Association of Rab3A with synaptic vesicles at late stages of the secretory pathway.

Rab3A is a small GTP-binding protein highly concentrated on synaptic vesicles. Like other small GTP-binding proteins it is thought to cycle between a soluble and a membrane-associated state. To determine at which stage of the life cycle of synaptic vesicles rab3A is associated with their membranes, the localization of the protein in neurons and neuroendocrine cells at different developmental and functional stages was investigated. In all cases, rab3A was colocalized with synaptic vesicle markers at the cell periphery, but was absent from the Golgi area, suggesting that rab3A associates with vesicles distally to the Golgi complex and dissociates from vesicle membranes before they recycle to this region. Immunofluorescence experiments carried out on frog motor end plates demonstrated that massive exocytosis of synaptic vesicles is accompanied by a translocation of rab3A to the cell surface. The selective localization of rab3A on synaptic vesicles at stages preceding their fusion with the plasmalemma suggests that the protein is part of a regulatory machinery that is assembled onto the vesicles in preparation for exocytosis.

Replication in plastic of three-dimensional fossils preserved in indurated clastic sedimentary rocks.

A new technique for replicating in plastic the fossils preserved in clastic rocks should now make available reliable morphologic and frequency data, comparable in quality to those derived from acid-prepared silicified faunas, for a major segment of the fossil record. The technique involves three steps: the dissolution of carbonate in fossiliferous rocks with hydrochloric acid, impregnation of resulting voids with liquid plastic, and dissolution of the rock matrix with hydrofluoric acid, leaving a concentrate of plastic-replaced fossils.

gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein.

The polymerization of actin filaments is involved in growth, movement, and cell division. It has been shown that actin polymerization is controlled by gelsolin, whose interactions with actin are activated by calcium ion (Ca2+) and inhibited by membrane polyphosphoinositides (PPI). A smaller Ca2(+)- and PPI-regulated protein, gCap39, which has 49% sequence identity with gelsolin, has been identified by cDNA cloning and protein purification. Like gelsolin, gCap39 binds to the fast-growing (+) end of actin filaments. However, gCap39 does not sever actin filaments and can respond to Ca2+ and PPI transients independently, under conditions in which gelsolin is ineffective. The coexistence of gCap39 with gelsolin should allow precise regulation of actin assembly at the leading edge of the cell.

Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins.

Synapsins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. Molecular cloning reveals that the synapsins comprise a family of four homologous proteins whose messenger RNA's are generated by differential splicing of transcripts from two genes. Each synapsin is a mosaic composed of homologous amino-terminal domains common to all synapsins and different combinations of distinct carboxyl-terminal domains. Immunocytochemical studies demonstrate that all four synapsins are widely distributed in nerve terminals, but that their relative amounts vary among different kinds of synapses. The structural diversity and differential distribution of the four synapsins suggest common and different roles of each in the integration of distinct signal transduction pathways that modulate neurotransmitter release in various types of neurons.

Chromosomal location and inheritance of stem rust resistance transferred from Hordeum bulbosum into cultivated barley (H. vulgare).

Stem rust, caused by Puccinia graminis f. sp. tritici, is an important disease on barley (Hordeum vulgare). Host resistance has effectively controlled stem rust, primarily through use of gene Rpg1. However, virulence to Rpg1 is present in North America, and a new race (TTKSK, or Ug99) from eastern Africa threatens barley production. A search for novel resistance was previously conducted, and an interspecific barley breeding line (212Y1) with introgressed chromatin from H. bulbosum was identified as carrying resistance to races MCCF and QCCJ. This study evaluated the inheritance of resistance in 212Y1 using populations from crosses to Morex (Rpg1 donor) and Q21861 (rpg4 donor) and the pathogen races MCCF (avirulent on Rpg1 and rpg4) and QCCJ (virulent on Rpg1 and avirulent on rpg4), and determined the chromosomal position of the introgression using genomic in situ hybridization (GISH) and chromosome-specific polymerase chain reaction (PCR)-based markers. Progeny from the 212Y1/Q21861 F(2) population segregated for resistant and susceptible plants, indicating different gene loci. Genetic analyses of Morex/212Y1 F(3) families fit a 7 homozygous resistant (HR):8 segregating:1 homozygous susceptible (HS) family segregation ratio to race MCCF, indicating that two genes controlled resistance. Plants in segregating families were in 3R:1S (Rpg1), 13R:3S (Rpg1+212Y1), and 1R:3S (212Y1) ratios. Genetic analyses of the same F(3) families fit a 1HR:2 segregating:1HS family segregation ratio to race QCCJ, indicating monogenic inheritance. Plants in segregating families were in a 1R: 3S ratio, indicating recessive inheritance in 212Y1. The introgression from H. bulbosum into H. vulgare was positioned on chromosome 6HS based on GISH and the PCR-based markers. No known stem rust resistance gene has previously been mapped to that region. Thus, it is proposed to name this novel gene from H. bulbosum as rpg6.

rab3A attachment to the synaptic vesicle membrane mediated by a conserved polyisoprenylated carboxy-terminal sequence.

rab3A is a small neuronal GTP-binding protein specifically localized to synaptic vesicles. Membrane-bound rab3A behaves like an intrinsic membrane protein in vitro, but reversibly dissociates from synaptic vesicles after exocytosis in vivo. Here we demonstrate that rab3A is attached to synaptic vesicle membranes by a carboxy-terminal Cys-X-Cys sequence that is posttranslationally modified. This modification is inhibited by compactin in a mevalonate-dependent manner, suggesting that the Cys-X-Cys sequence represents a novel polyisoprenylation sequence. Isolation of a rab3 homolog from D. melanogaster reveals high evolutionary conservation of rab3A, including its carboxy-terminal Cys-X-Cys sequence. The posttranslational modifications of soluble and membrane-bound rab3A are biochemically different, but both require the carboxy-terminal Cys-X-Cys sequence and are faithfully reproduced in nonneuronal cells. Our results suggest that the carboxy-terminal Cys-X-Cys sequence of rab3A is polyisoprenylated and is used as its regulatable membrane anchor. Furthermore, the hydrophobic modification of rab3A and its correct intracellular targeting to synaptic vesicles are independent, presumably consecutive events.

Prostaglandins mediate the vasodilatory effect of mannitol in the hypoperfused rat kidney.

We have previously reported that mannitol strikingly increases blood flow to rat kidneys hypoperfused at 35-40mm Hg. This vasodilator effect is not due to volume expansion or alterations in plasma osmolality. We have tested the hypothesis that the vasodilatory effect of mannitol in the ischemic rat kidney is mediated by one of the vasoactive renal hormone systems: renin-angiotension, kallikrein-kinin, or prostaglandins. Rats were infused with 5% mannitol in 0.9% saline to 3-5% of body weight. In agreement with our previous studies, RBF increased 1.3 +/- 0.1 ml/min despite maintenance of perfusion pressure at 35-40 mm Hg. The cyclooxygenase inhibitors, meclofenamate and indomethacin had no effect on renal blood flow (RBF) in hypoperfused kidneys. However, in rats pretreated with these inhibitors, expansion with mannitol increased RBF by only 0.37 +/- 0.02 ml/min, 28% of the response in the untreated group (p less than 0.001). Infusion of prostacyclin (PGI2) into the renal artery during reduced perfusion resulted in an increase in RBF of 1.0 +/- 0.1 ml/min. Subsequent expansion with mannitol increased RBF by only 0.5 +/- 0.1 ml/min more, less than one-half of the effect of mannitol in a concurrent group of rats not treated with PGI2. Unlike PGI2 prostaglandin E2 had only a minimal vasodilator effect during hyperperfusion. Imidazole, an inhibitor of thromboxane synthesis, did not alter RBF or renal vascular resistance during hypoperfusion. Treatment of rats during hypoperfusion. with the angiotensin-converting enzyme (kininase II) inhibitor teprotide increased RBF by 1.1 +/- 0.3 ml/min. However, teprotide did not alter the vascular response to mannitol: RBF increased 1.2 +/- 0.1 ml/min more when mannitol was infused into teprotide-treated rats. The renal vascular response to mannitol was not altered by treatment with aprotinin, an inhibitor of the kallikrein-kinin system. Aprotinin was ineffective whether given before or after the vascular response to mannitol was established. We conclude that the vasodilator response to mannitol in the ischemic rat kidney is mediated in large part by increased prostaglandin (PGI2) activity. The failure of converting enzyme inhibition and aprotinin to block the vasodilator response to mannitol is evidence against a role for the renin-angiotension or kallikreinkinin systems in mediating the vasodilator response.

Evidence for a concentration gradient favoring outward movement of sodium from the thin loop of Henle.

Recent models of the urinary concentrating mechanism have postulated that urea in the medullary interstitium creates a transtubular concentration gradient for sodium between fluid at the end of the descending limb of Henle's loop and the medullary interstitium, favoring the passive outward movement of sodium from Henle's thin ascending limb. These experiments were designed to determine whether such a gradient normally exists. Young nondiuretic Munich-Wistar rats were prepared for micropuncture of the exposed left renal papilla. Samples of loop of Henle fluid and vasa recta plasma (assumed to reflect the composition of interstitial fluid) were obtained from adjacent sites. Loop fluid values in 21 comparisons from 18 rats (mean +/- SE) were: sodium 344 +/- 12 meq/liter; potassium, 26 +/- 2 meq/liter; osmolality, 938 +/- 37 mosmol/kg H23. Vasa recta plasma values (in corresponding units of measurement) were: sodium, 284 +/- 11; potassium, 34 +/- 2; osmolality, 935 +/- 34. Mean values of paired differences (loop fluid minus vasa recta plasma) were: delta sodium, 60 +/- 11.1 (P less than 0.001); delta potassium, -8.0 +/- 2.1 (P less than 0.001); delta osmolality, 4 +/- 16 (NS). Corrected for plasma water, the loop fluid minus vasa recta differences (in milliequivalents per kilogram H2O) were: delta sodium, 40 +/- 11.4 (P less than 0.005); delta potassium, -9.7 +/- 1.9 (P less than 0.001). We interpret these findings to indicate that in the papilla of nondiuretic rats, a significant difference in sodium concentration exists across the thin loop of Henle favoring outward movement of sodium, which confirms a key requirement of the passive models. A concentration difference for potassium in the reverse direction was also observed.

Effect of volume expansion on hemodynamics of the hypoperfused rat kidney.

The hemodynamics of the rat kidney were studied during reduction of renal arterial pressure to 35-40 mm Hg (H), and after volume expansion at that pressure with 0.9% NaCl (IS), 1.7% NaCl (HS), 5% mannitol in 0.9% NaCl (MS), 5% mannitol in water (MW), or 50 mM mannitol + 125 mM NaCl. During H, left renal blood flow (RBF) was 0.8+/-0.1 ml/min. Expansion with IS did not alter RBF, but expansion with HS, MS, MW, and 50 + 125 mM NaCl elevated RBF to 200-250% of hypoperfusion values. Glomerular capillary pressure rose significantly from 15.7+/-0.7 mm Hg during H to 22.3+/-1.1, 24.4+/-0.7, and 26.6+/-0.7 mm Hg following expansion with HS, MS, or MW, respectively. Efferent arteriolar pressure also rose significantly to 6.9+/-0.5, 9.7+/-0.8, and 9.5+/-0.9 mm Hg, respectively. Preglomerular resistance fell to 18-24% of H values, and postglomerular resistance fell to 58-74% of H values after expansion with HS, MS, or MW. Glomerular filtration (GFR) could not be detected during H or after IS expansion. HS and mannitol-containing solutions restored GFR to 0.10+/-0.02-0.15+/-0.02 ml/min, and single nephron glomerular filtration to 6-12 nl/min. Papaverine, acetylcholine, and kinins had no effect on RBF or GFR at a perfusion pressure of 35-40 mm Hg. We conclude that mannitol and HS have the capacity to augment RBF during hypoperfusion by reducing arteriolar resistance. The mechanism of the rise in RBF is uncertain; it may be due to changes in effective osmolality of the extracellular fluid or to a direct action of mannitol on vascular smooth muscle. Other potent vasodilators were ineffective during hypoperfusion. Restoration of GFR occurs as a result of the combined effects of augmented RBF and elevated net filtration pressure.

Effect of chronic potassium loading on potassium secretion by the pars recta or descending limb of the juxtamedullary nephron in the rat.

Recently we demonstrated potassium secretion by the pars recta or by the descending limb of the juxtamedullary nephron. The purpose of this present investigation is to study the effect of a chronic high-potassium intake on this phenomenon. Fractional reabsorption of water and sodium by the juxtamedullary proximal nephron was decreased when compared to that in normal hydropenic rats. There was a striking increase in the fraction of filtered potassium at the end of the juxtamedullary descending limb from 94+/11% to 180+/18%, which was principally a result of enhanced potassium secretion. When the concentration of potassium in the collecting tubule fluid of potassium-loaded rats was reduced after the administration of amiloride, a sharp fall was observed in the amount of potassium which reached the end of the descending limb (64+/8%). A direct correlation was observed between the fraction of filtered potassium at the descending limb and the potassium concentration in the final urine (P less than 0.001). The findings suggest that potassium, like urea, normally undergoes medullary recycling, which is enhanced by chronic potassium loading.

Communication education in veterinary in the United Kingdom and Ireland: the NUVACS project coupled to progressive individual school endeavors.

This article reports on the coordination of communications skills training in veterinary schools in the United Kingdom and Ireland and describes the progress and status of training that is occurring in six of these schools.

Recent progress in barley improvement using wild species of Hordeum.

In this review we describe recent progress in barley (Hordeum vulgare) improvement through hybridisation with its wild relatives. We have focused on one species in the secondary genepool of cultivated barley, namely H. bulbosum. This wild species has desirable traits, such as disease resistance, that are worthwhile transferring to its cultivated relative. Progress has been achieved through developing partially fertile interspecific hybrids that have been selfed or backcrossed to barley. We present the results of cytogenetic and molecular analyses that have enabled us to characterise and produce agronomically useful recombinant lines obtained from the hybrids.

Increased plasma catecholamines and locomotor activity induced by centrally administered substance P in guinea-pigs.

The effects on locomotor activity and plasma catecholamines of substance P, 0.5 nmol, injected into each lateral ventricle (i.c.v.), or 1 nmol, injected into the cisterna magna of conscious guinea-pigs, were investigated. Locomotor activity was measured in cages fitted with an infra-red photocell and detector, and plasma catecholamines, were measured by HPLC with electrochemical detection. Substance P, given intraventricularly or into the cisterna magna, produced increased locomotor activity and a pattern of behavioural activity which mimicked the opiate withdrawal response, found in previous studies in this species. Levels of NA and AD in plasma were also significantly elevated after injection of substance P. These effects of substance P were relatively long-lasting, since they were present up to 1 hr after injection. The results show that the effects of centrally administered substance P in guinea-pigs are similar to those in rats. Furthermore, the results support the suggestion that substance P might be the mediator of the opiate withdrawal response in the central nervous system as has been proposed for the enteric nervous system.

Effects of intracerebroventricularly administered mu-, delta- and kappa-opioid agonists on locomotor activity of the guinea pig and the pharmacology of the locomotor response to U50,488H.

The effects of intracerebroventricular administration of morphine, the selective mu-agonist DAMGO, the delta-agonist DPDPE, the kappa-preferring peptide dynorphin A(1-13) and the kappa-agonist U50,488H on locomotor behaviour in the guinea pig were investigated. Morphine (total dose = 0.01, 0.1, 1, 10, 200 nmol), DAMGO and DPDPE (total dose = 0.1, 1, 10, 100 nmol of each) produced piloerection and sedation, indicating that the responses of guinea pigs to mu- and delta-opioid agonists differed from those of rats and mice. In contrast, U50,488H (total dose = 10, 100 nmol) and dynorphin A(1-13) (total dose = 100 nmol) produced increased locomotor activity which was attenuated by pretreatment with naloxone and norbinaltorphimine, thus confirming the involvement of kappa-opioid receptors. Furthermore, pretreatment with spantide, baclofen, muscimol, bicuculline, MK-801, raclopride and atropine also inhibited the U50,488H-induced locomotor activity, suggesting the involvement of GABA, dopamine, excitatory amino acids, substance P and acetylcholine in this response.

PCR detection of Hordeum bulbosum introgressions in an H. vulgare background using a retrotransposon-like sequence.

Retrotransposon-like sequences are ideal tools for initial screening assays to distinguish between closely related species because of their ubiquitous presence, high copy number, chromosome coverage and rapid sequence evolution. A retrotransposon-like sequence, pSc119.1, cloned from Secale cereale (rye) has been used to obtain PCR primers that are capable of detecting small introgressions of Hordeum bulbosum (bulbous barley grass) chromatin in a Hordeum vulgare (cultivated barley) background. Combining this PCR-based assay with a crude but effective high-throughput DNA extraction has enabled the rapid identification of plants possessing H. bulbosumintrogressions from large numbers of progeny from H. vulgarex H. bulbosumcrosses. These plants are then further characterized by more-refined cytological, molecular and pathological techniques to locate and map the introgressed chromatin and to evaluate their disease resistance.

Chronic treatment with ascorbic acid inhibits the morphine withdrawal response in guinea-pigs.

The effects of ascorbic acid (AA) were investigated on the morphine withdrawal response of guinea-pigs, a species which shares with man the inability to synthesize AA. Chronic pretreatment of guinea-pigs with AA, 1 g/l, in drinking water for 3 days, or AA 200 mg/kg subcutaneously (s.c.) 3 times daily for 3 days, markedly reduced the locomotor and behavioural withdrawal responses of guinea-pigs given naloxone hydrochloride, 15 mg/kg s.c. 2 h after a single dose of morphine sulphate, 15 mg/kg s.c. AA, 1 g/kg given intraperitoneally (i.p.) 30 min before morphine had no significant effect on morphine withdrawal. However, intracerebroventricular injection of AA, 1 mumol, 30 min before naloxone significantly enhanced morphine withdrawal. It is concluded that chronic but not acute administration of AA inhibits opiate withdrawal.

Tachykinin antagonists inhibit the morphine withdrawal response in guinea-pigs.

This study was an investigation of the effects of the tachykinin antagonists, spantide and (D-Pro4, D-Trp7,9,10)substance P 4-11, injected intracerebroventricularly (ICV), on the locomotor and behavioural responses of guinea-pigs to substance P (SP) injected ICV and to naloxone-induced morphine withdrawal. SP, 50 nmol, produced increased locomotor activity and behaviour that mimicked the response induced by injection of naloxone hydrochloride, 15 mg/kg, in guinea-pigs treated 2 h previously with morphine sulphate, 15 mg/kg. Spantide or (D-Pro4, D-Trp7,9,10)SP4-11, 10 nmol, reduced the locomotor and behavioural responses to SP and to morphine withdrawal. The results support the suggestion that SP or a related tachykinin might be a mediator of the opioid withdrawal response in the central nervous system as has been proposed for the enteric nervous system.

Organochlorines and mercury in pilot whale blubber consumed by Faroe islanders.

Some 22,000 pilot whales (Globicephala melaena) were taken in the Faroe Islands between 1970 and 1992. It is known that tissues from these animals are widely consumed by the islanders. The position of these animals at the apex of a direct marine food chain renders them liable to accumulate toxic chemicals, such as metals and organochlorines. Although the consumption of contaminating metals in pilot whale tissues has been studied, the significance of blubber as a dietary source of organochlorine compounds has not been fully considered. This study reports levels of organochlorine and mercury contamination in the blubber of pilot whales taken in two Faroese kills. Published estimates of pilot whale tissue consumption are used to evaluate dietary organochlorine intake in relation to established national and international guidelines and clinical studies conducted in the North American Great Lakes.

Organochlorine residues in fish oil dietary supplements: comparison with industrial grade oils.

The market for fish oils as dietary supplements is of global importance. Although it is widely recognised that lipophilic organic chemicals, particularly organochlorines, can accumulate in fish oils, dietary supplements are not routinely considered when estimating average daily intakes for these contaminants. This paper reports levels of organochlorine residues in 44 fish oils, collected from 15 countries between 1994 and 1995, including 38 purchased over the counter as dietary supplements. Despite controls on the use of persistent organochlorine substances, appreciable quantities are found in oils sold as dietary supplements. Levels are discussed in relation to the significance of fish oil dietary supplements as contributors to daily intake of PCBs and pesticide residues.

The effect of loop of Henle diuretics on the tubuloglomerular feedback mechanism.

Increases in the delivery of solute to the loop of Henle result in increased reabsorption, vasoconstriction of the afferent arteriole, and a reduction in the glomerular filtration. Although the details of this tubuloglomerular feedback (TGF) mechanism are not completely worked out, it appears certain that alterations in reabsorption by the loop of Henle are critical to its operation. In the following study, we assessed the effect of several different loop of Henle diuretics on the response of the TGF mechanism. The function of TGF was monitored by measuring the stop-flow pressure (SFP) in the early proximal tubule in response to alterations in perfusion rate through the loop of Henle. All drugs were given directly into the loop of Henle in a concentration of 10(-4) M. With control solutions, SFP fell in a sigmoidal fashion over a perfusion range of 5 to 45 nl/min. When furosemide was added to the perfusate at a dose of 10(-4) M, SFP did not change. Bumetanide had an effect similar to furosemide, but muzolimine failed to inhibit the reduction in SFP over the perfusion range. Three experimental compounds were tested. All three cause diuresis when administered orally to animals. MK447 had no effect on SFP, but its metabolite, MK447-SO4, had an effect similar to furosemide. Another compound with modest diuretic effects, ICI 207,828, actually increased the response in SFP. Two distal diuretics, hydrochlorothiazide and amiloride, had no effect on SFP. The response of SFP to all these compounds correlated with its measured effect on loop reabsorption of sodium. Furosemide, bumetanide and MK447-SO4 significantly reduced sodium reabsorption.(ABSTRACT TRUNCATED AT 250 WORDS)