Chromosomal deletion 4p15.32----p14 in a Treacher Collins syndrome patient: exclusion of the disease locus from and mapping of anonymous DNA sequences to this region.

Treacher Collins syndrome is an autosomal dominant condition of bilateral craniofacial abnormalities of structures derived from the first and second branchial arches. A patient with severe manifestations of Treacher Collins syndrome and a de novo chromosomal deletion in region 4p15.32----p14 was identified. Anonymous DNA sequences of loci D4S18, D4S19, D4S20, D4S22, and D4S23 were mapped to the deleted region. DNA probes previously mapped to loci on chromosome 4p (D4S10, D4S15, D4S16, D4S26, D4S35, D4S95, D4S144, RAF1P1, QDPR, and HOX7) were not deleted in this patient. Linkage analysis between the D4S18, D4S23, and QDPR loci and Treacher Collins syndrome in eight families excluded the Treacher Collins syndrome locus from the region of the deletion.     

Kinetic characterization of early immunoreactive intermediates during the refolding of guanidine-unfolded Escherichia coli tryptophan synthase beta 2 subunits.

This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded beta 2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of beta, F1 (N-terminal; Mw = 29,000) and F2 (C-terminal; Mw = 12,000). It is shown that, while selected as being "specific" for the native protein, these antibodies are all able to recognize early folding intermediates. The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed. On the contrary, the rate constants of appearance of two "native-like" epitopes, carried by F1, could be determined during the folding of beta chains. The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s-1 at 12 degrees C. This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured beta chains. Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured beta chains, the same early folding events seem to be involved in the appearance of this epitope. The rate constant was found to be significantly smaller (k = 0.02 s-1 at 12 degrees C) for the appearance of the epitope to antibody 9. This shows that the regain of immunoreactivity is not concerted within the F1 domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Role of Chloride in Acetylcholine Metabolism

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 m m -potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 m m -potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride-containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium-stimulated release of ACh was markedly reduced as compared with a chloride-containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 m m -potassium and was totally inhibited when the potassium concentration was increased to 35 m m . Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the V _max was markedly reduced when the potassium concentration was increased to 35 m m . The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.     

RELATIVE IMPORTANCE OF CHOLINE TRANSPORT TO SPONTANEOUS AND POTASSIUM DEPOLARIZED RELEASE OF ACh

—The importance of extracellular choline transport to spontaneous and K⁺ depolarized release of ACh was studied using mouse brain cortex minces. The results suggest that extracellular choline transport is not essential to spontaneously released ACh but is essential to K⁺ depolarized ACh release. Similar cumulative amounts of choline and ACh were found in the incubation media following incubation of minces in either Krebs or 35 mm -K⁺ Krebs suggesting the same production of free choline during both conditions. Double reciprocal plots of choline accumulation by non-depolarized cortex minces yield high and low affinity components. Conversely, similar analysis of choline accumulation by depolarized minces yields a single Michaelis constant (68 μm ) similar to the low affinity (50 μm ) Michaelis constant determined for choline accumulation by non-depolarized minces. Kinetic analysis of ACh release as a function of extracellular choline concentration during K⁺ depolarization also yields a Michaelis constant of 68 μm These data suggest a link between choline transport and ACh release during K⁺ depolarization.     

Transfer of habituation between stimulation sites of the siphon withdrawal reflex in Aplysia californica

Habituation of the siphon withdrawal reflex (SWR) can be evoked by iterative tactile stimuli presented to one of several sites, including the siphon and gill. The SWR evoked at an arbitrary "test" site did not habituate when stimuli were presented at 20-min intervals. However, there was a large decrease in the reflex evoked at the test site when the trial was preceded by 10 repetitive stimuli (interstimuli interval = 30 s) presented to the opposite "habituation" site. Transfer of habituation occurred from gill to siphon stimulation sites, and vice versa. There was a concomitant decrease in the excitatory input evoked in the central siphon motor neurons LDS1 and LDS3. Moreover, transfer of habituation occurred after the abdominal ganglion (central nervous system) was removed. There was little change in the magnitude of the control responses or transfer of habituation after deganglionation. Since transfer of habituation between stimulation sites of the SWR was similar to that reported previously for the gill withdrawal reflex, it was suggested that a common mechanism may underlie the two behaviors.     

Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin.

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

Effect of hypoproteinemia on lung fluid balance in awake newborn lambs

To study the influence of plasma protein concentration on fluid balance in the newborn lung, we measured pulmonary arterial and left atrial pressures, lung lymph flow, and concentrations of protein in lymph and plasma of eight lambs, 2-3 wk old, before and after we reduced their plasma protein concentration from 5.8 +/- 0.3 to 3.6 +/- 0.6 g/dl. Each lamb underwent two studies, interrupted by a 3-day period in which we drained protein-rich systemic lymph through a thoracic duct fistula and replaced fluid losses with feedings of a protein-free solution of electrolytes and glucose. Each study consisted of a 2-h control period followed by 4 h of increased lung microvascular pressure produced by inflation of a balloon in the left atrium. Body weight and vascular pressures did not differ significantly during the two studies, but lung lymph flow increased from 2.6 +/- 0.1 ml/h during normoproteinemia to 4.1 +/- 0.1 ml/h during hypoproteinemia. During development of hypoproteinemia, the average difference in protein osmotic pressure between plasma and lymph decreased by 1.6 +/- 2 Torr at normal left atrial pressure and by 4.9 +/- 2.2 Torr at elevated left atrial pressure. When applied to the Starling equation governing microvascular fluid balance, these changes in liquid driving pressure were sufficient to account for the observed increases in lung fluid filtration; reduction of plasma protein concentration did not cause a statistically significant change in calculated filtration coefficient. Protein loss did not influence net protein clearance from the lungs nor did it accentuate the increase in lymph flow associated with left atrial pressure elevation.(ABSTRACT TRUNCATED AT 250 WORDS)