The folding and mutual interaction of the domains of yeast 3-phosphoglycerate kinase

Analysis of the reversible unfolding of yeast phosphoglycerate kinase leads to the conclusion that the two lobes are capable of folding independently, consistent with the presence of intermediates on the folding pathway with a single domain folded. The domains have different free energies of stabilisation. Immunological cross-reactivity, circular dichroism and thiol reactivity provide evidence for cyanogen bromide peptide 1-173, which comprises five-sixths of the N-terminal domain, containing sufficient information to refold into a native-like structure which dimerises.     

Stimulation of rat uterine collagen synthesis by relaxin

Immature, ovariectomized, estrogen-primed rats respond to the administration of porcine relaxin by an increase in the incorporation of labeled amino acids ([14C]leucine, [14C]phenylalanine, [3H]proline) into uterine proteins in vitro. The maximum response occurs about 12 hr after a single injection of 0.1 mg relaxin in benzopurpurine 4B solution; subsequently, the relaxin effect declines but is still apparent after 24 hr. Smaller, but still significant increases in incorporation rates can be induced by relaxin in the absence of estrogen priming. Uterine collagen synthesis, as indicated by the incorporation of [3H]proline and its conversion to hydroxyproline, appears to be a primary target of the relaxin stimulus, since the effect of relaxin upon proline incorporation into uterine collagen is significantly greater than its effect upon labeling of noncollagen protein.     

Influence of chronic antigen exposure on the metabolism of PGH2 by microsomal preparations of airway and parenchymal tissues in the sheep

The effects of repeated antigen exposure on the synthesis of mediators by lung tissues are not well understood. To investigate the influence of antigen challenge on the synthesis of prostaglandins by central airway and peripheral lung tissues, fourteen sensitive sheep underwent biweekly exposure to aerosolized Ascaris suum antigen (7) or saline (7). Following the fifth exposure, microsomal and high speed supernatant fractions were prepared from trachealis muscle and lung parenchyma. Synthesis of thromboxane (TX) A2, prostaglandin (PG) D2 and PGI2 from the PG endoperoxide intermediate, PGH2, was assayed over a range of substrate concentrations from 3-200 microM. Synthesis of PGI2 by trachealis microsomes was approximately 5-fold greater than that of TXA2. PGI2 and TXA2 production was identical in tracheal preparations from Ascaris- and saline-exposed animals. In parenchymal tissues, where TXA2 production predominated over PGI2 by 9-fold, preparations from Ascaris-exposed animals synthesized 50% more TXA2 than controls at PGH2 concentrations of 25 microM and above, whereas synthesis of PGI2 and PGD2 were similar in preparations from both groups of animals. The density of pulmonary mast cells was decreased by 21% in the Ascaris group, whereas polymorphonuclear leukocyte density was unchanged. These results demonstrate the differential synthesis of TXA2 and PGI2 in central airways and peripheral lung regions of the sheep. They further indicate that repeated exposure of the airways to antigen selectively enhances TXA2 synthesis in the lung periphery of sensitized animals. The site of this increased enzymatic activity, whether in resident cells or newly-infiltrated cells, has not been determined.     

Myc oncogene activation in B and T lymphoid tumours

The chromosome translocations characteristic of certain B lymphoid tumours associate the myc oncogene and immunoglobulin loci. The typical t(12;15) in murine plasmacytomas and analogous t(14;8) in Burkitt lymphomas couple the myc coding region to one of the switch recombination regions within the immunoglobulin heavy (H) chain locus; hence the switch machinery may promote some translocations. Significantly, translocation induces constitutive myc expression, the untranslocated myc allele remaining silent. The predilection for breakpoints near the 5' end of the c-myc gene may reflect selection for altered myc regulation. In most tumours, the stimulatory effect of the H locus context is not understood, but an H locus enhancer participates in some tumours, including one displaying a novel transposition. The variant (6;15) translocations found in about 15% of plasmacytomas involve the myc band and the region of chromosome 6 where the kappa locus lies. The t(6;15) is shown here to represent an exchange between C kappa and a chromosome 15 locus (designated pvt-1) which lies unexpectedly far from c-myc. The association of myc expression with pvt-1 alterations suggest that myc can be activated at a distance. Myc has also been implicated in some T lymphomas by detection of proviral inserts near myc and also, surprisingly, within the pvt-1 locus. Inserts near myc appear to activate its expression via the retroviral enhancer.     

Some properties of acetylcholine receptors in human cultured myotubes

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]alpha-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 +/- 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 microM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22 degrees C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 microM). With desensitizing doses of ACh (10 microM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.     

Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G) contains 20 uncharged and mostly hydrophobic amino acids. We created DNAs specifying G proteins with shortened transmembrane domains, by oligonucleotide-directed mutagenesis. Expression of these DNAs showed that G proteins containing 18, 16, or 14 amino acids of the original transmembrane domain assumed a transmembrane configuration and were transported to the cell surface. G proteins containing only 12 or 8 amino acids of this domain also spanned intracellular membranes, but their transport was blocked within a Golgi-like region in the cell. A G protein completely lacking the membrane-spanning domain accumulated in the endoplasmic reticulum and was secreted slowly. These experiments indicate that the size of the transmembrane domain is critical not only for membrane anchoring, but also for normal cell surface transport.     

Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein.

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.     

Rapid measurement of creatine kinase activity in a coronary care unit using a portable benchtop reflectance photometer.

Rapid measurements of plasma creatine kinase activity using an inexpensive benchtop reflectance photometer (Ames Seralyzer) and disposable reagent strips were evaluated in the laboratory and coronary care unit. The system proved simple to use and capable of yielding rapid (four minutes per analysis), precise (coefficient of variation less than 9%), and accurate results (correlation with routine method 0.995) when used by medical staff. Creatine kinase values were available 6.5-102 hours earlier than routine laboratory data, depending on the time and day of sampling, thereby facilitating appropriate and economic patient management. This instrument might be used to supplement the routine enzyme service for selected admissions, resulting in greatly improved availability of results and hence contributing to the early discharge of patients from intensive care facilities.     

Potentiation of lung vascular response to endotoxin by superoxide dismutase

We studied the effects of superoxide dismutase (SOD), an enzyme that converts superoxide into peroxide, on the cardiopulmonary response to endotoxin in sheep. Sheep (n = 18) were prepared for chronic measurement of cardiopulmonary variables, including lung lymph flow, by surgically implanting catheters under halothane anesthesia. Nine of the animals were studied before and after the administration of endotoxin (0.75 microgram/kg) with and without SOD. An additional nine animals received SOD without the lipopolysaccharide. Endotoxin produced an increase in lung lymph flow that was initially associated with a marked pulmonary arterial (PA) hypertension and reduced lymph-to-plasma protein ratio (L/P). The lymph flow remained elevated later in the response, but there was only a mild increase in PA pressure, and the L/P was normal. There was also a fall in blood neutrophils and in cardiac index. SOD increased this secondary elevation in lung lymph flow, and the corresponding L/P was greater than the preendotoxin value. The fall in neutrophil count, cardiac output, and the elevation in PA pressure seen with endotoxin were not affected by SOD. When administered in the absence of endotoxin, SOD produced no perceptible change in the cardiopulmonary and lymph values. We conclude that peroxide, hydroxyl ion, and/or other free radicals formed by the action of SOD must be responsible for a portion of the endotoxin response rather than superoxide itself.     

The cytosol origin of macromolecules extruded by cultured chick embryo fibroblast cells

The DNA and protein extruded by chick embryo fibroblast cells has been analysed by chromatography. A high proportion of the DNA is in the form of a protein-complex of size around 5 X 10(5) dalton. The patterns of the DNA and protein extruded into the supernatant are closely similar in many respects to those found in the cell cytosol. It is concluded that the macromolecular material extruded by cells in culture is of cytosol origin: a possible function in terms of "information" carriage is proposed.