa and adult stages but not in the egg and larval stages. Upon bacterial challenge, however, the larvae expressed PxCec as early as 3 h post infection (PI) and maintained high expression levels at 12–24 h PI. By 48 h PI, its expression noticeably diminished. All tested tissues of bacteria-infected P. xylostella showed PxCec expression. However, other microbes, such as virus and fungus, did not induce the PxCec expression. Parasitization by C. plutellae suppressed the expression of PxCec in response to bacterial challenge. Among the parasitic factors of C. plutellae, its symbiotic virus (C. plutellae bracovirus: CpBV) alone was able to inhibit the expression of PxCec of P. xylostella challenged by bacteria. These results indicate that PxCec expression is regulated by both immune and developmental processes in P. xylostella. The parasitization by C. plutellae inhibited the expression of PxCec by the wasp’s symbiotic virus.     

Bacterial Growth Rate and Marine Virus–Host Dynamics

Abstract The dynamics of a marine virus–host system were investigated at different steady state growth rates in chemostat cultures and the data were analyzed using a simple model. The virus–host interactions showed strong dependence on host cell growth rate. The duration of the infection cycle and the virus burst size were found to depend on bacterial growth rate, and the rate of cell lysis and virus production were positively correlated with steady state growth rate in the cultures (r ² > 0.96, p < 0.05). At bacterial growth rates of 0.02 to 0.10 h⁻¹ in the chemostats the virus burst size increased from 12 ± 4 to 56 ± 4, and the latent period decreased from 2.0 to 1.7 h. Resistant clones of the host strain were present in the cultures from the beginning of the experiment and replaced the sensitive host cells following viral lysis in the cultures. Regrowth of resistant cells correlated significantly (r ²= 1.000, p < 0.02) with the lysis rate of sensitive cells, indicating that release of viral lysates stimulated growth of the non-infected, resistant cells. The constructed model was suitable for simulating the observed dynamics of the sensitive host cells, viruses and resistant clones in the cultures. The model was therefore used in an attempt to predict the dynamics of this virus–host interaction in a natural marine environment during a certain set of growth conditions. The simulation indicated that a steady state relationship between the specific viruses and sensitive and resistant bacterial clones may occur at densities that are reasonable to assume for natural environments. The study demonstrates that basic characterization and modeling of specific virus–host interactions may improve our understanding of the behavior of bacteria and viruses in natural systems. Received: 12 November 1999; Accepted: 2 May 2000; Online Publication: 11 August 2000     

Expression patterns of BMPRs in the developing mouse molar

During development, Bone Morphogenetic Proteins (BMPs) can induce apoptosis, cell growth or differentiation. These different effects are mediated by dimers of two types of BMP–receptors (BMPRs). To identify the responding cells during tooth development and search for possible tissue–or stage–specificities in the receptors involved, the distribution patterns of BMPR–IA, –IB and –II were investigated in the mouse molar, from bud to bell stage. At the bud stage, BMP–2 was suggested to be involved in the formation of an epithelial signaling center, the primary enamel knot (PEK), while BMP–4 would mediate the condensation of the mesenchyme. Immunostaining showed the presence of BMPR–IA and –II in the epithelium instead of BMPR–IB and –II in the mesenchyme. At the cap stage, BMPR–IB was detected in the epithelium but not BMPR–II, suggesting the existence of another type II receptor to form a functional dimer. At the late cap stage in the epithelium, BMP–4, BMPR–IA and –II were restricted to the internal part of the PEK and the stalk: two apoptotic areas. The three proteins were detected in the mesenchyme, showing a strong staining where cusps were about to form. At the late bell stage, BMP–2 or –4 may induce cell differentiation. BMPR–IB and –II were detected in odontoblasts instead of BMPR–IA and –II in ameloblasts. These results provide the first evidence of multiple type I and type II BMP–receptors, expressed in the dental epithelium and mesenchyme at different stages of development, to signal different cellular activities in a time– and tissue–specific way.     

Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells

Background  

During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the Drosophila embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.     

Bioluminescence in the symbiotic squid Euprymna scolopes is controlled by a daily biological rhythm

In most symbioses between animals and luminous bacteria it has been assumed that the bacterial symbionts luminesce continuously, and that the control of luminescent output by the animal is mediated through elaborate accessory structures, such as chromatophores and muscular shutters that surround the host light organ. However, we have found that while in the light organ of the sepiolid squid Euprymna scolopes, symbiotic cells of Vibrio fischeri do not produce a continuously uniform level of luminescence, but instead exhibit predictable cyclic fluctuations in the amount of light emitted per cell. This daily biological rhythm exhibits many features of a circadian pattern, and produces an elevated intensity of symbiont luminescence in juvenile animals during the hours preceding the onset of ambient darkness. Comparisons of the specific luminescence of bacteria in the intact light organ with that of newly released bacteria support the existence of a direct host regulation of the specific activity of symbiont luminescence that does not require the intervention of accessory tissues. A model encompassing the currently available evidence is proposed for the control of growth and luminescence activity in the E. scolopes/V. fischeri light organ symbiosis.Abbreviations CFU colony-forming-unit - LD light-dark     

Fusaric acid induces apoptosis in saffron root-tip cells: roles of caspase-like activity, cytochrome c, and H2O2

Programmed cell death (PCD), now known as apoptosis, is accompanied by specific morphological features. In this study, fusaric acid, a fusarium mycotoxin, was used to examine cell death in saffron (Crocus sativus Linnaeus) roots, using several apoptosis assays. Our results show that moderate FA doses (50–100 μM) induce apoptotic features while high FA doses (> 200 μM) stimulate necrosis. The apoptotic-like features induced by moderate doses of FA include chromatin condensation, formation of condensed chromatin spheres which bud from the nucleus, fragmentation of nucleosomal DNA into ∼ 180 bp fragments, exposure of phosphatidyl serine to the external membrane leaflet, delivery of cytochrome c to cytosol, and generation of H₂O₂. These apoptotic alterations in root cells are not observed in the presence of serine protease, caspase-1 or caspase-3 inhibitors. It is proposed that production of H₂O₂ and release of cytochrome c into the cytosol may activate caspase-like proteases and thus establish the apoptotic pathway. As nuclei budding spheres formed in plant root cells after exposure to 50–100 μM FA doses seem to be digested inside the cytosol, we suggest labeling them as internal apoptotic bodies (IAB) that may be more informative than previously used term, apoptotic-like bodies.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Epithelial cells release proinflammatory cytokines and undergo c-myc-induced apoptosis on exposure to filarial parasitic sheath protein-Bcl2 mediates rescue by activating c-H-ras

Summary Circulating filarial proteins elicit strong immunologic reactions in humans leading to the chronic manifestations in human lymphatic filariasis such as lymphatic occlusion, fibrosis, edema, and in some cases, tropical pulmonary eosinophilia. Our earlier studies, in vitro, conclusively prove that filarial parasitic sheath proteins induce apoptosis in HEp2 cells, an epithelial cell line, by a pathwa inhibitable by bcl2. The present findings provide evidence that c-myc activation triggers apoptosis in HEp2 cells and that it is also responsible for the burst of abortive proliferation at 6 d of treatment of HEp2 bcl2 cells that overexpress bcl2, with filarial parasitic sheath protein, demonstrating the interplay between the two genes c-myc and bcl2, wherein bcl2 acts by restoring the prosurvival signal to c-myc and keeping its apoptotic tendency in check. This study also indicates that bcl2 upregulates c-H-ras, engaring ras to bring about the suppression of apoptosis through protein tyrosine kinase elevation, thus promoting the survival of the HEp2 bcl2 cells. In addition to the activation of these “signal switches,” we also observe that these cells release cytokines like IL-6 and IL-8 through the upregulation of c-fos, when exposed to filarial parasitic sheath protein, reflecting on the immunomodulatory capacity of the epithelium to elicit a host immune response by setting up a chemotactic gradient, attracting inflammatory cells to the site of infection.     

Caspase-9/-3 activation and apoptosis are induced in mouse macrophages upon ingestion and digestion of Escherichia coli bacteria

A number of highly virulent, intracellular bacteria are known to induce cell death by apoptosis in infected host cells. In this work we demonstrate that phagocytosis of bacteria from the Escherichia coli laboratory strain K12 DH5alpha is a potent cell death stimulus for mouse macrophages. RAW264.7 mouse macrophages took up bacteria and digested them within 2-4 h as investigated with green fluorescent protein-expressing bacteria. No evidence of apoptosis was seen at 8 h postexposure, but at 24 h approximately 70% of macrophages displayed an apoptotic phenotype by a series of parameters. Apoptosis was blocked by inhibition of caspases or by forced expression of the apoptosis-inhibiting protein Bcl-2. Processing of caspase-3 and caspase-9 but not caspase-8 was seen suggesting that the mitochondrial branch of the apoptotic pathway was activated. Active effector caspases could be detected in two different assays. Because the adapter molecule myeloid differentiation factor 88 (MyD88) has been implicated in apoptosis, involvement of the Toll-like receptor pathway was investigated. In RAW264.7 cells, heat-treated bacteria were taken up poorly and failed to induce significant apoptosis. However, cell activation was almost identical between live and heat-inactivated bacteria as measured by extracellular signal-regulated kinase activation, generation of free radicals, and TNF secretion. Furthermore, primary bone marrow-derived macrophages from wild-type as well as from MyD88-deficient mice underwent apoptosis upon phagocytosis of bacteria. These results show that uptake and digestion of bacteria leads to MyD88-independent apoptosis in mouse macrophages. This form of cell death might have implications for the generation of the immune response.     

Vibrio fischeri lipopolysaccharide induces developmental apoptosis, but not complete morphogenesis, of the Euprymna scolopes symbiotic light organ

During initiation of the association between the squid host Euprymna scolopes and its bacterial partner Vibrio fischeri, the bacteria induce dramatic morphogenesis of the host symbiotic organ, a portion of which involves the signaling of widespread apoptosis of the cells in a superficial ciliated epithelium on the colonized organ. In this study, we investigated the role in this process of lipopolysaccharide (LPS), a bacterial cell-surface molecule implicated in the induction of animal cell apoptosis in other systems. Purified V. fischeri LPS, as well as the LPS of V. cholerae, Haemophilus influenzae, Escherichia coli, and Shigella flexneri, added in the concentration range of pg/ml to ng/ml, induced apoptosis in epithelial cells 10- to 100-fold above background levels. The absence of species specificity suggested that the conserved lipid A portion of the LPS was the responsible component of the LPS molecule. Lipid A from V. fischeri, E. coli, or S. flexneri induced apoptosis. In addition, strains of H. influenzae carrying a mutation in the htrB gene, which is involved in the synthesis of virulent lipid A, showed a diminished ability to induce apoptosis of host cells. Confocal microscopy using fluorescently labeled LPS indicated that the LPS behaves similar to intact bacterial symbionts, interacting with host cells in the internal crypt spaces and not directly with the superficial epithelium. Although LPS was able to induce apoptosis, it did not induce the full morphogenesis of the ciliated surface, suggesting that multiple signals are necessary to mediate the development of this animal-bacterial mutualism.     

Rapid cold-hardening blocks cold-induced apoptosis by inhibiting the activation of pro-caspases in the flesh fly <Emphasis Type="Italic">Sarcophaga crassipalpis</Emphasis>

Apoptosis plays important roles in the selective elimination of sub-lethally damaged cells due to various environmental stresses. The rapid cold-hardening (RCH) response protects insects from the otherwise lethal consequences of injury due to cold-shock. We recently demonstrated that cold shock induces apoptotic cell death in insects and that RCH functions to specifically block cold-shock-induced apoptosis. In the present study we used isolated fat body, midgut, muscle, and Malpighian tubules from adult flesh flies Sarcophaga crassipalpis to test the following hypotheses: (1) cold-induced apoptosis varies among different tissues and (2) RCH blocks the apoptotic pathway by preventing the activation of pro-caspases. Cold-shock induced substantial amounts of apoptotic cell death that matched with tissue damage as determined using vital dyes. RCH treatment significantly reduced apoptotic cell death in all tested tissues. Caspase-3 (executioner) activity was 2–3 times higher in the cold- and heat-shocked groups than in control and RCH groups. Likewise, the activity of caspase-9 (initiator) showed a similar trend as for caspase-3 in all tissues but midgut. In addition, cold-shock and heat-shock treatments also increased caspase-2 activity 2–3 folds in both soluble and membrane fractions of fat body and muscle extracts compared to controls.     

Acidification induces Bax translocation to the mitochondria and promotes ultraviolet light-induced apoptosis

It has been suggested that Bax translocation to the mitochondria is related to apoptosis, and that cytosol acidification contributes to apoptosis events. However, the mechanisms remain obscure. We investigated the effect of acidification on Bax translocation and on ultraviolet (UV) light-induced apoptosis. The Bax translocation assay in vitro showed that Bax translocated to the mitochondria at pH 6.5, whereas no Bax translocation was observed at pH 7.4. VHDBB cells expressing the GFP-Bax fusion protein were treated for 12 h with a pH 6.5 DMEM medium, nigericin (5 μg/ml) and UV light (50 J/cm²), separately or in combination, and Bax translocation to the mitochondria was determined by SDS-PAGE and Western blot, and apoptotic cell death was detected by flow cytometry. The results showed that some of the Bax translocated to the mitochondria in the cells treated with the normal medium, nigericin and UV in combination, whereas all of the Bax translocated to the mitochondria in the cells treated with the pH 6.5 medium, nigericin and UV in combination. In VHDBB cells treated for 12 h with nigericin, UV alone, and UV and nigericin in combination, the respective rates of apoptotic cell death were 25.08%, 33.25% and 52.88%. In cells treated with pH 6.5 medium and nigericin, pH 6.5 medium and UV, and pH 6.5 medium, nigericin and UV in combination, the respective rates of apoptotic cell death increased to 37.19%, 41.42% and 89.44%. Our results indicated that acidification induces Bax translocation from the cytosol to the mitochondria, and promotes UV lightmediated apoptosis. This suggests that there is a possibility of improving cancer treatment by combining acidification with irradiation or chemotherapeutic drugs.     

Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites

AIMS: To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS: After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS: Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.     

Vibrio fischeri‐derived outer membrane vesicles trigger host development

Outer membrane vesicles (OMV) are critical elements in many host‐cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage‐like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis.     

Bacterial Uptake by the Marine Sponge <Emphasis Type="Italic">Aplysina aerophoba</Emphasis>

Sponges (Porifera) are filter feeders that take up microorganisms from seawater and digest them by phagocytosis. At the same time, many sponges are known to harbor massive consortia of symbiotic microorganisms, which are phylogenetically distinct from those in seawater, within the mesohyl matrix. In the present study, feeding experiments were performed to investigate whether phylogenetically different bacterial isolates, hereafter termed “food bacteria,” microbial seawater consortia, and sponge symbiont consortia are taken up and processed differently by the host sponge. Aplysina aerophoba retained high numbers of bacterial isolates and microbial seawater consortia with rates of up to 2.76 × 10⁶ bacteria (g sponge wet weight)^–1 h^–1, whereas the retention of sponge symbionts was lower by nearly two orders of magnitude [5.37 × 10⁴ bacteria (g sponge wet weight)⁻¹ h^–1]. In order to visualize the processing of a food bacterium within sponge tissues, the green fluorescent protein-labeled Vibrio strain MMW1, which had originally been isolated from A. aerophoba, was constructed. Incubation of this strain with A. aerophoba and subsequent visualization in tissue cryosections showed its presence in the choanocytes and/or endopinacocytes lining the canals but, unlike latex beads, not in deeper regions of the mesohyl, which suggests digestion of the bacteria upon contact with the host. Denaturing gradient gel electrophoresis (DGGE) was performed on the incubation seawater to monitor the changes in phylogenetic composition after incubation of the sponge with either seawater or sponge symbiont consortia. However, the DGGE experiment provided no evidence for selective processing of individual lineages by the host sponge. In conclusion, this study extends early studies by Wilkinson et al. (Proc R Soc London B 220:519–528, 1984) that sponges, here A. aerophoba, are able to differentiate between food bacteria and their own bacterial symbionts.     

Population Dynamics of Vibrio fischeri during Infection of Euprymna scolopes

The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.     

The effects of benzene exposure on apoptosis in epithelial lung cells: Localization by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and the immunocytochemical localization of apoptosis-related gene products

Although benzene, a well-known human carcinogen, has been shown to induce apoptosis in vitro, no studies have been carried out to confirm and characterize its role in activating apoptosis in vivo. The present study investigated the effects of benzene inhalation on the epithelial cells lining the respiratory tract including bronchioles, terminal bronchioles, respiratory bronchioles and alveoli of male Sprague-Dawley rats. Inhalation of benzene 300 ppm for 7 days induced apoptotic changes in the parenchymal components in the lung that significantly exceeded the events of programmed cell death in normal control tissues. Apoptosis was confirmed by the electrophoretic analysis of internucleosomal DNA fragmentation of benzene-exposed lung tissues, which exhibited 180–200 bp laddering subunits indicative of genomic DNA degradation. Furthermore, semi-quantitative analysis of intracellular localization of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling TUNEL) showed a significant (p < 0.001) increase in the apoptotic index calculated for bronchiolar 73.5%, terminal bronchiolar (65%), and respiratory bronchiolar 60.8% segmental epithelial components as well as alveolar (55%) epithelia. Analysis of immunohistochemical expression of apoptosis-related gene products also supported the hypothesis that benzene can induce apoptosis in chemosensitive target cells in the lung parenchyma. Quantitative immunhistochemistry showed a statistically significant increase p < 0.001 in the immunoreactive staining index for cytochrome c, Apaf-1 (apoptosis activating factor-1), DNA fragmentation factor, and representative cysteine proteases including caspase-1, caspase-2L, caspase-8 and caspase-9. Thus this is the first study of the respiratory system that demonstrates that benzene inhalation induces lung cell apoptosis as confirmed by DNA electrophoresis, in situ nick end labeling, and the upregulation of apoptosis-related gene products that facilitate caspase-cleaved enzymes which lead to cell degradation via programmed cell death. These responses may represent an important defense mechanism within the parenchymal cells of the respiratory system that reduce mutational hazard and the potential carcinogenic effects of benzene-initiated pathogenesis.