Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.     

Solution structure of contryphan-R, a naturally occurring disulfide-bridged octapeptide containing D-tryptophan: comparison with protein loops.

Contryphan-R is a disulfide-constrained octapeptide containing a D-tryptophan that was isolated recently from venom of the cone shell Conus radiatus. The polypeptide is present in two forms in solution due to cis-trans isomerization at hydroxyproline 3. The solution structure of the major form of this unusual polypeptide, determined from NMR data, consists of a well-defined fold containing a non-hydrogen-bonded chain reversal from Gly1 to Glu5, which includes a cis-hydroxyproline and a D-Trp, and a type I beta-turn from Glu5 to Cys8. The presence of a putative salt bridge between the Glu5 carboxyl group and the N-terminal ammonium group is investigated by using various solvation models during energy minimization and is compared with the results of a pH titration. A comparison of the structure of contryphan-R with other cyclic peptide structures highlights some of the key structural determinants of these peptides and suggests that the contryphan-R fold could be exploited as a scaffold onto which unrelated protein binding surfaces could be grafted. Comparison with small disulfide-bridged loops in larger proteins shows that contryphan-R is similar to a commonly occurring loop structure found in proteins.     

Detection of nuclear lamin B epitopes in oocyte nuclei from mice, sea urchins, and clams using a human autoimmune serum

Somatic nuclei typically contain two or three major proteins, the lamins A, B, and C or their antigenically related equivalents, interspersed between the chromatin and its attachment site, the inner nuclear membrane. The late oocyte nuclear envelopes of the previously investigated Xenopus and Spisula germinal vesicles, however, have no chromatin attached and only one lamin-like protein. Since mouse and sea urchin germinal vesicles have chromatin attached, we tested them for the possible presence of more than one lamin. In both species we found two different lamins incorporated in their nuclear envelope structure. One lamin is recognized by anti-lamin B and the other by anti-lamin AC antibodies. Spisula germinal vesicles were found to contain not only the nuclear envelope-bound lamin (clamin), but also a 65-kDa protein cross-reactive with anti-lamin B antibodies. This protein is present unattached to any structure and is apparently soluble. Our findings provide a possible explanation of the early presence of lamin B in pronuclei of mouse and sea urchin contrary to the late appearance of a lamin B equivalent in amphibian embryos. In Spisula, as in Xenopus, the presence of a lamin B equivalent could not be documented in the nuclear envelopes of early embryos, indicating that a separate lamin B equivalent is not essential for chromatin binding to the envelope in these species during early embryogenesis. The results also indicate that the nuclear complement of structural proteins might vary substantially in the same cell type of different species.     

Muscle fiber structure in an aging long-lived seabird,the black-legged kittiwake (Rissa tridactyla)

Many long-lived animals do not appear to show classic signs of aging, perhaps because they show negligible senescence until dying from “catastrophic” mortality. Muscle senescence is seldom examined in wild animals, yet decline in muscle function is one of the first signs of aging in many lab animals and humans. Seabirds are an excellent study system for physiological implications of aging because they are long-lived animals that actively forage and reproduce in the wild. Here, we examined linkages between pectoralis muscle fiber structure and age in black-legged kittiwakes (Rissa tridactyla). Pectoralis muscle is the largest organ complex in birds, and responsible for flight and shivering. We obtained and fixed biopsies from wild black-legged kittiwakes of known age. We then measured muscle fiber diameter, myonuclear domain and capillaries per fiber area among birds of differing ages. All muscle parameters were independent of age. Number of nuclei per mm of fiber showed a positive correlation with muscle fiber cross-sectional area, and myonuclear domain increased with muscle fiber diameter. Thus, as muscle fibers increased in size, they may not have recruited satellite cells, increasing the protein turnover load per nuclei. We conclude that senescence in a long-lived bird with an active lifestyle, does not entail mammalian-like changes in muscle structure.     

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.     

Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells

Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12–18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.