Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.     

The Pharmacodynamic Characterization of an Antisense Oligonucleotide Against Monoamine Oxidase-B (MAO-B) in Rat Brain Striatal Tissue

1. The aim of our work was to pharmacodynamically characterize an antisense oligonucleotide sequence (5-GCC AAA CTT TTG CAT GAC-3) against MAO-B, using qualitative and quantitative analyses as assessment measures.2. Qualitative analysis using histochemical staining revealed that intracerebroventricular (ICV) administered antisense (100 picomoles twicedaily × 3.5 days) eliminated all visibly detectable histochemical staining for MAO-B throughout the striatum 1, 12, and 24 h after the last antisense treatment.3. Qualitative analysis using RT-PCR of the time course of MAO-B mRNA expression in the rat striatum following ICV administration of the antisense sequenceshowed that 12–24 h after the last administration there was a dramatic reduction in MAO-B mRNA expression in the striatum. The reverse and scrambled sequences generated no change in MAO-B mRNA at 1 or 24 h after the last treatment.4. Quantitative analysis using the MAO-B selective substrate 4-dimethylamino-phenethylamine (DMAPEA) showed that the antisense sequence reduced MAO-B activity by more than 40%, which was comparable to a single 2 mg/kg, ip dose of L-deprenyl.5. Quantitative analysis of neurotransmitter levels 24 h after the last treatment suggested that the antisense sequence did not produce any significant changes in neurotransmitter levels.6. Potential mechanisms for enhancing the antisense response and the speculated potential of an antisense against MAO-B for studying neurotoxicity, Parkinson's disease, and the aging process are also discussed.     

Characterization of human very low density lipoproteins containing two electrophoretic populations: double pre-beta lipoproteinemia and primary dysbetalipoproteinemia.

Two discrete populations of very low density lipoproteins, with fast and slow pre-beta electrophoretic mobility, were found in 50% of normolipemic and 30% of hyperlipemic individuals selected at random. The two populations were isolated by preparative electrophoresis from five hyperlipemic subjects. The particles comprising the slow component were smaller than those of the fast component and the slow component contained a larger proportion of cholesteryl esters, free cholesterol, B-apoprotein, and arginine-rich apoprotein and a smaller proportion of triglycerides and the two most anionic apoproteins (R-glutamic acid and R-alanine). The properties of the slow component thus closely resemble those of "remnant" very low density lipoproteins that accumulate in blood plasma of functionally hepatectomized rats. The chemical composition of the slow component was also similar to that of the very low density lipoproteins with beta mobility found in primary dysbetalipoproteinemia. However, the proportion of cholesteryl esters and argininerich apoprotein was much higher in the latter. The argininerich apoprotein from very low density lipoproteins of most normolipemic and hyperlipemic subjects separates into three or four major bands upon isoelectric focusing electrophoresis in polyacrylamide gels, with pI varying from 5.57 to 6.03. In very low density lipoproteins from individuals with primary dysbetalipoproteinemia, this protein uniquely contains little or none of the two most cationic bands. The number of bands was constant in all subjects studied. The pattern was the same in very low density lipoproteins with fast and slow pre-beta mobility as well as in the beta and pre-beta components in primary dysbetalipoproteinemia. These results suggest that many individuals have "remnant" very low density lipoproteins in their plasma. However, the beta-migrating "remnant" that accumulated in large amounts in individuals with primary dysbetalipoproteinemia contains much more arginine-rich protein and this protein is structurally abnormal.     

The Planktonic Index of Biotic Integrity (P-IBI): An approach for assessing lake ecosystem health

Using historical (1970) and more recent (1996) Lake Erie plankton and trophic status data, we developed a Planktonic Index of Biotic Integrity (P-IBI) to measure changes in lake ecosystem health. We used discriminant analysis to determine phytoplankton and zooplankton community characteristics (metrics) that distinguished among levels of impairment. Traditional measures of lake trophic status classes (i.e., oligotrophic, mesotrophic, eutrophic), such as chlorophyll a and total phosphorus concentrations, were used to classify sites on a gradient of impairment. We then judged the ability of plankton metrics to distinguish among trophic status classes. Because of the temporal variability found in plankton communities, we conducted analyses on a monthly basis (May–September). For June, July and August we found five unique metrics that could distinguish among trophic status classes. The P-IBI showed an increase in water quality in Lake Erie between 1970 (<3 = eutrophic) and the mid-1990s (1996 and 1997) (3–4 = mesotrophic) (which reflected mean (±standard error) total phosphorus concentrations (μg/L) 1970 > 1996; western basin (41.53 ± 2.68 > 29.75 ± 1.39), eastern basin (14.84 ± 0.82 > 7.74 ± 0.28) and mean (±standard error) chlorophyll a concentrations (μg/L) uncorrected for pheophytin 1970 > 1996; western basin (12.58 ± 1.82 > 5.40 ± 0.22), central basin (5.90 ± 0.36 > 3.17 ± 0.54), and eastern basin (5.17 ± 0.38 > 1.67 ± 0.18)), with declining water quality in the late 1990s (1998 and 1999) (3) and 2002 (<3). We recommend that the techniques used in creating the P-IBI be investigated for determining ecosystem health of other lakes.     

Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.     

Regulation of Vacuolar Proton-translocating ATPase Activity and Assembly by Extracellular pH

Vacuolar proton-translocating ATPases (V-ATPases) are responsible for organelle acidification in all eukaryotic cells. The yeast V-ATPase, known to be regulated by reversible disassembly in response to glucose deprivation, was recently reported to be regulated by extracellular pH as well (Padilla-López, S., and Pearce, D. A. (2006) J. Biol. Chem. 281, 10273–10280). Consistent with those results, we find 57% higher V-ATPase activity in vacuoles isolated after cell growth at extracellular pH of 7 than after growth at pH 5 in minimal medium. Remarkably, under these conditions, the V-ATPase also becomes largely insensitive to reversible disassembly, maintaining a low vacuolar pH and high levels of V₁ subunit assembly, ATPase activity, and proton pumping during glucose deprivation. Cytosolic pH is constant under these conditions, indicating that the lack of reversible disassembly is not a response to altered cytosolic pH. We propose that when alternative mechanisms of vacuolar acidification are not available, maintaining V-ATPase activity becomes a priority, and the pump is not down-regulated in response to energy limitation. These results also suggest that integrated pH and metabolic inputs determine the final assembly state and activity of the V-ATPase.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

Development of photochemical method for meniscal repair: a preliminary study

Visible light combined with naphthalimides has previously been shown to catalyze formation of physical bonds in avascular meniscal tissue. The first objective was to modify the existing in vitro testing method (i.e., adhesion testing using lap-jointed slices) to gain more sensitivity in detecting relative bonding strengths among candidate bonding agents. A repeated measures experimental design (RMED) was used to account for variability in properties among bovine menisci and was achieved by testing all treatments/controls on slices from each meniscus. Additionally, to make the method more clinically relevant in modeling a bucket-handle tear, the bovine meniscal slices were cut with collagen fibers parallel to the test slice's length. Peak stress was greater for the complete treatment group (light plus naphthalimide) than for the control or incomplete treatment groups (light only or napthalimide only). The second objective was to perform concentration and photoactivation time dose-response studies. In the concentration dose-response study, peak stress was greater for all treatments when compared with the control but not different among treatment groups; however, there was a trend of increased bonding strength with increased concentration. In the photoactivation time dose-response study, peak stress was greater for all treatments when compared with the control and greater for the 3-min treatment vs. the 6- and 9-min treatments. Peak stress was not different between the longer treatments. The RMED provided increased reproducibility and statistical sensitivity for detecting differences among treatments and will be used to test candidate bonding agents prior to in vivo testing.