Brief Communication: Role of Nuclear Background and in vivo Environment in Variable Segregation Behavior of the Aging-Dependent T414G Mutation at Critical Control Site for Human Fibroblast mtDNA Replication

Previous work had shown a large accumulation (up to 50% of mtDNA) of a noninherited T414G transversion at a critical control site for mtDNA replication in skin fibroblasts from the majority of human subjects above 65 years old, and its absence in younger individuals. In the present studies, long-term in vitro culture of several fibroblasts populations carrying the heteroplasmic T414G mutation revealed an outgrowth of the mutant cells by wild-type cells. This observation supported the previous conclusion that the mutation accumulation is an in vivo phenomenon, while, at the same time, indicating intrinsic physiological differences between mutant and wild-type cells. Furthermore, subcloning experiments revealed a striking mosaic distribution of the mutation in the original fibroblasts populations, as shown by its presence, in heteroplasmic or homoplasmic form, in a fraction (18–32%) of the fibroblasts, and its absence in the others. In other investigations, transfer of mitochondria from mutation-carrying fibroblasts into mtDNA-less 143B.TK^–0 206 cells revealed the persistence of the mosaic distribution of the mutation, however, with a near-complete shift to homoplasmy. The generality of the latter phenomenon would exclude a founder effect by one or few mitochondria in the transformation experiments, and would rather point to the important role of the nuclear background in the in vitro behavior of the T414G mutation. The stability of the homoplasmic mutation in ⁰ cell transformants provides a powerful tool for analyzing its biochemical effects.     

Study on the mechanism of the appearance of noise effects

Summary This study was undertaken to investigate the dose-response relationship between the biological effect and noise exposure, and to consider the mechanism of the appearance of noise effects. Rats were exposed to noise at intensities of 60 dB (A), 80 dB (A) and 100 dB (A) for 240 min and examined for the change of activities of dopamine--hydroxylase (DBH) in serum and adrenal glands. Plasma cyclic adenosine 3,5-monophosphate (c-AMP) levels were also measured. Some rats were given 6-hydroxydopamine (6-OHDA) as a chemical sympathectomyzing agent 20 h before noise exposure in order to consider the mechanism of the appearance of noise effects. By noise exposure, serum DBH activity was significantly (P<0.01) increased at each intensity compared with the control group, but there were no remarkable changes in adrenal DBH activity. Plasma c-AMP level was also significantly elevated in response to the noise stress. When the rats, which had been pretreated with 6-OHDA, were exposed to noise with an intensity of 100 dB (A), the response of serum DBH activity was no longer observed. Therefore it is suggested that the effect due to noise exposure appears through the post-ganglionic sympathetic nerve fiber.     

Changes in glutamate-related enzyme activities in the striatum of the rat following lesion of corticostriatal fibres

Summary The behaviour of enzymes putatively involved in glutamate/aspartate transmitter metabolism (glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase,-glutamyltranspeptidase) was studied in the striatum 3, 7, 14 days and 7 weeks after mechanical destruction of corticostriatal fibres. For a period of up to seven days after unilateral lesion, enzyme activities were significantly diminished (by up to 13% based on protein) in the ipsilateral striatum as compared to the striatum of the intact side. Later, the enzyme activities in the ipsilateral striatum recovered. After seven weeks, an increase was observed for glutamate dehydrogenase activity, whereas the activity of alanine aminotransferase showed a transient rise at the end of the second week. The decrease in enzyme levels is interpreted as being attributable to the destruction of nerve endings which are considered to be glutamatergic, interfering with various compensating processes (e.g. glial cell proliferation) which occur with advancing times after lesion.     

The appearance and role of gamma delta T cells in the peritoneal cavity and liver during primary infection with Listeria monocytogenes in rats.

We have previously reported that gamma delta T cells play important roles in protection during the early stage of infection with Listeria monocytogenes in mice. To generalize the protective roles of gamma delta T cells in listerial infection to different species, we examined the appearance of gamma delta T cells during infection with L. monocytogenes in Fisher F344 rats. The numbers of bacteria in the peritoneal cavity and liver increased to a maximum level on day 3 and then decreased to an undetectable level by day 10 after an intraperitoneal infection with a sublethal dose (1 x 10(8)) of viable L. monocytogenes in rats. CD3+ alpha beta- T cells in the peritoneal cavity and liver began to increase on day 3, reached a maximum level on day 6, and thereafter decreased gradually by day 10 after infection. Northern blot analysis confirmed that the CD3+ alpha beta- T cells expressed TCR delta and gamma gene messages. In vivo treatment with anti-TCR alpha beta mAb, which suppressed most of the alpha beta T cells in the periphery and impaired resistance during the late stage of listerial infection, did not affect the host defense by day 6 after infection. A significantly increased number of gamma delta T cells was detected in the peritoneal cavity of the TCR alpha beta-suppressed rats on day 6 after infection. These results suggest that the early appearing gamma delta T cells may contribute to the host defense at a relatively early stage during listeriosis in rats.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Correlation between soluble serum CD16 (sCD16) levels and disease stage in patients with multiple myeloma

CD16, the type III receptor for IgG, is expressed on neutrophils, natural killer cells, and some T lymphocytes, mast cells, and activated monocytes but not on cells of the B-lymphocyte lineage including plasma cells. It is also produced in a soluble form found in serum. We analyzed sera from 165 multiple-myeloma patients, 29 patients with monoclonal gammopathies of unknown significance, and 20 normal disease-free donors. We found that the level of soluble CD16 was significantly decreased in sera from patients with multiple myeloma compared to sera from healthy and monoclonal gammopathies of unknown significance donors (P=0.0001). In addition, a stage-dependent decrease in soluble CD16 was observed, with a highly significant difference (P=0.004) between stage I and stage II+III myeloma patients. The correlation between the myeloma stage and the serum level of soluble CD16, which is related to the host response, was found to be more sensitive than that of ₂-microglobulin, which reflects the tumor burden. The concomitant evaluation of the serum levels of these two markers allows better staging and therefore has a more precise prognostic value.     

The control of chloride conductance in rat parotid isolated acinar cells investigated by photorelease of caged compounds

The control of Cl^– conductance in rat parotid isolated acinar cells was studied by combined use of whole-cell recording and flash photolysis techniques. Cells were voltage-clamped either at a membrane potential of –40 mV or stepped between –85 mV and 0 mV. Bath-applied carbachol and noradrenaline evoked Cl^– current at –85 mV and K⁺ current at 0 mV. Similar current activations resulted from the photolytic release of either inositol trisphosphate (InsP ₃) or Ca²⁺ by a brief near-UV flash. The peak amplitudes of the Cl^– conductance (at –85 mV), measured relative to the K⁺ conductance (at 0 mV), evoked by application of carbachol, noradrenaline or direct manipulation of cytosolic free calcium ([Ca²⁺]_i), were very similar, being 0.56±0.09 (mean±SEM,n=9), 0.52 ± 0.01 (n=7) and 0.46±0.06 (n=7). In contrast, the relative amplitude of the Cl^– conductance evoked by InsP₃ was much larger: 1.49±0.24 (n=9). Neither bath application of isoprenaline nor photolysis of caged cAMP induced any detectable membrane current. The most probable interpretation of these results is that the observed activation of Cl^– conductance by agonists can be explained by the elevation of [Ca²⁺]_i alone. In addition, the present results provide further support for the previously reported suggestion that the Cl^– channels and the Ca²⁺-release sites are co-localised [10].     

Astrocytes and intracerebral immune responses

The astrocyte is the most abundant cell within the central nervous system (CNS). This cell subserves a multiplicity of important functions that contribute to the process of neural development as well as to the integrity of normal brain function. Adding to the already exhaustive list of capabilities, the astrocyte has now been demonstrated to function as an intracerebral antigen presenting cell. These findings are serving to revise our view of the brain as an immunoprivileged site and perhaps will shed some light on the pathogenetic mechanisms involved in a number of CNS disorders of immune dysregulation. In this review we provide some perspective on the regulatory mechanisms that influence astrocyte immune functions. Specifically, we address the role played by the major histocompatibility complex (MHC) antigens as well as adhesion molecules in the initiation of brain immune responses.