Changes in the incorporation of labeled amino acids into separate protein fractions of the cerebral tissue in rats after intrauterine hypoxia

Incorporation of 14C-amino acid mixture into the cortex and cerebellum protein was studied in 7, 15, 30 days old rats after prenatal hypoxia. Prenatal hypoxic rats was shown to have alteration of the pattern of incorporation of label predecessors into brain protein. Prenatal hypoxia led to significant decrease of incorporation value at 30,000 molecular weight fractions. It is assumed that prenatal hypoxia results in selective changes of the brain protein synthesis.     

P50 values in hypoxic albino rats of first and second generation.

We determined the "in vivo" (arterial pH and PCO2) and standard (pH = 7.4, PCO2 = 40 mm Hg) PO2 at 50% O2 saturation of hemoglobin (P50, vv and P50, st) in Wistar albino rats when living in a normobaric hypoxic environment. Two generations of hypoxic rats were observed for changes in their P50, vv, P50, st, (n50) 2,3-diphosphoglycerate (2,3-DPG), hemoglobin (Hb) and DPG-Hb ratio: the first generation (H1) and the second generation (H2). A few hours after birth, the H1 rats were placed and raised in a normobaric hypoxic environment (10% O2 in N2). The H2 rats were born from hypoxic parents of first generation and were raised in the same hypoxic environment. The control group had a normoxic environment. The P50, st was significantly higher in H1 rats than both H2 and controls. P50, st was similar in H2 and control rats. The P50, vv was significantly higher in H1 rats than both H2 and controls but it was significantly lower in H2 when compared with both controls and H1. Hb and 2,3-DPG had values significantly greater for both H1 and H2 when compared with their controls. However, the values of H2 were significantly lower than H1. The effectiveness of an increase in Hb-O2 affinity as an adaptive mechanism in H2 rats is discussed.     

Proteolytic generation of constitutive tyrosine kinase activity of the human insulin receptor.

We measured rates of protein synthesis in vivo in subcellular fractions (soluble, myofibrillar and stromal fractions) of the heart and the gastrocnemius from rats after fasting or under hypoxic conditions (i.e. atmospheres containing 5% or 10% O2). Such interventions are known to inhibit protein synthesis under some circumstances. The recovery of tissue protein after fractionation was 80-100%. The proportions of protein present in the soluble and stromal fractions were different in the two muscles. The rates of protein synthesis in the myofibrillar and stromal fractions were less than those for total mixed tissue protein, whereas the rate for soluble protein was greater. Both fasting and moderate hypoxia (10% O2 for 24 h) inhibited protein synthesis in the gastrocnemius. In this tissue, the synthesis of the myofibrillar fraction was apparently the most sensitive to inhibition, and this resulted in some significant increases in the soluble-fraction/myofibrillar-fraction protein-synthesis rate ratios. In the heart, fasting inhibited protein synthesis, but moderate hypoxia (10% O2 for 24 h) did not. The rate of protein synthesis in the cardiac myofibrillar fraction was again more sensitive to fasting than were the rates in the other fractions, but it was not as sensitive as that in the gastrocnemius. Under severely hypoxic conditions (5% O2 for 1 or 2 h), protein synthesis was decreased in all fractions in both tissues. These results suggest that the rates of protein synthesis in these relatively crude subcellular fractions vary.     

Hemoglobin and Hypoxic Acclimation in Maize Root Tips

Class 1 hemoglobins (Hbs) have a wide distribution in the plant kingdom and have been demonstrated in root, seed, stem, and leaf tissues. They are present at low concentrations in aerobic tissue, but their synthesis is rapidly induced by hypoxic stress. The pattern of expression of the maize Hb gene in roots of young maize plants exposed to hypoxia has been examined. Root Hb gene expression increased rapidly to a maximum within first two hours of hypoxia, then declining to prehypoxia levels within 48-h hypoxic exposure. Limiting oxygen supply to the roots by total plant immersion and darkness did not alter the time course of hemoglobin expression. Hb gene expression was about 20-fold higher in the stele than in the cortex of control, aerobically grown roots. Stele Hb expression increased about fourfold under hypoxic conditions, whereas its expression in the cortex increased about 60-fold. In these samples, alcohol dehydrogenase (Adh) gene expression increased about four- and ten fold in the stele and cortex, respectively. The effect of the state of the Hb on anoxic survival of maize root tips was assessed by exposing root tips to a carbon monoxide atmosphere to maximize the proportion of hemoglobin in the carbonmonoxy form. Carbon monoxide had no significant effect on the survival or the ATP levels in anoxic maize roots, regardless of whether they had been acclimated by exposure to a hypoxic pretreatment. This would suggest that the presence of oxyhemoglobin is not essential for the survival of anoxic root tips.     

The haemoglobin/nitric oxide cycle: involvement in flooding stress and effects on hormone signalling

BACKGROUND: Class 1 haemoglobins (Hbs) are induced in plant cells under hypoxic conditions. They have a high affinity for oxygen, which is two orders of magnitude lower than that of cytochrome oxidase, permitting the utilization of oxygen by the molecule at extremely low oxygen concentrations. Their presence reduces the levels of nitric oxide (NO) that is produced from nitrate ion during hypoxia and improves the redox and energy status of the hypoxic cell. SCOPE: The mechanism by which Hb interacts with NO under hypoxic conditions in plants is examined, and the effects of Hb expression on metabolism and signal transduction are discussed. CONCLUSIONS: The accumulated evidence suggests that a metabolic pathway involving NO and Hb provides an alternative type of respiration to mitochondrial electron transport under limited oxygen. Hb in hypoxic plants acts as part of a soluble, terminal, NO dioxygenase system, yielding nitrate ion from the reaction of oxyHb with NO. NO is mainly formed due to anaerobic accumulation of nitrite. The overall reaction sequence, referred to as the Hb/NO cycle, consumes NADH and maintains ATP levels via an as yet unknown mechanism. Hb gene expression appears to influence signal transduction pathways, possibly through its effect on NO, as evidenced by phenotypic changes in normoxic Hb-varying transgenic plants. Ethylene levels are elevated when Hb gene expression is suppressed, which could be a factor leading to root aerenchyma formation during hypoxic stress.     

Effect of chronic sodium cyanate administration on O2 transport and uptake in hypoxic and normoxic exercise.

Systemic O2 transport during maximal exercise at different inspired PO2 (PIO2) values was studied in sodium cyanate-treated (CY) and nontreated (NT) rats. CY rats exhibited increased O2 affinity of Hb (exercise O2 half-saturation pressure of Hb = 27.5 vs. 42.5 Torr), elevated blood Hb concentration, pulmonary hypertension, blunted hypoxic pulmonary vasoconstriction, and normal ventilatory response to exercise. Maximal rate of convective O2 transport was higher and tissue O2 extraction was lower in CY than in NT rats. The relative magnitude of these opposing changes, which determined the net effect of cyanate on maximal O2 uptake (VO2 max), varied at different PIO2: VO2 max (ml. min-1. kg-1) was lower in normoxia (72.8 +/- 1.9 vs. 81. 1 +/- 1.2), the same at 70 Torr PIO2 (55.4 +/- 1.4 vs. 54.1 +/- 1.4), and higher at 55 Torr PIO2 (48 +/- 0.7 vs. 40.4 +/- 1.9) in CY than in NT rats. The beneficial effect of cyanate on VO2 max at 55 Torr PIO2 disappeared when Hb concentration was lowered to normal. It is concluded that the effect of cyanate on VO2 max depends on the relative changes in blood O2 convection and tissue O2 extraction, which vary at different PIO2. Although uptake of O2 by the blood in the lungs is enhanced by cyanate, its release at the tissues is limited, probably because of a reduction in the capillary-to-tissue PO2 diffusion gradient secondary to the increased O2 affinity of Hb.     

Embryonic and fetal hemoglobin synthesis in K562 cell line

K562 cell line was grown in liquid suspension and in plasma clot cultures. Morphological studies revealed the presence of a minority of cells, which were identified as erythroblasts. However, the majority of the cells remained unidentified. Biochemical studies confirmed the synthesis of hemoglobin by K562 cells. The pattern of hemoglobin (Hb) production was of the embryonic type, with the presence of small amount of fetal Hb. The addition of several inducers, like Epo and butyrate, was unable to modify the pattern of Hb production of K562. In contrast, the addition of hemin increased the synthesis of Hb and stimulated the synthesis of fetal Hb and probably adult Hb.     

Reduction in hypertension-induced protein synthesis in the rat pulmonary trunk after treatment with teprotide (SQ 20881)

Angiotensin II has been previously implicated as a mediator of vasoconstriction during the development of hypoxic pulmonary hypertension. The effect of angiotensin-converting enzyme inhibition with teprotide (SQ 20881) on development of pulmonary hypertension was determined by measurement of the drug's ability to modify hypertension-induced protein synthetic changes in the rat pulmonary trunk. Rats were injected with either SQ 20881 (2 mg/kg body wt every 8 hr) or saline vehicle during exposure to chronic hypoxia at 0.5 atm for either 3 or 7 days. Comparisons were made of tissue weight, absolute protein content, and in vitro synthesis of collagen and noncollagen protein of the pulmonary trunks of SQ-treated hypoxic, SQ-treated normoxic, saline-treated hypoxic, and saline-treated normoxic rats. Treatment of hypoxic rats with SQ 20881 was found to significantly decrease right ventricular pressure, tissue weight, absolute protein content, and in vitro protein synthesis after 7 days compared to saline-treated hypoxic rats. Neither right ventricular hypertrophy nor the development of polycythemia was decreased by SQ 20881 treatment.     

High-altitude respiration of birds. Structural adaptations in the major and minor hemoglobin components of adult Rüppell's Griffon (Gyps rueppellii, Aegypiinae): a new molecular pattern for hypoxic tolerance

The primary structures of the hemoglobins Hb A, Hb A', Hb D and Hb D' of Rüppell's Griffon (Gyps rueppellii), which can fly as high as 11,300 m, are presented. The globin chains were separated on CM-Cellulose in 8M urea buffers, the four hemoglobin components by FPLC in phosphate buffers. The amino-acid sequences of five globin chains were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid-phase and gas-phase sequenators. The sequences are compared with those of other Falconiformes. A new molecular pattern for survival at extreme altitudes is presented. For the first time four hemoglobins are found in blood of a bird; they show identical beta-chains and differ in the alpha A- and alpha D-chains by only one replacement. These four hemoglobins cause a gradient in oxygen affinities. The two main components Hb A and Hb A' differ at position alpha 34 Thr/Ile. In case of Ile as found in Hb A' an alpha 1 beta 1-interface is interrupted raising oxygen affinity compared to Hb A. In addition the hemoglobins of the A- and D-groups differ at position alpha 38 Pro or Gln/Thr (alpha 1 beta 2-interface). Expression of Gln in Hb D/D' raises the oxygen affinity of these components compared to Hb A/A' by destabilization of the deoxy-structure. The physiological advantage lies in the functional interplay of four hemoglobin components. Three levels of affinity are predicted: low affinity Hb A, Hb A' of intermediate affinity, and high affinity Hb D/D'. This cascade tallies exactly with oxygen affinities measured in the isolated components and predicts oxygen transport by the composite hemoglobins over an extended range of oxygen affinities. It is contended that the mechanisms of duplication of the alpha-genome (creating four hemoglobins) and of nucleotide replacements (creating different functional properties) are responsible for this remarkable hypoxic tolerance to 11,300 m. Based on this pattern the hypoxic tolerances of other vultures are predicted.     

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.     

Isolated Hb Providence β82Asn and β82Asp fractions are more stable than native HbA(0) under oxidative stress conditions

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of β-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in βK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.     

Monitoring Hypoxia Induced Changes in Cochlear Blood Flow and Hemoglobin Concentration Using a Combined Dual-Wavelength Laser Speckle Contrast Imaging and Doppler Optical Microangiography System

A synchronized dual-wavelength laser speckle contrast imaging (DWLSCI) system and a Doppler optical microangiography (DOMAG) system was developed to determine several ischemic parameters in the cochlea due to a systemic hypoxic challenge. DWLSCI can obtain two-dimensional data, and was used to determine the relative changes in cochlear blood flow, and change in the concentrations of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) in mice. DOMAG can obtain three-dimensional data, and was used to determine the changes in cochlear blood flow with single vessel resolution. It was demonstrated that during a hypoxic challenge there was an increase in the concentrations of Hb, a decrease in the concentrations of HbO and cochlear blood flow, and a slight decrease in the concentration of HbT. Also, the rate of change in the concentrations of Hb and HbO was quantified during and after the hypoxic challenge. The ability to simultaneously measure these ischemic parameters with high spatio-temporal resolution will allow the detailed quantitative analysis of several hearing disorders, and will be useful for diagnosing and developing treatments.     

Protein metabolism by rat brain synaptosomes during learning

The synthesis and degradation of rat brain synaptosomal proteins were studied in three groups of animals: trained the behaviour pattern in the maze, "pseudo-trained" and control. These processes were assessed from protein specific radioactivity after 1, 3 days and after 1, 3, 6 and 9 weeks following intraventricular injection of 14C-lysine. The experiments showed three fractions differing in overall values of half-life (T50). An increase in specific radioactivity of brain proteins of trained animals was revealed as compared to that in "pseudo-trained" and control rats. T50 was recorded to rise for slow-metabolizing fractions of synaptosomal proteins of trained rats. Participation of synaptosomal proteins in the mechanisms of long-term memory is discussed.     

Ventilatory response to severe acute hypoxia in guinea-pigs and rats with high hemoglobin-oxygen affinity induced by cyanate

Baseline ventilation, hemoglobin concentration (Hb) and P50 were significantly lower in guinea-pigs than in rats. Chronic sodium cyanate (NaOCN) administration did not significantly increase hemoglobin concentration in either guinea-pigs or rats. It decreased the P50 significantly less in guinea-pigs than in rats. The high Hb-O₂ affinity experimentally induced did not modify the hypoxic ventilatory response (HVR) of guinea-pigs and rats. At the same level of acute hypoxia, HVR was significantly lower in NaOCN guinea-pigs than in NaOCN rats. Guinea-pigs, genotypically adapted animals to high altitude, displayed relatively minor ventilatory and Hb-O₂ affinity changes to NaOCN, and a relatively minor HVR to acute hypoxia. They probably use tissue and biochemical adaptive mechanisms, in addition to their limited extracellular responses to successfully tolerate ambient hypoxia.     

HbF-新晋[~Gy~I119(GH2)Gly→Arg]一种新的不稳定胎儿血红蛋白变异体

 1988年我室又发现一种新的慢泳异常胎儿血红蛋白(HbF)。先证者为一健康汉族女婴。醋纤膜电泳(pH8.6)示变异体区带位于HbF与Hb A_2之间,反相高效液相色谱(HPLC)示正常GγI峰前方出现一异常峰。异常Hb含量为Hb总量的16.8％。理化性质测定该变异体为一轻度不稳定Hb。结构分析证明该变异体为GγI链第119位(GH2)的甘氨酸(Gly)被精氨酸(Arg)取代。γ链GH2位于α_1γ_1接触面,此处的氨基酸置换可导致Hb的不稳定。此变异体被命名为HbF-新晋[~(GγI)119(GH2)Gly→Arg]。     

一氧化氮对急、慢性缺氧大鼠血流动力学及缺氧性肺血管收缩反应的影响

观察了吸入０．００４％的一氧化氮（ＮＯ）对急、慢性缺氧大鼠血流动力学、缺氧性肺血管收缩反应（ＨＰＶ）、血气及高铁血红蛋白（ＭｅｔＨｂ）的影响。结果表明：（１）常氧吸入ＮＯ时能明显降低慢性缺氧大鼠肺动脉平均压（Ｐｐａ）和肺血管阻力（ＰＶＲ）,但对正常大鼠的Ｐｐａ和ＰＶＲ无明显影响;（２）慢性缺氧大鼠急性缺氧时ＨＰＶ较正常大鼠弱,吸入ＮＯ不但降低两者的急性缺氧肺动脉高压,且完全逆转两者的ＨＰＶ;（３）吸入ＮＯ对急、慢性缺氧大鼠体循环血流动力学、血气及ＭｅｔＨｂ含量无明显影响。提示吸入ＮＯ能选择性降低急、慢性缺氧性肺动脉高压,且逆转ＨＰＶ。     

Hypoxia-induced inhibition of converting enzyme activity: role in vascular regulation

Systemic and pulmonary vascular reactivity to graded doses of angiotensin I (ANG I), angiotensin II (ANG II), and, as a control, phenylephrine were examined in 14- or 28-day hypoxia-exposed and air control rats. Hypoxic rats exhibited pulmonary hypertension that was reversible on return to room air, but systemic arterial pressure was not altered by hypoxia. Systemic pressor responses to ANG I and ANG II were significantly less in the hypoxic rats than in the control rats at 14 and 28 days but returned to control levels in hypoxic animals that were then returned to room air, demonstrating reversibility of the hypoxia-induced changes in vascular reactivity. Pulmonary pressor responses to ANG I were significantly less at 14 days, whereas responses to ANG II were significantly greater at 28 days, in hypoxic rats than in controls. There were no significant differences in systemic and pulmonary pressor responses to phenylephrine between the hypoxic and air control animals. The altered systemic and pulmonary pressor responsiveness to ANG I and ANG II in hypoxic rats is probably related to mechanisms specific to the renin-angiotensin system, such as inhibition of intrapulmonary angiotensin-converting enzyme activity and down regulation of ANG II receptors in the systemic circulation. Further study is needed to elucidate these mechanisms.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

A comparative analysis of the cerebral cortical proteins in rats with different sensitivities to hypoxia]

A comparative analysis of 339 protein fractions of cerebral cortex of rats both resistant and non-resistant to oxygen deficiency has been fulfilled by means of two-dimensional gel-electrophoresis. A specific group of 9 protein fractions with molecular weights in the range of 32-68 kD was found to be quantitatively changed under hypoxia influence. An activation of labile protein synthesis was a predominant response to acute hypoxia in the resistant rats, while the synthesizing processes in the non-resistant rats were rather weak. An adaptation to hypoxia mostly resulted in the decrease of quantitative representations of labile protein fractions and has been realizing in different ways in resistant and nonresistant rats. The data obtained seem to testify to the changes of protein synthesis under chronic hypoxia conditions in the cerebral cortex chiefly determined by fast adaptation mechanisms.     

THE EFFECT OF SLEEP DEPRIVATION UPON THE IN VIVO AND IN VITRO INCORPORATION OF TRITIATED AMINO ACIDS INTO BRAIN PROTEINS IN THE RAT AT THREE DIFFERENT AGE LEVELS

Abstract— The effect of sleep deprivation on the in vivo and in vitro tritiated amino acid incorporation into brain proteins was studied in the rat at three age levels. Sleep deprivation was induced either by water tank or handling methods. Three experimental groups of animals were used: control, sleep deprived and post deprivation sleeping rats.
A significant decrease of protein synthesis was found in the cerebellum, telencephalon and in crude subcellular fractions of brainstem of adult rats selectively deprived of paradoxical sleep. However, no alteration of protein synthesis was observed either in vivo or in vitro , in the same brain regions or in the liver after the rebound of paradoxical sleep following deprivation.
In four crude subcellular protein fractions a specific increase of the in vitro labelled amino acid incorporation was observed in the brain stem of 24-day-old rats allowed to recuperate after sleep deprivation as compared with the deprived rats. No significant changes were seen in the telencephalon.
No alteration of incorporation was found in 7-day-old rats deprived of sleep.
The possible functional significance of these results is discussed in relation to stress and to variations in the size of the precursor pool for protein synthesis.