Pediatric stress: hormonal mediators and human development

Stress activates the central and peripheral components of the stress system, i.e., the hypothalamic-pituitary-adrenal (HPA) axis and the arousal/sympathetic system. The principal effectors of the stress system are corticotropin-releasing hormone (CRH), arginine vasopressin, the proopiomelanocortin-derived peptides alpha-melanocyte-stimulating hormone and beta-endorphin, the glucocorticoids, and the catecholamines norepinephrine and epinephrine. Appropriate responsiveness of the stress system to stressors is a crucial prerequisite for a sense of well-being, adequate performance of tasks and positive social interactions. By contrast, inappropriate responsiveness of the stress system may impair growth and development, and may account for a number of endocrine, metabolic, autoimmune and psychiatric disorders. The development and severity of these conditions primarily depend on the genetic vulnerability of the individual, the exposure to adverse environmental factors and the timing of the stressful event(s), given that prenatal life, infancy, childhood and adolescence are critical periods characterized by increased vulnerability to stressors. The developing brain undergoes rapid growth and is characterized by high turnover of neuronal connections during the prenatal and early postnatal life. These processes and, hence, brain plasticity, slow down during childhood and puberty, and plateau in young adulthood. Hormonal actions in early life, and to a much lesser extent later, can be organizational, i.e., can have effects that last for long periods of time, often for the entire life of the individual. Hormones of the stress system and sex steroids have such effects, which influence the behavior and certain physiologic functions of individuals for life. Exposure of the developing brain to severe and/or prolonged stress may result in hyperactivity/hyperreactivity of the stress system, with resultant amygdala hyperfunction (fear reaction), decreased activity of the hippocampus (defective glucocorticoid-negative feedback, cognition), and the mesocorticolimbic dopaminergic system (dysthymia, novelty-seeking, addictive behaviors), hyperactivation of the HPA axis (hypercortisolism), suppression of reproductive, growth, thyroid and immune functions, and changes in pain perception. These changes may be accompanied by abnormal childhood, adolescent and adult behaviors, including excessive fear ('inhibited child syndrome') and addictive behaviors, dysthymia and/or depression, and gradual development of components of the metabolic syndrome X, including visceral obesity and essential hypertension. Prenatal stress exerted during the period of sexual differentiation may be accompanied by impairment of this process with behavioral and/or somatic sequelae. The vulnerability of individuals to develop varying degrees and/or components of the above life-long syndrome is defined by as yet unidentified genetic factors, which account for up to 60% of the variance. CRH has marked kindling and glucocorticoids have strong consolidating properties, hence both of these hormones are crucial in development and can alone produce the above syndrome. CRH and glucocorticoids may act in synergy, as in acoustic startle, while glucocorticoids may suppress or stimulate CRH, as in the hypothalamus and amygdala, respectively. A CRH type 1 receptor antagonist, antalarmin, inhibits both the development and expression of conditioned fear in rats, and has anxiolytic properties in monkeys. Profound stressors, such as those from sexual abuse, may elicit the syndrome in older children, adolescents and adults. Most frequently, chronic dysthymia and/or depression may develop in association with gastrointestinal complaints and/or the premenstrual tension syndrome. A lesser proportion of individuals may develop the classic posttraumatic stress disorder, which is characterized by hypocortisolism and intrusive and avoidance symptoms; in younger individuals it may present as dissociative personality disorder.     

Identification of repair enzymes for 5-formyluracil in DNA. Nth, Nei, and MutM proteins of Escherichia coli

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood. In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites. The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides. Extracts from strains defective in the nth, nei, or mutM gene lacked one of the proteins. All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant. The introduction of a plasmid carrying the nth, nei, or mutM gene into the E. coli triple mutant restored the formation of the corresponding protein-DNA complex. Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH(4). Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site. In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E. coli. The mutation frequency of the plasmid was significantly increased in the E. coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains. From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli.     

Establishment of an allergic rhinitis model in mice for the evaluation of nasal symptoms

The purpose of our study was to establish a new model of allergic rhinitis in mice, eliciting symptoms such as sneezing, infiltration of eosinophils into the nasal mucosa, and antigen-specific IgE production. One of the major human T-cell epitopes in Cry j 1, an allergen of Japanese cedar pollen, is also a major murine T-cell epitope in B10.S mice. Thus we tried to establish an allergic rhinitis model in B10.S mice with Cry j 1 as the antigen. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at intervals of 1 week. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally from the day after intranasal histamine pretreatment. Soon after, we counted the number of sneezes. We then evaluated the infiltration of eosinophils into the nasal tissues and also measured the serum levels of antigen-specific IgE antibody. In addition, we confirmed the effects of ketotifen fumarate and dexamethasone hydrochloride on these animals. In Cry j 1-sensitized B10.S mice, sneezes, eosinophil peroxidase (EPO) activity in nasal tissues, and Cry j 1-specific IgE clearly increased after intranasal histamine pretreatment and 5 days of continuous intranasal Cry j 1 challenge. Both ketotifen and dexamethasone inhibited the increase in sneezing, and dexamethasone also inhibited EPO activity and Cry j 1-specific IgE. Thus we succeeded in establishing a new model of allergic rhinitis in Cry j 1-sensitized B10.S mice, which exhibited sneezing, eosinophil infiltration into the nasal mucosa, and Cry j 1-specific IgE production.     

Enzymatic synthesis of alpha-anomer-selective D-glucosides using maltose phosphorylase

A maltose phosphorylase (EC 2.4.1.8; MPase) showed novel acceptor specificity and transferred the glucosyl moiety of maltose not only to sugars but also to various acceptors having alcoholic OH groups. Salicyl alcohol acted as acceptor for MPase from Enterococcus hirae, and the product, salicyl-O-alpha-D-glucopyranoside (alpha-SalGlc) was identified. The yield based on supplied salicyl alcohol was 86% (mol/mol).     

Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium

A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.