Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.     

NMR observation of individual molecules of hydration water bound to DNA duplexes: direct evidence for a spine of hydration water present in aqueous solution.

The residence times of individual hydration water molecules in the major and minor grooves of DNA were measured by nuclear magnetic resonance (NMR) spectroscopy in aqueous solutions of d-(CGCGAATTCGCG)2 and d-(AAAAATTTTT)2. The experimental observations were nuclear Overhauser effects (NOE) between water protons and the protons of the DNA. The positive sign of NOEs with the thymine methyl groups shows that the residence times of the hydration water molecules near these protons in the major groove of the DNA must be shorter than about 500 ps, which coincides with the behavior of surface hydration water in peptides and proteins. Negative NOEs were observed with the hydrogen atoms in position 2 of adenine in both duplexes studied. This indicates that a 'spine of hydration' in the minor groove, as observed by X-ray diffraction in DNA crystals, is present also in solution, with residence times significantly longer than 1 ns. Such residence times are reminiscent of 'interior' hydration water molecules in globular proteins, which are an integral part of the molecular architecture both in solution and in crystals.     

Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate theirin vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.     

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.     

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.     

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.     

Salivary gland of the tick vector (R. appendiculatus) of East Coast fever. I. Ultrastructure of the type III acinus

The brown ear tick Rhiplcephalus appendiculatus is the vector for East Coast fever, a disease that seriously limits livestock production in East Africa. The sporozoites of the infectious agent Theileria parva develop in the tick salivary gland. This paper describes the organization of the type III acinus of the gland and establishes unambiguous ultrastructural criteria for identification of the three secretory cell types: the d-cell, e-cell and f-cell. These observations are basic to exploration of possible cell-type specificity of the invading theileria and other aspects of host-parasite relations.