The study of a French family with two duplicated C4A haplotypes

Summary The finding of two duplicated C4A haplotypes in a normal French family led to a detailed study of their C4 polymorphism. The father had an extremely rare A^*6A^*11, B^* QO haplotype inherited by all of his children and the mother had the more common A^*3A^*2, B^*QO haplotype. Two HLA identical daughters only have four C4A alleles. The father's A11 allotype expresses Ch: 1 (Chido) rather than Rg:1 (Rodgers) and represents a new Ch phenotype Ch: 1,-2,-3,-4,-5,-6. In order to clarify the genetic background in this unusual family, DNA studies of restriction fragment length polymorphisms (RFLPs) were undertake. The father's rare haplotype, which expresses two C4A allotypes, results from a long and a short C4 gene normally associated with the A^*6, B^*1 that also exhibits the BglII RFLP. As it travels in an extended MHC haplotype HLA A2, B57 (17), C2^*C, BF^*S, DR7 that is most frequently associated with A^*6, B^*1, we postulate that the short C4B has been converted in the chain region to a C4A gene which produces a C4A protein. This report of a short C4A gene is the first example in the complex polymorphism of C4.     

Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants.

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.     

Diurnal periodicity of chalcone-synthase activity during the development of oat primary leaves

Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonio lyase Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged (G.W., We 630/9-7; We 630/10-1). Thanks are given to Dr. St. Kellam (Department of Plant Microbiological Sciences, University of Canterbury, New Zealand) for correcting the English.     

The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression

Complementary DNAs coding for four Desmodus rotundus salivary plasminogen activators (DSPAs) were isolated and characterized. The predicted amino acid sequences display structural features also found in tissue-type plasminogen activator. The largest forms (DSPA alpha 1 and -alpha 2) contain a signal peptide, a finger (F), an epidermal growth factor (EGF), a kringle, and a serine protease domain, whereas DSPA beta and -gamma lack the F and F-EGF domains, respectively. Additional differences between the four forms suggest that distinct genes code for the members of the DSPA family. Transfection of DSPA-encoding cDNAs, placed under the control of the simian virus 40 late promoter, into COS-1 cells resulted in the secretion of highly fibrin-dependent PAs.     

Respiratory mechanics in mechanically ventilated patients with respiratory failure

In 11 mechanically ventilated patients, respiratory mechanics were measured 1) during constant flow inflation and 2) following end-inflation airway occlusion, as proposed in model analysis (J. Appl. Physiol. 58: 1840-1848, 1985. During the latter part of inflation, the relationship between driving pressure and lung volume change was linear, allowing determination of static respiratory elastance (Ers) and resistance (RT). The latter represents in each patient the maximum resistance value that can obtain with the prevailing time constant inhomogeneity. Following occlusion, Ers and RT were also obtained along with RT (min) which represents a minimum, i.e., resistance value that would obtain in the absence of time constant inhomogeneity. A discrepancy between inflation and occlusion Ers and RT was found only in the three patients without positive end-expiratory pressure, and could be attributed to recruitment of lung units during inflation. In all instances Ers and RT were higher than normal. RT(min) was lower in all patients than the corresponding values of RT, indicating that resistance was frequency dependent due to time constant inequalities. Changes in inflation rate did not affect Ers, while RT increased with increasing flow.     

Respiratory responses to ventilatory loading following low cervical spinal cord injury

This study compared the respiratory responses to ventilatory loading in 8 normal subjects and 11 quadriplegic patients with low cervical spinal cord transection. Progressive hypercapnia was produced by rebreathing. Rebreathing trials were carried out with no added load and with inspiratory resistive loads of 5 and 16 cmH2O. l-1 X s. Measurements were made of ventilation and of diaphragmatic electromyographic activity. Base-line hypercapnic ventilatory responses were significantly lower than normal in the quadriplegic patients, but the effects of resistive loading on the ventilatory responses were comparable in the two groups. The change in peak moving-average diaphragmatic electrical activity (DI peak) for a given change in CO2 partial pressure (PCO2) and DI peak at PCO2 55 Torr increased significantly with resistive loading both in the normal subjects and the quadriplegic patients. In the normal subjects, but not in the quadriplegic patients, inspiratory duration increased progressively with increasing resistance. The increase in DI peak during ventilatory loading in the normal subjects was a consequence of inspiratory prolongation. In contrast, in the quadriplegic patients during breathing against the larger resistive load, there was a significant increase in the average rate of rise (DI peak divided by the time from onset to peak) of diaphragmatic activity. The change in DI rate of rise for a given change in PCO2 increased to 137 +/- 13% (SE), and the DI rate of rise at PCO2 55 Torr increased to 128 +/- 8% (SE) of control values. These results indicate that compensatory increases in diaphragmatic activation during ventilatory loading occur in quadriplegic patients in whom afferent feedback from rib cage receptors is disrupted.     

Interrupter technique for measurement of respiratory mechanics in anesthetized humans

Flow (V), volume (V), and tracheal pressure (Ptr) were measured throughout a series of brief (100 ms) interruptions of expiratory V in six patients during anesthesia (halothane-N2O) and anesthesia-paralysis (succinylcholine). For the latter part of spontaneous expiration and throughout passive deflation during muscle paralysis, a plateau in postinterruption Ptr was observed, indicating respiratory muscle relaxation. Under these conditions, passive elastance of the total respiratory system (Ers) was determined as the plateau in postinterruption Ptr divided by the corresponding V. The pressure-flow relationship of the total system was determined by plotting the plateau in Ptr during interruption against the immediately preceding V. Ers averaged 23.5 +/- 1.9 (SD) cmH2O X l-1 during anesthesia and 25.5 +/- 5.4 cmH2O X l-1 during anesthesia-paralysis. Corresponding values of total respiratory system resistance were 2.0 +/- 0.8 and 1.9 +/- 0.6 cmH2O X l-1 X s, respectively. Respiratory mechanics determined during anesthesia paralysis using the single-breath method (W.A. Zin, L. D. Pengelly, and J. Milic-Emili, J. Appl. Physiol. 52: 1266-1271, 1982) were also similar. Early in spontaneous expiration, however, Ptr increased progressively during the period of interruption, reflecting the presence of gradually decreasing antagonistic (postinspiratory) pressure of the inspiratory muscles. In conclusion, the interrupter technique allows for simultaneous determination of the passive elastic as well as flow-resistive properties of the total respiratory system. The presence of a plateau in postinterruption Ptr may be employed as a useful and simple criterion to confirm the presence of respiratory muscle relaxation.     

Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor.

To further our understanding of the protein folding process, it is desirable to examine the structural intermediates (equilibrium and kinetic) that are populated between the statistical coil state and the folded molecule. X-ray crystallography and NMR structural studies are unable to determine long-range distances in proteins under denaturing solution conditions. Nonradiative (F?rster) energy transfer, however, has been shown to be a spectroscopic ruler for the measurement of distance distributions and diffusion between selected sites in proteins under a range of different solution conditions. The distributions of distances between a donor probe at the N-terminal residue and an acceptor attached to one of the four lysine residues (15, 26, 41, 46) of reduced and unfolded (in 6 M guanidine hydrochloride and 20 mM dithiothreitol) bovine pancreatic trypsin inhibitor (BPTI) were measured as a function of temperature. Even in strong denaturant and reducing agent, BPTI does not exist as a statistical coil polypeptide. It appears that nonlocal (long-range) interactions are already beginning to "fold" the protein toward a more compact, native conformation. As the temperature is increased under these conditions, hydrophobic interactions lead to an even more compact structure consistent with the predictions of phase diagrams for globular proteins.