Aging of tubulin at neutral pH

The aging of calf brain tubulin in neutral solution has been investigated using the techniques of sedimentation velocity and equilibrium, microtubule assembly, fluorescence spectroscopy, circular dichroism and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that tubulin incubated at 4 °C undergoes a slow association process leading to the generation of a 9 S component. The fraction of 9 S component increases progressively with incubation time and appears to follow mono-molecular kinetics. The generation of the 9 S species is paralleled closely by inhibition of microtubule assembly and loss of colchicine-binding ability. Fluorescence spectroscopy and circular dichroism spectra indicate that tryptophan moieties are perturbed during the aggregation process and that the tubulin dimer undergoes a conformational change. There is no protein degradation up to an incubation period of 50 hours. Sedimentation equilibrium experiments in 6 m-guanidine hydrochloride, both in the presence and absence of 2-mercaptoethanol, indicate that the aggregates are stabilized by disulfide bonds and hydrophobic interactions. At periods of incubation >50 hours, the protein starts to be degraded.     

Respiratory behavior in the pond snail Lymnaea stagnalis

Summary This study describes the neural basis of respiratory behavior in a pulmonate mollusc, Lymnaea stagnalis. We describe and identify muscles of the respiratory orifice (pneumostome) and mantle cavity as well as relevant motor neurons innervating these muscles. All of these identified motor neurons are active during spontaneously occurring respiratory behavior and a sporadically occurring synaptic input, termed Input 3, controls the activities of these motor neurons. This spontaneous input can also be recorded from isolated brain preparations, suggesting that the respiratory motor program is generated centrally. However, evidence is also presented that in semi-intact preparations the role of peripheral feedback is important for the initiation and termination of respiratory behavior in Lymnaea.     

Respiratory behavior in the pond snail Lynmaea stagnalis

Summary Previously (Syed et al. 1991) we described the ventilatory behavior of the pond snail Lymnaea stagnalis and identified motor neurons that innervate various muscles involved in this behavior. In the present study we describe an interneuronal network that controls ventilatory behavior in Lymnaea. An identified interneuron, termed the input 3 interneuron (Ip.3.I), was found to be involved in the opening movement of the pneumostome (expiration), whereas another identified interneuron known as visceral dorsal 4 (V.D.4) caused its closure (inspiration). These cells have reciprocal inhibitory connections with each other, which accounts for their opposing effects on common follower motor neurons. In isolated brain preparations a third identified interneuron, right pedal dorsal 1 (R.Pe.D.1) initiated the respiratory cycle by the excitation of Ip.3.I. Whereas Ip.3.I in turn excited R.Pe.D.1, the connections between R.Pe.D.1 and V.D.4 were mutually inhibitory. Both Ip.3.I and V.D.4 were active during spontaneously occurring respiratory behavior as recorded from semi-intact preparations, and selective hyperpolarization of V.D.4 during such spontaneous activity disrupted the respiratory behavior. Regarding peripheral feedback, the mechanical stimulation of the pneumostome during its opening movements not only caused closure but also inhibited Ip.3.I in the middle of its discharge. Ip.3.I and V.D.4 were also found to be multifunctional, inhibiting both locomotor and whole body withdrawal neural networks. We conclude from these results that the rhythmic patterned activity underlying respiratory behavior in Lymnaea is generated centrally, and that the network described here therefore comprises a central pattern generator.     

Rhizotoxicity of aluminate and polycationic aluminium at high pH

Although monomeric Al species are often toxic in acidic soils, the effects of the aluminate ion (Al(OH) ₄ ⁻ ) on roots grown in alkaline media are still unclear. Dilute, alkaline (pH 9.5) nutrient solutions were used to investigate the effects of Al(OH) ₄ ⁻ on root growth of mungbean (Vigna radiata L.). Root growth was reduced by 13% after 3 d growth in solutions with an Al(OH) ₄ ⁻ activity of 16 μM and no detectable polycationic Al (Al₁₃). This decrease in root growth was associated with the formation of lesions on the root tips (due to the rupturing of the epidermal and outer cortical cells) and a slight limitation to root hair growth (particularly on the lateral roots). When roots displaying these symptoms were transferred to fresh Al(OH) ₄ ⁻ solutions for a further 12 h, no root tip lesions were observed and root hair growth on the lateral roots improved. The symptoms were similar to those induced by Al₁₃ at concentrations as low as 0.50 μM Al which are below the detection limit of the ferron method. Thus, Al(OH) ₄ ⁻ is considered to be non-toxic, with the observed reduction in root growth in solutions containing Al(OH) ₄ ⁻ due to the gradual formation of toxic Al₁₃ in the bulk nutrient solution resulting from the acidification of the alkaline nutrient solution by the plant roots.     

Proteolytic activity at neutral pH in bovine spleen

Proteolytic activity at neutral pH can be demonstrated in extracts from beef spleen. This activity is completely due to an aminopeptidase - or a number of aminopeptides - which is able to hydrolyse proteins completely to amino acids. No evidence was found for the presence of endopeptidases active at neutral pH. The enzyme resembles to some extent swine kidney aminopeptides.     

Effect of sub-lethal concentrations of aluminium on the filtration activity of the freshwater mussel Anodonta cygnea L. at neutral pH

Significant amounts of aluminium (Al) are commonly present in rivers and lakes, largely in particulate form in neutral waters. Freshwater bivalves, as filter feeders are therefore exposed to both particulate and dissolved metal and are potentially vulnerable to Al. The effect of Al on filtering behaviour of the freshwater mussel Anodonta cygnea L. was investigated during short (1 hour) and long-term (15 days) exposure to environmentally relevant concentrations (250 and 500 microg l(-1)) at neutral pH. Water flow through the outflow siphon was monitored as an indicator of pumping capacity. Short-term (1 hour) exposure to 500 microg l(-1) added Al produced an irreversible decrease in the duration of filtering periods, presumably as an avoidance response to the toxicant. One-hour exposure 250 microg l(-1) Al had no detectable effect. When mussels were exposed to 250 or 500 microg l(-1) added Al for 15 days, siphon activity measured in days 11-15 of exposure was inhibited by 50% and 65%, respectively, compared to pre-exposure levels. Recovery occurred following transfer of mussels to uncontaminated water. Interaction between Al and freshwater bivalves at neutral pH may affect both the performance of the mussels and the chemical speciation of the metal in the natural environment.     

An acid-cleavable linker stable at neutral pH that releases doxorubicin at lysosomal pH

A Kemp's triacid amide of doxorubicin (DOX), esterified at a second carboxyl, releases DOX at lysosomal pH at a reasonable rate but is extremely stable at neutral pH.     

Effect of enkephalins on the plasticity of pond snail

The effects of introduction of leu- and met-enkephalins (LE and ME) into the perfusion medium on the characteristics of electrical activity, spontaneous firing and habituation of single neurones to repeated intracellular electrical stimulation were studied on isolated CNS of molluscs. The character, speed, and degree of development of endoneuronal habituation changed significantly after application of LE in 67 per cent of the neurones studied and in 25 per cent of neurones after ME application. As a rule the changes of habituation dynamics occurred at constant levels of membrane potential, excitability, and reactivity of the neurone. LE and ME exerted different modulating effects on the initial electrical activity in 60 per cent of neurones. The obtained data on the independence of the effects of enkephalins on different parameters of activity of one and the same neurone give evidence of a mosaic character of excitable neuronal membrane. A suggestion is made about the possible role of the revealed opiate dependence of the endoneuronal functional plasticity in realization of opiate influences at the behavioural level.     

Predation and the distribution and abundance of a pulmonate pond snail

Summary The abundances of a freshwater pulmonate snail, Lymnaea elodes were studied in a temporary pond and a permanent, more productive pond in northeastern Indiana, USA. When snails from both populations were reared in each of the ponds in containers excluding predators, snails grew to be 1.3 to 2 times as large in the more productive pond, and laid 9 times as many eggs. However, field sampling data showed adults to be more abundant in the temporary pond. The only obvious difference between the two ponds was the presence of the molluscivorous central mudminnow (Umbra limi) in the permanent pond. These fish fed upon L. elodes when eggs and juvenile snails were abundant. In an experiment in the temporary pond, addition of mudminnows lowered egg and juvenile snail survival in pens where snail abundances had been increased. We suggest that vertebrate predators like the mudminnow can be significant sources of mortality for thin shelled species like L. elodes, possibly excluding them from habitats like lakes and rivers.Address for offprint requests     

A Kluyveromyces lactis mycocin active at neutral pH

A strain of Kluyveromyces lactis was found to secrete a fungicidal mycocin active in the pH range from 6 to 9 and exhibiting the highest activity at pH of approximately 7. A few yeast species of the families Saccharomycetaceae and Wickerhamomycetaceae were sensitive to the mycocin. Some genera and species were heterogeneous in this respect. UV treatment of the mycocinogenic strain resulted in loss of its antifungal activity. Although prokaryotes were not sensitive to the mycocin, the strain under study inhibited growth of some bacteria.     

Polyamines favor DNA triplex formation at neutral pH

The stability of triplex DNA was investigated in the presence of the polyamines spermine and spermidine by four different techniques. First, thermal-denaturation analysis of poly[d(TC)].poly[d(GA)] showed that at low ionic strength and pH 7, 3 microM spermine was sufficient to cause dismutation of all of the duplex to the triplex conformation. A 10-fold higher concentration of spermidine produced a similar effect. Second, the kinetics of the dismutation were measured at pH 5 in 0.2 M NaCl. The addition of 500 microM spermine increased the rate by at least 2-fold. Third, in 0.2 M NaCl, the mid-point of the duplex-to-triplex dismutation occurred at a pH of 5.8, but this was increased by nearly one pH unit in the presence of 500 microM spermine. Fourth, intermolecular triplexes can also form in plasmids that contain purine.pyrimidine inserts by the addition of a single-stranded pyrimidine. This was readily demonstrated at pH 7.2 and 25 mM ionic strength in the presence of 100 microM spermine or spermidine. In 0.2 M NaCl, however, 1 mM polyamine is required. Since, in the eucaryotic nucleus, the polyamine concentration is in the millimolar range, then appropriate purine-pyrimidine DNA sequences may favor the triplex conformation in vivo.     

The temperature dependence of the acute toxicity of copper to a freshwater pond snail,Viviparus bengalensis L.

Static bioassays with copper (as CuSO₄ · 5H₂O) were conducted in laboratory with a freshwater pond snailViviparus bengalensis, at different environmental temperatures. The 96 hr LC50 values in ppm of Cu were 0.060 at 32.5 °C; 0.066 at 24 °C; 0.09 at 27.3 °C and 0.39 at 20.3 °C. In higher copper concentrations some behavioural changes such as secretion of mucus, discharge of eggs and embryos were noted. The results indicate that toxicity to copper increases with temperature increase.     

Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo-microscopy

The three-dimensional structures of the complete haemagglutinin (HA) of influenza virus A/Japan/305/57 (H2N2) in its native (neutral pH) and membrane fusion-competent (low pH) form by electron cryo-microscopy at a resolution of 10 A and 14 A, respectively, have been determined. In the fusion-competent form the subunits remain closely associated preserving typical overall features of the trimeric ectodomain at neutral pH. Rearrangements of the tertiary structure in the distal and the stem parts are associated with the formation of a central cavity through the entire ectodomain. We suggest that the cavity is essential for relocation of the so-called fusion sequence of HA towards the target membrane.     

Bioaccumulation of cadmium: toxicity in Mugil cephalus

A series of 12 lethal and sublethal concentrations of Cd were applied in seawater to determine LC50 for two sizes of fish. Fries were found to be more sensitive to Cd toxicity than juveniles. During 2, 4, 6 and 8 week exposure periods to graded Cd concentrations, mortality increased but no significant effect on growth occurred. From subacute experiments, the MATC for fries and juveniles were 0.05-0.02 ppm Cd2+/1 and 2-0.1 ppm Cd2+/1, respectively. Liver was found to accumulate the highest concentration of Cd followed by gill, muscle, alimentary canal and finally the heart.     

Immunolabeled neuroactive substances in the osphradium of the pond snail Lymnaea stagnalis

The osphradium of molluscs is assumed to be a sensory organ. The present investigation in Lymnaea stagnalis has established two ultrastructurally different types of dendrites in the sensory epithelium. Cells immunoreactive to leucine-enkephalin and FMRFamide send processes to the sensory epithelium. These neurons of the osphradial ganglion are thus considered to be part of the sensory system, as are methionine-enkephalin-immunoreactive cells in the mantle wall in the vicinity of the osphradium. The complexity of the osphradial ganglion is further demonstrated by serotonin-immunoreactive neurons innervating the muscular coat around the osphradial canal and methionine-enkephalin-immunoreactive cells sending projections to the central nervous system.     

Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH

Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition. They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) supression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, but not of cytoplamic malate dehydrogenase. On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state. Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.     

Shell attachment in the pond snail Lymnaea stagnalis (L.)

The attachment of the body of the snail Lymnaea stagnalis to the shell was studied by histochemistry and light and electron microscopy. Muscles of the body wall insert into the connective tissue by way of long thin projections of sarcolemma. The muscle cells end under the basement membrane of a specialised area of the epidermis, the adhesive epithelium. The cells of this epithelium are filled with microfilaments and possess characteristic knob-like microvilli. The epithelium is attached to the shell by way of an adhesive substance containing proteins and mucopolysaccharides.This research was made possible by a grant from the Netherlands Organization for Pure Research (Z.W.O.)