Transmembrane Transport and Release of GABA in the Brain of Rats Subjected to Postnatal Hypoxia

We studied the effects of early postnatal hypoxia on the efficiency of active GABA transport through the plasma membrane of synaptic terminals (synaptosomes) isolated from the cerebral cortex, hippocampus, and thalamus of rats and on non-stimulated and Ca²⁺-stimulated GABA release. The state of hypoxia was induced by exposure of 10- to 12-day-old rats to a respiratory medium with low O₂ content (4% О₂ and 96% N₂) for 12 min (up to the initiation of clonico-tonic seizures). Animals were taken in the experiment 8 to 9 weeks after an episode of hypoxic stress. The intensity of transmembrane transport of GABA was estimated according to accumulation of [³Н]GABA in a coarse synaptosomal fraction. The process was characterized by calculation of the Michaelis constant K _m and also of the initial (within the 1st min) and maximum rates of accumulation of [³Н]GABA. The means of the initial rate of [³Н]GABA accumulation in preparations from the thalamus, cortex, and hippocampus were 205.5 ± 8.8, 266.2 ± 29.6, and 302.3 ± 31.2 pmol/min⋅mg protein, respectively. Hypoxic stress influenced the rates of accumulation of [³Н]GABA in synaptic terminals from the cortex and hippocampus but not in those from the thalamus. According to the characteristics of the response to hypoxic stress, all experimental animals could be classified into two groups. In some rats, accumulation of [³Н]GABA in both cortical and hippocampal synaptosomes decreased insignificantly (by about 15%), while in other animals this parameter increased significantly (by nearly 50%) for the cortex and decreased by 21.5%, on average, for the hippocampus. The affinity of the transporter with respect to [³Н]GABA in the cortex and hippocampus was nearly the same and showed no changes under the influence of hypoxia. The non-stimulated release of [³Н]GABA after the influence of hypoxia increased in all structures, while the depolarization-induced Ca²⁺-dependent release of [³Н]GABA was intensified only in synaptosomes from the cerebral cortex. The mechanisms of development of modifications of GABA-ergic processes under the influence of hypoxic stress in the course of the perinatal period are discussed. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 293–302, July–August, 2008.     

Role of Glutamate in the Mechanisms of Adaptation of the System of Respiratory Control in Rats to Intermittent Hypoxia

In experiments on Wistar rats, we studied the role of changes in the state of glutamatergic transmission in the course of adaptation of the system of respiratory control to intermittent hypoxia. The volume/temporal parameters of respiration were estimated according to characteristics of EMG activity (amplitude, integral intensity of EMG discharges) recorded from the diaphragmatic muscle. Changes in EMG activity of the diaphragm induced by acute hypoxia (breathing a 12% О₂-containing gas mixture) were estimated before and after of a 14-day-long course of intermittent hypoxia trainings and before and after inductions of a blocker of NMDA receptors, МK-801. The results prove that the glutamatergic transmitter system is significantly involved in the reaction of the respiratory system to presentation of a hypoxic stimulus within all stages of formation of the ventilatory response, both before and after the action of intermittent hypoxia. Blocking of NMDA receptors under conditions of adaptation to intermittent hypoxia exerted a more intense influence on the amplitude of respiratory EMG discharges of the diaphragm than on their frequency.     

Superoxide anion production by the mitochondrial respiratory chain of hepatocytes of rats with experimental toxic hepatitis

The progression of toxic hepatitis is accompanied by the activation of oxidative processes in the liver associated with an enhancement of the mitochondrial respiratory chain activity and superoxide anion production (О₂^˙-). The purpose of this study was to examine our previously formulated assumption concerning the predominant contribution of the complex I to О₂^˙- production increase by the mitochondrial respiratory chain of hepatocytes in toxic hepatitis (Shiryaeva et al. Tsitologiia, 49, 125–132 2007). Toxic hepatitis was induced by a combined application of ССl₄ and ethanol. Respiratory chain function analysis was executed with submitochondrial particles (SP) in the presence of specific inhibitors. It was shown that the rate of О₂^˙- production by SP of animals with toxic hepatitis, when NADH was delivered, was 2.5-fold higher as compared with the control. The rates of О₂^˙- production by SP of rats with toxic hepatitis in the presence of NADH or NADH + rotenone were similar. The О₂^˙- production rate by control SP in the presence of NADH + rotenone corresponded to the О₂^˙- production rate by toxic hepatitis SP when only NADH was delivered. When NADH + myxothiazol were delivered to the incubation system, О₂^˙- production by toxic hepatitis SP was 72% higher than for the control. Conversely, in the presence of antimycin A, the production of О₂^˙- by toxic hepatitis SP was lower compared to the control. Collectively, the presented data indicate that the О₂^˙- production rate was enhanced by the complex I of the hepatocyte mitochondrial respiratory chain in experimental toxic hepatitis. Complex III contribution to the production of О₂^˙- was insignificant. We assume that the increase in О₂^˙- production by the respiratory chain may be considered not only as the mechanism of pathology progression, but also as a compensatory mechanism preserving the electron transport function of the mitochondrial respiratory chain when complex I functioning is blocked in part.     

Changes of gas exchange parameters and of functional-biochemical properties of erythrocytes in dynamics of experimental anemia in rats

In experiments on male Wistar rats, in a specially constructed computerized installation, O₂ consumption by the animals in comparison with changes of hematological, biochemical, and rheological blood properties is studied after anemization—acute blood loss (12–15% of the total blood mass). An increase of the O₂ consumption by the organism and tissues by 18–28% has been revealed for the first 7 days after the blood loss, in spite of a pronounced decrease of hematocrit and of the amount of erythrocytes and hemoglobin in peripheral blood by 20–25% of the initial level. There was a 5–10-fold increase of the content of immature erythrocyte forms—reticulocytes and a progressive rise of cell acidic resistance, which is characteristic of young erythrocyte forms. An increase of O₂ consumption at a decrease of the blood oxygen capacity (a low hemoglobin level) seems to be due to the more efficient transport and yield of O₂ to tissues. At the 3rd and 7th day after the blood loss, activity of Na,K-ATPase has been found to increase by 60% and 20%, respectively. Analysis of the erythrocyte rheological properties has shown that the maximal firmness of aggregates (U_q) and the aggregation rate (1/T) decrease progressively beginning from 3 days after the blood loss; index of deformability (I_max) turned out to be elevated by 7–11%, probably due to an increase of the cell membrane elasticity. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed at optimization of blood circulation in large vessels and the capillary network.     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE₂ and in specified samples also of 6-keto-PGF_1α, as a measure of PGI₂, and PGF_2α were determined by radioimmunoassay after incubation at either 20% O₂ (normoxic) or 2% O₂ (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE₂ production was noted among independent cell lines. PGE₂ production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE₂ production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF_2α and 6-keto-PGF_1α increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

Effect of 7-Nitroindazole Sodium on the Cellular Distribution of Neuronal Nitric Oxide Synthase in the Cerebral Cortex of Hypoxic Newborn Piglets

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca⁺⁺-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.     

Disruption to the 5-HT<Subscript>7</Subscript> Receptor Following Hypoxia–Ischemia in the Immature Rodent Brain

It has become increasingly evident the serotonergic (5-hydroxytryptamine, 5-HT) system is an important central neuronal network disrupted following neonatal hypoxic–ischemic (HI) insults. Serotonin acts via a variety of receptor subtypes that are differentially associated with behavioural and cognitive mechanisms. The 5-HT₇ receptor is purported to play a key role in epilepsy, anxiety, learning and memory and neuropsychiatric disorders. Furthermore, the 5-HT₇ receptor is highly localized in brain regions damaged following neonatal HI insults. Utilising our well-established neonatal HI model in the postnatal day 3 (P3) rat pup we demonstrated a significant decrease in levels of the 5-HT₇ protein in the frontal cortex, thalamus and brainstem one week after insult. We also observed a relative decrease in both the cytosolic and membrane fractions of 5-HT₇. The 5-HT₇ receptor was detected on neurons throughout the cortex and thalamus, and 5-HT cell bodies in the brainstem. However we found no evidence of 5-HT₇ co-localisation on microglia or astrocytes. Moreover, minocycline treatment did not significantly prevent the HI-induced reductions in 5-HT₇. In conclusion, neonatal HI injury caused significant disruption to 5-HT₇ receptors in the forebrain and brainstem. Yet the use of minocycline to inhibit activated microglia, did not prevent the HI-induced changes in 5-HT₇ expression.     

Decreased GABA<Subscript>A</Subscript> Receptors Functional Regulation in the Cerebral Cortex and Brainstem of Hypoxic Neonatal Rats: Effect of Glucose and Oxygen Supplementation

Hypoxia in neonates can lead to biochemical and molecular alterations mediated through changes in neurotransmitters resulting in permanent damage to brain. In this study, we evaluated the changes in the receptor status of GABA_A in the cerebral cortex and brainstem of hypoxic neonatal rats and hypoxic rats supplemented with glucose and oxygen using binding assays and gene expression of GABA_Aα1 and GABA_Aγ5. In the cerebral cortex and brainstem of hypoxic neonatal rats, a significant decrease in GABA_A receptors was observed, which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with glucose and oxygen showed a reversal of the GABA_A receptors, andGABA_Aα1 and GABA_Aγ5 gene expression to control. Glucose acts as an immediate energy source thereby reducing the ATP-depletion-induced increase in GABA and oxygenation, which helps in encountering anoxia. Resuscitation with oxygen alone was less effective in reversing the receptor alterations. Thus, the results of this study suggest that reduction in the GABA_A receptors functional regulation during hypoxia plays an important role in mediating the brain damage. Glucose alone and glucose and oxygen supplementation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Respiratory and metabolic responses of the Australian Yabby Cherax destructor to progressive and sustained environmental hypoxia

The Australian Yabby, Cherax destructor, inhabits occasionally hypoxic water. The respiratory gas, acid-base, metabolite and energetic status of this crayfish was assessed during progressive hypoxia and during 3 h at a water PO₂ of 1.33 kPa. The O₂ affinity of haemocyanin from C. destructor was increased by lactate (Δlog P ₅₀/Δlog[lactate] = −0.111) and by Ca (Δlog P ₅₀/Δlog[Ca] = −0.62) but not by urate. While the non-bicarbonate buffering capacity was low (Δ[HCO₃ ⁻]/ ΔpH=−4.89) the haemocyanin had a low sensitivity to pH changes (ϕ = −0.33). The crayfish showed a compensatory hyperventilation, which induced a respiratory alkalosis, until the water O₂ partial pressure declined below 2.67 kPa, after which the O₂ uptake rate was approximately 10% of normoxic rates. The high haemocyanin-O₂ affinity maintained haemolymph O₂ content during progressive hypoxia despite the normally low arterial O₂ partial pressure of C. destructor. During severe hypoxia, pH decreased but increased lactate aided in maintaining haemocyanin-O₂ saturation. The importance of regulated haemocyanin-O₂ affinity in hypoxic C. destructor was reduced by lowered metabolism, including reduced cardiac output, and the consequent reduction in O₂ requirement. Anaerobiosis became important only at very low PO₂ but thereafter proceeded rapidly, supported by a marked hyperglycaemia. There was no depletion of adenylates, even after 3 h of severe hypoxia. The tail muscle of C. destructor held small amounts of glycogen which would sustain anaerobiosis for a only a few hours. Hypometabolism seems an important hypoxic response but severe hypoxia may encourage the crayfish to breathe air. Accepted: 26 February 1998     

Heart rate responses to altered ambient oxygen in early (days 3–9) chick embryos in the intact egg

Normal heart rate (HR), and the HR responses to hypoxia and hyperoxia during early heart development in chick embyros have not been studied in detail, particularly in undisturbed embryos within the intact egg. HR was measured in day 3–9 chick embryos at 38 °C using relatively noninvasive impedance cardiography. Embryos were exposed to air (control) and to hypoxic (10% O₂) or hyperoxic (100% O₂) gas for a 2-h or 4-h period, during which HR was continually monitored. Control (normoxic) HR increased from about 150 beats per min (bpm) on day 3 to about 240 bpm on days 7–9. HR in very early embryos showed a variety of moderate responses to hypoxia (all survived), but as development progressed beyond day 6, hypoxic exposure induced a profound bradycardia that frequently terminated in death before the end of the measurement period. In contrast to the marked developmental changes in hypoxic sensitivity, HR showed little response to hyperoxia throughout development, suggesting no “hypoxic drive” to HR. We speculate that hypoxia has little effect early in development because of the embryo's small absolute O₂ demand, but as the embryo grows, hypoxia represents a progressively more severe perturbation. Although general trends were identified, there was considerable variation in both HR and HR responses to ambient O₂ changes between individuals of the same developmental stage. Accepted: 16 December 1998     

Interactive effects of development and hypoxia on catecholamine synthesis and cardiac function in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

The rate-limiting enzyme in the biosynthetic pathway of catecholamines is tyrosine hydroxylase (TH), the activity of which is dependent on molecular oxygen. Zebrafish (Danio rerio) possess two non-allelic TH coding genes, TH1 and TH2. A principal goal of the present study was to determine if the expression of these genes is sensitive to environmental hypoxia. Additionally, we sought to determine if catecholamine content of larvae was changed by environmental hypoxia, and whether the hearts of hypoxic larvae were equally responsive to exogenous catecholamine (norepinephrine) exposure. After 2 days of exposure to hypoxia [5–7 days post-fertilization (dpf); PO₂ = 30 Torr] TH2 mRNA expression was significantly lower and dopamine β hydroxylase (DβH) mRNA was significantly higher in whole larvae. Whole body catecholamine levels were unchanged until after 4 days of hypoxic exposure (5–9 dpf), at which time there was a significant increase in epinephrine and norepinephrine contents. Norepinephrine content was significantly elevated in the hearts of adult fish after 2 and 4 days of hypoxic exposure, and TH1 mRNA expression was increased in the kidney of both groups. After 2 or 4 days of exposure to hypoxia, larvae displayed significantly lower heart rates than normoxic fish. However, application of exogenous norepinephrine caused similar increases in heart rate in both groups. Overall, it is concluded that the mRNA expression of TH1 and TH2 is differentially affected by hypoxia exposure in larvae and adults. Also, catecholamine biosynthesis appears to be activated by 2 dpf and although whole body catecholamine levels increase during hypoxia (possibly promoting downregulation of cardiac β-adrenergic receptors), there is no accompanying decrease in the response of the heart to adrenergic stimulation.     

Dietary Protein-induced Changes of the Erythropoietin Level in Rat Serum

Erythropoietin, a glycoprotein, is the primary regulator of erythropoiesis. The most convenient and sensitive assay for active erythropoietin is to measure its stimulatory effect on in vitro ³H-thymidine incorporation into DNA of erythropoietin-responsive cells. An attempt with this method to estimate the erythropoietin level in rat serum, however, was unsuccesful because of the presence of inhibitory substance(s) and non-erythropoietic factor(s) stimulating ³H-thymidine incorporation. Pretreatment of the serum by heating, extraction of erythropoietin from denatured-protein aggregates, and subsequent concentration of erythropoietin in the extract with alcohol precipitation made it possible to measure the serum erythropoietin levels. Rabbit anti-erythropoietin antibody was used for a quantitative estimation of erythropoietin in the concentrated extracts. Erythropoietin levels in sera of rats fed on varied amounts of casein for 7 days were measured with these procedures to find if the impairment of erythropoiesis upon protein deprivation was due to changes in the erythropoietin level. We found that the level in protein-deprived rats was less than 1/8 that of 20% casein-fed rats, a level undetectable by the present assay, and that the serum erythropoietin increased as the protein content in the diet was increased up to 20%, then leveled off. The erythropoietin in serum decreased rapidly after protein deprivation; the level at 12hr after deprivation began was about 1/5 that in 20% casein-fed rats. Thus, the depression of erythropoiesis upon protein deprivation is primarily caused by the lowered level of erythropoietin.     

Hoxd1 is Expressed by Oligodendroglial Cells and Binds to a Region of the Human Myelin Oligodendrocyte Glycoprotein Promoter in vitro

(1) Little information exists on the role of clustered Hox genes in oligodendrocyte (OG) development. This study examines the expression profile of Hoxd1 and identifies a potential downstream target in the OG lineage. (2) Immunocytochemical analysis of primary mixed glial cultures demonstrated Hoxd1 was expressed throughout OG development. (3) A human myelin protein gene, myelin oligodendrocyte glycoprotein (MOG), was identified as a putative downstream target of Hoxd1 through Genbank searches utilizing the Hoxd1 homeodomain consensus binding sequence. (4) The dissociation coefficient constant (K _D) and dissociation rate constant (k _d) of the Hoxd1–MOG complex, determined using electrophoretic mobility shift assays (EMSAs), were estimated to be 1.9 × 10⁻⁷ M and 1.3 × 10⁻³ s⁻¹, respectively. The DNA–Hoxd1 homeodomain complex had a half-life (t _1/2) of 15 min. (5) Mutational analysis of Hoxd1–MOG complexes revealed the binding affinity of M1 (with mutation from ⁻¹⁰⁵⁴5′-TAAT-3′⁻¹⁰⁵¹ to TACT within the consensus binding site) and M2 (with mutation from ^-10545′-TAATTG-3′^-1049 to TAATCC within the consensus binding site) probes to the MOG promoter was severely affected. Thus the TAATTG core of the binding sequence appears important for Hoxd1 specificity. (6) Analysis of the involvement of TAAT sites adjacent to the consensus sequence in Hoxd1 binding showed the binding affinity of the M3 probe was affected, but not as severely as the M1 and M2 probes. These in vitro results suggest the TTTAATTGTA sequence is involved in Hoxd1 binding to the MOG promoter but neighboring TAAT sites may also be needed. Thus, MOG may be a target of Hoxd1.     

An increased carbon dioxide production in transient hypoxia in healthy subjects at rest

Indices of pulmonary gas exchange and heart rate (HR) have been measured in 24 healthy subjects not adapted to hypoxia after hypoxic aerial mixture (HAM) (17, 15, 13 vol % of oxygen) respiration for 15 min. Using group data analysis, it has been shown that hypoxia under the conditions of inhalation of 17 and 15 vol % of O₂ caused no significant changes. Hypoxia under the conditions of 13 vol % of O₂ inhalation is a threshold one, when ventilation (SpO₂) drops below 85%. A significant increase in the lung ventilation (Ve) (10–14%, p < 0.05) and HR (11–15%, p < 0.05) have been observed in this case. Hyperpnea was accompanied by an increase in the oxygen uptake rate by 10% and carbon dioxide release rate (10–18%, p < 0.05). On the contrary, individual data analysis showed changes in the pulmonary gas exchange indices in 90% of subjects in the case of inhalation of 17 vol % of O₂ HAM. Four response types have been found: ventilation (increase in lung ventilation), hypoxic hypometabolism (decrease in oxygen consumption rate), and mobilization response (increase in oxygen utilization in the lungs), and anaerobic response, which is expressed in an increase in the carbon dioxide release rate along with an increase in the respiratory quotient. All these responses are of an individual type, but the ventilation response is developed in response to hypoxia caused by inhalation of 13 vol % of O₂ HAM and a decrease in SpO₂ below 85% in more than 60% of cases.     

Effect of hypoxia on the content and stoichiometry of cytochromes in muscle of the gray mullet Liza aurata

The maintenance of the gray mullet Liza aurata under conditions of hypoxia ( $ P_{O_2 } The maintenance of the gray mullet Liza aurata under conditions of hypoxia ( 55–82 GPa) for 1–2 days led to a decrease of oxygen tension in arterial blood by 47%, while in venous blood—by 28%. The mean muscle oxygen tension decreased by 19.2%. The number of hypoxic and anoxic zones in muscle tissue decreased significantly, in particular in red muscle. The maintenance of the fish in hypoxic waters for 15 days led to a rise of the content of cytochromes in muscle and a qualitative readjustment of the mitochondrial respiratory chain manifested in an increase of the content of cytochromes a and a3 and a decrease of cytochrome b. In discussion, literature data are presented which support the described regularity. Original Russian Text ? A. A. Soldatov, M. V. Savina, 2008, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2008, Vol. 44, No. 5, pp. 508–512.     

The emersion response of the Australian Yabby Cherax destructor to environmental hypoxia and the respiratory and metabolic responses to consequent air-breathing

The Australian Yabby Cherax destructor voluntarily emerges from water to breathe air with increased frequency as water PO₂ decreases. When the water PO₂ declined below 2.7 kPa the crayfish spent >50% of time breathing air. The respiratory gas transport, acid-base, ionic and energetic status were quantified in simulations of this emersion behaviour to determine the benefits that the crayfish may gain from switching to air-breathing. C. destructor initially showed an elevated O₂ uptake rate on emerging from hypoxic water, but after 1 h the O₂ uptake rate was not different from that of crayfish in normoxic water. During 3 h of air breathing, subsequent to 2.7 kPa aquatic hypoxia, the haemolymph PO₂ increased while oxygen content was essentially unchanged, although cardiac output increased 5-fold. The haemolymph PCO₂ increased from 0.44 to 1.21 kPa after 3 h while the CO₂ content increased from 3.47 to 8.66 mmol · l⁻¹ and the pH decreased from 7.73 to 7.57 after 1 h in air. In air C. destructor eventually achieved an O₂ uptake rate similar to that achieved in water. A general hyperglycaemia occurred without anaerobiosis. In air-breathing C. destructor, small changes in lactate appear to offset the decrease in haemocyanin-O₂ affinity caused by acid Bohr shift. During air-breathing, decreased haemocyanin-O₂ affinity assisted in maintaining O₂ diffusion into the tissues, but the ATP content of the tail muscle decreased so that after 3 h in air the energy charge was only 0.59. The data are consistent with a specific depression of the Emden-Meyerhof pathway, preventing either lactate formation or oxidative phosphorylation in the tail muscle, despite a concomitant glycogenolysis. Accepted: 26 February 1998     

Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimization of the sucrose and ion concentrations for saikosaponin production in hairy root culture ofBupleurum falcatum

Saikosaponin productivity was examined in aBupleurum falcatum L. BFHR2 hairy root culture in response to changes in the sucrose content (2≈8%), nitrogen content (0≈250 mM NH₄NO₃), phosphate content (0≈12 mM NaH₂PO₄), and the potassium content (0≈87.2 mM KCl) of the culture media. We found that the conditions for maximal saikosaponin production differed from those for optimal root growth. Highest saikosaponin yield was achieved for 8% sucrose, 62 mM NH₄NO₃, 1.2 mM NaH₂PO₄, and 0.5 mM KCl.     

Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage

The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption isotherms were determined at 23 and 45°C, and they fit the Guggenheim–Andersson–DeBoer (GAB) model well. Glass transition was determined by differential scanning calorimeter (DSC). The heat capacity changes of WPI and BLG during glass transition were small (0.1 to 0.2 Jg⁻¹ °C⁻¹), and the glass transition temperature (T _g) could not be detected for all samples. An increase in water content in the range of 7 to 16% caused a decrease in T _g from 119 down to 75°C for WPI, and a decrease from 93 to 47°C for WPH. Protein aggregation after 2 weeks’ storage was measured by the increase in insoluble aggregates and change in soluble protein fractions. For WPI and BLG, no protein aggregation was observed over the range of 0 to 85% RH, whereas for WPH, ∼50% of proteins became insoluble after storage at 23°C and 85% RH or at 45°C and ≥73% RH, caused mainly by the formation of intermolecular disulfide bonds. This suggests that, at increased water content, a decrease in the T _g of whey protein powders results in a dramatic increase in the mobility of protein molecules, leading to protein aggregation in short-term storage.