Gene/Protein Disease Symptom Drug Enzyme Compound
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We describe in this report that TEM-1 beta-lactamase has several desirable characteristics as a genetic reporter. First, it has no endogenous counterpart in eukaryotic cells and therefore provides a background-free measure of gene expression. Second, because of the uniqueness of the substrate cleavage reaction, a wide variety of substrates which are efficiently cleaved can be synthesized for beta-lactamase. Third, since the assays involve no more than addition of substrate to media, it is possible to continuously monitor a culture without destruction of the cells. Fourth, the enzyme is extremely versatile in that it can be fused to other proteins and retain activity. To demonstrate the versatility of beta-lactamase, we created three forms of the enzyme including secreted, intracellular, and membrane-bound forms of the enzyme, each form having distinct advantages as a reporter system. We also showed that levels of secreted beta-lactamase were proportional to both the levels of transfected DNA, beta-lactamase mRNA, as well as activity of the chloramphenicol acetyl transferase gene controlled by the same promoter, validating the reliability of this reporter. beta-Lactamase thus represents a novel and highly versatile genetic reporter.
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PMID:The development of beta-lactamase as a highly versatile genetic reporter for eukaryotic cells. 917 78

Ultrastructural changes of the tegument of adult liver flukes, Opisthorchis viverrini, after in vitro incubation in Minimal Essential Medium containing 0, 0.1, 1.0 and 10.0 micrograms/ml of anthelminthic praziquantel for 5, 15, 30, 45 and 60 minutes were investigated by scanning (SEM) and transmission (TEM) electron microscopy. SEM observations showed that the surface damage was composed of blebbing due to the swelling of microvilli, followed later by the disruption of these structures to form lesions that caused the erosion and desquamation of the surface. Sensory papillae, by contrast, appeared relatively unaffected. The surface changes could be observed at all doses but the extent of damage increased with increasing duration of incubation and concentration of the drug. The ventral as well as the dorsal surfaces exhibited similar change, whereas the anterior part tended to be damaged less than the posterior part. Under TEM observations, the earliest sign of changes was the depolymerization of the microtrabecular network in scattered foci, which resulted in the formation of non-membrane-bound vacuoles under microvilli. The basal infoldings also became dilated, and some turned into membrane-bound vacuoles in the basal zone. Subsequently, microvilli became enlarged, and eventually formed blebs that later rupture to form lesion spots as observed in the SEM. Finally, the microtrabecular network in all regions broke down, creating vacuoles of various sizes throughout the tegument, leading to its total disintegration and detachment. The sequence of morphological changes was generally similar at all doses; however, the changes occurred faster at the higher doses and the longer incubation times. In addition, at the longer durations myofilaments in most muscle cells also became depolymerized, while microtubules were unchanged by the drug. Therefore, it is possible that praziquantel, through its induction of Ca2+ influx, causes depolymerization of the microtrabecular network that leads to the vacuolization, swelling, blebbing, and eventually the disruption and detachment of the tegument, and the breakdown of myofilaments in the muscle cells.
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PMID:Opisthorchis viverrini: effect of praziquantel on the adult tegument. 927 94

Calf lungs were fixed with glutaraldehyde and examined by scanning (SEM) and transmission (TEM) electron microscopy to compare the ultrastructure of Clara cells in terminal bronchioles of neonatal calves and older cattle. In the neonatal calf, SEM revealed numerous smooth-surfaced Clara cells protruding above a similar number of ciliated cells, whereas in older animals the surface of Clara cells was lobulated. Thin sections examined by TEM revealed numerous cuboidal to columnar Clara cells with indented nuclei and a pale cytoplasm filled with faintly granular glycogen in the neonatal calf. Some cells were characterized by apical dense and/or pale membrane-bound granules or secretory droplets. Many cells had an apical tubular network of cisternae that were partly smooth and partly decorated with ribosomes. Ultrastructural comparison of Clara cells in a 2-day-old calf with those of 14- and 19-day-old, 4- and 5. 5-month-old, and 3.5-year-old cattle revealed a striking reduction in the amount of glycogen per cell after 14 days. The number of cells with apical granules was small at all ages, and the density of the secretory granules varied greatly in different cells. A variable amount of smooth endoplasmic reticulum (SER) was present but was less prominent than cisternae of ribosomal endoplasmic reticulum (RER). In older cattle, the limited amount of SER compared to the RER and secretory granules suggests that bovine Clara cells are more likely to be secretory than detoxifying.
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PMID:Comparative ultrastructure of Clara cells in neonatal and older cattle. 1076 Oct 52

Ultrastructural observations were made on colony forms of the protozoan parasite, Blastocystis hominis. Cross-sections of entire colonies were observed by TEM. Cells within a colony were heterogeneous in morphology, consisting of vacuolar, amoeboid, multivacuolar and unusual forms. Dying cells appeared to be in the process of fragmenting into numerous membranebound vesicles, giving rise to empty spaces within the colony. Interestingly, older colonies appeared to show cell fragmentation which resulted in larger, membranebound structures. Numerous cytoplasmic inclusions were present in the central vacuole of some cells, with many containing mitochondria. Amoeboid forms were observed to harbour small membrane-bound vesicles in endosome-like compartments. Other unusual features included margination of chromatin material and distinct blebbing of nuclei. These ultrastructural features suggest that B. hominis colony forms perhaps undergo a form of programmed cell death.
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PMID:Do Blastocystis hominis colony forms undergo programmed cell death? 1140 77

Beta-lactam antibiotics inhibit enzymes involved in the last step of peptidoglycan synthesis. These enzymes, also identified as penicillin-binding proteins (PBPs), form a long-lived acyl-enzyme complex with beta-lactams. Antibiotic resistance is mainly due to the production of beta-lactamases, which are enzymes that hydrolyze the antibiotics and so prevent them reaching and inactivating their targets, and to mutations of the PBPs that decrease their affinity for the antibiotics. In this study, we present a theoretical study of several penicillin-recognizing proteins complexed with various beta-lactam antibiotics. Hybrid quantum mechanical/molecular mechanical potentials in conjunction with molecular dynamics simulations have been performed to understand the role of several residues, and pK(a) calculations have also been done to determine their protonation state. We analyze the differences between the beta-lactamase TEM-1, the membrane-bound PBP2x of Streptococcus pneumoniae, and the soluble DD-transpeptidase of Streptomyces K15.
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PMID:Understanding the acylation mechanisms of active-site serine penicillin-recognizing proteins: a molecular dynamics simulation study. 1294 52

The ultrastructural characteristics of the mesothelial intracellular and extracellular lamellar bodies (LB) in norm, and especially in pathological conditions are still unknown. After routine fixation procedure, material from organs in the three serous cavities of Wistar rats, as control group were compared with animals following experimental hemothorax (EH) and experimental peritonitis (EP), using transmission and scanning electron microscopy (TEM, SEM). Different membrane-bound profiles, short strip-like structures and single LB characterize the control group. Five days after EH small groups of LB were observed. Single balloon-like profiles, numerous particles and larger groups of LB with wide varieties in size, form and membrane structure characterize the 8th day after EH. Thin and concentric membranes build single LB the 5th day of EP. Eight days after the same treatment, groups of LB, complex structures with several multilamellar centers, strip-like structures with rough granulo-filamentous material and uninterrupted covering over wide areas were observed. We conclude that constant components in the untreated rats are different membrane profiles, as initial lamellar formations and single typical LB with predominant intracellular position. The preferable sites for exocytosis of LB are the intercellular spaces. The present data demonstrate simultaneous findings of the two main groups of formations by using routine fixation in the control group and in the treated animals. The first represent LB as well as other round profiles, observed by TEM and particles, observed by SEM, which are widespread and probably have a basic role. The second ones are strip-like structures and its SEM-equivalents, i.e. uninterrupted covering. TEM-profiles and their SEM-images show significant organ differences and in some cases lack of correspondence between both electron microscopic techniques in the same treatment. Significant enlargement of the number of LB and the length of the strip-like structures characterize the mesothelium after pathologic conditions. LB with considerable differences in the structure and larger particles over the microvillous border predominate after EH. Strip-like profiles, continuous covering and smaller groups of LB with similar morphology and preferable submesothelial position characterize the mesothelium after EP.
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PMID:Mesothelial lamellar bodies in norm and experimental conditions. Transmission and scanning electron microscopic observations on the peritoneum, pleura and pericardium. 1519 89

Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003. A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found. Here we report investigations of this RLO in the tissues of the oyster C. ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C. ariakensis Gould. Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters. Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters. The shape, size, and color of inclusions from different tissues were polymorphic. Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic. RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size. The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid. Reproductive stages, including transverse binary fission, were observed by TEM. These stages were frequently observed within membrane-bound cytoplasmic vacuoles. Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.
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PMID:Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould. 1526 71

In this study we investigated the effect of uranium on the growth of the bacterium Thermus scotoductus strain SA-01 as well as the whole cell U(VI) reduction capabilities of the organism. Also, site-directed mutagenesis confirmed the identity of a protein capable of a possible alternative mechanism of U(VI) reduction. SA-01 can grow aerobically in up to 1.25 mM uranium and has the capability to reduce low levels of U(VI) in under 20 h. TEM analysis performed on cells exposed to uranium showed extracellular and membrane-bound accumulation of uranium. The reductase-like protein was surprisingly identified as a peptide ABC transporter, peptide-binding protein. This study showcases the concept of protein promiscuity, where this protein with a distinct function in situ can also have the unintended function of a reactant for the reduction of U(VI).
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PMID:Reduction of U(VI) by the deep subsurface bacterium, Thermus scotoductus SA-01, and the involvement of the ABC transporter protein. 2209 50

Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4-6 folds elongated, and formed intracytoplasmic membranes (ICM). To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP) transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare.
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PMID:Proteomic analysis of Ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection. 2238 64

Chlamydia trachomatis is an obligate intracellular bacterium responsible for one of the most common sexually transmitted diseases. In epithelial cells, C. trachomatis resides in a modified membrane-bound vacuole known as an inclusion, which is isolated from the endocytic pathway. However, the maturation process of C. trachomatis within immune cells, such as macrophages, has not been studied extensively. Here, we demonstrated that RAW macrophages effectively suppressed C. trachomatis growth and prevented Golgi stack disruption, a hallmark defect in epithelial cells after C. trachomatis infection. Next, we systematically examined association between C. trachomatis and various endocytic pathway markers. Spinning disk confocal time-lapse studies revealed significant and rapid association between C. trachomatis with Rab7 and LAMP1, markers of late endosomes and lysosomes. Moreover, pretreatment with an inhibitor of lysosome acidification led to significant increases in C. trachomatis growth in macrophages. At later stages of infection, C. trachomatis associated with the autophagy marker LC3. TEM analysis confirmed that a significant portion of C. trachomatis resided within double-membrane-bound compartments, characteristic of autophagosomes. Together, these results suggest that macrophages can suppress C. trachomatis growth by targeting it rapidly to lysosomes; moreover, autophagy is activated at later stages of infection and targets significant numbers of the invading bacteria, which may enhance subsequent chlamydial antigen presentation.
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PMID:Chlamydia trachomatis vacuole maturation in infected macrophages. 2280 27


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