A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II

A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage-clamp in Xenopus oocytes injected with the corresponding cDNA-derived mRNA.     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Direct segregation analysis of reciprocal translocations: a study of 283 sperm karyotypes from four carriers.

Using the technique of in vitro human-hamster fertilization, sperm of four men heterozygous for 4 reciprocal translocations--t(4;17),t(5;13),t(6;7), and t(9;18)--was studied. Frequencies of numerical abnormalities unrelated to the translocations range from 8.3% to 13.3%, and the incidence of imbalances ranges from 23.0% to 66.0%. Results are pooled with data from the nine other reciprocal translocations reported elsewhere, and the combined data demonstrate that male meiotic segregation is not random: whatever the type of translocation may be, the distribution of imbalances in sperm is constant, with approximately 72.0% adjacent 1, 18.5% adjacent 2, and 9.5% 3:1 segregations. The same prevalence of adjacent 1 segregations as that reported at term for translocations of paternal origin is observed. There is a strong postzygotic elimination process; for a given translocation it affects selectively the maximum-imbalance zygotes so that imbalanced segregations observed at term are always predetermined.     

Identification of thetrans-stilbene oxide-active glutathione transferase in human mononuclear leukocytes and in liver as GST1

A glutathione transferase from human mononuclear leukocytes with a high activity towardtrans-stilbene oxide (GT-tSBO) has been studied in liver and blood from fetus and adults and in blood from neonates. Using starch gel electrophoresis, different phenotypes of GST1 have been determined, GST1 0, GST1 1, and GST1 2. As judged from activity measurements and the fact that only those individuals who express the null allele of GST1, the GST1 0, which has a low activity towardtrans-stilbene oxide, it is concluded that the hepatic transferase GST1 is identical to GT-tSBO, as well as to hepatic transferase μ. In addition, it has been shown that the different genotypes of GST1 1 (GST1 1-1, GST1 1-0) and GST1 2 (GST1 2-2, GST1 2-0) can be separated by measuring the GT-tSBO activity in whole blood from the same individual. It is also demonstrated that GT-tSBO activity is much lower in fetal liver, approximately 10 times, compared with adult liver, while this activity seems to be unchanged in the blood from fetus and adults, as well as in neonates.     

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Herbicide effects on planktonic systems of different complexity

Bioassays of different complexity were compared with respect to their capability to predict the environmental impact of the herbicide atrazine in aquatic systems. Acute toxicity tests with Daphnia did not yield meaningful results. Sublethal tests with Daphnia (feeding inhibition, reduction of growth and reproduction) were more sensitive, but effective concentrations of atrazine were still rather high (2 mg/L). A relatively complicated artificial food chain system that incorporated direct and indirect effects on Daphnia yielded significant reduction of daphnid population growth at 0.1 mg/L. Enclosure experiments with natural communities were by far the most sensitive tools. Community responses could be measured at concentrations as low as 1 µg/L and 0.1 µg atrazine/L. At the lowest concentration, however, communities recovered after three weeks. We conclude that in complex systems indirect effects can be more important than direct effects, so that, contrary to the conditions in simple tests, non-target organisms may be the better indicators of herbicide stress to natural communities.     

Neuropeptide Y: presence in sympathetic and parasympathetic innervation of the nasal mucosa

Summary The occurrence of neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in the sympathetic and parasympathetic innervation of the nasal mucosa was studied in various species including man. A dense network of NPY-immunoreactive (IR) fibres was present around arteries and arterioles in the nasal mucosa of all species studied. NPY was also located in nerves around seromucous glands in pig and guinea-pig, but not in rat, cat and man. The NPY-IR glandular innervation corresponded to about 20% of the NPY content of the nasal mucosa as revealed by remaining NPY content determined by radioimmunoassay after sympathectomy. These periglandular NPY-positive fibres had a distribution similar to the VIP-IR and PHI-IR nerves but not to the noradrenergic markers tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH). The NPY nerves around glands and some perivascular fibres were not influenced by sympathectomy and probably originated in the sphenopalatine ganglion where NPY-IR and VIP-IR ganglion cells were present. The venous sinusoids were innervated by NPY-positive fibres in all species except the cat. Dense NPY and DBH-positive innervation was seen around thick-walled vessels in the pig nasal mucosa; the latter may represent arterio-venous shunts. Double-labelling experiments using TH and DBH, and surgical sympathectomy revealed that the majority of NPY-IR fibres around blood vessels were probably noradrenergic. The NPY-positive perivascular nerves that remained after sympathectomy in the pig nasal mucosa also contained VIP/PHI-IR. The major nasal blood vessels, i.e. sphenopalatine artery and vein, were also densely innervated by NPY-IR fibres of sympathetic origin. Perivascular VIP-IR fibres were present around small arteries, arterioles, venous sinusoids and arterio-venous shunt vessels of the nasal mucosa whereas major nasal vessels received only single VIP-positive nerves. The trigeminal ganglion of the species studied contained only single TH-IR or VIP-IR but no NPY-positive ganglion cells. It is concluded that NPY in the nasal mucosa is mainly present in perivascular nerves of sympathetic origin. In some species, such as pig, glandular and perivascular parasympathetic nerves, probably of VIP/PHI nature, also contain NPY.     

Purification of acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase from Catharanthus roseus leaves

The enzyme acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase which terminates vindoline biosynthesis has been isolated from Catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. Kinetic properties concerning the enzyme reaction are discussed. Five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. This enzyme is now the best characterized of the enzymes involved in vindoline biosynthesis.Abbreviations DTE dithiothreitol - EDTA ethylenediamine-tetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid - IEF isoelectric focusing - KP_i potassium phosphate - M_r rel.molecular mass - PEG polyethylene glycol - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

A practical test for in vitro evaluation of photosensitization: Assessment with hematoporphyrin derivative (HPD)

A simple procedure is described for the determination of the photosensitizing potency of drugs, using three leukemic cell lines, two of lymphocytic origin, L1210 and P388 and one of erythroid type, Friend-745. The procedure allows one to investigate several aspects of the photosensitization properties of tested compounds such as cellular localization and direct (trypan blue exclusion) or delayed (clonogenicity) photomediated toxicities.The method was assessed using crude hematoporphyrin derivative (HPD) as well as dihematoporphyrin ether (DHE) or commercially available Photofrin II. Results were compared to those obtained with normal cells, e.g spleen lymphocytes and erythropoietic stem cells (CFU-e), and discussed in the light of the relative response of normal versus transformed cells.Abbreviations DHE Dihematoporphyrin Ether - FCS Fetal Calf Serum - HPD Hematoporphyrin Derivative - PDT Photodynamic Therapy     

Histamine-like immunoreactivity in photoreceptors of the compound eyes and ocelli of the flies Calliphora erythrocephala and Musca domestica

Summary Antibodies to histamine were used for immunocytochemical studies of the visual system in the flies Calliphora erythrocephala and Musca domestica. Specific immunolabeling of photoreceptors was found both in the compound eyes and ocelli of both species. In the compound eyes histamine-like immunoreactivity (HA-IR) was found in all the short visual fibers (photoreceptors R1–6) and one type of long visual fiber (photoreceptor R8). In addition, the ocellar photoreceptors also show HA-IR. In view of earlier biochemical and pharmacological/physiological findings by Elias and Evans (1983) and Hardie (1987) it thus seems likely that histamine is a neurotransmitter in insect photoreceptors. Interestingly, the second type of long visual fiber (photoreceptor R7) has recently been found to be GABA-immunoreactive (Datum et al. 1986). The two types of long visual fibers may hence use different transmitters which act on different receptors of the postsynaptic neurons in the second visual neuropil, the medulla. In addition to the photoreceptors in the retina and ocelli, we found processes of HA-IR neurons in one of the optic lobe neuropils, the lobula. This finding indicates that histamine may also be a transmitter in certain interneurons in the visual system.Abbreviations HA histamine - GABA -amino butyric acid - GAD glutamic acid decarboxylase - 5-HT 5-hydroxytryptamine (serotonin) - HA-IR histamine-like immunoreactivity - R1-R6 class of short-axoned photoreceptors - R7 and R8 long-axoned photoreceptors - LMC large monopolar neuron of lamina - HSA human serum albumin - PBS phosphate-buffered saline - DEPC diethylpyrocarbonate