Functional interaction of auxiliary subunits and synaptic proteins with Ca(v)1.3 may impart hair cell Ca2+ current properties

We assessed the functional determinants of the properties of L-type Ca(2+) currents in hair cells by co-expressing the pore-forming Ca(V)1.3alpha(1) subunit with the auxiliary subunits beta(1A) and/or alpha(2delta). Because Ca(2+) channels in hair cells are poised to interact with synaptic proteins, we also co-expressed the Ca(V)1.3alpha(1) subunit with syntaxin, vesicle-associated membrane protein (VAMP), and synaptosome associated protein of 25 kDa (SNAP25). Expression of the Ca(V)1.3alpha(1) subunit in human embryonic kidney cells (HEK 293) produced a dihydropyridine (DHP)-sensitive Ca(2+) current (peak current density -2.0 +/- 0.2 pA/pF; n = 11). Co-expression with beta(1A) and alpha(2delta) subunits enhanced the magnitude of the current (peak current density: Ca(V)1.3alpha(1) + beta(1A) = -4.3 +/- 0.8 pA/pF, n = 10; Ca(V)1.3alpha(1) + beta(1A) + alpha(2delta) = -4.1 +/- 0.6 pA/pF, n = 9) and produced a leftward shift of approximately 9 mV in the voltage-dependent activation of the currents. Furthermore, co-expression of Ca(V)1.3alpha(1) with syntaxin/VAMP/SNAP resulted in at least a twofold increase in the peak current density (-4.7 +/- 0.2 pA/pF; n = 11) and reduced the extent of inactivation of the Ca(2+) currents. Botulinum toxin, an inhibitor of syntaxin, accelerated the inactivation profile of Ca(2+) currents in hair cells. Immunocytochemical data also indicated that the Ca(2+) channels and syntaxin are co-localized in hair cells, suggesting there is functional interaction of the Ca(V)1.3alpha(1) with auxiliary subunits and synaptic proteins, that may contribute to the distinct properties of the DHP-sensitive channels in hair cells.     

Sodium currents in mammalian muscle

1. A method is described which allows the approximate computation of membrane current from measurements with three electrodes in the mid-region of a muscle fibre.     

Determination of unsaturated end groups in low molecular weight poly(vinyl chloride) by 1H NMR spectroscopy of the hydroxyphenyl substituted product

Poly(vinyl chloride) of low molecular weight (the fraction extracted with a hexane/acetone mixture (vol.-ratio 3:2)) was substituted by hydroxyphenyl groups and then analyzed by ¹H NMR spectroscopy. It was established that the ¹H NMR spectra of hydroxyphenyl substituted poly(vinyl chloride) prepared by removal of HCl, allows to determine the number of unsaturated end groups and to distinguish between the structures ? CHCl? CH₂? CH?CH? CH₂Cl and ? CH₂? CHCl? CH?CH? CH₂Cl, and even between their trans and cis forms. In addition the ¹H NMR spectra of hydroxyphenyl substituted poly(vinyl chloride) prepared without removal of HCl allow to determine the total number of unsaturated end groups.     

γδ cells regulate autoimmunity

γδ cells are attractive candidates for mediators of autoimmune disease. They can expand in germ-free mice, probably through recognition of autoantigens, and γδ-cell-deficient mice, unlike mice deficient in αβ T cells or B cells, show no severe defects in the immune response to foreign antigen challenge. A capacity of γδ cells to effect or regulate tissue damage is also plausible, given their ready localization to tissues, and their myriad of effector functions. Added to this, attempts to reconstruct the physiological course of autoimmune diseases with only autoreactive αβ T cells seem invariably to fall short for lack of other unidentified players. γδ cells and their putative ligands have been linked to autoimmune conditions, and recent experiments confirm that γδ cells play a significant role in autoimmune disease in vivo.     

Infection of the reproductive tract and eggs with Salmonella enterica serovar pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity

Salmonella enterica serovar Pullorum causes persistent infections in laying hens. Splenic macrophages are the main site of persistence. At sexual maturity, numbers of bacteria increase and spread to the reproductive tract, which may result in vertical transmission to eggs or chicks. In this study we demonstrate that both male and female chickens may develop a carrier state following infection but that the increases in bacterial numbers and spread to the reproductive tract are phenomena restricted to hens, indicating that such changes are likely to be related to the onset of egg laying. The immunological responses during the carrier state and through the onset of laying in hens were determined. These indicate that chickens produce both humoral and T-cell responses to infection, but at the onset of laying both the T-cell response to Salmonella and nonspecific responses to mitogenic stimulation fall sharply in both infected and noninfected birds. The fall in T-cell responsiveness coincided with the increase in numbers of Salmonella serovar Pullorum and its spread to the reproductive tract. Three weeks after the onset of egg laying, T-cell responsiveness began to increase and bacterial numbers declined. Specific antibody levels changed little at the onset of laying but increased following the rise in bacterial numbers in a manner reminiscent of a secondary antibody response to rechallenge. These findings indicate that a nonspecific suppression of cellular responses occurs at the onset of laying and plays a major role the ability of Salmonella serovar Pullorum to infect the reproductive tract, leading to transmission to eggs. The loss of T-cell activity at the point of laying also has implications for Salmonella enterica serovar Enteritidis infection and transmission to eggs, along with its control by vaccination offering a "window of opportunity" in which infection may occur.     

The calcium ion dependence of scallop myosin ATPase activity

Summary The ATPase activity of scallop (Pecten maximus) striated adductor myosin and heavy meromyosin (HMM) have been investigated as a function of [Ca²⁺] using formycin triphosphate (FTP) as a fluorescent ATP analogue. The FTPase activity of the regulated fraction of these preparations was activated steeply over the range of 0.1 to 1M [Ca²⁺ ], implying the existence of a form of cooperativity that is intrinsic to the myosin heads. In addition to the previously characterised heterogeneity with respect to an unregulated fraction, the regulated fraction of HMM was resolved into two populations whose activities showed a slightly different dependency on [Ca²⁺ ]. This was revealed unambiguously at intermediate levels of activation where, in some experiments, the product release rate constants differed for the two populations by more than fivefold. At maximum relaxation or maximum activation, these rate constants differed by two-to three-fold and were not clearly resolved by the multiexponential fitting procedure. The populations might arise as a consequence of isoenzymes, modification during preparation or slowly interconverting conformers; Ca²⁺ binding itself being a rapid equilibrium process in both populations. FTP turnover by myosin could not be analysed in such detail because of the technical problems of measuring the fluorescence of a suspension of filaments, but the rates of the elementary steps appeared similar to those of HMM. The fraction of unregulated molecules in myosin preparations was comparable to that of HMM indicating that if it is a consequence of preparative damage, the modification must occur prior to tryptic digestion.     

A novel mitochondria-targeting method using special staining for the detection of apoptotic hepatocytes

ABSTRACT

Early detection of apoptotic cells on histological slides is of major importance for both diagnostic and research areas. In the current study, the aim was to propose a convenient method to stain the mitochondria and establish whether hepatocytes undergoing apoptosis can be identified in tissue sections using the proposed method. Liver tissue from five adult chinchillas was fixed with 10% neutral buffered formalin for Goldner’s trichrome (GT) and Groat’s iron hematoxylin and eosin (HE) stains and with Kolster’s fixative for the Heidenhain’s iron hematoxylin procedure. The HE and GT-stained sections showed the morphological features consistent with apoptosis i.e., homogenous intensely acidophilic cytoplasm, cell shrinkage with an irregular outline, nuclear shrinkage with cloudy karyoplasm, and karyopyknosis in the late stage. Sections stained with Heidenhain’s iron hematoxylin method was used to pinpoint mitochondria and revealed cells which were undergoing the first stages of the apoptosis process i.e., disappearance of mitochondria from the cell, chromatin condensation and margination, paracentral localization of nucleoli, and vacuolated nuclei. In more advanced stages of apoptosis, cells presented significant nuclear and cytoplasmic changes. It was concluded that this is the first report targeting the mitochondria, by performing inexpensive histological staining techniques, in order to assess dead cells in situ.     

Detection of Haemophilus influenzae type b by real-time PCR

A real-time PCR assay targeting the capsulation locus of Haemophilus influenzae type b (Hib) was developed. The linear detection range was from 1 to 10(6) microorganisms per reaction mixture. No H. influenzae other than Hib or any other control bacteria typically found in the upper respiratory tract was detected.     

The interface between tapasin and MHC class I: identification of amino acid residues in both proteins that influence their interaction

Prior to the binding of antigenic peptide, a complex of chaperone proteins associates with the Major Histocompatibility Complex (MHC) class I heavy chain/β₂m heterodimer. Although each dornain of the MHC class I heavy chain contains amino acid resid uses that influence chaperone binding, there are several pieces of evidence that point to an interaction between the MHC clas 1α2/α3 domains and tapasin. In egard to the site on tapasin involved in the tapasin/MHC interface, we have found that a particular region of tapasin (containing amino acid residues 334–342) is necessary for the binding of tapasin to the MHC class I heavy chain. Our results also indicate that amino acids in this region of tapasin also affect the proportion of MHC class I open forms expressed at the cell surface and MHC class I egress from the endoplasmic reticulurn. Based on these results and those obtained by other laboratories, a model for MHC class I/tapasin interaction is proposed.