Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic,ionic, contractile and electrophysiological studies

Summary The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37°C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22°C and 27°C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37°C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24±2.49 IU·g dry wt^-1, and the total amount of protein lost was 70.37±0.73 mg·g dry wt^–1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37°C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.     

Alterations in the energy metabolism of the isolated perfused frog heart during calcium depletion and subsequent repletion

Summary The changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, a-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism.Abbreviations ADP Adenosine diphosphate - AMP Adenosine monophosphate - ATP Adenosine triphosphate - EDTA Ethylene-diamino-tetraacetic acid - NAD ⁺ Nicotinamide-adeninedinucleotide - NADH Nicotinamide-adenine-dinucleotide (reduced form) - TRA Triethanolamine     

Binding of estradiol to horseradish peroxidase and horse heart microperoxidase

Horseradish peroxidase and horse heart microperoxidase can bind estradiol to human or bovine serum albumin in the presence of hydrogen peroxide. However, we have shown here that, in the absence of serum albumin, the hormone was fixed by the enzyme molecule itself. Evidence is presented that (a) the hormone is transformed into a water-soluble and dialysable derivative of estradiol; (b) this new product is easily separated from the enzyme by gel filtration chromatography. It appears to have a high affinity for the chromatographic gel. The implications of the binding of an estradiol derivative to peroxidases are discussed.     

Genetic studies in Paraguay: blood group, red cell, and serum genetic patterns of the Guayaki and Ayore Indians, Mennonite settlers, and seven other Indian tribes of the Paraguayan Chaco

Genetic studies of 540 Paraguayan Indians from nine tribal groups and 51 Mennonites are presented for ABO, MNSs, P₁, Rh, Kell, Lewis, Duffy, Diego; for serum immunoglobulins and haptoglobins, G6PD-deficiency, and thalassemia trait. Group O gene frequencies for all Indian groups were 1.00; for r (cde), 0.00. Tapiete, Lengua, Toba, and Sanapana R_z (CDE) frequencies were among the highest ever reported. N frequencies were high for Ache Kwera (Guayaki), Lengua, Cheroti, Guarayu, Tapiete; N and s low for Ayore. MS frequencies were high for Sanapana, Lengua, Ayore; Ns for Tapiete. Diego was notably absent for Toba, Lengua, Guarayu, Tapiete, Ayore. Homogeneous frequencies for Fy^a (1.000) occurred among Guarayu and Tapiete, and for P₁ among Guayaki. Inv(a) frequencies were low for Cheroti, Chulupi, Guayaki. Hp 1 among Guayaki (Ache Kwera 0.15) is lowest ever reported. G6PD deficiency and abnormal hemoglobins were uniformly absent from all groups. Mennonite results were homogeneous and point toward Dutch origins. Differences among groups studied, and between Paraguayan and other Amerinds emphasize importance of genetic drift and founder principle. Abandonment of their tribes by mixed-blood offspring is partly responsible for apparent genetic purity and homogeneity of groups.     

Shifts in specific serum protein concentrations: apolipoprotein-B and albumin in the southern elephant seal during the breeding and molting periods in Antarctica

Serum protein values in Southern elephant seals (Mirounga leonina) were analyzed during the breeding and molting periods at the 25 de Mayo Island (King George Island), Antarctica, during the 1999/2000 and 2000/2001 field seasons, in order to study the reaction of adults to fasting, and of pups to nursing and fasting. The following analyses were carried out: Total Proteins (TP) and Albumin (Alb) were analyzed by a colorimetric technique, and Apolipoprotein B (Apo-B) was determined by the immunodiffusion technique on agarose gel plates. The general ranges of average values for total proteins and specific fractions during the breeding period were: TP (g/dl) = 5.12–9.83; Alb (g/dl) = 1.72–5.71 and Apo-B (mg/dl) = 10–266, and during the post-breeding period: TP = 4.85–9.45; Alb = 2.06–4.20 and Apo-B = 13–232. The essential obtained data were: (a) fasting does not impact adult males, except for a significant decrease of Apo-B during breeding; (b) fasting does not impact adult females, except for a significant decrease in TP during molting; (c) suckling increases significantly TP and Apo-B in pups; (d) post-weaning fast decreases significantly all measured serum components in pups. We can conclude that the adults are adapted to long term fasting, without any metabolic downside they maintain homeostasis during this period, as shown by the serum data. The pups clearly react to both suckling and the post-weaning fast, which are periods driven almost exclusively by lipid chemistry. These impacts may be seen in the chemistry of serum proteins.     

罗格列酮和血清脂对绵羊前体脂肪细胞分化的影响

目的探讨罗格列酮(rosiglitazone,Ros)和血清脂(serum lipid,Lip)对绵羊前体脂肪细胞分化的影响及不同组织来源的前体脂肪细胞分化影响的差异。方法用不同浓度的Ros和(或)Lip培养绵羊皮下前体脂肪细胞和肾周前体脂肪细胞,通过测量3-磷酸甘油脱氢酶(GPDH)活性和油红O染色萃取液A值分析前体脂肪细胞的分化程度和脂肪细胞充脂量的变化,应用实时荧光定量PCR检测PPARγ和LPL mRNA的表达水平。结果 Ros和Lip提高细胞GPDH活性和脂滴的沉积量(P<0.05),上调LPL mRNA表达(P<0.05),最佳浓度分别为100nmol/L和20μL/mL;最佳浓度条件下Ros的诱导作用强于Lip(P<0.05),Ros显著提高了PPARγmRNA表达量(P<0.05),而Lip对PPARγmRNA的表达没有明显影响(P>0.05);Ros和Lip共同诱导与Ros单独作用之间没有明显差异(P>0.05);在相同诱导分化条件下,皮下前体脂肪细胞的分化程度高于肾周前体脂肪细胞(P<0.05)。结论研究结果表明Ros和Lip可促进绵羊前体脂肪细胞的分化,在相同条件下,皮下前体脂肪细胞的分化能力强于肾周前体脂肪细胞。     

辣椒素对离体大鼠胃平滑肌收缩性的影响

目的:考察辣椒素对离体大鼠胃平滑肌收缩性的影响。方法:本研究以大鼠的离体胃平滑肌条为模型,首先考察在正常钙克氏液、高钙克氏液和低钙克氏液中辣椒素对胃平滑肌收缩作用的影响。然后正常克氏液中,分别观察辣椒素对乙酰胆碱、新斯的明、阿托品分别存在下的胃平滑肌收缩性的影响。结果:辣椒素在2.5μmol/L-40μmol/L浓度范围内可剂量依赖性显著抑制高钙溶液(Ca2 终浓度5μmol/L)引起的大鼠胃平滑肌强烈收缩,在5μmol/L-40μmol/L浓度范围内可显著抑制正常克氏液中大鼠胃平滑肌条的运动,且具有明显剂量依赖性,在10μmol/L-40μmol/L浓度范围内可显著抑制低钙克氏液中大鼠胃平滑肌条的运动,且具有剂量依赖性。辣椒素(10μmol/L)可拮抗乙酰胆碱和新斯的明引起的收缩作用(P<0.01)。辣椒素(10μmol/L)的作用与阿托品具有相加作用(P<0.01)。结论:辣椒素对胃平滑肌的收缩具有明显的抑制作用。     

Fine structure and its functional properties of the ependymal cell in the frog median eminence

Summary Intercellular canaliculi surrounded by several ependymal cells, having numerous microvilli and a few cilia on the apical surface, are present throughout the frog median eminence. The intercellular canaliculi penetrate deeply near the portal vessel from the third ventricle. They are separated from the pericapillary space only by the thin cytoplasm of the ependymal cell.The cytoplasmic protrusions containing a large number of clear vesicles are often found at the apical surface of ependymal cells facing the third ventricle or the lumen of intercellular canaliculus. The ependymal cell shows well developed Golgi apparatus and well developed rough endoplasmic reticulum in its cytoplasm. Dense granules of about 1200–1500 A diameter suggesting secretory materials are found in small number near the Golgi apparatus and abundantly in the ependymal process lying around the portal vessel.Synaptic contacts between the ependymal cell and two different types of the nerve endings, monoaminergic and peptidergic, are frequently observed. A few small flasklike caveolae suggesting micropinocytosis are found in the post-synaptic membrane as well as in the lateral and basal plasma membranes of the ependymal cell. The author consideres that the ependymal cell in this region has secretory and transport (absorption) activities.     

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (T_c) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [¹⁴C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T_c of the lipid and increases with temperature above the T_c. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.     

Determination on the binding of chlortetracycline to bovine serum albumin using spectroscopic methods

In this work, the interaction of chlortetracycline with bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. Results indicated that chlortetracycline quenches BSA fluorescence mainly by a static quenching mechanism. The quenching constants (K_SV) were obtained as 5.64 × 10⁴, 4.49 × 10^4/, and 3.44 × 10^4/ M_?1 at 283, 295, and 307 K, respectively. The thermodynamic parameters of enthalpy change Δ H°, entropy change Δ S°, and free energy change Δ G° were ?5.12 × 10^4/ J mol?1, ?97.6 J mol?1 K?1, and ?2.24 × 10^4/ J mol?1 (295 K), respectively. The association constant (K_A) and the number of binding sites (n) were 9.41 × 10^3/ M?1 and 0.86, respectively. The analysis results suggested that the interaction was spontaneous, and van der Waals force and hydrogen‐bonding interactions played key roles in the reaction process. In addition, CD spectra proved secondary structure alteration of BSA in the presence of chlortetracycline. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:331–336, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21424     

Effects of GABA-mediated inhibition on direction-dependent frequency tuning in the frog inferior colliculus

Earlier studies from our laboratory have shown that the frequency selectivity of neurons in the frog inferior colliculus is direction dependent. The goal of this study was to test the hypotheses that gamma-aminobutyric acid or GABA (but not glycine)-mediated synaptic inhibition was responsible for the direction-dependence in frequency tuning, and that GABA acted through creation of binaural inhibition. We performed single unit recordings and investigated the unit's free-field frequency tuning, and/or the unit's response to the interaural level differences (under dichotic stimulation), before and during local applications of antagonists specific to gamma-aminobutyric acid a and glycine receptors. Our results showed that application of bicuculline produced a broadening of free-field frequency tuning, and differential changes in free-field frequency tuning depending on sound direction, i.e., more pronounced at azimuths at which the unit exhibited narrower frequency tuning under the pre-drug condition, thereby typically abolishing direction dependence in tuning. Application of strychnine produced no change in frequency tuning. The results from dichotic stimulation further revealed that bicuculline typically elevated and/or flattened the unit's interaural-level-difference response function, indicating a reduction in the strength of binaural inhibition. Our study provides evidence that gamma-aminobutyric acid-mediated binaural inhibition is important for direction dependence in frequency tuning. Accepted: 24 July 1998     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Spectroscopic investigation on the interaction of Cr(VI) with bovine serum albumin

The interaction of potassium dichromate (Cr(VI)) with bovine serum albumin (BSA) was investigated by fluorescence, synchronous fluorescence, resonance light scattering (RLS), ultraviolet-visible absorption, and circular dichroism (CD) spectroscopies under simulated physiological conditions. The experimental results showed that Cr(VI) could quench the intrinsic fluorescence of BSA following a static quenching process, which indicates the formation of a Cr(VI)-BSA complex. The binding constant (KA) and binding site (n) were measured at different temperatures. The spectroscopic results also revealed that the binding of Cr(VI) to BSA can lead to the loosening of the protein conformation and can change the microenvironment and skeleton of BSA.     

Novel antimicrobial peptides isolated from the skin secretions of Hainan odorous frog, Odorrana hainanensis

Long time geographical isolation of Hainan Island from the China continent has resulted in appearance of many novel frog species. As one of them, Hainan odorous frog, Odorrana hainanensis possesses some special antimicrobial peptides distinct from those found in other Odorrana. In this study, three antimicrobial peptides have been purified and characterized from the skin secretion of O. hainanensis. With the similarity to the temporin family, two peptides are characterized by amidated C-terminals, so they are named as temporin-HN1 (AILTTLANWARKFL-NH₂) and temporin-HN2 (NILNTIINLAKKIL-NH₂). The third antimicrobial peptide belongs to the brevinin-1 family which is widely distributed in Eurasian ranids, and thus, it is named as brevinin-1HN1 (FLPLIASLAANFVPKIFCKITKKC). Furthermore, after sequencing 68 clones, eight cDNAs encoding antimicrobial peptide precursors were cloned from the skin-derived cDNA library of O. hainanensis. These eight cDNAs can encode seven mature antimicrobial peptides including the above three, as well as brevinin-1V, brevinin-2HS2, odorranain-A6, and odorranain-B1. Twelve different species of microorganisms were chosen, including Gram-positive, Gram-negative and fungi, to test the antimicrobial activities of temporin-HN1, temporin-HN2, brevinin-1HN1, brevinin-1V, and brevinin-2HS2. The result shows that, in addition to their activities against Gram-positive bacteria, temporin-HN1 and temporin-HN2 also possess activities against some Gram-negative bacteria and fungi. However, the two antimicrobial peptides, brevinin-1HN1 and brevinin-1V of the brevinin-1 family have stronger antimicrobial activities than temporin-HN1 and temporin-HN2 of the temporin family. Brevinin-1HN1 possesses activity against Staphylococcus aureus (ATCC25923), Rhodococcus rhodochrous X15, and Slime mould 090223 at the concentration of 1.2 μM.     

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations.With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.     

Study on the binding of cerium to bovine serum albumin

The interaction of Ce(3+) to bovine serum albumin (BSA) has been investigated mainly by fluorescence spectra, UV-vis absorption spectra, and circular dichroism (CD) under simulative physiological conditions. Fluorescence data revealed that the quenching mechanism of BSA by Ce(3+) was a static quenching process, the binding constant is 6.70 × 10(5) , and the number of binding site is 1. The thermodynamic parameters (ΔH = -29.94 kJ mol(-1) , ΔG = -32.38 kJ mol(-1) , and ΔS = 8.05 J mol(-1) K(-1) ) indicate that electrostatic effect between the protein and the Ce(3+) is the main binding force. In addition, UV-vis, CD, and synchronous fluorescence results showed that the addition of Ce(3+) changed the conformation of BSA.     

Effects of dietary selenium supplementation on parasitemia,anemia and serum proteins of Trypanosoma brucei brucei infected rats

Trypanosomosis has been associated with immunosuppression, anemia and oxidative damage while selenium possesses both immunostimulatory and antioxidative effects. This study was designed to assess the effect of dietary selenium supplementation on parasitemia, anemia, survival pattern and serum protein profiles of trypanosome-infected rats. Twenty five rats, divided into five groups (A–E) of 5 each, were treated as follows: 4, 8 and 16 ppm (ppm) of selenium in their feed, respectively throughout the experimental period and were infected with Trypanosoma brucei brucei on day 14 post supplementation, infected not supplemented and the negative control. Supplementation at 4 and 8 ppm increased the packed cell volume (PCV) and hemoglobin (Hb) concentration on day 7 of supplementation (PS) when compared with the unsupplemented groups. Following infection on day 14 PS, the PCV, Hb of 16 ppm and infected not supplemented groups were significantly (P < 0.05) lower than other groups on days 28 and 35 PS. Supplementation did not lead to significant (P > 0.05) changes on the total protein, albumin and globulin by day 14 PS. Infection, however, caused significant (P > 0.05) decrease in the total protein and albumin from day 28. The supplementation did not significantly (P > 0.05) increase the pre-patent period but caused a significant reduction in the parasitemia levels and increased survival intervals. Dietary selenium supplementation, from the results, may show promise in the management of African trypanosomosis as the supplementation was able to: reduce anemia and parasitemia and increase survival intervals of trypanosome infected rats.     

Effects of zinc supplementation on serum zinc and leptin levels,BMI, and body composition in hemodialysis patients

ProjectThe aim of this study was to determine the effects of zinc supplementation on serum zinc and leptin levels as well as on anthropometric status and some biochemical parameters in hemodialysis (HD) patients.ProcedureIn this randomized, double-blind, and placebo-controlled trial, sixty HD patients were randomly divided into groups to receive a daily supplement of 100 mg elemental Zn (supplemented group) or placebo (control group) for 60 days. Anthropometric measurements were taken using standard calibrated instruments. Serum zinc and leptin levels were determined by atomic absorption and ELISA method respectively before and after intervention.ResultsZinc supplementation resulted in significant increase in the mean serum zinc level in the experimental group while changes observed in the placebo group were not significant. The mean serum leptin in women part of the experimental group was decreased significantly after supplementation. After adjusting for age, BMI, body fat (%), serum zinc and dietary Zn intake, a negative and significant association was observed between serum zinc and leptin levels in all subjects (β = −0.33, P = 0.03) as a result of Zn supplementation.ConclusionsMore studies are needed to clarify the mechanisms by which serum leptin level is influenced as a result of zinc supplementation in HD patients.     

Action of caerulein,gastrin 17, pentagastrin,and secretin on the active transport of sodium by the frog skin

Summary Frog skin was mounted in an Ussing chamber and the actions of caerulein, gastrin, pentagastrin, and secretin on the active transport of sodium were studied using the short-circuit current method. All polypeptides exerted their effect when placed in the solution bathing the outside surface of the skin. The response was a transient dose-related increase in the transepithelial electrical potential difference and in the short-circuit current. Analysis of the response indicated that at submaximal doses the effect was due to an increase in the rate of entry of sodium through the outer barrier to active sodium transport. At supramaximal doses the passive permeability of the skin was also increased. Th ED50 concentrations of the hormones were: caerulein, 50pm; gastrin, 53pm; pentagastrin, 440pm; and secretin, 30pm. It is argued that the large quantity of caerulein or caerulein-like peptides stored in the skin may be required either to control the entry of sodium when the amphibian is undergoing maximum stress in a freshwater environment, or that it may have a protective function for the amphibian as it could elicit a noxious hypersecretion in the gastrointestinal tract of the predator together with a marked hypotension.