Photoaffinity labeling and characterization of isolated inositol 1,3,4,5-tetrakisphosphate- and inositol hexakisphosphate-binding proteins.

We have isolated high affinity inositol (1,3,4,5)-tetrakisphosphate (IP4)- and inositol hexakisphosphate (IP6)-binding proteins from detergent-solubilized rat brain membranes using a P1-tethered IP4 derivative linked to an Affi-Gel support. To determine the identity, binding characteristics, and distribution of the individual IP4 recognition sites, we have synthesized an IP4 photoaffinity label probe, 125I-(D,L)-1-O-[N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]- IP4 (125I-ASA-IP4). Two apparently distinct IP4-binding proteins (IP4BP), isolated with the IP4 affinity column, display high affinity and selectivity for IP4 over inositol trisphosphate (IP3), inositol pentakisphosphate (IP5), and IP6. The first IP4-binding protein (IP4BP1) which has a KD for IP4 of 4 nM, is comprised of a protein at 182 kDa which is specifically photolabeled with high affinity by 125I-ASA-IP4. The second, IP4BP2, has an affinity for IP4 of 1.5 nM and contains proteins at 84 and 174 kDa, both of which are specifically photoaffinity labeled. A putative IP6-binding protein (IP6BP), also isolated with the IP4 affinity column, binds IP6 with a KD of 14 nM and comprises three proteins of 115, 105, and 50 kDa. The 115- and 105-kDa subunits, but not the 50-kDa subunit, specifically incorporate the photolabel. The IP4BP (182, 174, and 84 kDa) and IP6BP (115 and 105 kDa) proteins are specifically photolabeled in the crude membrane, partially purified, and purified fractions. These receptor-binding proteins vary in inositol phosphate specificity and in the effects of pH, Ca2+, and heparin on IP4 photoaffinity labeling. In addition, IP4BP and IP6BP are enriched in the brain but differ in their regional localizations within the brain.     

In situ localization of enzymes and mucin in normal rat colon embedded in plastic

Summary Several enzymes were investigated histochemically in the colons of normal male F344 rats in order to understand the function of different types of cells in this tissue. Serial methacrylate-embedded sections (2–4 µm) allowed the precise localizations of several enzymes including acid phosphatase, alkaline phosphatase, -glutamyl transpeptidase,N-acetyl--d-glucosaminidase (hexosaminidase), -naphthyl butyrate esterase and 5-nucleotidase. Sites reactive with periodic acid-Schiff were also localized. Gradients of enzyme activity were observed between caecum and rectum and/or from the luminal surfaces to the bases of the crypts for hexosaminidase, esterase and -glutamyl transpeptidase. To our knowledge this is the first histochemical demonstration of -glutamyl transpeptidase in normal rat colonic epithelial cells. The utilization of the methacrylate-embedding technique has revealed previously undescribed gradients of enzyme activity and has allowed the localization of enzyme activities not previously reported in normal rat colonic mucosa.     

Improved preparation of amyloid-beta peptides using DBU as Nalpha-Fmoc deprotection reagent.

Previous studies have shown the amyloid peptides, Abeta 1-40/42, to be exceptionally difficult to assemble by Fmoc-solid phase peptide synthesis due to the high hydrophobicity of the C-terminal segment and resulting on-resin aggregation. We found that the use of the stronger and more efficient base, DBU, at a concentration of 2% in DMF for Nalpha-Fmoc deprotection allowed substantially improved continuous flow solid phase assembly of the model peptide Abeta 29-40/42 fragments. This suggested that, at least for these sequences, incomplete deprotection was a greater problem than incomplete amino acid acylation. This base was then used during the synthesis of both Abeta 1-40 and Abeta 1-42, up to and including Ser8, from which point 20% piperidine in DMF was utilized so as to avoid potential aspartimide formation at Asp7. By this means, the deprotection efficiency through the difficult C-terminal portion of the sequence was much improved and resulted in increased availability of terminal amino groups for acylation. This simple strategy that obviates the need for special conditions significantly improved crude peptide quality and allowed considerable facilitation of subsequent purification.     

Mannitol metabolism of the epiphytic mangrove red alga Caloglossa leprieurii (Ceramiales): A long-term field study

The concentrations of mannitol and floridean starch were determined in a 1–year field study of the epiphytic red alga Caloglossa leprieurii (Montagne) J. Agardh from warm temperate waters of eastern Australia. Seasonal environmental data for air and water temperature, day length and rainfall were recorded. The mannitol content and the floridean starch content varied significantly between collections but no seasonal responses were observed, nor were the contents correlated with any of the abiotic factors. A possible function of the starch pool as a supply for respiratory substrates under emergent conditions is discussed. All data indicate that productivity and biomass of C. leprieurii are affected by short-term abiotic and/or biotic conditions rather than controlled directly by seasonally fluctuating environmental factors. In addition, the activity of three highly specific enzymes (mannitol-1–phosphate dehydroge-nase, mannitol-1–phosphatase, mannitol-dehydroge-nase) and one non-specific enzyme (hexokinase), all of which are involved in the mannitol cycle, were measured in cell-free extracts every 2 weeks. All enzymes showed marked changes in activity over the year, but again no clear patterns emerged, neither with season nor in relationship to one another. On the basis of the results here, C. leprieurii is considered to be a 'season responded rather than a ‘season anticipator’.     

Reduction of Experimental Salmonella and Campylobacter Contamination of Chicken Skin by Application of Lytic Bacteriophages

Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log₁₀ unit. Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log₁₀ units over 48 h. When the level of Salmonella contamination was low (< log₁₀ 2 per unit area of skin) and the MOI was 10⁵, no organisms were recovered. By increasing the number of phage particles applied (i.e., MOI of 10⁷), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach.     

First isolation and structural determination of cyclic beta-(1-->2)-glucans from an alga, Chlorella pyrenoidosa

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.     

The Specimen Dealer: Entrepreneurial Natural History in America's Gilded Age

The post-Civil War American natural history craze spawned a new institution – the natural history dealer – that has failed to receive the historical attention it deserves. The individuals who created these enterprises simultaneously helped to promote and hoped to profit from the burgeoning interest in both scientific and popular specimen collecting. At a time when other employment and educational prospects in natural history were severely limited, hundreds of dealers across the nation provided encouragement, specimens, publication outlets, training opportunities, and jobs for naturalists of all motivations and levels of expertise. This paper explores the crucial role that specimen dealers played in the larger natural history community. After briefly examining the development of local taxidermy shops in the mid-nineteenth century, it then traces the history of four large natural history dealerships established in the United States during the latter half of the century: Ward's Natural History Establishment, Frank Blake Webster's Naturalists' Supply Depot, Southwick & Jencks' Natural History Store, and Frank H. Lattin & Co. By the early twentieth century, changing tastes in interior design, the growth of the Audubon movement, and the dramatic expansion of alternate training and job opportunities for naturalists led many specimen dealers either to shift their emphasis or to shut their doors. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vasoactive intestinal peptide (VIP) prevents killing of virulent and phoP mutant Salmonella typhimurium by inhibiting IFN-gamma stimulated NADPH oxidative pathways in murine macrophages

Vasoactive intestinal peptide is an immunomodulator with great potential in the treatment of inflammatory pathology. In this study, we have examined the effect of VIP on the growth dynamics of virulent Salmonella enterica. Serovar typhimurium (S. typhimurium) 14028 and 4/74 and an avirulent mutant (14028 phoP) in a murine, macrophage cell line (J774.2). In contrast to standard growth dynamics, in which phoP mutants do not survive in macrophages, we show that VIP (10(-10) M) significantly enhances phoP growth over a 24 h post-infection period even when the cells are co-cultured with IFN-gamma. We examined the effect of VIP on the generation of NADPH-induced reactive oxygen species (ROS) in Salmonella-infected/IFN-gamma cultured J774 cells. VIP inhibited gp91 mRNA levels, gp91 protein and subsequent ROS. The importance of ROS in killing of Salmonella by J774 cells was highlighted by experiments in which ROS production by J774 cells was inhibited using a conventional inhibitor, N-acetyl-L-cysteine captopril (ACC) and in which Salmonella growth significantly increased. Our findings suggest that although VIP inhibits inflammatory pathways in myeloid cells it also promotes the growth of avirulent (phoP) mutants.