Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.     

Contamination of red-meat carcasses by Campylobacter fetus subsp. jejuni.

Campylobacter fetus subsp. jejuni was commonly present in the feces of unweaned calves (2 to 3 weeks old) and from two of four groups of sheep. One new season lamb (12 to 16 weeks old) carried the organism, but the bacteria were not isolated from cattle. With unweaned calves, the fractions of animals infected and carcasses contaminated were similar. Contamination of carcasses usually involved low densities of C. fetus subsp. jejuni (ca. 1 to 10/cm2), which were isolated from flank but not rump areas. The organism was recovered less frequently from chilled carcasses and deboned veal. Small numbers of C. fetus subsp. jejuni could be recovered from equipment during the processing of unweaned calves but not after routine cleaning.     

Induction of innate immunity against herpes simplex virus type 2 infection via local delivery of Toll-like receptor ligands correlates with beta interferon production

Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta interferon (IFN-beta) but not production of IFN-alpha, IFN-gamma, or tumor necrosis factor alpha (TNF-alpha) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-beta in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-beta in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-alpha or IFN-gamma in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-alpha(-/-) and IFN-gamma(-/-) mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly IFN-beta, mediates innate protection, mice unresponsive to type I interferons (IFN-alpha/betaR(-/-) mice) and mice lacking IFN regulatory factor-3 (IRF-3(-/-) mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-alpha/betaR(-/-) or IRF-3(-/-) mice. Local delivery of murine recombinant IFN-beta protected C57BL/6 and IRF-3(-/-) mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-beta production and innate antiviral immunity against HSV-2.     

Activation of 5-HT1A receptors in the paragigantocellularis lateralis decreases shivering during cooling in the conscious piglet

Activation of 5-HT(1A) receptors in the medullary raphé decreases sympathetic outflow to thermoregulatory mechanisms, including brown adipose tissue (BAT), thermogenesis, and peripheral vasoconstriction when these mechanisms are previously activated with leptin, prostaglandins, or cooling. These same mechanisms are also inhibited during rapid eye movement (REM) sleep. It is not known whether shivering is also modulated by medullary raphé neurons. We previously showed in the conscious piglet that activation of 5-HT(1A) receptors with 8-OH-DPAT (DPAT) in the paragigantocellularis lateralis (PGCL), a medullary region lateral to the midline raphé that contains 5-HT neurons, decreases heart rate, body temperature and muscle activity during non-rapid eye movement (NREM) sleep. We therefore hypothesized that activation of 5-HT(1A) receptors in the PGCL would also attenuate shivering and peripheral vasoconstriction during cooling. During REM sleep in a cool environment, shivering, carbon dioxide production, and body temperature decreased, and ear capillary blood flow and ear skin temperature increased. Shivering associated with rapid cooling was attenuated after dialysis of DPAT into the PGCL. In animals maintained in a continuously cool environment, dialysis of DPAT into the PGCL attenuated shivering and decreased body temperature, but there were no significant increases in ear capillary blood flow or ear skin temperature. We conclude that both naturally occurring REM sleep and exogenous activation of 5-HT(1A) receptors in the PGCL are associated with a suspension of shivering during cooling. Our data are consistent with the hypothesis that 5-HT neurons in the PGCL facilitate oscillating spinal motor circuits involved in shivering but are less involved in modulating sympathetically mediated thermoregulatory mechanisms.     

Genomes and characterization of phages Bcep22 and BcepIL02, founders of a novel phage type in Burkholderia cenocepacia

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.     

X-ray and model-building studies on the specificity of the active site of proteinase K

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca²⁺ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.     

5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.     

There are two gene sequences for human apolipoprotein CI (apo CI) on chromosome 19, one of which is 4 kb from the gene for apo E

A cDNA probe corresponding to the mRNA sequence for apolipoprotein E (apo E) was used to screen two independently-constructed human genomic libraries. Two recombinants (lambda E-2, and lambda E2-1), isolated using the apo E cDNA probe, also contain part or all of the apo CI gene. Hybridisation studies using both apo E and apo CI cDNA probes show that these two genes are in the same orientation and separated by 4 kb.     

Tryptamine-gallic acid hybrid prevents non-steroidal anti-inflammatory drug-induced gastropathy: correction of mitochondrial dysfunction and inhibition of apoptosis in gastric mucosal cells

We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.