Termination of inspiration by phase-dependent respiratory vagal feedback in awake normal humans.

Imperceptible levels of proportional assist ventilation applied throughout inspiration reduced inspiratory time (TI) in awake humans. More recently, the reduction in TI was associated with flow assist, but flow assist also reaches a maximum value early during inspiration. To test the separate effects of flow assist and timing of assist, we applied a pseudorandom binary sequence of flow-assisted breaths during early, late, or throughout inspiration in eight normal subjects. We hypothesized that imperceptible flow assist would shorten TI most effectively when applied during early inspiration. Tidal volume, integrated respiratory muscle pressure per breath, TI, and TE were recorded. All stimuli (early, late, or flow assist applied throughout inspiration) resulted in a significant increase in inspiratory flow; however, only when the flow assist was applied during early inspiration was there a significant reduction in TI and the integrated respiratory muscle pressure per breath. These results provide further evidence that vagal feedback modulates breathing on a breath-by-breath basis in conscious humans within a physiological range of breath sizes.     

Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21.

Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron-density-difference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-D-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.     

Purification of bovine liver histidyl-tRNA synthetase, the Jo-1 antigen of polymyositis: size of the whole enzyme and its characteristic proteolytic fragments

We have recently reported the marked increase in frequency which can be achieved in the detection of the anti-Jo-1 antibody of polymyositis in serum samples by replacing commercial mixtures of cytoplasmic and nuclear antigens with the purified antigen, histidyl-tRNA synthetase. The present paper describes a method for purifying this antigen and an investigation of its size. Molecular masses previously reported for the enzyme have varied from 85-154 kDa and subunit molecular masses varying from 40-77 kDa have been observed. Several of these fragments are of sizes similar to those of a number of other autoantigens commonly observed in connective tissue diseases. Since the clinical identification of these autoantigens often relies exclusively on size determination by Western blotting, we have characterized the commonly occurring fragments of histidyl-tRNA synthetase lest they confuse such identification. It is concluded that histidyl-tRNA synthetase, like many other aminoacyl-tRNA synthetases, is subject to severe proteolysis during extraction procedures. Several characteristic fragments (Mr = 80, 75, 61, 55, 50 and 45 kDa) result, a finding that provides a satisfactory explanation of the various values previously reported. The intact bovine enzyme is a dimer of molecular mass close to 160 kDa.     

Derivation and characterization of POJ cells, transformed human fetal glial cells that retain their permissivity for JC virus.

The study of the medically important polyomavirus JC virus is limited to only a few laboratories, primarily because the permissive cell system most often used, primary human fetal glial cells, is difficult to obtain and propagate. We have introduced mutations at the origin of DNA replication of JC virus and transformed glial cells with the replication-defective genomes. Although normal glial cell cultures rapidly lose their permissivity for the virus after subculture, the transformed cells (designated POJ) had a greatly expanded life span and remained permissive for JC virus even after 30 passages in vitro. POJ cells constitutively express a functional T protein that complements the replication defect of lethal early-region mutations in JC virus. We expect that these cells will greatly facilitate the study of this human virus.     

Influence of adrenergic receptors on ovarian progesterone secretion in the pseudopregnant cat and oestradiol secretion in the oestrous cat

The infusion of isoprenaline or propranolol into the abdominal aorta of the pseudopregnant cat caused an increase or decrease respectively in the ovarian progesterone secretion rate. These observations suggest that the sympathetic innervation of the ovary has a physiological influence on normal progesterone secretion, and this mechanism may explain stress-related increases in progesterone concentrations. The infusion of isoprenaline or propranolol after the stimulation of follicular growth had no consistent or convincing effect on oestradiol secretion.     

Serum-mediated suppression of nonspecific B-cell activation. III. Selective inhibition of the polyclonal B-cell response by normal mouse serum

Normal, nonimmune adult serum is known to inhibit in vitro immune responses when present in sufficient amounts. The significance of inhibition of the immune response by serum, however, is not known. Previous work suggested that normal mouse plasma or serum (NMS) was selectively more inhibitory to nonantigen-specific (e.g., polyclonal) as compared to antigen-specific responses. This led to the hypothesis that constituents of serum (or plasma) may serve naturally to minimize the polyclonal type of antibody response, preserving immune specificity. The present study further examined the effect of NMS on polyclonal versus antigen-specific antibody responses. Under the in vitro assay conditions used, 0.5% NMS supported bacterial endotoxin (ET)-induced mitogenic and polyclonal B lymphocyte responses, antigen (SRBC, TNP-KLH)-specific antibody (IgM, IgG) responses, and antigen-induced or -specific T-lymphocyte proliferative responses, while 5% NMS inhibited all of these responses. However, antigen-specific T-lymphocyte responses could be restored by a 10-fold increase in the antigen concentration and antigen-specific antibody responses could be restored by the addition of ET (10 micrograms/ml) as adjuvant. On the other hand, the mitogenic response to ET remained suppressed regardless of ET concentration. Thus, despite significant reduction of the mitogenic and polyclonal properties of ET in 5% NMS (greater than 70% suppression), sufficient antigenic stimuli permitted optimal specific T- and B-cell responses. Many naturally occurring antigens, e.g., bacterial, fungal, and viral, have inherent B-cell mitogenic and polyclonal activity in addition to adjuvanticity and the presence of the serum inhibitory factor may serve to minimize their indiscriminate polyclonal stimulation of antibody.     

Chromatin structure. Further evidence against the existence of a beaded subunit for the 30-nm fiber

The size distribution of chromatin fragments released by micrococcal nuclease digestion of liver chromatin at various ionic strengths was examined. Below 20 mM ionic strength, gradient profiles with a peak centered at 6 nucleosomes are generated, whereas between 20 and 50 mM the peak is always centered on 12 nucleosomes, and above 50 mM ionic strength the 30-nm fiber becomes less accessible to the nuclease and there is a corresponding increase in the size distribution of fragments in the gradients. However, extensive digestions always give profiles with a peak of 12 nucleosomes as nuclease-resistant dodecamers accumulate. All of these observations are consistent with the winding of the 10-nm polynucleosome chain into a helical coil commencing at about 20 mM ionic strength. The helical turns are stabilized by histone H1 interactions between 20 and 50 mM ionic strength producing stable dodecamers. Above 50 mM ionic strength the coil condenses longitudinally and the profiles are consistent with a random attack of this fiber by the nuclease. Consequently it is not necessary to invoke the existence of a subunit bead to explain the profiles. We further define the conditions at which specific structural transitions take place and provide methodology for the preparation of chromatin at various levels of condensation.