The Interaction of Polyene Antibiotics with Thin Lipid Membranes

Optically black, thin lipid membranes prepared from sheep erythrocyte lipids have a high dc resistance (R_m ≅ 10⁸ ohm-cm²) when the bathing solutions contain NaCl or KCl. The ionic transference numbers (T_i) indicate that these membranes are cation-selective (T _Na ≅ 0.85; T _Cl ≅ 0.15). These electrical properties are independent of the cholesterol content of the lipid solutions from which the membranes are formed. Nystatin, and probably amphotericin B, are cyclic polyene antibiotics containing ≈36 ring atoms and a free amino and carboxyl group. When the lipid solutions used to form membranes contained equimolar amounts of cholesterol and phospholipid, these antibiotics reduced R_m to ≈10² ohm-cm²; concomitantly, T _Cl became ≅0.92. The slope of the line relating log R_m and log antibiotic concentration was ≅4.5. Neither nystatin (2 x 10^-5 M) nor amphotericin B (2 x 10^-7 M) had any effect on membrane stability. The antibiotics had no effect on R_m or membrane permselectivity when the lipids used to form membranes were cholesterol-depleted. Filipin (10^-5 M), an uncharged polyene with 28 ring atoms, produced striking membrane instability, but did not affect R_m or membrane ionic selectivity. These data suggest that amphotericin B or nystatin may interact with membrane-bound sterols to produce multimolecular complexes which greatly enhance the permeability of such membranes for anions (Cl^-, acetate), and, to a lesser degree, cations (Na⁺, K⁺, Li⁺).     

Chytrid epidemics may increase genetic diversity of a diatom spring-bloom

Contrary to expectation, populations of clonal organisms are often genetically highly diverse. In phytoplankton, this diversity is maintained throughout periods of high population growth (that is, blooms), even though competitive exclusion among genotypes should hypothetically lead to the dominance of a few superior genotypes. Genotype-specific parasitism may be one mechanism that helps maintain such high-genotypic diversity of clonal organisms. Here, we present a comparison of population genetic similarity by estimating the beta-dispersion among genotypes of early and peak bloom populations of the diatom Asterionella formosa for three spring-blooms under high or low parasite pressure. The Asterionella population showed greater beta-dispersion at peak bloom than early bloom in the 2 years with high parasite pressure, whereas the within group dispersion did not change under low parasite pressure. Our findings support that high prevalence parasitism can promote genetic diversification of natural populations of clonal hosts.     

Neuromagnetic evidence for early auditory restoration of fundamental pitch

Background

Understanding the time course of how listeners reconstruct a missing fundamental component in an auditory stimulus remains elusive. We report MEG evidence that the missing fundamental component of a complex auditory stimulus is recovered in auditory cortex within 100 ms post stimulus onset.

Methodology

Two outside tones of four-tone complex stimuli were held constant (1200 Hz and 2400 Hz), while two inside tones were systematically modulated (between 1300 Hz and 2300 Hz), such that the restored fundamental (also knows as “virtual pitch”) changed from 100 Hz to 600 Hz. Constructing the auditory stimuli in this manner controls for a number of spectral properties known to modulate the neuromagnetic signal. The tone complex stimuli only diverged on the value of the missing fundamental component.

Principal Findings

We compared the M100 latencies of these tone complexes to the M100 latencies elicited by their respective pure tone (spectral pitch) counterparts. The M100 latencies for the tone complexes matched their pure sinusoid counterparts, while also replicating the M100 temporal latency response curve found in previous studies.

Conclusions

Our findings suggest that listeners are reconstructing the inferred pitch by roughly 100 ms after stimulus onset and are consistent with previous electrophysiological research suggesting that the inferential pitch is perceived in early auditory cortex.     

Gender affects calf venous compliance at rest and during baroreceptor unloading in humans

Leg venous compliance is a determinant of peripheral venous pooling during orthostatic stress such that high venous compliance could contribute to reduced orthostatic tolerance. We tested the hypotheses that 1) calf venous compliance is reduced during baroreceptor unloading, and 2) calf venous compliance is greater in women than men. Twelve men (27 +/- 2 yr) and 12 women (25 +/- 2 yr) were studied in the supine posture. Calf venous compliance was determined by inflating a thigh venous collecting cuff to 60 mmHg for 8 min and then decreasing cuff pressure at a rate of 1 mmHg/s to 0 mmHg. The slope of the pressure-compliance relation (compliance = beta(1) + 2.beta(2).cuff pressure), which is the first derivative of the quadratic pressure-volume relation [(Deltalimb volume) = beta(0) + beta(1).(cuff pressure) + beta(2).(cuff pressure)(2)] during the reduction in collecting cuff pressure, was used to assess venous compliance at baseline and during one-legged lower body negative pressure (LBNP; -50 mmHg). At baseline, calf venous compliance was 48% lower (P < 0.001) in women than men and decreased in men (Delta-25 +/- 8%; P < 0.05) but not women (Delta1 +/- 11%) during LBNP. Rhythmic ischemic handgrip (Delta6 +/- 9%) and cold pressor testing (Delta-9 +/- 7%) did not alter calf venous compliance in a subgroup of men (n = 6). These data indicate gender-dependent effects on calf venous compliance under conditions associated with low sympathetic outflow (i.e., rest) and high sympathetic outflow (i.e., LBNP). However, they cannot explain gender-associated differences in orthostatic tolerance.     

Oxygen transfer in a pulse bioreactor

Oxygen transfer in a novel pulse bioreactor has been evaluated. The agitator consists of a series of alternately fixed and movable parallel plates mounted so that the movable plates vibrate at 30 Hz causing a pulsating fluid motion. Pure oxygen, at pressures up to 5 atm, diffuses through silicone rubber tubing that also vibrates at 30 or 60 Hz. The main feature of this bioreactor is high oxygen transfer with low shear to prevent damage to fragile animal cell membranes. We estimate that sufficient oxygen can be supplied to support over 10(8) cells/mL of human diploid foreskin cells growing on microcarriers. (c) 1993 John Wiley & Sons, Inc.     

Expression, Purification, and Characterization of Recombinant α-N-Acetylgalactosaminidase Produced in the YeastPichia pastoris

α-N-Acetylgalactosaminidase (αNAGAL, EC 3.2.1.49) purified from chicken liver has been used in seroconversion of human erythrocytes. Blood group A, defined by the terminal α-linkedN-acetylgalactosamine, can be cleavedin vitroby αNAGAL, resulting in the underlying penultimate blood group H (O) epitope structure. In order to produce sufficient quantities of recombinant αNAGAL (rαNAGAL) for such studies, we expressed the cDNA encoding chicken liver αNAGAL inPichia pastoris,a methylotrophic yeast strain. The αNAGAL coding sequence was cloned into theEcoRI site of the vector pPIC 9 such that the protein was in the same reading frame as the secretion signal of yeast α-mating factor derived from the vector. AfterP. pastoristransformation, colonies were screened for high-level expression of rαNAGAL based on enzyme activity. As a result of methanol induction of high-density cell cultures in a fermentor, enzymatically active rαNAGAL was produced and secreted into the culture medium. The recombinant enzyme was purified over 150-fold by chromatography on a cation exchange column followed by an affinity column. Its homogeneity was confirmed by Coomassie blue-stained SDS–PAGE, Western blot, and N-terminal sequencing. The purified rαNAGAL has a molecular mass of approximately 50 kDa while its native counterpart has a molecular mass of 43 kDa. This discrepancy in size was eliminated by endoglycosidase treatment, suggesting that the recombinant protein was hyperglycosylated by the hostP. pastoriscells. rαNAGAL was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability by comparing with αNAGAL purified from chicken liver. The data presented here suggest that by overexpressing rαNAGAL inP. pastorisand purifying with affinity chromatography one can readily obtain the quantity of enzyme needed for seroconversion studies.     

Early events in the cellular formation of proparathyroid hormone

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.     

Phencyclidine

Phenycyclidine (PCP) produces many profound effects in the central nervous system. PCP has numerous behavioral and neurochemical effects such as inhibiting the uptake and facilitating the release of dopamine, serotonin, and norepinephrine. PCP also interacts with sigma, mu opioid, muscarinic, and nicotinic receptors. However, the psychotomimetic effects induced by PCP are believed to be mediated by specific PCP receptors, where PCP binds with greater potency than sigma compounds. Electrophysiological, behavioral, and neuro-chemical evidence strongly suggests that at least some of the many PCP actions result from antagonism of excitatory amino acid-induced responses via PCP receptors. The recent isolation and partial characterization of the alpha and beta endopsychosins and the identification of other endogenous ligands for the PCP and sigma receptors, is another promising area of research in the elucidation of the physiological role of an endogenous PCP and sigma system.