Localization of lysosomal antigens in activated T-lymphocytes

Summary The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel, electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme -glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that -glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density.Fractionation of homogenates from lymphocytes on Percoll gradients revealed that -glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of -glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.     

Myosin light chain 2 modulates calcium-sensitive cross-bridge transitions in vertebrate skeletal muscle.

We investigated the mechanism of the Ca2+ sensitivity of cross-bridge transitions that limit the rate of force development in vertebrate skeletal muscle. The rate of force development increases with Ca2+ concentration in the physiological range. We show here that at low concentrations of Ca2+ the rate of force development increases after partial extraction of the 20-kD light chain 2 subunit of myosin, whereas reconstitution with light chain 2 fully restores native sensitivity to Ca2+ in skinned single skeletal fibers. Furthermore, elevated free Mg2+ concentration reduces Ca2+ sensitivity, an effect that is reversed by extraction of the light chain but not by disruption of thin-filament activation by partial removal of troponin C, the Ca2+ binding protein of the thin filament. Our findings indicate that the Ca2+ sensitivity of the rate of force development in vertebrate skeletal muscle is mediated in part by the light chain 2 subunit of the myosin cross-bridge.     

Clues in diagnosing congenital heart disease.

A number of practical office and bedside clues to cardiac disease in infants and children have been passed on through the years. They relate to the history, to the inspection and palpation components of the physical examination, and to knowledge of the specific cardiac defects that are likely to be associated with certain clinical syndromes. With the possible exception of coarctation of the aorta, the clues are not diagnostically specific. In many instances, however, they serve to narrow a broad array of diagnostic possibilities to 2 or 3 and, with the aid of other clues and auscultation, they can often be distinguished from one another. When a primary care physician is confronted with a child who has an incidental murmur that is "probably" innocent but could be organic, useful clues favoring an organic murmur are a history of congenital heart disease in a first-degree relative; a history of maternal rubella syndrome, alcohol use, or teratogenic drug use during pregnancy; a history of inappropriate sweating; a history of syncope, chest pain, or squatting; maternal diabetes mellitus; premature birth; birth at a high altitude; cyanosis; abnormal pulsations; recurrent bronchiolitis or pneumonia; chronic unexplained hoarseness; asymmetric facies with crying; and a physical appearance suggestive of a clinical syndrome.     

Anesthetic inhibition of firefly luciferase, a protein model for general anesthesia, does not exhibit pressure reversal.

The surprising observation that pressures of the order of 150 atmospheres can restore consciousness to an anesthetized animal has long been central to theories of the molecular mechanisms underlying general anesthesia. We have constructed a high-pressure gas chamber to test for "pressure reversal" of the best available protein model of general anesthetic target sites: the pure enzyme firefly luciferase, which accounts extremely well for animal potencies (over a 100,000-fold range). We found no significant pressure reversal for a variety of anesthetics of differing size and polarity. It thus appears that either firefly luciferase is not an adequate model for general anesthetic target sites or that pressure and anesthetics act at different molecular sites in the central nervous system.     

Effect of temperature shifts on gliding motility, adhesion, and fatty acid composition of Cytophaga sp. strain U67.

Gliding motility and flipping of 25 degrees C-adapted Cytophaga sp. strain U67 were inhibited when the bacteria were shifted to a less than or equal to 12 degrees C environment; motility was not blocked by a shift to 13 degrees C. Bacteria adapted to 4 degrees C were motile over the entire 4 to 25 degrees C temperature range tested. U67 adhesion to the substratum appeared to be unaffected by temperature shifts. Bacteria adapted to 4 degrees C had higher proportions of unsaturated and branched-chain fatty acids than did those grown at 25 degrees C. When 25 degrees C-adapted bacteria were subjected to a gradual temperature decline, the time of reappearance of gliding competence at 4 to 5 degrees C was correlated with these changes in fatty acid composition.     

Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.     

Guanine nucleotide-dependent ADP-ribosylation of soluble rho catalyzed by Clostridium botulinum C3 ADP-ribosyltransferase. Isolation and characterization of a newly recognized form of rhoA

Two C3 ADP-ribosyltransferase substrates with different characteristics were isolated from bovine brain cytosol. Amino acid sequences of tryptic peptides from the two substrates were identical to rhoA and rhoB; hence, the purified proteins are referred to as rhoA* and rhoB*, respectively. Soluble rhoA* exhibits properties different from those previously reported for rho proteins. In contrast to other C3 substrates, rhoA* behaved as a 77-80-kDa protein on gel filtration, although on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the ADP-ribosylated moiety had a mobility consistent with a 21.5-kDa protein. Furthermore, C3-catalyzed ADP-ribosylation of rhoA* was dependent on guanine nucleotides in the presence of 1 mM Mg2+ or 1 mM EDTA (0.19 microM free Mg2+). Half-maximal stimulation by GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), guanylyl-imidodiphosphate (Gpp(NH)p), and GDP was observed at 16, 20, 220, and 380 nM, respectively; guanosine 5'-O-(2-thiodiphosphate), GMP, and adenine nucleotides were ineffective. In the presence of GTP gamma S, the rate and extent of ADP-ribosylation was enhanced by dimyristoylphosphatidylcholine and/or cholate. This increase in ADP-ribosylation was specific for rhoA*; it was not observed with rhoB* and has not been reported for other C3 substrates. These distinct properties suggest that rhoA* is a newly recognized type of C3 substrate, differing from the rhoA-like proteins previously reported. rhoB*, on the other hand, has properties similar to those reported for membrane-associated rhoB and its ADP-ribosylation was independent of guanine nucleotides in the presence of 1 mM Mg2+ and not affected by dimyristoylphosphatidylcholine and/or cholate.     

Modulation of collagen production by fibroblasts. Effects of chronic exposure to agonists that increase intracellular cyclic AMP.

Cultured human lung fibroblasts were evaluated for their responsiveness to isoprenaline (isoproterenol) or prostaglandin E2 before and after chronic incubation with the agonist. Cells incubated for 6 h with either agonist were suppressed in terms of collagen production and exhibited increased intracellular cyclic AMP. Cells incubated for 72 h with the agonist and then re-challenged for 6 h with the same agonist did not demonstrate suppressed collagen production or increased cyclic AMP. Cells incubated for 72 h with isoprenaline still responded to prostaglandin E2 when challenged for 6 h; however, when the order of agonist exposure was reversed, cells incubated with prostaglandin E2 did not respond to a challenge by isoprenaline. If cells were allowed to recover for 48 h without the agonist after a 72 h chronic incubation, they recovered their responsiveness to the agonist. The results indicate that, although cultured fibroblasts may become desensitized to one agonist, they may retain their sensitivity to a second agonist and chronic suppression of collagen production may be achieved by alternate exposure to isoprenaline and prostaglandin E2.