Estimation of domain length of chicken erythrocyte chromatin

Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease. The length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy. These results were fitted with a theoretical distribution which was an outcome of the domain model proposed by Igo-Kemenes and Zachau (Igo-Kemenes, T. and H.G. Zachau (1977) Cold Spring Harbour Symp. Quant. Biol. 42, 109–118). A domain length of 45 kbp was obtained.     

Impaired aldosterone secretion from dispersed adrenal capsular cells of chronically alpha-MSH-treated rats

To investigate the chronic effects of alpha-melanocyte-stimulating hormone (alpha-MSH) on aldosterone secretion, synthetic alpha-MSH (8 micrograms/day) was infused in Sprague-Dawley rats by miniosmotic pumps for 5 days. Saline was infused in equivalent volume for 5 days using the same type of pumps in the control group of rats. Aldosterone secretion from the capsular cells of the two groups was examined in the basal state and in response to various stimuli of aldosterone secretion. Aldosterone secretion in vitro from the alpha-MSH-treated rats was significantly impaired in response to all stimuli tested including cyclic AMP, suggesting an intracellular defect in aldosterone synthesis in that group. These results are similar to those observed after chronic adrenocorticotropin administration.     

Increased variation in sex organ positions can increase reciprocity and pollination success in heterostylous plant populations

The reciprocal position of sexual organs in complementary floral morphs is central to our understanding of heterostyly. Reciprocity indices are used to quantify the spatial match between complementary sex organs, but previous indices fail to appropriately account for intra-population variation in sex organ positions. In this study, we examine how an increase in intra-population variation in sex organ heights affects reciprocity and consequently reproductive success. We formulated a reciprocity index that incorporates this variation and asked if estimates of reciprocity can predict reproductive success in naturally occurring heterostylous populations. We developed a reciprocity index that assumed pollen transfer success equalled one for a perfectly matched stigma–anther pair, and decreased to zero with increasing mismatch. We examined the relationship between intra-population variation in organ position and reciprocity, compared previously proposed indices using simulated populations and empirical data from natural populations, and tested the ability of the indices to predict reproductive success. We observed that when differences between mean complementary sex-organ heights are small, increasing intra-population variation in heights resulted in a decrease in reciprocity. However, when this difference is larger, reciprocity increased, reached a peak, and then decreased with increasing variation. Previous indices failed to capture this behavior. Seed set was positively related to reciprocity for our index. These results challenge the current understanding that increasing variation in sex-organ heights will always decrease reciprocity in heterostylous populations. This may help explain why heterostylous systems exhibit and tolerate high amounts of intra-population variation in sex organ heights.     

The Bordetella pertussis Bps polysaccharide enhances lung colonization by conferring protection from complement‐mediated killing

Bordetella pertussis is a human‐restricted Gram‐negative bacterial pathogen that causes whooping cough or pertussis. Pertussis is the leading vaccine preventable disease that is resurging in the USA and other parts of the developed world. There is an incomplete understanding of the mechanisms by which B. pertussis evades killing and clearance by the complement system, a first line of host innate immune defence. The present study examined the role of the Bps polysaccharide to resist complement activity in vitro and in the mouse respiratory tract. The isogenic bps mutant strain containing a large non‐polar in‐frame deletion of the bpsA–D locus was more sensitive to serum and complement mediated killing than the WT strain. As determined by Western blotting, flow cytometry and electron microscopic studies, the heightened sensitivity of the mutant strain was due to enhanced deposition of complement proteins and the formation of membrane attack complex, the end‐product of complement activation. Bps was sufficient to confer complement resistance as evidenced by a Bps‐expressing Escherichia coli being protected by serum killing. Additionally, Western blotting and flow cytometry assays revealed that Bps inhibited the deposition of complement proteins independent of other B. pertussis factors. The bps mutant strain colonized the lungs of complement‐deficient mice at higher levels than that observed in C57Bl/6 mice. These results reveal a previously unknown interaction between Bps and the complement system in controlling B. pertussis colonization of the respiratory tract. These findings also make Bps a potential target for the prevention and therapy of whooping cough.     

A Pit-1 Binding Site Adjacent to E-box133 in the Rat PRL Promoter is Necessary for Pulsatile Gene Expression Activity

Recent evidence reveals that prolactin gene expression (PRL-GE) in mammotropes occurs in pulses, but the molecular process(es) underlying this phenomenon remains unclear. Earlier, we have identified an E-box (E-box133) in the rat PRL promoter that binds several circadian elements and is critical for this dynamic process. Preliminary analysis revealed a Pit-1 binding site (P2) located immediately adjacent to this E-box133 raising the possibility that some type of functional relationship may exist between these two promoter regions. In this study, using serum shocked GH₃ cell culture system to synchronize PRL-GE activity, we determined that Pit-1 gene expression occurred in pulses with time phases similar to that for PRL. Interestingly, EMSA analysis not only confirmed Pit-1 binding to the P2 site, but also revealed an interaction with factor(s) binding to the adjacent E-box133 promoter element. Additionally, down-regulation of Pit-1 by siRNA reduced PRL levels during pulse periods. Thus, using multiple evidences, our results demonstrate clearly that the Pit-1 P2 site is necessary for PRL-GE elaboration. Furthermore, the proximity of this critical Pit-1 binding site (P2) and the E-box133 element coupled with the evidences of a site-to-site protein interactions suggest that the process of PRL-GE pulse activity might involve more dynamic and intricate cross-talks between promoter elements that may span some, or all, of the proximal region of the PRL promoter in driving its pulsatile expression.     

Novel tricyclic aminoacetyl and sulfonamide inhibitors of Ras farnesyl protein transferase

Novel tricyclic FPT inhibitors with submicromolar FPT activity are described. Greatly enhanced FPT activity is realized with phthaloyl derivatized amino compound 2k, which showed FPT inhibitory activity of IC₅₀ = 0.66 μM. Sulfonamides 5g and 50 were also found to be potent FPT inhibitor. SAR resulting from a variety of tricyclic amino acids and sulfonamide derivatives is discussed.