Transient Receptor Potential (TRP) and Cch1-Yam8 Channels Play Key Roles in the Regulation of Cytoplasmic Ca2+ in Fission Yeast

The regulation of cytoplasmic Ca2+ is crucial for various cellular processes. Here, we examined the cytoplasmic Ca2+ levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca2+ level and its change caused by extracellular stimulants such as CaCl2 or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca2+ caused a dose-dependent increase in the cytoplasmic Ca2+ level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca2+ peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca2+ increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca2+ increase is abolished by the addition of the Ca2+ chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca2+ channel complex, indicating that the synergistic increase is caused by the Ca2+ influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca2+ influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state

Augmented water binding and low cellular water content in erythrocytes of camel and camelids.

We investigated a link between hemoglobin primary structure, hemoglobin hydrophobicity-hydrophilicity, and erythrocyte water content in various mammalian species. Some hemoglobin molecules, particularly those of the camel and camelids, contain more charged amino acid residues and are more hydrophilic than the hemoglobins of human and a number of other mammalian species. To test the in vivo significance of these alterations of hemoglobin primary structure, we determined the osmotically unresponsive erythrocyte water fractions in mannit solutions of various osmolarities at 4 degreesC. Among the species investigated, the size of the osmotically unresponsive erythrocyte water fraction relates in a positive linear way to hemoglobin hydrophilicity. The extreme low total erythrocyte water content of camel erythrocytes (1.1-1.3 g water/g dry mass) may be explained by a comparatively high osmotically unresponsive erythrocyte water fraction. It is proposed that alterations of hemoglobin sequences of camel and camelids may be the part of a natural selection process aimed at protecting these animals against osmotic dehydration in arid environments

Effect of non-lytic concentrations of Brij series detergents on the metabolism-independent ion permeability properties of human erythrocytes.

Subcritical micellar concentrations (sub-CMC) of Brij-series detergents alter ion movements between human erythrocytes and their environment when metabolism has been slowed down by incubation at zero degrees centigrade. The effect of nonhemolytic concentrations of detergents on the erythrocyte K+ and Na+ movements is described. Results indicate a significant difference in monovalent cation movements, depending on the number of hydrophilic polyoxyethylene units (n). There is an increasing loss of K+ and gain of Na+ as n increases from 4 to 20. Where n > or = 21, ion movements are not significantly different from those found in erythrocytes not exposed to detergents. The carbon chain length of the detergent fatty acid residue (10-18 carbons) appears to be relatively unimportant, but detergents with unsaturated (oleic acid) hydrophobic regions potentiate K+ release and Na+ uptake when compared to the corresponding saturated fatty acid (stearic acid). The erythrocyte stabilizing effect of detergents against hypo-osmotic shock correlates well with the increase of monovalent ion traffic and the mobility of membrane lipids revealed by fluorescence anisotropy measurements

Co-infections with Hepatitis G and TT Virus in Patients with Chronic Hepatitis C in Hungary

The significance of co-infections with novel hepatitis viruses Hepatitis G (GBV-C, HGV) and TT virus (TTV) in chronic hepatitis C is not clear. We determined the prevalence of HGV RNA and TTV DNA in chronic hepatitis C patients and in asymptomatic hepatitis C virus (HCV) carriers, and assessed the influence of these agents on the course of HCV infection.  Seventy-seven patients with chronic hepatitis C - 50 of them treated with interferon (IFN) - and 33 HCV carriers with normal alanine aminotransferase have been investigated. Previous HBV infection was detected by testing serum HBsAg and aHBc. HGV RNA and TTV DNA were detected by PCR. In the healthy population, the prevalence of anti-HCV was 0.3%, HGV RNA 8.0% and TTV DNA 18.5%. In chronic hepatitis C HGV RNA occurred in 9.09% and TTV DNA in 40.25% of cases. In IFN-treated patients with sustained remission, the frequency of TTV was 20% vs. 45.7% found in non-responders. Among asymptomatic HCV-carriers, the prevalence of HGV RNA was 9.09% and TTV DNA 75.7%. Neither HGV RNA nor TTV DNA had apparent effect on the HCV infection. TTV was detected with the lowest frequency in persons with sustained remission due to IFN, suggesting antiviral effect of IFN on TTV

Insulin Therapy of Nondiabetic Septic Patients Is Predicted by para

Hydroxyl radical converts Phe to para-, meta-, and ortho-Tyr (p-Tyr, m-Tyr, o-Tyr), while Phe is converted enzymatically to p-Tyr in the kidney and could serve as substrate for gluconeogenesis. Pathological isoforms m- and o-Tyr are supposed to be involved in development of hormone resistances. Role of Phe and the three Tyr isoforms in influencing insulin need was examined in 25 nondiabetic septic patients. Daily insulin dose (DID) and insulin-glucose product (IGP) were calculated. Serum and urinary levels of Phe and Tyr isoforms were determined using a rpHPLC-method. Urinary m-Tyr/p-Tyr ratio was higher in patients with DID and IGP over median compared to those below median (P=0.005 and P=0.01, resp.). Urinary m-Tyr and m-Tyr/p-Tyr ratio showed positive correlation with DID (P=0.009 and P=0.023, resp.) and with IGP (P=0.004 and P=0.008, resp.). Serum Phe was a negative predictor, while serum p-Tyr/Phe ratio was positive predictor of both DID and IGP. Urinary m-Tyr and urinary m-Tyr/p-Tyr, o-Tyr/p-Tyr, and (m-Tyr+o-Tyr)/p-Tyr ratios were positive predictors of both DID and IGP. Phe and Tyr isoforms have a predictive role in carbohydrate metabolism of nondiabetic septic patients. Phe may serve as substrate for renal gluconeogenesis via enzymatically produced p-Tyr, while hydroxyl radical derived Phe products may interfere with insulin action

Breast Milk for Term and Preterm Infants—Own Mother’s Milk or Donor Milk?

Hormones are important biological regulators, controlling development and physiological processes throughout life. We investigated pituitary hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL) and total protein levels during the first 6 months of lactation. Breast milk samples were collected every fourth week of lactation from mothers who gave birth to preterm (n = 14) or term (n = 16) infants. Donor milk is suggested when own mother’s milk is not available; therefore, we collected breast milk samples before and after Holder pasteurization (HoP) from the Breast Milk Collection Center of Pécs, Hungary. Three infant formulas prepared in the Neonatal Intensive Care Unit of the University of Pécs were tested at three different time points. Our aim was to examine the hormone content of own mother’s milk and donor milk. There were no significant changes over time in the concentrations of any hormone. Preterm milk had higher PRL (28.2 ± 2.5 vs. 19.3 ± 2.3 ng/mL) and LH (36.3 ± 8.8 vs. 15.9 ± 4.1 mIU/L) concentrations than term milk during the first 6 months of lactation. Total protein and FSH concentrations did not differ between preterm and term breast milk. Holder pasteurization decreased the PRL concentration (30.4 ± 1.8 vs. 14.4 ± 0.6 ng/mL) and did not affect gonadotropin levels of donor milk. Infant formulas have higher total protein content than breast milk but do not contain detectable levels of pituitary hormones. Differences were detected in the content of pituitary hormones produced for preterm and term infants. Divergence between feeding options offers opportunities for improvement of nutritional guidelines for both hospital and home feeding practices