Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss ) coronary system

The effects of l-arginine, and its analogues N ^ω-nitro-l-arginine methyl ester and N ^ω-nitro-l-arginine on vascular resistance were investigated in the intact coronary system of an isolated non-working trout heart preparation. l-Arginine, at 10^–8 mol · l^–1induced a slight vasodilatory effect (max 10%). N ^ω-nitro-l-arginine methyl ester and N ^ω-Nitro-l-arginine in the range 10^–8–10^–4 mol · l^–1 caused dose-dependent increases in coronary resistance. The vasodilatory action of l-arginine was abolished when the preparation was pretreated with 10^–4 mol · l^–1 N ^ω-nitro-l-arginine or N ^ω-nitro-l-arginine methyl ester. Nitroprusside alone at 1 mmol · l^–1 induced a maximum vasodilation (30%) of the coronary system. Methylene blue a known inhibitor of guanylate cyclase, induced a strong vasoconstriction (already significant at 10^–5 mol · l^–1) and was able to overcome the vasodilative effect of nitroprusside. The endothelial nitric oxide agonists acetylcholine and serotonin, established in mammalian vessels, also mediate vasodilation in trout coronary system. In 50% of preparations, acetylcholine induced a biphasic response with vasodilation at low concentration (max 15% at 10^–8 mol · l^–1). Serotonin displayed a dose-response vasodilation in the range 10^–8–10^–4 mol · l^–1 (max 20%). These vasodilative effects were reduced or abolished by 10^–4 mol · l^–1 l-NA. These data support the existence of NO-mediated vasodilation mechanisms in the trout coronary system. Accepted: 1 July 1996     

Ontogeny of deep-body cold sensitivity in Pekin ducklings Anas platyrhynchos

The ontogeny of deep-body cold sensitivity was studied in 1 to 12 days old Pekin ducklings Anas platyrhynchos. Deep-body cold sensitivity was determined by means of thermodes implanted in the abdominal cavity. The thermodes were perfused with cold water for 15-min periods to lower the core temperature. Cooling of the body core elicited increases in metabolic rate and vasoconstrictions in the legs of all the ducklings. From the changes induced in metabolic rate and core temperature, deep-body cold sensitivity values of between −5.17 and −6.36 W · kg⁻¹ · °C⁻¹, were estimated. These values, which are in the range of those reported previously for adult Pekin ducks, did not change with age, and it is concluded that deep-body cold sensitivity is fully developed at hatching. Our next aim was to investigate whether the autonomic responses elicited by exposure of ducklings to cold ambient conditions could be explained by temperature changes within the body core. During cold exposure, the increase in metabolic rate was not accompanied by a concomitant decrease in core temperature. On the contrary, deep-body temperature increased slightly during the initial phase of cold exposure. The ducklings attained a metabolic rate amounting to 85–90% of their peak metabolic rate before the core temperature fell below the regulated level measured at thermoneutrality. Thus, despite the findings that Pekin ducklings have a highly-developed deep-body cold sensitivity, their metabolic cold defence under natural conditions seems to be mediated primarily by peripheral thermoreceptors. Accepted: 7 January 1997     

Cold induced vasodilatation and cardiovascular responses in humans during cold water immersion of various upper limb areas

To study the physiological responses induced by immersing in cold water various areas of the upper limb, 20 subjects immersed either the index finger (T1), hand (T2) or forearm and hand (T3) for 30 min in 5°C water followed by a 15-min recovery period. Skin temperature of the index finger, skin blood flow (Q_sk) measured by laser Doppler flowmetry, as well as heart rate (HR) and mean arterial blood pressure (ˉBP_a) were all monitored during the test. Cutaneous vascular conductance (CVC) was calculated as Q_sk / ˉBP_a. Cold induced vasodilatation (CIVD) indices were calculated from index finger skin temperature and CVC time courses. The results showed that no differences in temperature, CVC or cardiovascular changes were observed between T2 and T3. During T1, CIVD appeared earlier compared to T2 and T3 [5.90 (SEM 0.32) min in T1 vs 7.95 (SEM 0.86) min in T2 and 9.26 (SEM 0.78) min in T3, P < 0.01]. The HR was unchanged in T1 whereas it increased significantly at the beginning of T2 and T3 [+13 (SEM 2) beats · min⁻¹ in T2 and +15 (SEM 3) beats · min⁻¹ in T3, P < 0.01] and then decreased at the end of the immersion [−12 (SEM 3) beats · min⁻¹ in T2, and −15 (SEM 3) beats · min⁻¹ in T3, P < 0.01]. Moreover, ˉBP_aincreased at the beginning of T1 but was lower than in T2 and T3 [+9.3 (SEM 2.5) mmHg in T1, P < 0.05;  +20.6 (SEM 2.6) mmHg and 26.5 (SEM 2.8) mmHg in T2 and T3, respectively, P < 0.01]. The rewarming during recovery was faster and higher in T1 compared to T2 and T3. These results showed that general and local physiological responses observed during an upper limb cold water test differed according to the area immersed. Index finger cooling led to earlier and faster CIVD without significant cardiovascular changes, whereas hand or forearm immersion led to a delayed and slower CIVD with a bradycardia at the end of the test. Accepted: 26 November 1996     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

Walking performance and economy in chronic heart failure patients pre and post exercise training

The effect of a 3-week exercise programme on performance and economy of walking was analysed in 16 male patients with chronic heart failure [mean age 51.8 (SD 6.9) years, height 174.9 (SD 6.3) cm, body mass 75.3 (SD 11.5) kg, ejection fraction 20.8 (SD 5.0)%]. They were submitted to a cardiopulmonary exercise test on a cycle ergometer and a 6-min walking test on a treadmill before and after the period of exercise training. The training programme consisted of interval cycle (five times a week for 15 min), and treadmill ergometer training (three times a week for 10 min) at approximately 70% cycling peak oxygen uptake (O_2peak) and supplementary exercises (three times a week for 20 min). Compared to the pre values cycling O_2peak [11.9 (SD 2.9) vs 14.0 (SD 2.3) ml ·  kg^–1 · min^–1], maximal self paced walking speed [0.68 (SD 0.33) vs 1.16 (SD 0.30) m · s^–1], and net walking power [2.16 (SD 0.89) vs 2.73 (SD 0.91) W · kg^–1] had increased (P < 0.01) while net energy cost [3.31 (SD 0.66) vs 2.33 (SD 0.38) J · kg^–1 ·  m^–1] had decreased (P < 0.001) after the training period. Approximately 42% of the increase of walking speed resulted from a higher walking power output, whereas approximately 58% corresponded to a positive effect on walking economy. The improvement in walking economy was a function of an increase in walking velocity itself and a result of a more efficient walking technique. These results would indicate that in patients with marked exercise intolerance, adequate exercise training programmes could contribute to favourable metabolic changes with positive effects on the economy of motion. Accepted: 29 August 1996     

Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold

The effect of exposure to low temperatures (5 °C) on lymphocyte proliferation, leukocyte populations, and serum complement levels was examined in the northern leopard frog, Rana pipiens. Proliferation of T lymphocytes in response to phytohemagglutinin stimulation was significantly decreased in frogs kept for 2, 3, and 5 months at 5 °C compared to that of animals kept at 22 °C. A significant increase in the average percentage of neutrophils and a decrease in the mean percentage of eosinophils was observed in the blood of frogs held for 5 months in the cold compared to animals held at 22 °C for the same length of time. Mean serum complement activity after 1 month at 5 °C was significantly reduced in comparison to animals held at 22 °C and was not detectable after 5 months in the cold. Recovery of complement levels at room temperature (22 °C) was also examined after cold exposure. Complement levels were significantly higher than controls (at 22 °C) in frogs returned to 22 °C for 7 and 14 days after 5 months in the cold. After frogs were held at 5 °C for 1 month, serum complement levels increased significantly within 2 days after returning to 22 °C and continued to rise 5 and 9 days after warming. Injections with Aeromonas hydrophila following a 5-week exposure to 5 °C failed to cause death or observable symptoms of disease in frogs that were returned to 22 °C. Accepted: 20 November 1996     

Influence of soil hydric parameters on the winter cold hardiness of a burrowing beetle, Leptinotarsa decemlineata (Say)

This investigation examined the influence of soil moisture and associated parameters on the cold hardiness of the Colorado potato beetle (Leptinotarsa decemlineata Say), a temperate-zone species that overwinters in terrestrial burrows. The body mass and water content of adult beetles kept in sand at 4 °C varied over a 16-week period of diapause according to the substratum's moisture content. Changes in body water content, in turn, influenced the crystallization temperature (range −3.3 to −18.4 °C; n = 417), indicating that environmental moisture indirectly determined supercooling capacity, a measure of physiological cold hardiness. Beetles held in dry sand readily tolerated a 24-h exposure to temperatures ranging from 0° to −5 °C, but those chilled in sand containing as little as 1.7% water (dry mass) had elevated mortality. Thus, burrowing in dry soils not only promotes supercooling via its effect on water balance, but may also inhibit inoculative freezing. Mortality of beetles exposed to −5 °C for 24 h was lower in substrates composed of sand, clay and/or peat (36–52%) than in pure silica sand (78%) having an identical water content (17.0% dry mass). In addition to moisture, the texture, structure, water potential, and other physico-chemical attributes of soil may strongly influence the cold hardiness and overwintering survival of burrowing insects. Accepted: 10 September 1996     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

Rhythmic components of photoperiodic time measurement in the pitcher-plant mosquito, Wyeomyia smithii

Photoperiodic time measurement regulating larval diapause in the pitcher-plant mosquito, Wyeomyia smithii, varies in a close relationship with latitude. The critical photoperiod mediating the maintenance and termination of diapause is positively correlated with latitude (r ² = 0.977) among six populations from southern (30–31° N), intermediate (40° N), and northern (46–49° N) latitudes in North America. The developmental response to unnaturally short and to unnaturally long photoperiods declines with increasing latitude, so that longer critical photoperiods are associated with a downward rather than a lateral shift in the photoperiodic response curve. Exotic light and dark cycles of varying period (T) with a short (10 h) photophase and a scotophase ranging from 14 (T = 24) to 62 (T = 72) h, reveal two geographic patterns: a decline in perturbability of the photoperiodic clock with increasing latitude, and no change with latitude in the 21-h period of rising and falling development with increasing T. These results show (1) that there is a rhythmic component to photoperiodic time measurement in W. smithii, (2) that the period of this rhythm is about 21 h in all populations, and (3) that more northern populations show decreasing responsiveness to photoperiod and increasing stability against perturbation by exotic period lengths (T > 24). Previous studies on W.␣smithii indicate that this single temperate species of a tropical and subtropical genus has evolved from south to north. We therefore conclude that the evolution of increasing critical photoperiod in W. smithii during its adaptive radiation into North America has more likely involved the amplitude and not the period of the underlying circadian pacemaker. Received: 22 July 1996 / Accepted: 30 September 1996     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

Phenotypic variation in seasonal adjustments of testis size, body weight, and food intake in deer mice: role of pineal function and ambient temperature

We investigated pineal function as well as reproductive and energetic characteristics in male deer mice (Peromyscus maniculatus) that differentially respond to short photoperiod with full, partial or no gonadal regression. In mice at both high (23 °C) and low temperature (1 °C), these phenotypic differences in reproductive responses to short days were not reflected by differences in urinary excretion of 6-sulphatoxy-melatonin, the main metabolite of pineal melatonin. Neither duration nor amplitude or phase-angle of nocturnal peaks in 6-sulphatoxymelatonin significantly differed between reproductive phenotypes at either temperature. Differences in testis size were, however, associated with different energy requirements. In gonadally regressed males only, food intake and body weight were significantly (P < 0.01) reduced by up to 29% and 13% respectively. Chronic cold exposure (5 °C) had no effect on the proportion of males undergoing testicular regression under short days, but caused a general elevation in body weights among all mice (P < 0.05). Phenotypic differences in body weight and food intake were maintained in the cold. Together, these results suggest that within-population variation of reproductive responses in male deer mice is based on post-pineal differences in the regulation of gonadal function, and that phenotypic characteristics in reproductive and energetic responses to short days are largely unaffected by ambient temperature. Accepted: 2 October 1995     

Indices of skeletal muscle damage and connective tissue breakdown following eccentric muscle contractions

 Indirect indices of exercise-induced human skeletal muscle damage and connective tissue breakdown were studied following a single bout of voluntary eccentric muscle contractions. Subjects (six female, two male), mean (SD) age 22 (2) years performed a bout of 50 maximum voluntary eccentric contractions of the knee extensors of a single leg. The eccentric exercise protocol induced muscle soreness (P < 0.05 Wilcoxon test), chronic force loss, and a decline in the 20:100 Hz percutaneous electrical myostimulation force ratio [P < 0.01, repeated measures analysis of variance (ANOVA)]. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities were elevated (P < 0.01, repeated measures ANOVA) following the bout. The mean (SD) CK and LDH levels recorded 3 days post-exercise were 2815 (4144) IU · l^–1 and 375 (198) IU · l^–1, respectively. Serum alkaline phosphatase activity showed no changes throughout the study, and a non-significant increase (P = 0.058, repeated measures ANOVA) in pyridinoline was recorded following the bout. Urinary hydroxyproline (HP) and hydroxylysine (HL) excretion, expressed in terms of creatinine (Cr) concentration, increased after exercise (P < 0.05 and P < 0.01, respectively, repeated measures ANOVA). An increased HP:Cr was recorded 2 days post-exercise and HL:Cr was increased above baseline on days 2, 5, and 9 post-exercise. This indirect evidence of exercise-induced muscle damage suggests that myofibre disruption was caused by the eccentric muscle contractions. Elevated urine concentrations of indirect indices of collagen breakdown following eccentric muscle contractions suggests an increased breakdown of connective tissue, possibly due to a localised inflammatory response. Accepted: 9 October 1996     

Delayed recovery of β-glucuronidase activity driven by an Arabidopsis heat shock promoter in heat-stressed transgenic Nicotiana plumbaginifolia

 The expression of the Arabidopsis heat shock protein (HSP) 18.2 promoter-β-d-glucuronidase (GUS) chimera gene was investigated in transgenic Nicotiana plumbaginifolia plants during the recovery phase at normal temperatures (20–22  °C) after a heat shock (HS) treatment. GUS activity increased during the recovery phase after HS at 42  °C for 2 h, and maximal GUS activity was observed after 12 h at normal temperatures, at levels 50–100 times higher than the activity immediately after HS. After HS at 44  °C, little GUS activity was observed during the first 20–24 h at normal temperatures, but the activity increased gradually thereafter, to reach a maximum at 40–50 h. After HS at 45  °C, no GUS activity was observed throughout the experimental period. RT-PCR analysis showed that GUS mRNA remained for 10 h after a 2-h HS at 42  °C and for 40 h after a 2-h HS at 44  °C. These findings demonstrate that brief HS treatment, especially at a sublethal temperature, induces a long-term accumulation of HSP-GUS mRNA during the recovery phase. Received: 31 July 1998 / Revision received: 4 November 1998 / Accepted: 19 February 1999     

Light response characteristics of a morphologically diverse group of southern hemisphere conifers as measured by chlorophyll fluorescence

Unlike northern hemisphere conifer families, the southern family, Podocarpaceae, produces a great variety of foliage forms ranging from functionally broad-, to needle-leaved. The production of broad photosynthetic surfaces in podocarps has been linked qualitatively to low-light-environments, and we undertook to assess the validity of this assumption by measuring the light response of a morphologically diverse group of podocarps. The light response, as apparent photochemical electron transport rate (ETR), was measured by modulated fluorescence in ten species of this family and six associated species (including five Cupressaceae and one functionally needle-leaved angiosperm) all grown under identical glasshouse conditions. In all species, ETR was found to increase as light intensity increased, reaching a peak value (ETR_max) at saturating quantum flux (PPFD_sat), and decreasing thereafter. ETR_max ranged from 217 μmol electrons · m⁻² · s⁻¹ at a PPFD_sat of 1725 μmol photons · m⁻² · s⁻¹ in Actinostrobus acuminatus to an ETR of 60 μmol electrons · m⁻² · s⁻¹ at a PPFD_sat of 745 μmol electrons · m⁻² · s⁻¹ in Podocarpus dispermis. Good correlations were observed between ETR_max and both PPFD_sat and maximum assimilation rate measured by gas-exchange analysis. The effective quantum yield at light saturation remained constant in all species with an average value of 0.278 ± 0.0035 determined for all 16 species. Differences in the shapes of light response curves were related to differences in the response of non-photochemical quenching (q _n), with q _n saturating faster in species with low PPFD_sat. Amongst the species of Podocarpaceae, the log of average shoot width was well correlated with PPFD_sat, wider leaves saturating at lower light intensities. This suggests that broadly flattened shoots in the Podocarpaceae are an adaptation to low light intensity. Received: 15 April 1996 / Accepted: 30 September 1996