FV blood group and haemoglobin type versus haematological and blood chemical parameters in young Swiss bulls

A relationship between the FV blood group phenotype and 4 out of 45 haematological and blood chemical parameters--red cell number, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), and serum iron--has been demonstrated in young bulls of three Swiss cattle breeds. There was also a relationship between haemoglobin type and 7 out of 45 haematological and blood chemical parameters (haemoglobin concentration, red cell number, MCV, MCHC and red cell concentrations of K+ and Na+ and their sum). In addition to expanding the species in which there is an effect of haemoglobin phenotype on MCV to include cattle, these data also demonstrate a significant correlation between their FV phenotype and MCV.     

Bumetanide-sensitive potassium transport and volume regulation in turkey erythrocytes

The bumetanide-sensitive (K+ + Na+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by either of two treatments: addition of epinephrine or an increase in osmolarity. At elevated (20 mM) K+ concentration, cotransport activity induced by epinephrine slowly (within 90 min) declines to background level again. This time-dependent inactivation has been linked to bumetanide-sensitive cell swelling. We have compared both the initial rate of cotransport activity and its time dependence after induction by either epinephrine, increased osmolarity or a combination of the two treatments. As a measure of cotransport activity we took the bumetanide-sensitive fraction of 86Rb+ influx. Immediately after activation, several kinetic characteristics of this flux (Vmax; Km towards K+; Ki towards bumetanide; pH profile) were identical in cells activated by either treatment. By contrast, cotransport activated by hypertonicity was significantly more resistant towards subsequent inactivation. We show this to be due to the increase in intracellular ion concentrations brought about by hypertonic cell shrinkage. This tended to reverse the driving force for cotransport, and thereby prevented the bumetanide-sensitive swelling associated with inactivation. Our data support the notion that cell volume plays a key role both in the activation and in the time-dependent inactivation of bumetanide-sensitive transport.     

Studies on lung surfactant replacement in respiratory distress syndrome. Rapid film formation from binary mixed liposomes

Binary mixed liposomes were prepared from dipalmitoylphosphatidylcholine (DPPC) and a minor compound, e.g., egg phosphatidylglycerol (PG) at a ratio of 9:1. Using different preparative techniques, large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) or multilamellar vesicles (MLV) were obtained and were studied with an electron microscope for morphology, with a Wilhelmy balance for spreading and surface tension lowering potential, and in the surfactant-depleted isolated rat lung for their ability to restore expiratory lung capacity. Only the simultaneous investigation of phospholipids by negative staining and thin sectioning allows unequivocal classification of liposomes. The surface-active structures prepared with the technique of Bangham et al. (Bangham, A.D., Hill, M.W. and Miller, N.G.A. (1974) in Methods in Membrane Biology (Korn, E., ed.), Vol. 1, pp. 1-68, Plenum Press, New York) at room temperature are LUV. LUV containing DPPC:PG at a ratio of 9:1 rapidly spread to a film with high surface tension lowering potential. Within 5 min after injection into the subphase they rise to the surface and form a film at the air/liquid interface able to lower the surface tension to less than 1 mN/m at compression. SUV of the same chemical composition, however, are immediately surface-active only when spread directly onto the surface. MLV exhibit poor surface activity. LUV or pure DPPC, applied onto the surface, are weakly surface active within 5 min. DPPC vesicles injected into the subphase at 37 degrees C do not adsorb to any film with surface tension lowering potential in this time. The minor compounds PE, PI, PS, PA, lysoPC enable DPPC to form surface-active films after application on saline at 37 degrees C. Removal of surfactant decreases the expiratory lung capacity of the isolated rat lung from 49.7 to 12.4% at 4 cmH2O. After substitution with natural surfactant, the expiratory lung capacity is twice that of the washed lung (25.9%), but the original distensibility of the native lung is not restituted. The effect of LUV containing DPPC:PG at a ratio of 9:1 is also remarkable (21.2%).     

Isolation and characterization of an inter-alpha-trypsin inhibitor-IgG complex from human serum

Inter-alpha-trypsin inhibitor is a human serum protease inhibitor of Mr 180 000 which may release physiological derivatives. A complex between IgG and an inter-alpha-trypsin inhibitor derivative of Mr 30 000 has been recently detected in human serum and was found to be inactive against trypsin, in contrast with the known inhibitory activity of the free 30-kDa derivative. The present study deals with detailed characterization of an inter-alpha-trypsin inhibitor-IgG complex following its purification by affinity chromatography techniques (anti-inter-alpha-trypsin inhibitor immunoadsorbent and Protein A-Sepharose) in mild conditions. The resulting product reacted simultaneously with anti-IgG and anti-inter-alpha-trypsin inhibitor antibodies. This complex contained Mr 180 000 inhibitor at least to some extent. It migrated in the beta-gamma zone in agarose; its molecular weight was estimated to be 1 500 000 or more; part of it displayed covalent bonding between inter-alpha-trypsin inhibitor and IgG; it had a trypsin inhibitor activity. Immunoelectrophoresis allowed one to demonstrate the native complex in serum owing to the use of anti-inter-alpha-trypsin inhibitor and anti-gamma radioactively labelled antibodies. The double immunoreactivity thus evidenced proved to be heterogeneous with respect to its level and location in the native as well as in the purified complex.     

Evidence that cyclic nucleotides activating rabbit muscle protein kinase I interact with both types of cAMP binding sites associated with the enzyme

Eighty different adenine-modified cAMP analogs were tested as activators of rabbit muscle protein kinase I (cAKI) in an in vitro phosphotransferase assay. All the analogs tested were able to activate completely the kinase. The affinities of the cAMP derivatives for the two types (A and B) of binding sites associated with the regulatory moiety of cAKI were determined under conditions similar to those used in the phosphotransferase assay. The potency of the cAMP analogs as cAKI activators was found to correlate with the mean affinity for sites A and B, rather than to the affinity for only one of the sites. This was true whether cAKI was assayed at low or near physiological ionic strength, whether the concentration of cAKI binding sites was 0.2 or 400 nM, and whether the kinase substrate was mixed histones or homogeneous phenylalanine-4-monooxygenase. Furthermore, site A-selective and site B-selective cAMP analogs activated cAKI synergistically. Finally, it was shown that the degree of synergism between cAMP analogs in activating cAKI correlated with their degree of site selectivity. It is concluded that cyclic nucleotides interact with both types of binding sites in the process of cAKI activation.     

25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria

Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

Effect of alpha particles on bacteriophage T4

Exponential survival curves were obtained for a dry film culture of bacteriophage T4 Br+ after exposure to both alpha-particles and gamma-quanta. Relative biological effectiveness of alpha-particles was 4.68 with respect to survival. The mutation spectrum after alpha-irradiation slightly differed from that produced by gamma-radiation.     

Intralipid and free plasmatic tryptophan in vitro

In an attempt to investigate the role of the lipidic emulsion Intralipid in the development of metabolic encephalopathy in a patient showing high free tryptophan levels, the relationship between lipidic emulsion and free tryptophan was examined in in vitro experiments. The addition of intralipid to normal serum produces an immediate increase in non-esterified fatty acids and a parallel rise in free tryptophan. Moreover, when serum with intralipid is incubated at 37 degrees C, the lipases release new non-esterified fatty acids and the free tryptophan increases proportionally. The non-esterified fatty acid content of intralipid was found to be 12 +/- 2 mEq X 1(-1). An inverse correlation was seen between free tryptophan and different serum albumin concentrations. It is concluded that intralipid causes an increase in free tryptophan levels. It is known that in vivo free tryptophan modulates 5-hydroxytryptamine synthesis and thus may be considered a possible causal agent for encephalopathy.     

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.