Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate.

1. A Hepes-based medium has been devised which allows rapid Pi exchange across the plasma membrane of the human erythrocyte. This allows the metabolically labile phosphate pools of human erythrocytes to come to equilibrium with [32P]Pi in the medium after only 5 h in vitro. 2. After 5-7 h incubation with [32P]Pi in this medium, only three phospholipids, phosphatidic acid (PtdOH), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) are radioactively labelled. The concentrations of PtdIns4P and PtdIns4,5P2 remain constant throughout the incubation, so this labelling process is a reflection of the steady-state turnover of their monoester phosphate groups. 3. During such incubations, the specific radioactivities of the monoesterified phosphates of PtdIns4, PtdIns4,5P2 and PtdOH come to a steady value after 5 h that is only 25-30% of the specific radioactivity of the gamma-phosphate of ATP at that time. We suggest that this is a consequence of metabolic heterogeneity. This heterogeneity is not a result of the heterogeneous age distribution of the erythrocytes in human blood. Thus it appears that there is metabolic compartmentation of these lipids within cells, such that within a time-scale of a few hours only 25-30% of these three lipids are actively metabolized. 4. The phosphoinositidase C of intact human erythrocytes, when activated by Ca2+-ionophore treatment, only hydrolyses 50% of the total PtdIns4,5P2 and 50% of 32P-labelled PtdIns4,5P2 present in the cells: this enzyme does not discriminate between the metabolically active and inactive compartments of lipids in the erythrocyte membrane. Hence at least four metabolic pools of PtdIns4P and PtdIns4,5P2 are distinguishable in the human erythrocyte plasma membrane. 5. The mechanisms by which multiple non-mixing metabolic pools of PtdOH, PtdIns4P and PtdIns4,5P2 are sustained over many hours in the plasma membranes of intact erythrocytes are unknown, although some possible explanations are considered.     

Effects of experimental acidification on a lotic macroinvertebrate community

A three month experimental acidification was carried out on lotic bottom communities. Experiments were conducted under semi-natural conditions in plasticized wooden channels. Acidified communities (pH 4.0), with or without added aluminum, were compared with a reference community (pH 6.3–6.9). Added aluminum concentrations were respectively 0.2 and 0.4 mg 1^–1 in experiments performed in 1982 and 1983. Water chemistry and taxonomic composition of the macroinvertebrate communities were monitored. Under acidified conditions, results were similar, with or without added aluminum. Mean abundances of all groups of organisms were lowered. Mayflies nearly completely disappeared from the acidified channels. The only organism not affected by the acidification was Microtendipes sp. Differences in the organism response were observed: Orthocladiinae (Rheocricotopus, Parametriocnemus, Corynoneura, Thienemanniella, Nanocladius, Cricotopus) and Ephemeroptera (Baetis, Habrophlebia, Habrophlebiodes, Paraleptophlebia, Ephemerella), especially early instars, were very sensitive to low pH, Chironomini and Tanypodinae were much less sensitive, while Tanytarsini were intermediate; Oligochaeta and Nematoda were difficult to classify, their response being different from one year to another. Organisms inhabiting the surface of artificial substrates disappeared very rapidly from the system, while those buried inside had a delayed reaction to acidification. Aluminum which was mainly in the monomeric form was not responsible for community modifications. Direct action of hydrogen ions through a physiological stress seems a more credible explanation. These results, induced by a continuous experimental acidification, suggest that if this small headwater stream undergoes acidification, the resulting invertebrate community will be very simplified, with only resistant species able to cope with the acid conditions.     

Effects of α2-Adrenergic Agonists and Antagonists on Photoreceptor Membrane Currents

Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presynaptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (greater than 100 microM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cell's resting potential as well as after a light step. Clonidine reduces voltage-dependent outward K+ currents (IA, an early current, ICa2+-K+, a late Ca2+-dependent current) that control the type B cell's excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the alpha 2-receptor antagonist, yohimbine (0.5 microM), but not the alpha 1-blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde-induced fluorescence or Falck-Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine-like synaptic effect on type B cells, therefore, could amplify conditioning-specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.     

Low-Level Microwave Irradiations Affect Central Cholinergic Activity in the Rat

Sodium-dependent high-affinity choline uptake was measured in various regions of the brains of rats irradiated for 45 min with either pulsed or continuous-wave low-level microwaves (2,450 MHz; power density, 1 mW/cm2; average whole-body specific absorption rate, 0.6 W/kg). Pulsed microwave irradiation (2-microseconds pulses, 500 pulses/s) decreased choline uptake in the hippocampus and frontal cortex but had no significant effect on the hypothalamus, striatum, and inferior colliculus. Pretreatment with a narcotic antagonist (naloxone or naltrexone; 1 mg/kg i.p.) blocked the effect of pulsed microwaves on hippocampal choline uptake but did not significantly alter the effect on the frontal cortex. Irradiation with continuous-wave microwaves did not significantly affect choline uptake in the hippocampus, striatum, and hypothalamus but decreased the uptake in the frontal cortex. The effect on the frontal cortex was not altered by pretreatment with narcotic antagonist. These data suggest that exposure to low-level pulsed or continuous-wave microwaves leads to changes in cholinergic functions in the brain.     

Electrophoretic confirmation of the intergeneric hybrid ×Ruttyruspolia (Acanthaceae)

A plant collected in South Africa in the early 1960's has been considered an intergeneric hybrid with the parental taxa beingRuspolia hypocrateriformis (Vahl)Milne-Redhead var.australis Milne-Redhead andRuttya ovata Harv. The intermediate morphology of the plant provided the strongest evidence of its hybrid origin. The natural hybrid, named formally as ×Ruttyruspolia A. Meeuse & de Wet, is highly sterile. Crosses between the two presumed parental taxa produced two plants that are very similar to the putative natural hybrid. We had examined the presumed parental species and the natural and artificial hybrids using enzyme electrophoresis. The two parental species are highly differentiated at genes specifying soluble enzymes; they have a genetic identity of 0.51. They have no common alleles at two genes, and contain alternative alleles in very different frequencies at two loci.Ruttya andRuspolia exhibit both unique and common alleles at two additional genes. The natural and artificially produced plants of ×Ruttyruspolia are identical electrophoretically and contain alleles unique to each of the parental species at two genes. In addition, individuals of ×Ruttyruspolia combine alternative high frequency alleles from each parent at two loci. Allozymes provide strong confirming evidence for the hybrid origin of naturally occurring ×Ruttyruspolia because the products of specific alleles either unique to or highly characteristic of the two putative parental taxa are found combined in ×Ruttyruspolia.     

Fertile heteroallelic combinations of mutant alleles of the otu locus of Drosophila melanogaster

Summary This paper describes the ovarian pathologies observed when 108 different heteroallelic combinations were made involving 17 independent mutations at the ovarian tumor (otu) locus. Most of the mutant phenotypes can be explained as graded responses by individual germ cells to different levels of functionally active otu gene product (OGP) synthesized by the mutant cells themselves. The lowest and highest levels of OGP appear to be produced by otu ¹⁰ and otu ¹⁴, respectively. In most heteroallelic ovaries the alleles have additive effects, and hybrid germ cells reach a developmental stage more advanced than the weaker homozygote but less advanced than the stronger homozygote. However, examples of both positive and negative complementation also have been found, and these suggest that the products encoded by different mutant alleles can combine to form dimers or multimers which may be superior or inferior to the homodimers. In flies homozygous for otu ¹¹ most ovarioles contain tumors, but some germ cells are able to develop further than those in otu ¹⁴ homozygotes. This suggests that, while otu ¹¹ produces intermediate levels of OGP, it also produces a second product (which otu ¹⁴ cannot make) that is utilized at the period in oogenesis when development in cells homozygous for otu ¹⁴ is blocked. When otu ¹¹ is combined with any one of eight specific alleles, it allows oocyte/nurse cell syncytia to differentiate that can complete development and undergo embryogenesis, if fertilized. The endopolyploid nurse cells of these hybrids have giant polytene chromosomes, and the presence of GPCs in functionally active, germ-line derived cells provides an interesting new system for experimental study. Analysis of the characteristic ovarian pathologies produced by flies of different genotypes leads to the conclusion that the products of the otu ⁺ gene are utilized during at least six different periods in Drosophila oogenesis.     

Microbial degradation of oxalate in the gastrointestinal tracts of rats.

Rates of oxalate degradation by mixed bacterial populations in cecal contents from wild rats ranged from 2.5 to 20.6 mumol/g (dry weight) per h. The oxalate-degrading activity in cecal contents from three strains of laboratory rats (Long-Evans, Wistar, and Sprague-Dawley) from four commercial breeders was generally lower, ranging from 1.8 to 3.5 mumol/g (dry weight) of cecal contents per h. This activity did not increase when diets were supplemented with oxalate. When Sprague-Dawley rats from a fifth commercial breeder were fed an oxalate diet, rates of oxalate degradation in cecal contents increased from 2.0 to 23.1 mumol/g (dry weight) per h. Obligately anaerobic, oxalate-degrading bacteria, similar to ruminal strains of Oxalobacter formigenes, were isolated from the latter group of laboratory rats and from wild rats. Viable counts of these bacteria were as high as 10(8)/g (dry weight) of cecal contents, which was less than 0.1% of the total viable population. This report presents the first evidence for the presence of anaerobic oxalate-degrading bacteria in the cecal contents of rats and represents the first direct measurement of the concentration of these bacteria in the large bowel of monogastric animals. We propose that methods used for the maintenance of most commercial rat colonies often preclude the intestinal colonization of laboratory rats with anaerobic oxalate-degrading bacteria.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.