Isolation of peroxisomes from frozen human liver samples.

This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen at -70 degrees C. Purification of these peroxisomes was obtained by a combination of two subcellular fractionation techniques: differential centrifugation and isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency measurements and by ultrastructural observation. The procedure described here may be useful for the isolation of other subcellular organelles from frozen human samples.     

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.     

Structures of poly(dA-dT, ip5dU) containing various small amounts of the antiherpetic 5-isopropyl-2'-deoxyuridine.

Three different concentrations of the antiherpetic agent 5-isopropyl-2'-deoxyuridine (ip5dU) were introduced into the synthetic DNA poly(dA-dT) to analyze resulting copolymers by electron microscopy, UV absorption and CD spectroscopy. The poly(dA-dT, ip5dU) containing 1.3 and 4.3% ip5dU did not much differ from the parent poly(dA-dT) but poly (dA-dT, ip5dU) with 7.1% ip5dU behaved in an unusual way. Results are explained by the notion that if bulky isopropyls occur sufficiently close to each other then stable hairpins protruding from the double helix are formed, presumably to accommodate the ip5dU-s into the loops.     

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum bv. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.