Regulation of increase in anastomotic blood flow following pulmonary arterial occlusion

We have previously shown that there is an acute increase in anastomotic bronchial blood flow (Qbr) after pulmonary arterial obstruction in dogs. We examined the role of arachidonic acid metabolites in mediating this increase. The left lower lobe (LLL) was isolated and perfused (zone 2) with autologous blood in open-chested anesthetized dogs (n = 19). Qbr was measured from the amount of blood that overflowed from the closed vascular circuit of the suspended LLL and changes in its weight. In the control animals, there was a prompt and significant increase in Qbr following pulmonary arterial obstruction. Pretreatment with indomethacin (n = 6) or sodium salicylate (n = 4) almost completely blocked this rise in Qbr. Following pulmonary arterial occlusion, there was a rise in both thromboxane and a prostacyclin metabolite (6-keto-PGF1 alpha) in the blood of the pulmonary circulation of the LLL, although the 6-keto-PGF1 alpha rose relatively more. Pretreatment with indomethacin caused a fall in both thromboxane and prostacyclin levels (n = 3), which no longer rose after pulmonary arterial occlusion. These findings suggested that the balance of the vasodilator (prostacyclin) and vasoconstrictor (thromboxane) prostaglandins may play an important role in mediating the rise in Qbr that follows pulmonary arterial obstruction.     

Stable transfection of rat preporinsulin II gene into rat hematopoietic stem cells via recombinant adeno-associated virus

We investigated the ability of recombinant adeno-associated virus (rAAV), to mediate the transfer of rat preproinsulin II (rI2) gene into rat hematopoietic stem cells in vitro and expression of rI2 following intra-venous (i.v.) injection of infected stem cells into syngeneic rats. The pLP-1 recombinant plasmid containing rI2 was engineered as follows: rI2 with RSV-promoter was released from pBC 12BI (ATCC), purified, and inserted into BamH1 site of rAAV vector plasmid pWP-19. Plasmid pLP-1, together with pAAV?AD (Somatix Corp.), was used to co-transfect cell line 293 (ATCC). The rAAV genome was rescued using helper adenovirus and packaged into mature rAAV virions (vLP-1). Bone-marrow from female Wistar-Furth rats was enriched for stem cells by using plastic adherence and negative selection with monoclonal anti-rat CD3 and CD45RA to deplete T and B cells. The remaining cells were exposed to vLP-1 (moi=50:1) for 2 hours. Transfection was confirmed by PCR of neomycin resistance gene (neoR) after 8 days in culture. For in vivo studies, ten million exposed stem cells were injected i.v. into syngeneic rats (n=3). The results represent 3 identical experiments. Expression of neoR and rI2 was analyzed by RT-PCR. At week 1, neoR and rI2 were expressed in liver, spleen, thymus, peripheral blood lymphocytes and bone marrow. At week 2, neoR was expressed in spleen and brain, while at week 6, thymus, lymph nodes, bone-marrow, liver, spleen, and brain expressed neoR. rI2 was not detected after week 1. In summary, we showed that rAAV was efficient for transferring neoR and rI2 into rat hematopoietic stem cells.     

Misunderstanding the preorganization concept can lead to confusions about the origin of enzyme catalysis

Understanding the origin of the catalytic power of enzymes has both conceptual and practical importance. One of the most important finding from computational studies of enzyme catalysis is that a major part of the catalytic power is due to the preorganization of the enzyme active site. Unfortunately, misunderstanding of the nontrivial preorganization idea lead some to assume that it does not consider the effect of the protein residues. This major confusion reflects a misunderstanding of the statement that the interaction energy of the enzyme group and the transition state (TS) is similar to the corresponding interaction between the water molecules (in the reference system) and the TS, and that the catalysis is due to the reorganization free energy of the water molecules. Obviously, this finding does not mean that we do not consider the enzyme groups. Another problem is the idea that catalysis is due to substrate preorganization. This more traditional idea is based in some cases on inconsistent interpretation of the action of model compounds, which unfortunately, do not reflect the actual situation in the enzyme active site. The present article addresses the above problems, clarifying first the enzyme polar preorganization idea and the current misunderstandings. Next we take a specific model compound that was used to promote the substrate preorganization proposal and establish its irrelevance to enzyme catalysis. Overall, we show that the origin of the catalytic power of enzymes cannot be assessed uniquely without computer simulations, since at present this is the only way of relating structure and energetics.     

Papillary cystic neoplasm of the pancreas. Report of a case diagnosed by fine needle aspiration cytology

Fine needle aspiration (FNA) performed on a young woman who presented with a mass in the left hypochondrium yielded fluid. Smears and Cytospin preparations of the fluid showed good cellularity, consisting of relatively monomorphic cells forming a perivascular papillary pattern. FNA cytology thus suggested a diagnosis of papillary cystic neoplasm of the pancreas. Surgical removal of the pancreatic tumor and detailed histologic study confirmed the cytologic diagnosis.     

A Rhodanine Derivative CCR-11 Inhibits Bacterial Proliferation by Inhibiting the Assembly and GTPase Activity of FtsZ

A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 μM. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 [(E)-2-thioxo-5-({[3-(trifluoromethyl)phenyl]furan-2-yl}methylene)thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 ± 0.3 μM. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen-oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC(50)) of 1.2 ± 0.2 μM and a minimal inhibitory concentration of 3 μM. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC(50) value of 18.1 ± 0.2 μM (～15 × IC(50) of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents.     

Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure

Drug-induced liver injury (DILI) limits the development and application of many therapeutic compounds and presents major challenges to the pharmaceutical industry and clinical medicine. Acetaminophen-containing compounds are among the most frequently prescribed drugs and are also the most common cause of DILI. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and acetaminophen-induced hepatotoxicity. We demonstrate that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation and death caused by liver-toxic drugs. We identify a small-molecule inhibitor of Cx32 that protects against liver failure and death in wild-type mice when co-administered with known hepatotoxic drugs. These findings indicate that gap junction inhibition could provide a pharmaceutical strategy to limit DILI and improve drug safety.     

Assessment of Napier millet (Pennisetum purpureumx P. glaucum) and sorghum (Sorghum bicolor) trap crops for the management of Chilo partellus on maize

Two Napier millet (Pennisetum purpureumxP. glaucum) hybrids, namely PBN 83 and PBN 233 and one sorghum (Sorghum bicolor) variety, SL 44, were assessed for their potential role as a trap crop in the management of the stem borer, Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) on maize. Oviposition preference and larval survival and development were determined for different test plants under laboratory and screen house conditions. Further, field dispersal of C. partellus larvae was assessed between Napier millet and maize crops. Results from no-choice and dual-choice tests indicated that Napier millet hybrids were preferred for oviposition over maize by C. partellus moths. Sorghum was, however, not preferred over maize in this respect. Napier millet hybrids were poor larval hosts, and a rapid decline in larval numbers was noticed within the first five days after hatching and virtually no larvae survived to pupation. Leaf area eaten by the borer larvae was significantly less on these hybrids than on maize or sorghum. Plant damage was more severe in maize and sorghum than Napier millet hybrids. No appreciable larval shift was noticed from Napier millet hybrids to the adjoining maize crop. The evaluated Napier millet hybrids, therefore, had potential for use as trap crop in C. partellus management. Sorghum, however, did not hold promise in this respect.     

A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.     

Enzymatic and anatomical changes in abscission zone cells of apple fruits induced by Ethephon

The enhancement of fruit abscission zone formation with ethephon treatment caused an increase in soluble proteins, endo-cellulase, exo-polygalacturonase and peroxidase activities. Exo-cellulase and endo-polygalacturonase did not show any relationship with apple abscission. The separation of cells initiated in the cortex region and progressed towards vascular tissue. Cell separation in the cortex appeared to be due to dissolution of middle lamella but vascular tissues ruptured mechanically.