Frequency selectivity of hearing in the green treefrog,Hyla cinerea

Frequency selectivity of hearing was measured in the green treefrog, Hyla cinerea. A psychophysical technique based on reflex modification was used to obtain masked threshold estimates for pure tones (300-5,400 Hz) presented against two levels of broadband masking noise. A pure tone (S-1) presented 200 ms prior to a reflex-eliciting stimulus (S-2) inhibited the motor reflex response to S-2. The magnitude of this reflex modification effect varied systematically with the sound pressure level (SPL) of S-1, and threshold was defined as the SPL of S-1 at which the reflex modification effect disappeared. Masked thresholds were used to calculate critical ratios, an index of the auditory system's frequency selectivity. The frequency selectivity of the treefrog's hearing is greatest and critical ratios are lowest (22-24 dB) at about 900 and 3,000 Hz, the two spectral regions dominant in the male treefrog's species-specific advertisement call. These results suggest that the treefrog's auditory system may be specialized to reject noise at biologically-relevant frequencies. As in other vertebrates, critical ratios remain constant when background noise level is varied; however, the shape of the treefrog's critical ratio function across frequencies differs from the typical vertebrate function that increases with increasing frequency at a slope of about 3 dB/octave. Instead, the treefrog's critical ratio function resembles its pure tone audiogram. Although the shape of the treefrog's critical ratio function is atypical, the critical ratio values themselves are comparable to those of many other vertebrates in the same frequency range. Critical ratio values here measured behaviorally do not match critical ratio values previously measured physiologically in single eighth nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective regulation by pertussis toxin of insulin-induced activation of particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes

Incubation of 3T3-L1 adipocytes with insulin or isoproterenol for 10 min increased particulate "low Km" cAMP phosphodiesterase activity by 42% and 50%, respectively. Pertussis toxin catalyzed the [32P]-ADP ribosylation of a 41,000 dalton protein in adipocyte particulate fractions; prior incubation of adipocytes with toxin markedly reduced incorporation of radiolabel. Exposure of adipocytes to pertussis toxin (0.3 microgram, 18 hr) increased glycerol production and inhibited activation of cAMP phosphodiesterase by insulin, but not by isoproterenol. These results suggest that pertussis toxin can interfere with receptor-mediated processes that stimulate cAMP hydrolysis as well as those that inhibit cAMP formation.     

Reactivity of proteins in ribosomes from Saccharomyces cerevisiae with trypsin

The susceptibility of 40S and 60S ribosomal subunits from Saccharomyces cerevisiae to digestion with varying concentrations of trypsin was studied by two-dimensional electrophoresis and quantitative measurements of the protein remaining with the ribosomal particles after trypsin treatment. Proteins from both subunits can be classified into three groups according to their rate of digestion by trypsin. These results are in good agreement with those obtained on the order of ribosomal assembly in vivo, i.e., proteins which are most susceptible to trypsin digestion have been shown to associate with the ribosomal particles at a relatively late stage of ribosome assembly.     

Exogenous gangliosides enhance the interaction of fibronectin with ganglioside-deficient cells

The major cell-surface glycoprotein fibronectin mediates a variety of cellular adhesive interactions that have been reported to be competitively inhibited by gangliosides. These effects suggest a possible function of gangliosides as receptors for fibronectin. To test this hypothesis more directly, we examined the interaction of endogenous fibronectin with a ganglioside-deficient cell line, NCTC 2071. These cells, which grow in serum-free medium, synthesized fibronectin. The fibronectin did not bind to these cells, but instead bound diffusely to the culture substratum. When the cells were cultured in medium containing ganglioside, the fibronectin became bound to the cell surface in fibrillar strands. The order of effectiveness of purified gangliosides was GT1b greater than GD1a greater than GM1 greater than GM2 greater than GM3. The effect with mixed gangliosides was accompanied by a restoration of cellular capacity to bind and to respond to cholera toxin. Treatment of the cells with several phospholipids did not alter fibronectin binding. Our results support the hypothesis that gangliosides can help mediate the binding of fibronectin to fibroblasts.     

Studies on sphingomyelinase and beta-glucosidase activities in Niemann-Pick disease variants. Phosphodiesterase activities measured with natural and artificial substrates

Cultured fibroblasts were studied from 12 cases of Niemann-Pick disease group C. In 11, sphingomyelinase and glucocerebrosidase (and beta-glucosidase) activities were reduced to around 50% of those of controls. On isoelectric focusing, all 12 strains lacked sphingomyelinase activity in the major cathodic region (pI 8.0). The defect was also demonstrated with the artificial phosphodiester substrates bis(4-methylumbelliferyl) phosphate and 4-methylumbelliferyl pyrophosphate diester. In control fibroblasts and those heterozygous for types A or B or group C Niemann-Pick disease, the major sphingomyelinase peak electrofocused at pI 8.0. No direct interaction could be demonstrated by mixing experiments between group C Niemann-Pick extracts and those of type A disease or Gaucher disease. Profiles for beta-glucosidase activity appeared normal in Niemann-Pick group C fibroblasts. No reduction of sphingomyelinase or glucocerebrosidase activities was found in Niemann-Pick group C liver, nor any attenuation of cathodic sphingomyelinase activity in the affected tissue. Results suggest that sphingomyelinase expression differs in fibroblasts and liver. Enzyme defects associated with Niemann-Pick disease group C were only observed in cultured cells.     

Amino acid-specific ADP-ribosylation

[adenine-U-14C]ADP-ribose-agmatine and [adenine-U-14C ))ADP-ribose-histone were synthesized by an NAD:arginine ADP-ribosyltransferase from [14C]NAD and agmatine and histone, respectively. The pseudo-first order rate constants for breakdown of the two components either in 0.4 N NaOH or in 0.4 M neutral hydroxylamine were identical. Hydroxylamine treatment of [14C]ADP-ribose-agmatine or [32P]ADP-ribose-histone yielded a single radioactive product which was separated by high pressure liquid chromatography and identified as ADP-ribose-hydroxamate by the formation of a ferric chloride complex. Hydrolysis of ADP-ribose-hydroxamate with snake venom phosphodiesterase resulted in the formation of 5'-AMP, consistent with the presence of a pyrophosphate bond. Incubation of ADP-ribose-[14C]agmatine, synthesized by the ADP-ribosyltransferase from NAD and [14C]agmatine, with 0.4 M neutral hydroxylamine resulted in the release of [14C]agmatine rather than phosphoribosyl[14C]agmatine. In addition, neither NAD nor ADP-ribose reacts with hydroxylamine; i.e. there was no evidence of nucleophilic attack by hydroxylamine at the pyrophosphate bond. The ADP-ribosyl-protein linkage formed by the NAD:arginine ADP-ribosyltransferase is considerably more stable to hydroxylamine than is the ADP-ribose-glutamate bond. The presence of ADP-ribose-arginine and ADP-ribose-glutamate synthesized by the ADP-ribosyltransferase and poly(ADP-ribose) synthetase, respectively, may be the chemical basis for the "hydroxylamine-stable" and "hydroxylamine-labile" bonds described by Hilz (Hilz, H. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, 1415-1425).     

Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells

In neuroblastoma-glioma (NG108-15) hybrid cells, opiates inhibit adenylate cyclase and stimulate a low Km GTPase. It has been postulated that the stimulation of GTPase plays a role in opiate inhibition of adenylate cyclase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4185-4189). Treatment of NG108-15 cells with pertussis toxin attenuates receptor-mediated inhibition of adenylate cyclase. The toxin acts by catalyzing the ADP-ribosylation of a 41,000-dalton substrate believed to be a part of the receptor-adenylate cyclase complex. We have found that toxin treatment of NG108-15 results in inhibition of the opiate-stimulated GTPase. The concentration of toxin required for inhibition of this GTPase was similar to that needed for both attenuation of opiate inhibition of adenylate cyclase and ADP ribosylation of the 41,000-dalton substrate. Inhibition of the opiate-induced GTPase by pertussis toxin in isolated membranes required NAD, consistent with the hypothesis that this effect of the toxin resulted from ADP ribosylation of a protein component of the system. Since the opiate-stimulated GTPase is believed to play a role in the receptor-mediated decrease in adenylate cyclase activity, inhibition of this GTPase may be an important part of the mechanism by which the toxin interferes with opiate action on adenylate cyclase.     

Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle

F?rster energy-transfer techniques have been applied to labeled myosin subfragment 1 from rabbit skeletal muscle to determine an intramolecular distance and whether this distance changes during magnesium-dependent ATPase activity. The alkali one light chain was labeled at Cys-177 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and then exchanged into subfragment 1. High specificity of labeling was indicated by high-performance liquid chromatography analysis of a tryptic digest of the labeled light chain. 2'(3')-O-(2,4,6-Trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was bound to the labeled protein at the ATPase active site. The efficiency of energy transfer between the probes was 0.09 when measured by both steady-state and time-resolved fluorescence. Anisotropy measurements of the bound AEDANS indicated considerable freedom of motion of the probe. The probable distance between the probes was 57 A. This distance was unchanged during triphosphatase activity. Two further sites of TNP-ADP interaction with subfragment 1 were found. The effect of these interactions on the energy-transfer measurements was reduced to a minimum by careful choice of reaction conditions.