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.     

Phagotrophy and NH4+ regeneration in a three-member microbial food loop

In a series of batch experiments we compared the efficiencyof nitrogen regeneration of a two- and three-member microbialfood loop consisting of a mixed bacterial assemblage, a small(3–5 µm) heterotrophic flagellate (Paraphysomonassp.), and a large (7–12 µm) heterotrophic flagellate(Paraphysomonas imperforata). In the two-member system the nitrogenregeneration efficiency for NH₄⁺ (R_n) was 41% and the grossgrowth efficiency (GGE) was 57% during active grazing by thesmall flagellate on bacteria. Regeneration of NH₄⁺ continuedduring the stationary phase so that R_n was 75% after 6 daysincubation. When the larger flagellate was introduced at theend of exponential growth of the smaller grazer in the three-membersystem, initially there was rapid regrowth of bacteria, tyingup 15% of the nitrogen originally in the bacteria. The largerflagellate grazed the smaller one with a GGE of 55%. Total nitrogenregeneration efficiency through exponential growth of the largerflagellate was 73%. Because microbial food loops in naturalwaters are far more complicated and with more grazing stepsthan portrayed in this study, we would expect the bulk of nutrientswithin these systems to be recycled with little transfer tohigher trophic levels.     

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.     

PHENOTYPIC PLASTICITY IN THE SAILFIN MOLLY,POECILIA LATIPINNA (PISCES: POECILIIDAE). I. FIELD EXPERIMENTS

Sailfin mollies (Poecilia latipinna) display marked interdemic variation in body size. We employed “common-garden” experiments in field enclosures to explore the potential role of environmental factors in determining the interdemic phenotypic variation in growth rate, age at maturity, and size at maturity. The largest single, consistent source of variation for all traits was family identity within populations. Environmental effects acted predominantly through family x environment interactions. There was little evidence for any intrinsic variation among populations once family heterogeneity had been accounted for. In general, when statistically significant differences existed, fish raised in a saltwater pond grew faster than their broodmates raised in a freshwater pond. Both males and females tended to mature at a smaller size and later in the freshwater pond than in the saltwater pond. The effects of the environmental conditions differed among the three years in which we performed these studies. In only one year was there a substantial difference between fish raised under the two environmental conditions. These results indicate that direct environmental effects are not strong enough to account for the differences in body size among natural populations and that intrinsic differences among natural populations are due to different frequency distributions of genotypes that are present in all populations.     

PHENOTYPIC PLASTICITY IN THE SAILFIN MOLLY,POECILIA LATIPINNA (PISCES: POECILIIDAE). II. LABORATORY EXPERIMENT

Field studies indicate that the influence of environmental factors on growth rate and size and age at maturity in sailfin mollies (Poecilia latipinna) is inconsistent over time and suggest that the marked interdemic variation in male body size in this species is the result of genetic variation. However, the role of specific environmental factors in generating phenotypic variation must be studied under controlled conditions unattainable in nature. We raised newborn sailfin mollies from four populations in laboratory aquaria under all possible combinations of two temperatures, three salinities, and two food levels to examine explicitly the influence of these environmental factors. Males were much less susceptible than females to temperature variation and were generally less plastic than females in terms of all three traits. Members of both sexes matured at larger sizes and at later ages in less saline and in cooler environments. Food levels were not sufficiently different to affect the traits we studied. The effects of temperature and salinity were not synergistic. Males from different populations exhibited different average ages and sizes at maturity, but females did not. The magnitudes of the effects we found were not substantial enough to account for the consistent interdemic differences in male and female body size that have been observed previously. Our results also indicate that no single environmental factor is solely responsible for the environmental effects observed in field experiments on growth and development. These studies, together with other work, indicate that the strongest sources of interdemic variation are genetic differences in males and differences in postmaturation growth and survivorship in females.     

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.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.     

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.