Diversity and characterization of oxytetracycline-resistant bacteria associated with non-native species, white-leg shrimp (Litopenaeus vannamei), and native species, black tiger shrimp (Penaeus monodon), intensively cultured in Thailand.

AIMS: This study aimed at surveying prevalence of oxytetracycline (OTC)-resistant bacteria in the white-leg shrimp Litopenaeus vannamei, and the black tiger shrimp Penaeus monodon, intensively cultured in Thailand. We investigated the phylogenetic diversity of the bacterial isolates, as well as the minimum inhibitory concentration (MIC) of OTC, the occurrence of major OTC-resistant genes and multiple-antibiotic resistance in the isolates. METHODS AND RESULTS: Shrimps were collected from culture ponds, and the homogenates of whole bodies were plated on tryptic soy agar supplemented with or without OTC. Percentages of OTC-resistant bacteria were 0·3-52·1% in white-leg samples and 0·008-22·3% in black tiger samples. Analyses of 16S rDNA sequences indicated that most OTC-resistant isolates were closely related to Aeromonas spp. and Lactococcus garvieae. MICs of OTC were 4-128 µg ml(-1) in the OTC-resistant aeromonads and 128-256 µg ml(-1) in OTC-resistant L. garvieae. OTC resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S), detected either singly or in pairs. No resistance to ceftazidime, imipenem or chloramphenicol was observed in any isolate. CONCLUSIONS: Both species of shrimp are associated with OTC-resistant bacteria, occasionally at high densities exceeding 10(6) cfu g(-1). The associated bacteria, predominantly Lactococcus and Aeromonas genera, are potential pathogens and are reservoirs of a variety of OTC-resistant genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Cultured shrimps can be vehicle to carry OTC-resistant bacteria to domestic and foreign consumers via the food chain. Very low populations of OTC-resistant bacteria observed in the several ponds suggest that levels of the resistant bacteria are artificially high and should be reduced in farmed shrimps.

Excitation-contraction coupling in isolated locomotor muscle fibres from the pelagic tunicate Doliolum which lack both sarcoplasmic reticulum and transverse tubular system.

In the locomotor muscle of the pelagic tunicate Doliolum, both the sarcoplasmic reticulum (SR) and the transverse-tubular (T-tubular) system are absent. The mechanism of excitation-contraction (E-C) coupling was studied in single muscle fibres enzymatically dissociated from Doliolum denticulatum. Whole cell voltage clamp experiments demonstrated an inward ionic current associated with membrane depolarisation. This current was blocked by 5 mmol.l(-1)Co(2+), a calcium current blocker, and suppressed by nifedipine, a specific L-type calcium channel blocker. An increase in the external K(+) concentration to 200 mmol.l(-1) (K(+)-depolarisation) induced a rise in the intracellular Ca(2+) level detected with fluo-3, a Ca(2+)-sensitive dye. However, when 5-10 mmol.l(-1) Co(2+) or 10-15 micro mol.l(-1) nifedipine was present in the external solution, K(+)-depolarisation did not induce a rise in the intracellular Ca(2+) level. Externally applied 5-10 mmol.l(-1) caffeine or 20 micro mol.l(-1) ryanodine had no effect on the intracellular Ca(2+) level. K(+)-depolarisation induced a rise in the intracellular Ca(2+) level in the presence of caffeine or ryanodine. Replacement of external Na(+) with Li(+) increased intracellular Ca(2+) levels. Our results show that contraction of the locomotor muscle in Doliolum is solely due to the influx of Ca(2+) through L-type calcium channels, and that relaxation is due to extrusion of Ca(2+) by Na(+)/Ca(2+) exchange across the sarcolemma.

Regulation of the H+ pump activity in the plasma membrane of internally perfused Chara corallina.

The role of cytoplasm for the maintenance of the H+ pump activity in Chara corallina internodal cells was examined by the intracellular perfusion technique. Cytoplasm-rich and -poor states were obtained by changing the perfusion time, short-term (less than 2 min) and long-term (more than 5 min), respectively. A large portion of cytoplasm was left by short-term perfusion but most of the cytoplasm was removed by long-term perfusion. The activities of the H+ pump of these two different conditions were examined by measuring current-voltage relation (I-V curve) and conductance-voltage relation (G-V curve) under voltage clamp conditions. The H+ pump conductance decreased to 37%, 9% and zero by short-term, long-term and hexokinase perfusion, respectively, whereas the passive channel conductance decreased to 71%, 39% and 73% by short-term, long-term and hexokinase perfusion, respectively. On the other hand, the electromotive-force of the H+ pump (approximately -260 mV) and the passive channel (approximately -130 mV) were not affected by either short- or long-term perfusion. It is indicated that the cytoplasm plays an essential role to regulate the activity of both the H+ pump and the passive channel together with ATP.

Cyclic AMP-mediated inhibition of noradrenaline-induced contraction and Ca2+ influx in guinea-pig vas deferens.

The relaxation effects of forskolin and methylxanthines on noradrenaline (NA)-induced contractions were investigated by measuring isotonic contraction and intracellular calcium concentration ([Ca2+]i) in the epididymal side of guinea-pig vas deferens. NA (100 microM) and high K+ (55 mM) induced a biphasic contraction; fast, transient (phasic) and slow, sustained (tonic) phases. Both phases in either NA or high K+ stimulation were abolished in Ca2+-free solution. Pretreatment with 10 microM nifedipine, an L-type Ca2+ channel blocker, reduced both phasic and tonic contractions induced by high K+. In the case of NA-induced contraction, however, nifedipine reduced the phasic contraction but not the tonic contraction. The nifedipine-insensitive tonic contraction was relaxed by the application of polyvalent cations (Mn2+, Co2+, Cd2+ and La3+). These findings indicate that NA-induced biphasic contraction is mainly due to nifedipine-insensitive Ca2+ influx, especially in the tonic phase. Cyclic AMP-increasing agents such as forskolin (0.5-10 microM), IBMX (5-500 microM) and caffeine (1-20 mM) relaxed the NA-induced contraction extensively in a concentration-dependent manner. However, these agents only partially relaxed the high K+-induced contraction. Forskolin (10 microM) and IBMX (100 microM) reduced the [Ca2+]i response to NA, but had no effect on the [Ca2+]i response to high K+. These results suggest that an increase in intracellular cAMP may relax the NA-induced contraction by attenuating a nifedipine-insensitive Ca2+ influx and by a mechanism independent of a reduction in [Ca2+]i.

Activation of locomotor and grasping spine muscle fibres in chaetognaths: a curious paradox.

Chaetognath muscle fibres resemble vertebrate muscle fibres in having an abundant sarcoplasmic reticulum (SR) and analogues of the transverse (T) tubular system. but contraction is regulated differently. In intact chaetognaths electrically-evoked contractions of the striated locomotor muscles were largely or totally blocked by d-tubocurarine, by surgical removal of the ventral ganglion and by Co2 +. Contractions of single cells enzymatically dissociated from locomotor muscles were likewise blocked by Co2+, they twitched once only after calciseptine, showed neither contractures nor elevated intracellular Ca2+ with caffeine, and ryanodine did not block contractions. Whole cell voltage-clamped locomotor muscle cells displayed a typical inward rectified Ca2 + current that was sensitive to the Ca2+ channel blockers nifedipine and calciseptine and showed voltage-dependent activation with a threshold at approximately-25 mV and a peak inward current at approximately + 10 mV. In contrast, whole cell voltage-clamped cells from the muscles operating the grasping spines of the head showed an initial very rapid and rapidly-inactivating inward current abolished by tetrodotoxin (TTX), followed by a slower and slowly-inactivating inward current blocked by calciseptine. The relation between these observations and the unusual 'vertebrate-like' structure of the muscle cells is discussed.

Contraction and relaxation in the absence of a sarcoplasmic reticulum: muscle fibres in the small pelagic tunicate Doliolum.

Previous ultrastructural observations suggested that Doliolum muscle fibres apparently lacked both sarcoplasmic reticulum and transverse tubular-system. External Ca2+ is required for contraction, caffeine does not evoke contraction, nor does it increase intracellular Ca2+ level. Ryanodine at 50 microM has no effect on electrically-evoked contractions. Further, electrical stimulation in external solutions lacking Na+ leads to sustained contracture. We conclude that intracellular Ca2+ stores are absent in these rapid obliquely-striated fibres, and that reduction in internal Ca2+ levels following contraction depends upon Na(+)-Ca2+ exchange across the sarcolemma.

K+ accumulation and K+ conductance inactivation during action potential trains in giant axons of the squid Sepioteuthis.

1. During action potential trains in giant axons from the squid Sepioteuthis, decline of the peak level of the undershoot potential was observed. The time course of the decline of the undershoot could be fitted with a three-exponential function with time constants of approximately 25, approximately 400 and approximately 7,000 ms, respectively. 2. When the osmolarity of the external solution was doubled by adding glucose (1.2 M), the fast component of undershoot decline, but not the medium and slow components, was significantly reduced. 3. Under voltage clamp in high osmolarity solutions where K+ accumulation was completely removed, repeated depolarizing pulses at 40 Hz (designed to mimic a train of action potentials) elicited K+ currents whose peak value declined. The decline is consistent with inactivation of the K+ conductance (gK). The decline of gK was fitted by a two-exponential function with time constants of approximately 400 and approximately 7,000 ms, respectively. 4. Interventions designed to modify Schwann cell physiology, such as high frequency stimulation (100 Hz, 2 min), externally applied ouabain (100-500 microM), L-glutamate (100 microM), ACh (100 microM), Co2+ (5mM), Ba2+ (2mM), or removal of external Ca2+ by EGTA, had no significant effects on the fast, medium or slow components of undershoot decline. 5. The results suggest that the fast component of undershoot decline represents K+ accumulation in the space between Schwann cell and axolemma. The medium and slow components are the result of axonal gK inactivation. Schwann cells appear to be involved in K+ clearance only to the extent that they provide an efficient physical pathway for the clearance of K+ by extracellular diffusion.

Regional differences in the contractile and intracellular Ca2+ responses of the guinea-pig vas deferens to neurotransmitters and excess K+.

The contractile responses to various concentrations of noradrenaline (NA), adenosine triphosphate (ATP), acetylcholine (ACh) and excess external K+ in the epididymal, middle and prostatic portions of the guinea-pig vas deferens were investigated by measuring the isotonic contraction, and the intracellular Ca2+ concentration ([Ca2+]i) using fura-2 fluorescence. In the epididymal portion, the contractions evoked by each of these agonists were biphasic, comprising a transient followed by a tonic phase. In the middle portion, NA and ACh evoked biphasic contractions, whereas the ATP-induced contraction was an almost monophasic transient. In contrast, in the prostatic portion, only transient contractions were evoked by ACh and ATP, while the NA-induced contraction was oscillatory. The responses to each of the neurotransmitters in the three portions were not affected by pretreatment with TTX. The maximal responses of tonic contraction in response to each of the neurotransmitters were largest in the epididymal portion, decreased in the middle and were almost absent in the prostatic portion. These regional differences in the contractile properties of the vas deferens were also evident upon stimulation with excess external K+. As with the contractile responses, regional differences in the [Ca2+]i increases were observed during exposure to all the stimulants. It is suggested that the regional differences in the contractile responses of the vas deferens to various neurotransmitters and excess external K+ may involve variation in the mechanisms of Ca2+ homeostasis and in the sensitivity of the contractile apparatus to intracellular Ca2+ ions.

Emergence of a chloride permeability after internal perfusion with calcium in squid giant axons.

In the resting condition, the membrane of the squid giant axon is highly permeable to K+, and shows large changes in membrane potential following an increase in external or internal K-concentration. When 3-5 mM Ca2+ was applied internally for 3 min, the membrane became highly permeable to Cl- as compared to K+. The permeability ratio PCl/PK was about 0.77 in control experiments, rising to a ratio of 5.5 after Ca2+ administration. The membrane conductance also increased 6-fold over controls. The permeability ratios for other anions, Cl-, F-, methanesulfate (MS), and HEPES (PCl, PF, PMS, and PHEPES) were 1.00, 0.54, 0.23, and 0.13, respectively.

Electrical tolerance (breakdown) of the Chara corallina plasmalemma: II. Inductive property of membrane and effects of pHo and impermeable monovalent cations on breakdown phenomenon.

Changes in the chord conductance G and the membrane electromotive force Em in the so-called breakdown region of large negative potential of the Chara plasmalemma were analyzed in more detail. In addition to the increase in G, the voltage sensitivity of the change in G increased, which was the cause of marked inductive current in the breakdown region. The break-down potential, defined as a critical potential at which both low and high slope conductances of the I-Vm relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pHo in a range between 5 and 9. The Chara plasmalemma was electrically most tolerant around pHo 7. In some cells Em shifted to a positive level as large as +50 approximately +70 mV during the breakdown phenomenon. Such a large positive shift of Em is caused mainly by the increase in conductance of Cl- and partly Ca2+ and K+.

Electrical tolerance of the Chara corallina plasmalemma.

[Ca2+]o controls at least three distinct electrical phenomena in the Chara plasmalemma. 1). Increase of the electrical tolerance. 2). Decrease of the G without a marked change of the Gp/Gd ratio. 3). Elevation of the threshold for excitation (data not shown). On the other hand, [Mg2+]o shows quite opposite effects. Thus, optimum [Ca2+]o and [Mg2+]o are expected in vivo. Voltage dependencies of various conductances in the Chara plasmalemma are schematically shown in Fig. 4.

Effect of enrichment of infusion solutions with branched chain amino acids in parenteral nutrition of rats.

The effect of enrichment of the branched chain amino acids (BCAAs) leucine, isoleucine and valine on total parenteral nutrition was studied in rats. Experimental infusion solutions with a sufficient, marginal or deficient level of glucose contained either the conventional amino acid composition (22.6% BCAAs) or a BCAA-enriched amino acid composition (36% BCAAs). Rats were infused with experimental solutions for 4 days and several parameters of protein metabolism were evaluated in various tissues. Under conditions of sufficient energy supply, BCAA-enriched and conventional groups showed similar body weight gains and muscle protein degradations as measured by urinary 3-methylhistidine excretion. Polysome profiles in the liver and gastrocnemius muscle of the BCAA-enriched group were more heavily aggregated than those of the conventional group. Under the conditions of marginal or deficient energy supply, beneficial effects of BCAA enrichment over the conventional amino acid composition became more evident in terms of better body weight retention, higher RNA/DNA ratio and heavier polysome profile in both liver and muscle, and reduced protein catabolism in muscle. The present study suggests that enrichment of BCAAs, particularly valine and isoleucine, may be useful for nutritional support under hypercatabolic or stressed conditions.

[The role of brain-stem vasomotor centers on the neurogenic control of cerebrovascular tonus. Part 2: The effect of stimulation of the medullary reticular formation on cerebrovascular tonus].

Acute brain swelling has been proposed to be caused by vasomotor paralysis secondary to disruption of metabolic and/or neurogenic control of cerebrovascular tone. In previous experiments, acute brain swelling was produced by stimulation of the reticular formation of the medulla oblongata (MORF) with destruction of the dorsomedial nucleus of the hypothalamus (DM) and the reticular formation of the midbrain (MBRF) in 2 (out of 56) animals. The purpose of the present study is to clarify the effect of stimulation of the MORF on cerebrovascular tone. Using 32 cats, blood pressure (BP), intracranial pressure (ICP) and cerebral blood volume (CBV) were continuously measured. The animals were divided into 4 groups: those in which the ICP was normal (Group A), those in which the ICP increased after subarachnoid hemorrhage (Group B), those which underwent superior cervical ganglionectomy (Group C) and those in which the spinal cord was transected at the C2 level (Group D). In all animals, the MORF was stimulated after the DM and MBRF were destroyed. Thirty minutes after cessation of stimulation, the BP was increased again by injection of angiotensin II until it reached the same level as with MORF stimulation. The vasomotor index (VI), defined by delta ICP/delta BP, was calculated and compared between the two situations. BP, ICP and CBV increased simultaneously immediately after MORF stimulation. In 29 out of the 32 animals, ICP increased abruptly from 2 to 48 mmHg and after ten seconds or more decreased gradually. The BP rose about 50 mmHg more slowly than the ICP. After cessation of MORF stimulation, BP, ICP and CBV returned to the control value.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmitter release at the mouse neuromuscular junction stimulated by cadmium ions.

Cadmium ion (Cd2+) was examined for effects on frequency of miniature end-plate potentials (m.e.p.p.s) under several conditions in mouse diaphragm muscles. Cd2+ depressed potassium (K+)-stimulated rise in m.e.p.p. frequency, acting antagonistically to Ca2+, whereas in the absence of K+ stimulation Cd2+ produced a time- and concentration-dependent rise in m.e.p.p. frequency without affecting the resting membrane potential of muscle fibers. Such a presynaptic effect was observed with or without external Ca2+ [( Ca]0). In the absence of [Ca]0, Cd2+ frequently produced twitching in several fibers, which caused dislodgement of the micro-electrode. Therefore, 10 mM KCl was added to the Ca-free solution to overcome such difficulty. The following experiments were performed in the Ca-free 10 mM KCl solution. Prolonged exposure to Cd2+ led to an eventual decline in m.e.p.p. frequency within 20-30 min. After decline of the effect, m.e.p.p. frequency in the presence of Cd2+ was not restored by adding Ca2+ combined with elevated K+ and with A23187 or by carbonyl cyanide p-trifluoromethoxyphenyl hydrazine and hypoxic condition, each of which was separately effective in enhancing transmitter release. Exposure to 50 microM Cd2+ for 60 min decreased acetylcholine content in diaphragm muscle to approximately 50% of control. Tetrodotoxin and caffeine did not modify the presynaptic stimulatory effect of Cd2+. The effect of Cd2+ on m.e.p.p. frequency was partially reduced by trifluoperazine and phenytoin each of which depressed m.e.p.p. frequency rise induced by Ca2+ with elevated K+. The possible mechanism of Cd2+ action and of its interaction with intracellular Ca2+ stores is discussed.