Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.     

Regulatory role of B cells in a murine model of allergic airway disease

Mice sensitized to OVA and subjected to acute OVA aerosol exposures develop allergic airway disease (AAD). However, chronic continuous Ag exposure results in resolution of AAD and the development of local inhalational tolerance (LIT). Because we have previously observed the persistence of B cells in the bronchoalveolar lavage (BAL) and hilar lymph nodes (HLN) at the resolution stage of this model, we investigated the role of B cells in the modulation of AAD. Although B cell-deficient mice developed LIT, adoptive transfer of HLN B cells from LIT mice to OVA-sensitized recipients resulted in attenuated AAD following subsequent OVA aerosol exposure, as determined by reduced BAL leukocytosis and eosinophilia, decreased tissue inflammation, and absent methacholine hyper-responsiveness. In similar adoptive transfer studies, HLN B cells from AAD mice were without effect. The protection transferred by LIT HLN B cells was Ag specific and was associated with accumulation of Foxp3(+) T regulatory cells regionally in BAL and HLN, but not systemically in the spleen. Fluorescent labeling of LIT HLN B cells before adoptive transfer demonstrated that these cells had the capacity to migrate to local inflammatory sites. In vitro assessment demonstrated that the LIT HLN B cells exerted this regulatory effect via TGF-beta induced conversion of CD4(+)CD25(-) T effector cells into functionally suppressive CD4(+)CD25(+)Foxp3(+) T regulatory cells. These findings illustrated a novel regulatory role for regional B cells in AAD and suggested a possible contributory role of B cells, along with other cell types, in the establishment of LIT.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

Chinch bug-resistant buffalograss: an investigation of tolerance, antixenosis, and antibiosis

Choice and no-choice studies were conducted to determine the categories (antibiosis, antixenosis, and tolerance) of resistance of four buffalograsses (NE91-118, 'Bonnie Brae', 'Cody', and 'Tatanka') previously identified as resistant to the western chinch bug, Blissus occiduus Barber. Antibiosis studies found no significant differences in western chinch bug fecundity, nymphal development, or survival among the resistant and susceptible buffalograsses. Tolerance studies indicated that NE91-118, Cody, and Tatanka exhibited moderate-to-high levels of tolerance based on western chinch bug damage ratings and plant height, whereas Bonnie Brae exhibited moderate-to-low levels of tolerance. Choice studies indicated the presence of antixenosis in NE91-118, whereas Cody and Tatanka showed little or no antixenosis. Scanning electron microscopy was used to disclose morphological differences between NE91-118 (resistant) and '378' (susceptible). The epicuticular wax structures and trichome densities were similar between 378 and NE91-118, suggesting that morphological structures do not contribute to NE91-118 antixenosis.     

Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases

For DNA polymerases to proofread a misincorporated nucleotide, the terminal 3-4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. Genetic and biochemical studies of the bacteriophage T4 DNA polymerase revealed that a prominent beta-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. We present here further mutational analysis of the loop region of the T4 DNA polymerase beta-hairpin structure, which provides additional evidence that residues in the loop, namely, Y254 and G255, are important for DNA replication fidelity. The mechanism of strand separation was probed in in vitro reactions using the fluorescence of the base analogue 2-aminopurine (2AP) and mutant RB69 DNA polymerases that have modifications to the beta hairpin, to the exonuclease active site, or to both. We propose from these studies that the beta hairpin in the exonuclease domain of the T4 and RB69 DNA polymerases functions to facilitate strand separation, but residues in the exonuclease active center are required to capture the 3' end of the primer strand following strand separation.     

Ranging behavior of Mahale chimpanzees: a 16 year study

We have analyzed the ranging patterns of the Mimikire group (M group) of chimpanzees in the Mahale Mountains National Park, Tanzania. During 16 years, the chimpanzees moved over a total area of 25.2 or 27.4 km², as estimated by the grid-cell or minimum convex polygon (MCP) methods, respectively. Annually, the M group used an average of 18.4 km², or approximately 70 %, of the total home-range area. The chimpanzees had used 80 % of their total home range after 5 years and 95 % after 11 years. M group chimpanzees were observed more than half of the time in areas that composed only 15 % of their total home range. Thus, they typically moved over limited areas, visiting other parts of their range only occasionally. On average, the chimpanzees used 7.6 km² (in MCP) per month. Mean monthly range size was smallest at the end of the rainy season and largest at the end of the dry season, but there was much variability from year to year. The chimpanzees used many of the same areas every year when Saba comorensis fruits were abundant between August and January. In contrast, the chimpanzees used several different areas of their range in June. Here range overlap between years was relatively small. Over the 16 years of the study we found that the M group reduced their use of the northern part of their range and increased their frequency of visits to the eastern mountainous side of their home range. Changes in home-range size correlated positively with the number of adult females but not with the number of adult males. This finding does not support a prediction of the male-defended territory model proposed for some East African chimpanzee unit-groups.