Effect of varying deep stop times and shallow stop times on precordial bubbles after dives to 25 msw (82 fsw).

In our previous research, a deep 5-min stop at 15 msw (50 fsw), in addition to the typical 3-5 min shallow stop, significantly reduced precordial Doppler detectable bubbles (PDDB) and "fast" tissue compartment gas tensions during decompression from a 25 msw (82 fsw) dive; the optimal ascent rate was 10 msw (30 fsw/min). Since publication of these results, several recreational diving agencies have recommended empirical stop times shorter than the 5 min stops that we used, stops of as little as 1 min (deep) and 2 min (shallow). In our present study, we clarified the optimal time for stops by measuring PDDB with several combinations of deep and shallow stop times following single and repetitive open-water dives to 25 msw (82 fsw) for 25 mins and 20 minutes respectively; ascent rate was 10 msw/min (33 fsw). Among 15 profiles, stop time ranged from 1 to 10 min for both the deep stops (15 msw/50 fsw) and the shallow stops (6 msw/20 fsw). Dives with 2 1/2 min deep stops yielded the lowest PDDB scores--shorter or longer deep stops were less effective in reducing PDDB. The results confirm that a deep stop of 1 min is too short--it produced the highest PDDB scores of all the dives. We also evaluated shallow stop times of 5, 4, 3, 2 and 1 min while keeping a fixed time of 2.5 min for the deep stop; increased times up to 10 min at the shallow stop did not further reduce PDDB. While our findings cannot be extrapolated beyond these dive profiles without further study, we recommend a deep stop of at least 2 1/2 mins at 15 msw (50 fsw) in addition to the customary 6 msw (20 fsw) for 3-5 mins for 25 meter dives of 20 to 25 minutes to reduce PDDB.

Nutrient absorption by Aphidius ervi larvae.

It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.

Absorption of sugars and amino acids by the epidermis of Aphidius ervi larvae.

Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of several aphid species of economic importance, widely used in biological control. The definition of a suitable artificial diet for in vitro mass production of this parasitoid is still an unresolved issue that, to be properly addressed, requires a deeper understanding both of its nutritional needs and of the functional properties of the larval epithelia involved in nutrient absorption. The experimental evidence presented in this paper unequivocally demonstrates that the uptake of sugars and amino acids takes place through the body surface of the larval stages of A. ervi. These nutrients are efficiently absorbed by the larval epidermis, but the transport rate progressively declines over time. The epidermis exhibits a cross-reactivity to antibodies raised against the mammalian facilitative glucose transporter GLUT2 and the sodium cotransporter SGLT1. The analysis of sugar transport sensitivity to specific inhibitors indicates the involvement of GLUT2-like transporters, while a role for SGLT1-like transporters is not supported. The peculiar pathways of nutrient absorption in A. ervi larvae further corroborate the general idea that the pre-imaginal stages of endophagous koinobiont Hymenoptera, like Metazoan parasites, show a high degree of physiological integration with their hosts.

Leucine methyl ester is a powerful allosteric activator of the neutral amino acid cotransport system in Bombyx mori larval midgut.

We have identified three methyl esters that have a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids in the silkworm Bombyx mori. L-Leucine methyl ester, the most powerful activator, determined a large dose-dependent, K(+)-independent increase of leucine uptake into midgut brush border membrane vesicles. Kinetic experiments revealed non-essential mixed-type activation, with K(a) values of 27+/-2 and 47+/-8 microM in the presence and in the absence of K(+), respectively. The activation increased K(m) twofold, and V(max) up to 18-fold depending upon the experimental conditions. Leucine uptake mediated by the amino acid uniport appears to be unaffected by the activator.

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch.

Modulation of leucine absorption in the larval midgut of Bombyx mori (Lepidoptera, Bombycidae).

In the larval midgut of Bombyx mori a K(+)-dependent transporter for leucine and amino acids with a hydrophobic side chain is responsible for the absorption of most essential amino acids. We investigated if a modulation of its activity occurred as a result of starvation or after hormonal treatments. We measured amino acid uptake in brush border membrane vesicles (BBMV) purified from the anterior-middle (AM) and posterior (P) regions of the midgut in fifth instar larvae. Silkworms were either starved or topically treated with low dosages of fenoxycarb, a molecule often used as a juvenile hormone mimic. The maximal uptake value of K(+)-driven leucine transport was increased in BBMV of AM- and P-midgut regions of starved larvae. The initial uptake rates of serine and glutamine, two amino acids transported by the same cotransporter as leucine, were also increased. Leucine kinetics proved that V(max) was the kinetic parameter modified by starvation in both midgut regions. Topical applications of fenoxycarb at a dose of 2.5 fg/larva immediately after the fourth ecdysis, induced an increase of leucine initial uptake rates and of intravesicular accumulation of leucine in both AM- and P-BBMV. Kinetic analysis of leucine uptake indicated again that V(max) was increased in BBMV from both midgut regions in treated larvae.

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori.

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K(+), a low-affinity saturable transport of arginine in both AM- and P-BBMV (K(m) 1.01 mM, V(max) 4.07 nmol/7s/mg protein and K(m) 1.38 mM, V(max) 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Deltapsi) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N(G)-monomethylarginine, N(G)-nitroarginine>lysine>>ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K(+), the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (K(m) 0.035 mM, V(max) 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Deltapsi and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N(G)-monomethylarginine>>cysteine, lysine>>N(G)-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K(+)/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH(out)) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.

Larval anatomy and structure of absorbing epithelia in the aphid parasitoid Aphidius ervi Haliday (Hymenoptera, Braconidae).

The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and development, focusing on time-related changes of body structure and cell ultrastructure, especially of the epithelial layers involved in nutrient absorption. Newly hatched 1st instar larvae of A. ervi are characterised by gut absence and a compact cluster of cells makes up their body. As the parasitoid larva develops, the central undifferentiated cell mass becomes hollowed out, leading to the formation of gut anlage. This suggests that absorption of nutrients at that stage may take place through the body surface, as more directly demonstrated by the occurrence on the epidermis of proteins associated with transepithelial transport, such as Na(+)/K(+)-ATPase and alkaline phosphatase (ALP). Second instar larvae show the presence of the gut with a well-differentiated brush border and a peritrophic membrane. Gut cells are filled by masses of glycogen granules and lipid droplets. The tracheal system starts to be visible. The haemocoel becomes evident in late 2nd instar, and contains large silk glands. Mature 3rd instar larvae are typically hymenopteriform. The midgut accounts for most of the body volume and is actively involved in nutrient absorption, as indicated by the well developed brush border and by the presence of Na(+)/K(+)-ATPase and ALP on the basolateral and luminal membrane respectively. At this stage, large lipid droplets have gradually replaced the cellular glycogen stores in the midgut cells. The tracheae are completely differentiated, but their internal lumen still contains fibrillar material, suggesting that they are not functional as long as host fluids bath the parasitoid larva. In late 3rd instar larvae, silk glands, structurally similar to Malpighian tubules, show a very intense vesicular traffic toward the internal lumen, which, eventually, results in being filled by secretion products, suggesting the possible recycling of metabolic waste products during mummy formation.

Substrate specificity of the brush border K+-leucine symport of Bombyx mori larval midgut.

L-leucine uptake into membrane vesicles from Bombyx mori larval midgut was tested for inhibition by 55 compounds, which included sugars, N-methylated, alpha-, beta-, gamma-, delta-, epsilon-amino acids, primary amines, alpha-amino alcohols, monocarboxylic organic acids and alpha-ketoacids. Based on cis-inhibition experiments performed at the high pH (10.8) characteristic of the midgut luminal content in vivo, we find that the carrier binding site interacts with molecules which possess a well-defined set of structural features. Amino acids are preferentially accepted as anions and the ideal inhibitor must have an hydrophobic region and a polar head constituted by a chiral carbon atom bearing two hydrophilic groups, a deprotonated amino-group and a dissociated carboxylic group. Binding is reduced if one of the two hydrophilic groups is removed. Lowering the pH to less alkaline value (8.8) only affects the affinity of delta- and epsilon-amino acids, which are excluded from binding because of their positively charged side-chain. Modifications of the potassium electrochemical gradient increased the affinity constant values of the molecules, but have little effect on the rank of specificity. Physiological implications of the data reported are discussed.

Integumental amino acid uptake in a carnivorous predator mollusc (Sepia officinalis, Cephalopoda).

The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cation-independent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed.

K(+)-neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions.

The K(+)-dependent symporter for leucine and other neutral amino acids expressed along the midgut of the silkworm Bombyx mori operates with best efficiency in the presence of a steep pH gradient across the brush-border membrane, with external alkaline pH values up to 11, and an electrical potential difference (delta psi) of approximately 200 mV. Careful determinations of leucine kinetics as a function of external amino acid concentrations between 50 and 1,000 microM, performed with brush-border membrane vesicles (BBMV) obtained from the middle and posterior midgut regions, revealed that the kinetic parameter affected by the presence of a delta pH was the maximal rate of transport. The addition of delta psi caused a further marked increase of the translocation rate. At nonsaturating leucine concentrations in the solution bathing the external side of the brush-border membrane, leucine accumulation within BBMV and midgut cells was not only driven by the gradient of the driver cation K+ and delta psi but occurred also in the absence of K+. The ability of the symporter to translocate the substrate in its binary form allows the intracellular accumulation of leucine in the absence of K+, provided that a pH gradient, with alkaline outside, is present. The mechanisms involved in this accumulation are discussed.

Evidence for a low-affinity, high-capacity uniport for amino acids in Bombyx mori larval midgut.

We investigated the kinetics of leucine influx as a function of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K(+)-dependent symporter for neutral amino acids, of a K(+)-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol.7 s-1.mg protein-1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.

Bacillus thuringiensis CrylAa delta-endotoxin affects the K+/amino acid symport in Bombyx mori larval midgut.

We have examined the type of inhibition exerted by an activated preparation of the Bacillus thuringiensis delta-endotoxin CrylAa on K+-dependent leucine transport into midgut brush border membrane vesicles or epithelial cells of the isolated midgut from Bombyx mori to study its possible interaction with the amino acid symporter. K+ permeability and the cation-dependent amino acid translocation into brush border membrane vesicles were evaluated by monitoring the fluorescence of the voltage-sensitive cyanine dye 3, 3'-dipropylthiadicarbocyanine iodide. The symporter ability to accept Na+ instead of K+ was exploited and the dissipation of an imposed inside-negative potential (K+ gradient in>out and valinomycin) was registered in the presence of a Na+ gradient (out>in) and of the amino acid. The fluorescence quenching dissipated more rapidly when the amino acid was present. Preincubation of brush border membrane vesicles with CrylAa caused a significant decrease of the amino acid-dependent recovery of fluorescence, whereas K+ permeability was sparely affected. In the isolated midgut, CrylAa inhibits leucine uptake as well as the transepithelial electrical potential difference. The strong inhibition exerted by the delta-endotoxin was observed also in the absence of potassium and the transepithelial electrical potential difference. The results obtained strongly suggest a direct interaction of CrylAa delta-endotoxin with the K+/amino acid symporter.

Alanine transport in rat liver plasma membrane vesicles during the acute-phase response in young and old rats.

The Na(+)-dependent transport of L-alanine into liver plasma membrane vesicles isolated from young, 2-month-old, and aged, 24-month-old, normal and turpentine-treated rats, to induce an aseptic inflammatory condition and the acute-phase response, was studied. In old rats, alanine transport maintained the same features observed in young controls, being strictly Na(+)-and electrical potential-dependent. However, old rats showed a twofold increase in the Vmax value for alanine uptake compared with young controls, the affinity constant (Km) remaining unmodified. Four hours after turpentine treatment, the Vmax value of alanine transport in young rats was 2.5-fold over that observed in untreated controls. In contrast, old turpentine-treated rats demonstrated only slight increases in this value, compared to untreated old controls. In both young and old turpentine-treated rats, the Km values of alanine transport remained almost unmodified. Since dysregulation of interleukin 6 (IL-6) gene expression, a multifunctional cytokine that is proving to be a major contributor to the acute phase response, occurs with advancing age in different animal species, the increased alanine transport across liver plasma membrane vesicles observed in old "normal" rats might be related to an increased IL-6 production, inducing an acute phase protein synthetic activity, in these animals.

Theoretical analysis of cotransport: its use in alanine uptake in plasma membrane vesicles.

A theoretical model of a cotransport system in plasma membrane vesicles has been utilized for the analysis of the Na(+)-dependent L-alanine transport into plasma membrane vesicles purified from Yoshida ascites hepatoma (AH 130) cells in the exponential and stationary phases of growth. The analysis was performed by comparing the experimental curves with computer simulations. In particular we considered the differences in alanine uptake observed in the two preparations and we tried to ascribe them to changes of some parameters of the transport model. The simulations indicate that sodium, alanine or water passive permeability changes cannot explain the experimental data which are consistent, on the contrary, with a relevant enhancement of the Vmax of the transport agency. The involvement of the membrane electrical potential difference is also discussed.

Changes in alanine transport in plasma membrane vesicles from rat liver during the early stages of diethylnitrosamine-induced hepatocarcinogenesis.

The transport of L-alanine, a natural substrate of system A, across liver plasma membrane vesicle preparations was modified during the early stages of rat DENA hepatocarcinogenesis. Kinetic studies indicated an increase of the Vmax, with normal Km values, at 30 h in rats undergoing a partial hepatectomy. Normal Vmax and drastically reduced Km values were present using membrane preparations from liver tissue showing enzyme-altered hyperplastic foci and/or preneoplastic nodules. The results suggest that alanine transport is differently affected by initiating and promoting stimuli during rat DENA hepatocarcinogenesis. The changes of the Vmax could be related to the promoting effect of partial hepatectomy on cell proliferation whereas the changes of the affinity constant (Km) could be the result of intrinsic modifications of the transporter in initiated cells.

Amino acid uptake in plasma membrane vesicles isolated from proliferating tumor cells and tissues.

The transport of L-alanine, a natural substrate of system A, across plasma membrane vesicle preparations has been studied in the early stages of rat DENA-PH hepato-carcinogenesis and in a very undifferentiated rat ascites hepatoma cell line (Yoshida AH-130) in the exponential and stationary phase of growth.Kinetic analyses indicated an increase of the Vmax value in DENA-PH-treated rats 30 h after partial hepatectomy as well as in exponential growing Yoshida ascites cells. In DENA-PH-treated rats the Km value was drastically reduced 7 and 60 days after surgery, when enzyme-altered hyperplastic and preneoplastic lesions were present in rat liver. Drastically reduced Km values were also found in Yoshida ascites cells.The results suggest that an altered alanine transporter might take place in liver plasma membranes from carcinogen-treated rats. This appears to occur also in an established tumor cell line, grown in vivo.

Alanine transport in plasma membrane vesicles from Yoshida ascites hepatoma cells (AH 130) in the exponential and stationary phase of growth.

The Na(+)-dependent transport of L-alanine into plasma membrane vesicles from Yoshida ascites hepatoma (AH-130) cells in the exponential and stationary phase of growth has been studied. A transient accumulation of the amino acid occurred in the presence of an inwardly directed sodium gradient, in both conditions. However, the height and the shape of the overshoot curve differed noticeably in the two preparations. The accumulation ratio increased three-fold and the maximal uptake value occurred at an earlier time in plasma membrane vesicles from exponential growing rather than stationary phase cells. This might suggest that one of the two systems, A or ASC, serving hepatocytes, is fully expressed only in the exponential phase of growth or, alternatively, that the kinetic parameters of a possibly unique transport system are modified. Inhibition, countertransport as well as adaptive stimulation experiments and kinetic studies suggested the presence of a unique carrier-mediated transport of alanine in both phases of growth. The Vmax value was drastically reduced in the stationary phase of growth whereas the Km value was almost the same in both preparations. Therefore, the differences in time courses observed could be related to changes of the Vmax of a single transport agency rather than to the appearance/disappearance of an additional transport system (e.g. system A) in the conditions studied.

Histidine transport in plasma membrane vesicles from rat liver.

The transport of L-histidine, a selective substrate for system N, across liver plasma membrane vesicles has been studied. The amino acid is accumulated sevenfold within the intravesicular space in the presence of a sodium gradient. Lithium can replace sodium to some extent in the cotransport mechanism. The amino acid translocation is not influenced by the transmembrane electrical potential difference. Histidine kinetics involves a saturable component plus a linear one both in the presence and in the absence of sodium. Sodium affects mainly the affinity of the translocator and to a lesser extent its mobility. Histidine uptake is competitively inhibited by glutamine and it is affected by alanine in a noncompetitive manner.

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo.

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.