lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.     

Storage of marine fish erythrocytes in liquid nitrogen

The preservation of erythrocytes from cod ( Gadus morhua ), saithe ( Pollachius virens ) and mackerel ( Scomber scombrus ) at −196° C was studied using dimethyl sulphoxide (DMSO) as a cryoprotectant. Erythrocyte recoveries of greater than 90% were obtained from all species and cod erythrocytes were stored for eighteen months with insignificant lysis. Larger quantities of blood were stored by removal of plasma from citrated blood prior to the addition of DMSO solution, and by storage of pelleted frozen blood in aluminium canisters in liquid nitrogen. Maximum recoveries of washed intact erythrocytes required thawing of pellets in 125% DMSO solution and washing with buffer containing decreasing concentrations of DMSO. Washed erythrocytes kept at 4° for at least two days showed little haemolysis, were morphologically similar to fresh erythrocytes and equally susceptible to the δ-haemolysin of Staphylococcus aureus .     

An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells

A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.     

Kininogen and Kinin in Experimental Spinal Cord Injury

Activation of the kallikrein-kinin system has been implicated in the pathogenesis of vasogenic brain edema and posttraumatic vascular injury. We determined the levels of kininogen and kinin in an experimental spinal cord injury model in the rat. Kininogen content in traumatized cord segments increased in a time-dependent manner. Western blot analysis showed that the kininogen in traumatized cord comigrates with 68K low-molecular-weight kininogen or T-kininogen. Trypsin treatment of the kininogen in traumatized cord released both bradykinin and T-kinin, which were separated by HPLC and quantified with a kinin radioimmunoassay. Endogenous kinin levels in the frozen spinal cord also increased up to 40-fold 2 h after injury as compared with controls. The results demonstrate an increased accumulation of kininogen and its conversion to vasoactive kinins in experimental spinal cord injury.     

Inhibition of rat tissue kallikrein gene family members by rat kallikrein-binding protein and alpha 1-proteinase inhibitor.

The regulation of tissue kallikrein activity by plasma serine proteinase inhibitors (serpins) was investigated by measuring the association rate constants of six tissue-kallikrein family members isolated from the rat submandibular gland, with rat kallikrein-binding protein (rKBP) and alpha 1-proteinase inhibitor (alpha 1-PI). Both these serpins inhibited kallikreins rK2, rK7, rK8, rK9 and rK10 with association rate constants in the 10(3)-10(4) M-1.s-1 range, whereas only 'true' tissue kallikrein rK1 was not susceptible to alpha 1-PI. This results in slow inhibition of rK1 by plasma serpins, which could explain why this kallikrein is the only member of the gene family identified so far that induces a transient decrease in blood pressure when injected in minute amounts into the circulation.     

Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor.

In order to determine the effect of calcium mobilization on mitogen-activated protein (MAP) kinase activation, we have treated human foreskin fibroblasts (HSWP cells) and human epidermal carcinoma (A431) cells with thapsigargin. Intracellular free calcium was monitored by single cell image analysis using fura-2 and correlated with MAP kinase stimulation as assessed by immunoprecipitation, kinase renaturation assays and immunoblotting. Thapsigargin stimulated the 44- and 42-kDa MAP kinase isozymes in both cell types with kinetics that were slightly delayed relative to enzyme stimulated by epidermal growth factor. Removal of external calcium did not significantly affect the activation of the MAP kinases by thapsigargin, indicating that intracellular calcium mobilization is sufficient to stimulate the enzymes. However, treatment of cells with EGTA under conditions which deplete both intra- and extracellular calcium inhibited stimulation by thapsigargin but not epidermal growth factor. Stimulation of the MAP kinases by the calcium ionophore ionomycin paralleled the activation observed with thapsigargin in both calcium-containing and calcium-free conditions. These results indicate that there are at least two independent pathways for stimulation of MAP kinase: one that is dependent on intracellular calcium mobilization, and one that is mediated by the tyrosine kinase epidermal growth factor receptor and is calcium-independent.     

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.     

Arachidonic acid inhibits Cl secretion in cultures of dog tracheal epithelium.

We studied the effects of arachidonic acid (AA) on Cl secretion across primary cultures of dog tracheal epithelium. Cell sheets showed mean values for baseline short-circuit current (Isc) and transepithelial resistance of 33.8 muA/cm2 and 360 omega.cm2 (n = 44). AA (5 x 10(-5) M) added to both sides increased Isc by 27.8 +/- 5.2 muA/cm2 (mean +/- SE, n = 8), and elevated intracellular cAMP levels. In the presence of 5 x 10(-6) M of both indomethacin (INDO) and nordihydroguaiaretic acid (NDGA) (inhibitors of cyclooxygenase and lipoxygenase, respectively), AA reduced Isc by 4.4 +/- 0.6 muA/cm2 (n = 10) without changing cAMP. Both INDO and NDGA were necessary to abolish the Isc increase in response to AA. The effects of AA on Isc were unaffected by amiloride. In the presence of INDO and NDGA, isoproterenol (ISO) raised cAMP and increased Isc by 27.6 +/- 4.3 (transient) and 12.8 +/- 3.2 muA/cm2 (sustained) (n = 9). With AA present as well as INDO and NDGA, the transient and sustained responses to ISO were significantly reduced to 13.2 +/- 2.4 and 3.9 +/- 0.8 muA/cm2 (n = 10), respectively; the increase in cAMP was unaltered. AA approximately halved baseline efflux of 125I from confluent cell sheets in high K medium and reduced the isoproterenol-induced increase in efflux to 20% of control. These data are consistent with the reported inhibitory effect of AA on apical membrane chloride channels.