Crystals of an antibody FV fragment against an integral membrane protein diffracting to 1.28 Å resolution

The F_v fragment of a monoclonal antibody, 7E2 (IgG₁, κ, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for highresolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 51.51 Å, b = 56.15 Å, c = 99.86 Å (1 Å = 0.1 nm) and contain one F _v fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 Å resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. © 1995 Wiley-Liss, Inc.     

Constituents of human muscle in isometric fatigue.

Three subjects performed five successive isometric contractions to fatigue; the tension in any one experiment was constant at tensions varying from 20 to 80% of the maximal voluntary contraction (MVC). The interval between contractions was held constant at 11 min. Muscle biopsy specimens were obtained at the start of the experiment, after the first, fourth, and fifth, and before the second and fifth of the successive contractions. The concentrations of ATP, CP, glycogen, and lactate were measured in each sample of muscle. Changes in ATP and glycogen were insufficient to be held accountable for the development of isometric fatigue. Changes in CP and lactate were large after fatigue at intermediate tensions, but those of CP were considered unlikely to be responsible for the fatigue. At tensions of 30-50% MVC the increase in lactate could be responsible for fatigue either directly or by indirect changes in pH; at higher and lower tensions the possibility that lactate is directly implicated in the development of fatigue seems remote.     

Serotonin-mediated cardiovascular responses to electrical stimulation of the raphe nuclei in the rat.

The dorsal and median raphe nuclei in rats were electrically stimulated and blood pressure and heart rate were recorded. Stimulation of each raphe nucleus caused an increase in blood pressure without affecting heart rate. The size of the increase in blood pressure depended upon the stimulus-intensity.Significant increases were already obtained with 5 sec. trains of 0.3 msec., 200 μA stimuli given at a frequency of 50 Hz. The increases in blood pressure could be obtained with electrodes within the raphe nuclei.Pretreating rats with para-chlorophenylalanine (pCPA, 100 mg/kg.day for 3 days) significantly diminished the increases in blood pressure obtained during electrical stimulation of the median raphe nucleus. However, similar pretreatment did not affect blood pressure rises induced by dorsal raphe stimulation.These data are discussed in relation to the role of central serotoninergic mechanisms in cardiovascular control.     

Evaluation of the social bond: a new method tested in Mus spicilegus

Innovative and fruitful studies of social bonds have been developed in recent years, although the methods used to establish the existence of a social bond between two individuals have not evolved significantly. Two types of paradigms have been currently used: the separation-reunion paradigm, which evaluates the distress caused by the disruption of the social bond, and choice paradigms, which test the specificity of the bond to a given individual. We have developed a new paradigm based on the idea that the cost an individual was ready to pay in order to gain access to a conspecific depended on the strength of the social bond between the two individuals. To test our paradigm we used mound-building mice, Mus spicilegus that present, in both males and females, a level of tolerance that differs greatly according to the degree of familiarity between the individuals. Our new method for testing social bond revealed unsuspected differences between males and females. Our results suggested that, at least in Mus spicilegus, strong social bonds were not necessary to the development of a high level of tolerance between individuals.     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.     

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.     

Slowing of synapse elimination by alpha-bungarotoxin superfusion of the neonatal rabbit soleus muscle

To examine the role of postsynaptic activity in regulating the rate of neuromuscular synapse elimination, contractile activity of neonatal rabbit soleus muscles was decreased by chronic superfusion of alpha-bungarotoxin (alpha-BGT) over their surfaces. Superfusion was begun at 6 days postnatal and continued for a variable duration (2 to 5 days) before muscles were analyzed. The percentage of polyinnervated fibers was assessed both physiologically and anatomically for alpha-BGT-treated muscles and their contralateral muscles, in addition to normal and control muscles of the same age. Within muscles exposed to alpha-BGT, polyinnervation was significantly greater than that for muscles from each of the control groups. The anatomical assay further revealed that the retention of polyinnervation in alpha-BGT-treated muscles was most pronounced near the muscle's surface, although end plates at the center were also affected. This finding, coupled with evidence that only a small percentage of the muscle fibers were completely inactivated, suggests that the activity block was also most pronounced near the surface and relatively low at the muscle's center. The percentage of end plates at which synapse elimination was delayed was greater than the estimated percentage whose activity was completely blocked, suggesting that synapse loss was slowed even in muscle fibers retaining some postsynaptic activity. These observations indicate that the rate of synapse elimination depends on the levels of functional acetylcholine receptors. This process could be mediated in a graded fashion by changes in postsynaptic activity (subthreshold or suprathreshold) or by a nonelectrical effect of blocking postsynaptic receptors.