Enhanced post-ischemic liver injury in iNOS-deficient mice: a cautionary note.

The objective of this study was to assess the role of inducible nitric oxide synthase (iNOS) in ischemia- and reperfusion (I/R)-induced liver injury. We found that partial hepatic ischemia involving 70% of the liver resulted in a time-dependent increase in serum alanine aminotransferase (ALT) levels at 1-6 h following reperfusion. Liver injury at 1, 3, and 6 h post-ischemia was not due to the infiltration of neutrophils as assessed by tissue myeloperoxidase (MPO) activity and histopathology. iNOS-deficient mice subjected to the same duration of ischemia and reperfusion showed dramatic and significant increases in liver injury at 3 but not 6 h following reperfusion compared to their wild type controls. Paradoxically, iNOS mRNA expression was not detected in the livers of wild type mice at any point during the reperfusion period and pharmacological inhibition of iNOS using L-N(6)(iminoethyl)-lysine (L-NIL) did not exacerbate post-ischemic liver injury at any time post-reperfusion. These data suggest that iNOS deficiency produces unanticipated genetic alterations that renders these mice more sensitive to liver I/R-induced injury.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239Δnef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients

The live attenuated simian immunodeficiency virus (SIV) SIVmac239Δnef is the most effective SIV/human immunodeficiency virus (HIV) vaccine in preclinical testing. An understanding of the mechanisms responsible for protection may provide important insights for the development of HIV vaccines. Leveraging the uniquely restricted genetic diversity of Mauritian cynomolgus macaques, we performed adoptive transfers between major histocompatibility complex (MHC)-matched animals to assess the role of cellular immunity in SIVmac239Δnef protection. We vaccinated and mock vaccinated donor macaques and then harvested between 1.25 × 10⁹ and 3.0 × 10⁹ mononuclear cells from multiple tissues for transfer into 12 naive recipients, followed by challenge with pathogenic SIVmac239. Fluorescently labeled donor cells were detectable for at least 7 days posttransfer and trafficked to multiple tissues, including lung, lymph nodes, and other mucosal tissues. There was no difference between recipient macaques'' peak or postpeak plasma viral loads. A very modest difference in viral loads during the chronic phase between vaccinated animal cell recipients and mock-vaccinated animal cell recipients did not reach significance (P = 0.12). Interestingly, the SIVmac239 challenge virus accumulated escape mutations more rapidly in animals that received cells from vaccinated donors. These results may suggest that adoptive transfers influenced the course of infection despite the lack of significant differences in the viral loads among animals that received cells from vaccinated and mock-vaccinated donor animals.     

Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis

Proximal tubular epithelial cells from mice which develop autoimmune interstitial nephritis were found to express the nephritogenic target antigen, 3M-1. Anti-3M-1 mAbs (alpha 3M-1-Ab) were used to positively select for 3M-1-secreting tubular epithelium and, after stabilization in culture, this new cell line (MCT) was examined for the production of several moieties important to either immune interactions or to the development of extracellular matrix. Alkaline phosphatase-staining MCT cells also express epithelial growth factor receptors with a Kd of 0.87 nM and an epithelial growth factor receptor constant (Ro) of 2.1 X 10(4) receptors/cell. MCT culture supernatants contain greater amounts of laminin, and types IV and V procollagens compared to types I and III procollagens, and growing MCT cells on type I collagen matrix causes them to preferentially secrete even more type IV and V procollagen. The 30,000-Mr 3M-1 antigen could be immunoprecipitated from biosynthetically labeled MCT cell supernatants with alpha 3M-1-Ab. An identical-sized moiety was isolated by immunoaffinity chromatography from collagenase-solubilized mouse kidney tubular basement membranes. The 3M-1 antigen can be found on the MCT cell surface by radioimmunoassay, or deposited in a linear array in the extracellular matrix surrounding the MCT cells in culture by immunofluorescence. Mature messenger RNA species for both class I and class II major histocompatibility complex (MHC) molecules were detected by Northern hybridization, and their corresponding cell surface gene products were detected by cytofluorography of MCT cells stained with haplotype-specific antibodies. Both the cell surface 3M-1 and the small amounts of detected class II MHC molecules appear to be biologically functional, as MCT cells can support the proliferation of 3M-1-specific, class II MHC-restricted helper T cells in culture. These findings suggest that MCT cells provide all the necessary biological parameters for interfacing both as the target of a nephritogenic immune response, and as a potential source for new extracellular matrix which develops as a fibrogenic response to interstitial nephritis.     

Removal of blood group determinants from bovine erythrocyte membranes. 2. Degradation of ghosts by butanol and pyridine.

Ghosts produced from erythrocytes collected from six different cattle were degraded with butanol and pyridine. Of a total of 38 different antigenic determinants available for investigation among the six cows, F, V, J and L were the only specificities detected in the subfractions resulting from either method of degradation. After butanol degradation V, J and L antigens were found in the soluble protein fraction, while F was found in the insoluble protein. Pyridine digestion resulted in all four determinants being detected in the sialoprotein layer, while J was found in the lipoprotein as well. All antigens were relatively weak, being detected in inhibition strengths of 10.0 to 1.25 mg/ml.     

Linkage of bovine erythrocyte antigen loci B, C, L, S, Z, R' and T' and the serum protein loci post-transferrin 2 (PTF 2), vitamin D binding protein (GC) and albumin (ALB) to DNA microsatellite markers

Seven bovine erythrocyte antigen loci and three serum protein loci were tentatively assigned to chromosomes or synteny groups by linkage analysis to previously assigned microsatellite DNA markers. The erythrocyte antigen locus EAB was mapped to synteny group U27; EAC to chromosome 18, synteny group U9; EAL to chromosome 3, synteny group U6; EAS to chromosome 21, synteny group U4; EAZ to chromosome 10, synteny group U5; EAR' to chromosome 16, synteny group U1; and EAT' to chromosome 19, synteny group U21. The vitamin D binding protein (GC) and albumin (ALB) loci were assigned to chromosome 6, synteny group U15 and post-transferrin 2 (PTF 2) to chromosome 19, synteny group U21.     

Assessing the role of body mass and sex on apparent adult survival in polygynous passerines: a case study of cetti's warblers in central Portugal

Adult survival, an important fitness component, is usually 1) lower in lighter individuals due to their reduced ability to survive winter conditions compared to heavier ones, especially in resident species at northern temperate latitudes and 2) lower in females compared with males due to higher reproductive costs incurred by females. In this paper, a capture–mark–recapture dataset of 649 cetti's warblers Cettia cetti ringed seasonally at two wetlands in central Portugal over an 11‐yr period (2000–2010) was modelled in a multi‐state framework to examine the influence of these individual covariates on apparent adult survival, while controlling for the presence of transient individuals in our study area. The probability of change in mass state (ψ_{Light→Heavy}, ψ_{Heavy→Light}) during the annual cycle was also estimated. Overall, birds survived better during spring–summer (breeding/moulting periods) compared with autumn–winter, but there was no effect of body mass on apparent adult survival probability. The modelling detected a significant interaction between sex and season, in which resident females survived better than resident males in spring–summer (?_RF= 0.857 ± 0.117 and ?_RM= 0.698 ± 0.181) while the opposite pattern was found in autumn–winter (?_RM= 0.440 ± 0.086 and ?_RF= 0.339 ± 0.084). In addition, cetti's warblers had a tendency to lose mass in spring–summer (ψ_{Heavy → Light}= 0.560 ± 0.063) and to regain mass in autumn–winter (ψ_{Light→Heavy}= 0.701 ± 0.069). This pattern of body mass change during the annual cycle may reflect energetic costs to reproduction and moulting, and/or a response to increased starvation risk during winter. High body mass, however, did not increase adult survival in this population presumably due to the relatively mild winter weather prevailing in central Portugal. Survival estimates are more likely to be explained by important ecological and behavioural differences between the two sexes in polygynous passerines. Our results highlight that studies aiming to identify the main factors shaping survival and individual fitness in polygynous species should be conducted during different phases of their annual cycle.