Synergic Effects of β-Estradiol and Erythromycin on hERG Currents

The incidence rates of long QT syndrome (LQTS) and drug-induced torsades de pointes (TDP) are higher in women than men. Although gonadal steroids are assumed to play an important role in the gender-based differences in cardiac electrophysiological properties, the underlying mechanisms of the gender-based differences are not fully understood. In particular I _Kr, which comprises the repolarization phase of the action potential, has not been well understood in its modulation by sex hormones. To assess this, we examined the effects of the female sex hormone β-estradiol on the human ether-a-go-go-related gene (hERG)-encoded potassium current stably expressed in human embryonic kidney-293 (HEK) cells. We demonstrated that hERG currents were inhibited by β-estradiol maximally to 62% of control with an IC₅₀ of 1.3 μM and a Hill coefficient of 0.87, which might account for the sex-related differences in LQTS. We also examined whether estrogen modulated drug-induced blocking effects on hERG currents or not. With simultaneous application of 10 μM erythromycin, which is known to block hERG currents but not in low doses, the blocking effects of β-estradiol on hERG currents were enhanced. Namely, hERG currents were inhibited maximally to 45.8% of control with an IC₅₀ of 59 nM (P < 0.02) by β-estradiol with 10 μM erythromycin. We conclude here that a significant block of hERG currents by β-estradiol may account for the sex-related differences in LQTS and the synergic effects of β-estradiol and erythromycin indicate a higher risk of drug-induced TDP in women than men.     

Effects of 17β-Estradiol and Vitamin E Treatments on Blood Trace Element and Antioxidant Enzyme Levels in Ovariectomized Rats

We investigated the effect of 17β-estradiol (E₂) alone and separately vitamin E treatment on trace element status of rats following an ovariectomic operation. Forty rats were equally divided into four groups: Group 1, control, non-ovariectomized rats; Group 2, (OVX) rats, ovariectomized under general anesthesia; Group 3, (OVX+E₂) rats, the group received a 40 μg kg⁻¹ subcutan dose of E₂ per day after ovariectomy; and Group 4, (OVX + E₂ + vitamin E) rats, received the same E₂ treatment, but with an additional 100 mg kg⁻¹ intraperitoneal dose of vitamin E per day after ovariectomy. At the end of the 30-day experiment, the rats were sacrificed and their blood was collected for the measurement of zinc, copper, iron, phosphorus, selenium, magnesium, calcium, manganese, and chromium; copper–zinc superoxide dismutase (SOD); manganese-superoxide dismutase (Mn-SOD); glutathione peroxidase (Se-GSH-Px); and catalase (CAT). The levels of zinc, copper, iron, phosphorus, selenium, calcium, chromium, and manganese and activities of SOD, Mn-SOD, Se-GSH-Px, and CAT were lower in the OVX than in the control group, but magnesium level was unaffected. However, zinc, copper, iron, phosphorus, selenium, calcium, chromium, and manganese levels and SOD, Mn-SOD, Se-GSH-Px, and CAT activities were higher under separate E₂ and E₂ + vitamin E treatments. The level of magnesium in the treated-OVX groups was not different than in the OVX group. In conclusion, E₂ treatment has an ameliorating effect on the trace element status in OVX, and this effect may be enhanced with the addition of vitamin E.     

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.     

17 β-Estradiol Enhances the Outgrowth and Survival of Neocortical Neurons in Culture

Results of this investigation demonstrate that exposure to 17 -estradiol differentially and significantly regulates cortical nerve cell outgrowth depending on the cortical region. Parietal and occipital neurons treated with 1 nM 17 -estradiol showed a greater magnitude of neuronal outgrowth whereas outgrowth of temporal cortex neurons was decreased in the presence of 1 nM 17 -estradiol. Frontal cortex neurons showed a consistent enhancement of neuronal outgrowth that did not reach statistical significance. The dose response profile for 17 -estradiol regulation of the macromorphological features exhibited a bimodal dose response relationship whereas the dose response profile for 17 -estradiol regulation of the micromorphological features exhibited a dose response more characteristic of an inverted V-shaped function. An antagonist to the NMDA receptor antagonist, AP5, abolished the growth promoting effect of 17 -estradiol whereas the nuclear estrogen receptor antagonist ICI 182,780 did not. Lastly, neocortical neurons exposed to 17 -estradiol exhibited greater viability and survival than control neurons over a two week period. These data indicate that 17 -estradiol can enhance the growth and viability of select populations of neocortical neurons and that the growth promoting effects of 17 -estradiol can be blocked by an antagonist to the NMDA glutamate receptor and not by an antagonist to the estrogen nuclear receptor.     

Changes in Fatty Acid Composition and Lipid Content Occurring in Potato Leaves during Cold Hardening: the Role of ∆12-Acyl-Lipid Desaturase

Russian Journal of Plant Physiology - The qualitative composition and changes in the total fatty acid (FA) content in leaves of wild-type potato (Solanum tuberosum L., cv. Desnitsa) plants and...     

Effects of β-alanine supplementation on the onset of neuromuscular fatigue and ventilatory threshold in women

Summary. This study examined the effects of 28 days of β-alanine supplementation on the physical working capacity at fatigue threshold (PWC_FT), ventilatory threshold (VT), maximal oxygen consumption ( O_2-MAX), and time-to-exhaustion (TTE) in women. Twenty-two women (age ± SD 27.4 ± 6.1 yrs) participated and were randomly assigned to either the β-alanine (CarnoSyn™) or Placebo (PL) group. Before (pre) and after (post) the supplementation period, participants performed a continuous, incremental cycle ergometry test to exhaustion to determine the PWC_FT, VT, O_2-MAX, and TTE. There was a 13.9, 12.6 and 2.5% increase (p < 0.05) in VT, PWC_FT, and TTE, respectively, for the β-alanine group, with no changes in the PL (p > 0.05). There were no changes for O_2-MAX (p > 0.05) in either group. Results of this study indicate that β-alanine supplementation delays the onset of neuromuscular fatigue (PWC_FT) and the ventilatory threshold (VT) at submaximal workloads, and increase in TTE during maximal cycle ergometry performance. However, β-alanine supplementation did not affect maximal aerobic power ( O_2-MAX). In conclusion, β-alanine supplementation appears to improve submaximal cycle ergometry performance and TTE in young women, perhaps as a result of an increased buffering capacity due to elevated muscle carnosine concentrations.     

Changes in the Mutagenic and Estrogenic Activities of 17β-Estradiol after Treatment with Nitrite

We determined the changes in the mutagenic and estrogenic activities of 17β-estradiol after a nitrite treatment. Nitrite-treated 17β-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17β-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17β-estradiol to have been formed in the reaction mixture. 2-Nitro-17β-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17β-estradiol was found to be lower than that of 17β-estradiol. These data suggest that a daily oral intake of 17β-estradiol and nitrite might induce the formation of mutagenic compounds in our body.     

Effects of Turn Stability and Side-Chain Hydrophobicity on the Folding of β-Structures

Key elements of β-structure folding include hydrophobic core collapse, turn formation, and assembly of backbone hydrogen bonds. In the present folding simulations of several β-hairpins and β-sheets (peptide 1, protein G B1 domain peptide, TRPZIP2, TRPZIP4, 20mer, and 20mer^DP6D), the folding free-energy landscape as a function of several reaction coordinates corresponding to the three key elements indicates apparent dependence on turn stability and side-chain hydrophobicity, which demonstrates different folding mechanisms of similar β-structures of varied sequences. Turn stability is found to be the key factor in determining the formation order of the three structural elements in the folding of β-structures. Moreover, turn stability and side-chain hydrophobicity both affect the stability of backbone hydrogen bonds. The three-stranded β-sheets fold through a three-state transition in which the formation of one hairpin always takes precedence over the other. The different stabilities of two anti-parallel hairpins in each three-stranded β-sheet are shown to correlate well with the different levels of their hydrophobic interactions.     

Effects of β-alanine treatment on the taurine and DNA content of the rat heart and retina

Experimental evidence has suggested that the high endogenous levels of taurine found in the rat heart and retina are maintained to a large extent by transport processes out of the blood, rather than by endogenous biosynthesis. When these high levels are depleted, dysfunction ensues. In vitro studies have shown that -alanine is a good antagonist of these transport processes. The current studies were done to evaluate the feasibility of depleting heart and retinal taurine levels in vivo through treatment of adult rats either orally or with injections of -alanine. None of the treatments had significant effects on retinal taurine content; ventricular taurine was reduced in some situations, but the effects were not maintained, nor as large as with another transport antagonist. No functional changes were observed. Oral treatment with -alanine had fewer obvious side effects than injections, but all treated rats had body weights less than age-matched controls.     

Effects of Electromagnetic Fields on the Levels of Biogenic Amine Metabolites,Quinolinic Acid,and β-Endorphin in the Cerebrospinal Fluid of Dairy Cows

Eight multiparous non-lactating pregnant Holstein cows at 198 ± 35 d of gestation, weighing 608 ± 24 kg, were confined to wooden metabolic cages in an electric and magnetic field chamber with a 12:12 h light:dark cycle. Subarachnoidal catheters were installed 5 d before the activation of the electric and magnetic fields. The cows were exposed to electric and magnetic fields (60 Hz, 10 kV/m and 30 T) continuously except for the feeding and cleaning time for an average of 21.44 ± 1.4 h per day for a period of 30 d. Cerebrospinal fluid samples were collected on three consecutive days before an exposure period of 30 d, on the last 3 d of the exposure period, and for 3 d starting 5 d after the exposure period. The concentrations of -endorphin, tryptophan, 5-hydroxyindoleacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylethyleneglycol and quinolinic acid in cerebrospinal fluid were determined. There was a significant increase in quinolinic acid, and a trend towards an increase in tryptophan, findings consistent with a weakening of the blood-brain barrier due to exposure to the electric and magnetic fields.     

Effects of pH on β-hydroxybutyrate transport in rat erythrocytes and thymocytes

Entry of β-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or α-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [β-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ from 29 to 16 to 10 mM, entry $\text{V}$ being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external β-hydroxybutyrate enhanced efflux slightly. At high external pH, external β-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal β-hydroxybutyrate (βHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled βHB^?/OH^? exchange, βHB^? and OH^? competing for influx, C : OH^? moving faster than C : βHB^?, empty C being immobile. They are also compatible with coupled βHB^?/H⁺ copermeation, empty C moving inward faster than H⁺ : C : βHB^?, H⁺ : C being immobile, and C : βHB^? (without H⁺) being so unstable as not to be formed in significant amounts (relative to C, H⁺ : C, and H⁺ : C : βHB^?).     

Effects of the Bgs locus on mouse β-galactosidase

The Bgs locus determines tissue levels of β-galactosidase in the mouse, so that enzyme levels are twice as high in mice carrying the Bgs ^hallele as in mice carrying the Bgs ^dallele (Felton et al., 1974). By immunotitration with antiserum to purified β-galactosidase, we have found that the Bgs locus influences the amount of enzyme protein present in the tissues. We have utilized recombinant inbred lines derived from a cross between C57BL/6J and DBA/2J mice to confirm the location of the Bgs locus on chromosome 9. The inhibition of mouse β-galactosidase by the active-site-directed reagent N-bromoacetyl-β-d-galactosylamine has been investigated. β-Galactosidase from the high and low Bgs strains has identical affinity for this inhibitor.     

Role of β-alanine in pupal cuticle coloration in the tobacco hornworm,Manduca sexta: Effects of β-alanine analogues on melanization and catecholamine levels

Hydrazino and aminooxy derivatives of β-alanine were found to cause blackening of Manduca sexta pupal cuticle when they were injected into pharate pupae at the onset of pre-ecdysial tanning. One of these compounds, ethyl hydrazinoacetate (EHA), was used for further study. It was effective if injected up to about 4 hr before pupal ecdysis. These melanized cuticles contained excessive amounts of dopamine and decreased amounts of N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Furthermore, EHA induced elevated dopamine and lowered β-alanine levels in the hemolymph. Similar blackening occurred when 20 mg/animal dopamine was injected. Injection of excess β-alanine rescued the normal brown color, irrespective of the concentration of EHA. Also, EHA caused melanization in vitro in the presence of dopamine, whereas the addition of β-alanine and NBAD allowed normal pupal coloration in vitro. These hydrazino and aminooxy compounds likely interfere with β-alanine synthesis or mobilization and thus with N-acylation of the catecholamines to form NBAD and N-β-alanylnorepinephrine.     

Effects of nandrolone and resistance training on the blood pressure,cardiac electrophysiology,and expression of atrial β-adrenergic receptors

AimsThis study was performed to assess isolated and combined effects of nandrolone and resistance training on the blood pressure, cardiac electrophysiology, and expression of the β₁- and β₂-adrenergic receptors in the heart of rats.Main methodsWistar rats were randomly divided into four groups and submitted to a 6-week treatment with nandrolone and/or resistance training. Cardiac hypertrophy was accessed by the ratio of heart weight to the final body weight. Blood pressure was determined by a computerized tail-cuff system. Electrocardiography analyses were performed. Western blotting was used to access the protein levels of the β₁- and β₂-adrenergic receptors in the right atrium and left ventricle.Key findingsBoth resistance training and nandrolone induced cardiac hypertrophy. Nandrolone increased systolic blood pressure depending on the treatment time. Resistance training decreased systolic, diastolic and mean arterial blood pressure, as well as induced resting bradycardia. Nandrolone prolonged the QTc interval for both trained and non-trained groups when they were compared to their respective vehicle-treated one. Nandrolone increased the expression of β₁- and β₂-adrenergic receptors in the right atrium for both trained and non-trained groups when they were compared to their respective vehicle-treated one.SignificanceThis study indicated that nandrolone, associated or not with resistance training increases blood pressure depending on the treatment time, induces prolongation of the QTc interval, and increases the expression of β₁- and β₂-adrenergic receptors in the cardiac right atrium, but not in the left ventricle.     

Autolysis of glycoproteins in rat kidney lysosomes in vitro. Effects on the isoelectric focusing behaviour of glycoproteins, arylsulphatase and β-glucuronidase

1. Rat kidney lysosomal glycoproteins, prelabelled in the N-acetylneuraminic acid and polypeptide portions with N-acetyl[(3)H]mannosamine and [(14)C]lysine, or with N-acetyl-[(14)C]glucosamine, were incubated under various conditions. Autolytic cleavage of labelled N-acetylneuraminic acid and peptide was maximum at pH5.0. 2. N-Acetylneuraminic acid was released more rapidly than peptide during incubation at 37 degrees or 4 degrees C at pH5. p-Nitrophenyloxamic acid, an inhibitor of bacterial neuraminidase (Edmond et al., 1966), inhibited the cleavage of N-acetylneuraminic acid and peptide, and also inhibited cathepsin D activity. 3. Galactono-, mannono-, and glucono-lactone, inhibitors of the corresponding glycosidases, blocked the autolytic cleavage of N-acetyl[(14)C]glucosamine and protein without inhibiting beta-N-acetylhexosaminidase or cathepsin D activity. These findings suggest that the carbohydrate side chains protect the polypeptide portion of the lysosomal glycoproteins against proteolytic attack by lysosomal cathepsins. 4. In electrofocusing experiments, autolysis was minimized by adding 0.1% p-nitrophenyloxamic acid to the media used for extraction and electrofocusing, and by maintaining an alkaline pH (pH8.8-9) during extraction and dialysis. Arylsulphatase occurred in two forms with pI values of 4.4 and 6.4-6.7, and beta-glucuronidase in two forms with pI values of 4.4 and 6.1. When [(14)C]lysine and N-acetyl[(3)H]mannosamine were given to rats 1.5 and 1 h before killing, (14)C and (3)H were largely restricted to highly acidic glycoprotein species with pI values of 2.1-5.1. 5. When a lysosomal extract was adjusted to pH5 and incubated at 20 degrees C for 16h and then at 37 degrees C for 1 h before electrofocusing, 32 and 58% of the labelled peptide and N-acetylneuraminic acid was cleaved and the pI values of the labelled glycoproteins were markedly increased. About 80% of the acidic form of arylsulphatase and beta-glucuronidase was recovered with the basic form, and the pI of the basic form of both enzymes rose to 7.0. Similar, though less marked changes, were observed when a lysosomal extract was kept at pH5 for 2h at 4 degrees C before electrofocusing. 6. When an acidic lysosomal fraction (pI4.2-4.6) was incubated at pH5 for 2.5h and refocused, 80% of the arylsulphatase now occurred in two forms with pI values of 5 and 6.4. When a basic lysosomal fraction (pI5.8-6.4) was similarly incubated, the pI of arylsulphatase increased from 6.4 to 7.2. The relative increase in pI of arylsulphatases was accompanied by a proportional loss of N-acetylneuraminic acid from the glycoprotein associated with these forms. 7. These experiments show that lysosomal glycoproteins and two representative hydrolases, when exposed to a mildly acidic pH, readily undergo autolytic degradation and their pI values increase. These observations may have a bearing on the origin of the molecular heterogeneity of the lysosomal enzymes.     

Studies on Some Properties of the Apoplastic β-N-Acetyl-D-Hexosaminidase from Tomato Leaves

Some properties of the β-N-acetyl-D-hexosaminidase purified from intercellular fluid of tomato leaves after the plant was systematically infected by TMV (tobacco mosaic virus) were studied. When pNP β-D-GlcNAc (p nitrophenyl-N-aeetyl β-D-glucosaminide) or pNP β-D- GalNAc (p-nitrophenyl-N-acetyl-β-D galactosaminide) was used as the substrate, it showed the optical pH between 4. 8--5.0 and optical temperature between 44— 47℃. Studies of thermostabillty indicated that the enzyme had a biphasic denaturation curve. Using pNP-β-D-GIcNAc or pNP-β-D GalNAc as the substrate, the Km value of the enzyme was 0. 36 and 0. 67 mmol/L respectively. N acetyi-D glucosamine and N acetyl-D-galactosamine were competitive inhibitors of the enzyme activities. Ag+ and Hg2+ were sensitive inhibitors and Fe2+ . Fe3+ and Cu2+ were also inhibitors enzyme activities.     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.