Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH(4))(2)SO(4) precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen-CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor-plasminogen complex was determined to be approx. 3mum at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme-inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26mum) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor-plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.     

Hyperparathyroidism in pregnancy: case report and review of the literature.

The apparent incidence of hyperparathyroidism (HPT) is low in pregnancy but will likely increase now that more asymptomatic HPT is being diagnosed. However, since the serum calcium levels are decreased in pregnant women, mild primary HPT may go unrecognized. In untreated cases of HPT, complications during pregnancy or during the neonatal period have included spontaneous abortion, stillbirth, neonatal death, neonatal tetany and hypercalcemia. A review of the literature indicates a substantial improvement in fetal outcome when parathyroidectomy is done during pregnancy, as in the case reported here. Therefore, parathyroidectomy is the treatment of choice when the diagnosis is made during pregnancy, although oral phosphate therapy may be an alternative if surgery is contraindicated.     

A biometric study of some hybridizing Crataegus populations in Denmark

Crataegus curvisepala Lindm., C. laevigata (Poiret) DC. and C. monogyna Jacq. (Rosaceae) form hybrid complexes in Denmark due to introgression. C. palmstruchii Lindm. seems to be variously introgressed individuals of C. laevigata. C. eremitagensis Raunk., C. raavadensis Raunk. and C. schumacheri Raunk. apparently belong to C. curvisepala x laevigata. The delimitation between C. curvisepala x laevigata and C. laevigata x monogyna is discussed.     

Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards

Determination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time-consuming and inaccurate. Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and the sample and used to indicate the DNA content (DNA ratio) is, however, very sensitive to changes in the zero level adjustment of the flow cytometer. If two internal standards are used the DNA ratio becomes independent of the zero level. Rainbow trout red blood cells (TRBC) have a DNA content of 80% of human diploid cells. A mixture of CRBC and TRBC was prepared and stored in small aliquots at -80 degrees C. This mixture was added to the sample before staining. The day-to-day variation of the DNA ratio obtained by use of the two standards was smaller than that obtained by CRBC alone. The possibility of sex related differences in DNA content of CRBC and TRBC was examined. The results indicated that a new batch of standards should be tested against the old batch to avoid the introduction of a systematic error.     

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.     

Effect of the organophosphorous insecticide, chlorpyrifos (Dursban), on growth, fecundity and mortality of Biomphalaria alexandrina and on the production of Schistosoma mansoni cercariae in the snail.

Exposure of Biomphalaria alexandrina to sublethal concentrations (0.125, 0.25 and 0.05 ppm) of the organophosphorous insecticide, chlorpyrifos (Dursban), induced a reduction in egg production and egg hatchability. Exposure of Schistosoma mansoni miracidia to the insecticide (60 min, 0.50 ppm) prior to infection of B. alexandrina did not affect the subsequent production of cercariae. However, exposure of S. mansoni-infected snails to the insecticide until day 55, from day 20 to day 62 and from day 35 to 62 following infection resulted in blockage of cercarial shedding. Cercarial shedding commenced in some snails when the treatment stopped. Exposure to the insecticide in concentrations of 0.125 and 0.25 ppm during the first 20 days following infection did not affect the subsequent production of cercariae, but exposure to 0.5 ppm during the first 20 days affected markedly the production of cercariae due to a high snail mortality. The findings indicate that the cercaria is the target stage for the activity of chlorpyrifos on the intramolluscan larval development. It is suggested that S. mansoni cercarial production in B. alexandrina may be a useful system for monitoring the effect of low concentrations of pesticides on the aquatic environment, and that the ability by chemical means to interrupt the cercarial production might be a useful tool in further analyses of important aspects of the snail/parasite relationship.     

Renal tubule gp330 is a calcium binding receptor for endocytic uptake of protein.

gp330, a large glycoprotein located in renal proximal tubules, has sequence similarities with the low-density lipoprotein receptor and the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. The 40 KD human alpha 2-macroglobulin receptor-associated protein is a newly discovered heparin binding protein homologous to a major rat Heymann nephritis factor and exhibiting high affinity binding to the alpha 2-macroglobulin receptor. The present study shows by ligand blotting, light and electron microscopic autoradiography, and cytochemistry that gp330 located in coated apical membrane regions of the rat proximal tubule strongly binds the 40 KD protein. Furthermore, 45Ca2+ blotting experiments disclosed gp330 as a quantitatively important Ca2+ binding protein in renal cortex. Binding of 125I-labeled 40 KD protein to electroblotted gp330 and to coated apical membrane regions in sections of renal proximal tubules was abolished by excess unlabeled 40 KD protein, heparin, and EDTA. The endocytic properties of gp330 were investigated by in vivo microperfusion of rat proximal tubules. After 6 min, 125I-labeled 40 KD protein was mainly found in endocytic vacuoles and later accumulated in lysosomes. These data demonstrate that gp330 is a Ca2+ binding receptor for endocytosis of protein and is functionally related to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Furthermore, our results demonstrate the usefulness of semi-thin and ultra-thin cryosections in studies of ligand binding and subcellular localization of receptors with autoradiographic techniques.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Ammonia volatilization after injection of anhydrous ammonia into arable soils of different moisture levels

Laboratory experiments have shown appreciable losses of ammonia after injection of anhydrous ammonia into dry and wet soils. In this study losses of ammonia injected into a moist (tension 10 kPa), dry (tension 160 kPa) and a wet (tension 1.6 kPa) sandy loam were measured under field conditions using wind tunnels. Losses were insignificant from a moist soil. However losses from a dry and a wet soil were 20% and 50% of injected ammonia, respectively. From the dry soil, losses of gaseous ammonia took place within the first hours after injection, which indicates a rapid transport through cracks and voids. From the wet soil, 20% of the injected ammonia was lost more gradually between 6 h and 6 d. This indicates that upward movement of water due to evaporation may be the cause of these ammonia losses which proceeded for longer periods.