Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Dynamic compression counteracts IL-1β induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs

Background  

Nitric oxide and prostaglandin E₂ (PGE₂play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1β and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Cytoplasmic organization and quantitation of microtubules in bovine mammary epithelial cells during lactation and involution

Summary Ultrastructural examination of milk secretory cells from lactating bovine mammary gland revealed presence of numerous microtubules in the apical and paranuclear cytoplasm, particularly in the vicinity of Golgi components. Most microtubules were oriented perpendicular to the apical plasma membrane and appeared to form a framework around Golgi dictyosomal elements and secretory vesicles. In comparison, non-secretory cells obtained from involuting glands displayed few microtubules and these were randomly located throughout the cytoplasm with no particular orientation.     

Analysis of responses to human synthetic adrenomedullin and calcitonin gene-related peptides in the hindlimb vascular bed of the cat

Vasodilator responses to human adrenomedullin (hADM), a newly discovered hypotensive peptide, human calcitonin gene-related peptide- (hCGRP-) and hCGRP-, which share structural homology with hADM, were compared in the hindlimb vascular bed of the cat under constant flow conditions. Injections of hADM (0.003-1 nmol), hCGRP-, and hCGRP- (0.003-0.3 nmol) into the perfusion circuit caused dose-related decreases in hindlimb perfusion pressure. Vasodilator responses to hCGRP- and hCGRP- were similar in potency and duration, and the doses of hCGRP- and hCGRP- required to reduce hindlimb perfusion pressure 40 mm Hg (ED40 mm Hg) were significantly lower than the ED40 mm Hg for hADM. The duration of the hindlimb vasodilator responses to hCGRP- and hCGRP- were significantly longer than the duration of the response to hADM. Amylin, a peptide that shares structural homology with ADM and with CGRP, had no significant effect on hindlimb perfusion pressure when injected in doses up to 1 nmol. Decreases in hindlimb perfusion pressure in response to hADM, hCGRP-, and hCGRP- were not altered by L-N5-(1-iminoethyl)-ornithine (L-NIO) in a dose of the nitric oxide synthase inhibitor that decreased the vasodilator response to acetylcholine or by the cyclooxygenase inhibitor, meclofenamate, in a dose that decreased the vasodilator response to archidonic acid. The present data demonstrate that hADM, hCGRP-, and hCGRP- have potent, but relatively short-lasting, vasodilator activity, and that vasodilator responses are not dependent on the release of nitric oxide or vasodilator prostaglandins in the hindlimb vascular bed of the cat.     

Primary culture of bovine mammary acini on a collagen matrix

Lactating bovine mammary epithelial acini were isolated and primary culture on rat tail attached collagen gels are described. Acini rapidly attach to the gels and morphologically change little over days of culture under the culture conditions described herein. Cells release lactose, alpha-lactalbumin and alpha-s1 casein over a 6-day period. A new HPLC method for measuring lactose in mammary cell culture media is described. Comparisons of acini cultures with individual cell cultures show acini to be 1.5-5 times more productive than cells in secreting lactose and casein, respectively.     

Effect of supplemental light on growth,prolactin, progesterone and luteinizing hormone in water buffalo (Bubalus bubalis)

Fifty non-pregnant Surti buffalo heifers aged between 17 and 42 months (n=24, <24 months;n=26, >24 months) were randomly assigned to groups subject to either natural daylight +4h supplemental light (n=25) or natural day light (n=25), to study changes in growth, serum prolactin (Prl), progesterone (P₄) and luteinizing hormone (LH) to supplemental lighting. Ambient temperatures (T) and relative humidity (RH) generally were >27° C and <70% during the day-time, respectively. Light-supplemented heifers had 16.2 kg net body weight (BW) gain at 9 weeks compared to 20.8 kg for controls, but higher mean Prl after 6.5 weeks (P<0.01), and higher P₄ (0.41 vs 0.19 ng/ml;P<0.06) than control heifers. Older heifers had 39.7% greater BW (P<0.01), but a net 4.3% BW gain compared to a 10.1% gain for younger heifers at 10 weeks. Older, light-supplemented heifers had higher mean P₄ (0.63 vs 0.19 ng/ml;P<0.07) than the other groups. These weight and hormonal changes suggest that 4 h supplemental light can alter growth and endocrine function in buffaloes under similar planes of nutrition. While light supplementation did not have a positive effect on body wieght during the 10 week study, body weight and endocrine changes due to supplemental light may be important factors for initiation of reproductive cyclicity.     

Activation of Gelatinase A (72-kDa Type IV Collagenase) Induced by Monensin in Normal Human Fibroblasts

In monolayer culture, fibroblasts secrete all matrix metalloproteinases, including gelatinase A (72-kDa type IV collagenase), as inactive zymogens. Whereas limited proteolysis by plasmin or other matrix metalloproteinases (MMPs) can accomplish the extracellular activation of other proenzymes in this family, gelatinase A proenzyme is uniquely refractory to cleavage by such proteinases. Previously it has been shown that fibroblasts cultured in the presumably more physiologic culture milieu of a type I collagen lattice can be induced to secrete active gelatinase A. In monolayer culture, however, the plant lectin concanavalin A will induce gelatinase A activation. Here we show that in monolayer culture activation of gelatinase A by normal fibroblasts is also induced by the sodium ionophore monensin. The monensin response is dose-dependent, time-dependent, requires protein synthesis, and is specific to gelatinase A among the secreted matrix metalloproteinases. The activator appears to be associated with cell membranes and may be membrane-type matrix metalloproteinase 1(MT-MMP1). Both mRNA and immunodetectable protein of MT-MMP1 are increased with monensin treatment while message for the protein inhibitor of gelatinase A, TIMP-2, is unchanged. The monensin-induced signal transduction pathway leading to gelatinase activation in monolayer culture appears to be different from the integrin-mediated pathway operative in the collagen lattice system. The tyrosine kinase inhibitor genistein blocks monensin activation of gelatinase A in monolayer culture. In contrast, genistein has no effect on proenzyme activation in the collagen lattice. Likewise, the cyclooxygenase inhibitor indomethacin abrogates the monensin effect in monolayer culture and can be reversed by addition of exogenous prostaglandin E₂(PGE₂). Neither indomethacin nor PGE₂affects activation of gelatinase A in the collagen lattice.     

A molecular rotor as viscosity sensor in aqueous colloid solutions

BACKGROUND: Molecular rotors exhibit viscosity-dependent quantum yield, allowing non-mechanical determination of fluid viscosity. We analyzed fluorescence in the presence of viscosity-modulating macromolecules several orders of magnitude larger than the rotor molecule. METHOD OF APPROACH: Fluorescence of aqueous starch solutions with a molecular rotor in solution was related to viscosity obtained in a cone-and-plate viscometer. RESULTS: In dextran solutions, emission intensity was found to follow a power-law relationship with viscosity. Fluorescence in hydroxyethylstarch solutions showed biexponential behavior with different exponents at viscosities above and below 1.5 mPa s. Quantum yield was generally higher in hydroxyethylstarch than in dextran solutions. The power-law relationship was used to backcalculate viscosity from intensity with an average precision of 2.2% (range of -5.5% to 5.1%). CONCLUSIONS: This study indicates that hydrophilic molecular rotors are suitable as colloid solution viscosity probes after colloid-dependent calibration.     

Presynaptic modulation of evoked NE release contributes to sympathetic activation after pressure overload

Activation of the sympathetic nervous system is well documented in heart failure. Our previous studies demonstrated an increase in evoked norepinephrine (NE) release from left ventricle (LV) slices at 10 days of pressure overload. The purpose of this study was to test the hypothesis that presynaptic modulation of NE release contributes to sympathetic activation after pressure overload. We examined the functional status of the presynaptic alpha(2)- and beta(2)-receptors and ANG II subtype 1 (AT(1)) receptors in LV slices from 10-day aortic constricted (AC) and sham-operated (SO) rats. Evoked (3)H overflow from LV slices preloaded with [(3)H]NE was increased in AC rats. The alpha(2)-agonist UK-14,304 decreased evoked (3)H overflow with no differences between groups. The beta(2)-agonist salbutamol increased evoked (3)H overflow with greater sensitivity in slices from AC rats. The beta-antagonist propranolol decreased evoked (3)H overflow from LV slices of AC rats but not controls. ANG II increased evoked (3)H overflow with greater sensitivity in slices from AC rats. These data support the hypothesis that aberrant presynaptic modulation of catecholamine release contributes to sympathetic activation after pressure overload.