Proteins of the kidney microvillar membrane. Purification and properties of the phosphoramidon-insensitive endopeptidase (''endopeptidase-2'') from rat kidney.

A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3' position. The relationship of this membrane metalloendopeptidase to mouse meprin and human 'PABA peptidase' is discussed.     

Effects of airway tone and volume history on maximal expiratory flow in asthma

We assessed the difference between isovolumic maximal expiratory flows (Vmax) using maneuvers begun at mid-lung volumes, so-called partial expiratory flow-volume curves (P), vs. those begun at full inflation, so-called maximal expiratory flow-volume curves (M), in 10 asthmatic subjects before and following obstruction induced by isocapnic hyperpnea with cold air and before and after bronchodilation with a beta-agonist or antimuscarinic agent. Volume history effects were quantitated as an M-to-P ratio of Vmax at 30% vital capacity (M/P V30). Although M/P V30 was variable among patients at base line, there was a uniform increase in M/P V30 during constriction and a consistent decrease below base line after dilation. Blunting of induced obstruction with beta-agonists also diminished the increase in M/P V30. Antimuscarinics, despite equivalent bronchodilation, failed to alter the degree of obstruction induced by cold air or the increase in M/P V30 seen during obstruction. The level of airway tone, as indicated by specific resistance, related directly to the M/P V30. We conclude that the response of the asthmatic lung to a deep inhalation is relatively predictable when acute changes in airway tone are produced.     

Characterization of microsatellite loci for the Argentine ant,Linepithema humile,and their potential for analysis of colony structure in invading Hawaiian populations

We developed primers for five polymorphic microsatellite loci to analyse the genetic structure of colonies in an invading Argentine ant population located in Haleakala National Park on the island of Maui, Hawaii. Microsatellite loci were isolated using both a polymerase chain reaction‐based and a cloning‐based method. With a range of 3–18 alleles and expected levels of heterozygosity of 0.46–0.77, these loci provide useful markers for the detection of colony and population structure in new or expanding populations of this species.     

Vomiting as a diagnostic aid in acute ischaemic cardiac pain.

The incidence of vomiting before the administration of analgesics was studied in 109 patients admitted to hospital as emergencies with prolonged ischaemic cardiac pain. In transmural myocardial infarction (58 patients) the incidence was 43% (anterior infarction 58%, inferior infarction 41%). Of the 23 patients with myocardial necrosis but without transmural infarction (that is, those with diffuse or subendocardial necrosis) and the 28 with coronary insufficiency but no necrosis, only one patient in each group experienced vomiting. When vomiting occurs early in association with cardiac pain transmural infarction may be expected in 90% of patients.     

Selective Inhibition of Deoxyribonucleic Acid Synthesis by 2-Deoxyadenosine in the Blue-Green Bacterium Agmenellum quadruplicatum

Concentrations of deoxyadenosine which have little effect on net ribonucleic acid (RNA) synthesis or on increase in cell mass selectively inhibit deoxyribonucleic acid (DNA) synthesis in Agmenellum quadruplicatum. Exogenously supplied deoxyadenosine, at concentrations above 10 mug/ml, stimulates DNA degradation. These results are correlated with a rapid loss in viability. Over a narrow concentration range (6-15 mug/ml), deoxyadenosine impairs the division process and induces the formation of elongated cells. Low exogenous concentrations of deoxyadenosine are readily incorporated into both DNA and RNA, with the major portion as DNA.