A model of the peripheral auditory system

Summary Recent electrophysiological data obtained from auditory nerve fibers of cats have made possible the formulation of a model of the peripheral auditory system that relates the all-or-none activity of these fibers to acoustic stimulation. The components of the model are intended to represent the major functional components of the peripheral system. These components are: (i) a linear mechanical system intended to represent the outer, middle, and mechanical parts of the inner ear; (ii) a transducer intended to represent the action of the sensory cells; and (iii) a model neuron whose properties are intended to represent the nerve excitation process. A general-purpose digital computer has been used to determine the response of the model to a variety of acoustic stimuli. These results have been compared with data obtained from auditory nerve fibers.This work was supported in part by the Joint Services Electronics Program (Contract DA 36-039-AMC-03200(E); and in part by the National Science Foundation (Grant GP-2495), the National Institutes of Health (Grant MH-04737-05), the National Aeronautics and Space Administration (Grant NsG-496); and by Research Grant NB-01344, National Institute of Neurological Diseases and Blindness of the National Institutes of Health, Public Health Service.     

Role of the renin-angiotensin system in the adaptation to high salt intake in immature rats

The response of the renin-angiotensin system, extracellular fluid volume, plasma volume, plasma sodium and mean arterial blood pressure to an increase in salt intake (8% NaCl in the diet for 10 days) was compared in immature (20 days) and adult (80 days) rats which were either sham-operated or uninephrectomised. Salt feeding induced a significant increase in plasma sodium in immature animals, and a greater suppression of the renin-angiotensin system in immature than in adult rats, although extracellular fluid volume, plasma volume and blood pressure remained unchanged. Following uninephrectomy, however, the renin-angiotensin system was maximally suppressed in both age groups and in younger animals extracellular fluid volume, plasma volume and blood pressure were significantly increased. It is concluded that (i) the renin-angiotensin system in immature rats is more responsive to a chronically increased salt intake, (ii) this greater responsiveness partly compensates for the lower natriuretic efficiency of the kidneys of immature rats, which becomes evident after reduction of renal mass, and (iii) these events bear a relation to the higher susceptibility of prepubertal rats to the hypertensive effect of a chronically increased salt intake.     

Scanning Electron Microscopy of Cell-Surface Changes in Methylnitrosurea (MNU)-Treated Rat Bladders in vivo and in vitro

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals.
There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface.
In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.     

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.     

Effect of metal ions on the adaptive response induced by N-methyl-N-nitrosourea in Escherichia coli

Induction of the adaptive response was quantified by analysis of beta-galactosidase released after the treatment of Escherichia coli CHS26/pYM3 (ada'-lacZ') with N-methyl-N-nitrosourea (MNU). Of the 15 metal ions examined, only Cd++ and Hg++ inhibited induction of the adaptive response with neither severe suppression of cell growth nor inhibition of the induction of the SOS response by MNU. Mutagenicity of MNU was potentiated by the presence of these metal ions in an E. coli strain. These results suggest that the inhibition mechanism involves a specific interaction of Cd++ or Hg++ with O6-methyl-guanine-DNA methyltransferase.     

A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes

For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.     

Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma.

We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week-old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S.     

Regulation of the bacterial cell wall: isolation and characterization of peptidoglycan mutants of Staphylococcus aureus

Temperature-sensitive mutants of Staphylococcus aureus H which require 1.0 m NaCl for growth at 42 C can be divided into two major classes. Most of the mutants (class A) do not accumulate nucleotide precursors of cell wall biosynthesis in the absence of salt at the nonpermissive temperatures, whereas the class B mutants accumulate these precursors. The most extensively studied mutant RUS 1 (carrying peg-1) is defective in biosynthesis of peptidoglycan at the nonpermissive conditions as evidenced by: (i) reduced incorporation of cell wall precursors into peptidoglycan; (ii) accumulation of the nucleotide, uridine diphosphate (UDP) muramyl-l-alanyl-d-glutamic acid; (iii) reduced specific activity of UDP N-acetylmuramyl (MurNAc)-l-alanyl-d-glutamate: l-lysine ligase (EC 6.3.2.7); and (iv) an increased susceptibility to lysis with sodium dodecyl sulfate. Addition of 1.0 m NaCl reverses these defects with the exception of the specific activity of UDP-MurNAc-l-alanyl-d-glutamate: l-lysine ligase. Nevertheless, the structure of the cell wall is normal at the nonpermissive conditions if 1.0 m NaCl is present. An alteration in the binding of a fluorescent dye, 8-anilino-1-napthalene-4-sulfonic acid at the nonpermissive conditions in the absence of 1.0 m NaCl suggests that there may also be defects in the membrane in this strain.     

Characterization of the effects of direct alkylators on in vitro immune responses

Although their mechanism of degradation may differ, both the SN1 alkylators, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-nitroso-N-methylurea (MNU), and the SN2 alkylators, dimethyl sulfate (DMS) and methyl methanesulfonate (MMS), spontaneously decompose under aqueous conditions to the methyldiazonium ion or a direct methylating intermediate, respectively. Thus, these agents serve as useful probes to investigate the immunosuppressive potential of the putative primary reactive intermediate of dimethylnitrosamine (DMN) metabolism, the methyldiazonium ion. The effects of these direct alkylating agents on the in vitro immune response were characterized. Direct addition of both the SN1 and SN2 alkylators to naive B6C3F1 murine splenocytes produced a dose-dependent suppression of the in vitro antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocytes (sRBC), T-independent antigen, dinitrophenyl (DNP)-Ficoll, and the polyclonal activator, lipopolysaccharide (LPS). The T-dependent and T-independent responses proved to be more sensitive than the polyclonal response to the effects of these compounds, except for MNNG in which all 3 antibody responses were equally affected. The suppression of the AFC response for all antigens was unaffected by the addition of 2-ME, and was observed at concentrations below those affecting viability, although at the highest concentrations an effect on viability was often observed. The addition of MNNG to the T-dependent AFC response at any time within the first 96 h produced a marked suppression, while the addition of DMS to cultures was only effective in suppressing the AFC response if added within the first 24 h. MNNG and DMS suppressed the proliferative responses to both B-cell (LPS) and T-cell (Concanavalin A; Con A) mitogens, as well as in the mixed lymphocyte response (MLR). In addition, a positive correlation between immunosuppression and DNA damage, as measured by single-strand breaks, was observed. Although these compounds produced suppression of in vitro immune responses, their profile of activity on immunocompetence and DNA damage was different from that associated with DMN and thus, the direct alkylators may not prove to be useful models to elucidate the mechanism of the DMN-induced immunosuppression.     

Effect of inhibitors of oxygen radical and nitric oxide formation on UV radiation-induced erythema, immunosuppression and carcinogenesis

We investigated whether supplementation of a sunscreen containing the UVB absorber 2-ethyl-hexyl-methoxycinnamate (cinnamate) with oxygen radical inhibitors (ORI) would improve protection from sunburn, immunosuppression and carcinogenesis. Mice were exposed to solar-simulated UV radiation (ssUV) containing a mixture of UVB and UVA. In initial studies, the ORI 2,2'-dipyridyl and N(G)-monomethyl-L-arginine acetate (L-NMMA) were shown to prevent UVA-induced suppression of contact sensitivity (CS) in mice. Addition of these inhibitors to the sunscreen did not affect the sun protection factor (SPF), but lowered the level of edema when mice were exposed to ssUV. Combination of both inhibitors with the sunscreen, however, increased the SPF from 5 to 5.5. The immune protection factor (IPF) of the sunscreen was only 1.18, but addition of neither dipyridyl nor L-NMMA singly or in combination measurably improved immune protection. However, the ORI improved the ability of the sunscreen to prevent carcinogenesis. The results indicate that reactive oxygen or nitrogen species produced in response to UV radiation are important for erythema, immunosuppression and carcinogenesis, and addition of inhibitors improves the protective capacity of sunscreens.     

The effect of 3,4-disuccinyldianhydrogalactitol, N-nitrosourea and their combination on DNA synthesis in normal and mouse P388 tumor cells

The rates of incorporation of 2-14C-thymidine into DNA of leukemia P388, bone marrow, gastrointestinal mucosa and spleen cells at various time after administration of 3,4-disuccinyldianhydrogalactitol (DisuDAG), 1-methyl-1-nitrosourea (MNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea (HECNU) and their combinations at different doses to mice with leukemia P388 (solid form) were studied. DisuDAG (80 mg/kg) induced the deep and the stable inhibition in DNA synthesis of leukemia P388, bone marrow and spleen cells. The combination of DisuDAG and HECNU at small doses induced the deep and the stable suppression of DNA synthesis in tumor cells, however DNA synthesis in normal dividing cells was shown to recover more rapidly than in leukemia P388 cells. Administration of the combination of DisuDAG with MNU to tumor-bearing mice induced more stable inhibition of DNA synthesis in tumor cells in comparison with MNU and DisuDAG. In vivo inhibition of DNA synthesis in leukemia P388 cells with DisuDAG and HECNU was not due to damage in pool of precursors (TCA soluble fraction).     

Effects of a glucokinase activator on hepatic intermediary metabolism: study with 13C-isotopomer-based metabolomics

GKAs (glucokinase activators) are promising agents for the therapy of Type 2 diabetes, but little is known about their effects on hepatic intermediary metabolism. We monitored the fate of (13)C-labelled glucose in both a liver perfusion system and isolated hepatocytes. MS and NMR spectroscopy were deployed to measure isotopic enrichment. The results demonstrate that the stimulation of glycolysis by GKA led to numerous changes in hepatic metabolism: (i) augmented flux through the TCA (tricarboxylic acid) cycle, as evidenced by greater incorporation of (13)C into the cycle (anaplerosis) and increased generation of (13)C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) lowering of hepatic [Pi] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) stimulation of glycogen synthesis from glucose, but inhibition of glycogen synthesis from 3-carbon precursors; (iv) increased synthesis of N-acetylglutamate and consequently augmented ureagenesis; (v) increased synthesis of glutamine, alanine, serine and glycine; and (vi) increased production and outflow of lactate. The present study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis, but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.     

Effect of inhibitors of oxygen radical and nitric oxide formation on UV radiation-induced erythema,immunosuppression and carcinogenesis

Abstract

We investigated whether supplementation of a sunscreen containing the UVB absorber 2-ethyl-hexyl-methoxycinnamate (cinnamate) with oxygen radical inhibitors (ORI) would improve protection from sunburn, immunosuppression and carcinogenesis. Mice were exposed to solar-simulated UV radiation (ssUV) containing a mixture of UVB and UVA. In initial studies, the ORI 2,2′-dipyridyl and N^G-monomethyl-L-arginine acetate (L-NMMA) were shown to prevent UVA-induced suppression of contact sensitivity (CS) in mice. Addition of these inhibitors to the sunscreen did not affect the sun protection factor (SPF), but lowered the level of edema when mice were exposed to ssUV. Combination of both inhibitors with the sunscreen, however, increased the SPF from 5 to 5.5. The immune protection factor (IPF) of the sunscreen was only 1.18, but addition of neither dipyridyl nor L-NMMA singly or in combination measurably improved immune protection. However, the ORI improved the ability of the sunscreen to prevent carcinogenesis. The results indicate that reactive oxygen or nitrogen species produced in response to UV radiation are important for erythema, immunosuppression and carcinogenesis, and addition of inhibitors improves the protective capacity of sunscreens.     

Bidirectional regulation of mitochondrial gene expression during developmental neuroplasticity of visual cortex

The first several months of life are a critical period for neuronal plasticity in the visual cortex, during which anatomical and physiological development depends upon visual experience. Rearing in darkness slows the time course of this critical period, such that at 5 weeks normal cats are more plastic than dark-reared cats, while at 20 weeks dark-reared cats are more plastic. This study reports the identification of a subset of mitochondrial genes that are regulated in this manner. Opponent patterns of bidirectional expression were found: several genes (ATPase 6, cytochrome b, NADH dehydrogenase subunits 4 and 2) showed elevation in normal cats at 5 weeks and in dark-reared cats at 20 weeks ("plasticity" genes); others (NADH dehydrogenase subunits 3 and 5) showed the opposite ("anti-plasticity" genes). These findings add a new dimension to the growing evidence that changes in mitochondrial gene expression are involved in the neuroplastic response.     

Cardiovascular system of normal aged subjects. Cardiovascular senescence

The cardiovascular system of the elderly is characterised by (i) an increased characteristic impedance of the big vessels, without hypertension; (ii) a cardiac index unchanged at rest and during submaximal exercising, despite a diminished VO2 max; (iii) a diastolic dysfunction with reduced rapid early filling (E wave at echo) and enhanced atrial systole (A wave); (iv) benign arrhythmias; (v) a reduced coronary reserve and an increased sensitivity to ischemia; (vi) an efficacy of converting enzyme inhibitors remaining unchanged, despite an altered renin-agiotensin system.     

Ontogeny of the immune system inSebastiscus marmoratus: Histogenesis of the lymphoid organs and effects of thymectomy

Synopsis The ontogenetic development of the immune system in a marine teleostSebastiscus marmoratus was studied by histological examination and removal of the thymus. The pronephros and the spleen had been differentiated at the time of birth and contained small numbers of haemopoietic cells. In contrast to most vertebrates, the rudiments of the thymus were first visible 1 week post-birth in the dorsoposterior part of the pharynx, the same location as in the adults. However, small lymphocytes first appeared in the thymus of fish at 3 weeks of age, followed by the pronephros at 4 weeks and the spleen at 6 weeks. Complete or partial suppression of the antibody response to sheep red blood cells (SRBC) occurred in fish that were thymectomized at 1.5 months of age and immunized 2 weeks later, and a marked decrease in lymphocytes was observed in the pronephros and spleen. The thymectomy of adult fish also caused reduced serum antibody titres in fish immunized 1 month after the operation. These results suggest that the thymus plays an essential role in the development of the immune system and its functions continue into adult life.     

Immunotoxicity of phosphamidon following subchronic exposure in albino rats

Effect of subchronic doses of phosphamidon exposure on humoral and cell mediated immune (CMI) responses were studied in male albino rats using SRBC, ovalbumin and KLH as antigens. Humoral immune responses were assessed by estimating antibody titre against antigen and splenic plaque forming cells (PFC) assay. CMI responses were studied by using leucocyte migration inhibition (LMI), macrophage migration inhibition (MMI) and delayed type hypersensitivity (DTH) response. Results obtained in the present study revealed marked suppression of humoral and CMI responses in a dose dependent pattern. Hence, suppression of immune responses by phosphamidon even at subchronic doses is clearly an important aspect for its safety evaluation.     

Neonatal exposure to UVR alters skin immune system development, and suppresses immunity in adulthood

Neonates have a developing immune response, with a predisposition towards induction of tolerance. As the immune system develops, immunity rather than tolerance is induced, with this development of immunity occurring in response to external factors such as the environment. As ultraviolet radiation (UVR) suppresses immunity, it is likely that the effect of UVR on the neonatal immune system would be augmentation of the suppressive response. In support, childhood exposure to UVR has been linked with an increased incidence of melanoma; consistent with an increase in suppression. To address this, phenotypic and functional immune system studies were undertaken at 8 weeks after one single exposure of solar-simulated UVR to mice, when mice had reached adulthood. Subtle changes were observed in cell populations resident in the skin-draining lymph nodes (LNs) and there also appeared to be a subtle, but not statistically significant, increase in the production of interleukin-10 and interferon-γ. Importantly, these changes also corresponded with significant suppression of the contact hypersensitivity response in irradiated mice compared with their control counterparts. This suppression was apparent when antigen sensitisation occurred during the neonatal or adult period, and thus did not appear to be analogous to UVR-induced suppression in adults. Although the percentage of T regulatory cells was increased in the skin-draining LNs, they were induced in a different manner to those induced following adult UVR exposure, with no increase in function on a per-cell basis. It therefore appears that one single neonatal exposure to UVR alters development of the immune system, leading to long-term implications for induction of immunity.