Superoxide production contributes to osteoclastic bone resorption. Evidence strongly indicates that NADPH oxidase is an enzyme system responsible for superoxide generation in osteoclasts. A membranebound subunit, p91, is the catalytic domain of NADPH oxidase. However, osteoclasts from p91 knockout mice still produce superoxide at a rate similar to that observed in wild type mice. This unexpected phenomenon prompted us to examine the osteoclasts for an alternative to the p91-containing oxidase. In this study, the cloning of a NADPH oxidase subunit (Nox 4) with 578 amino acids is reported. Nox 4 has 58% similarity in amino acids with the known p91 subunit of NADPH oxidase. Nox 4 is present and active in osteoclasts. Antisense oligonucleotides of Nox 4 reduced osteoclastic superoxide generation as well as resorption pit formation by osteoclasts. This new oxidase complex was present and functional in osteoclasts from p91 knockout mice, explaining the normal resorptive activity seen in the osteoclasts where no p91 is present.

Mutagens, distinguishable from benzo[a]pyrene and from mutagenic amino acid and protein pyrolysis products, are formed when ground beef is cooked in a home hamburger cooking appliance or when beef stock is concentrated, by boiling, to a paste known commercially as beef extract. "Well-done" hamburgers contain about 0.14 part per million of the mutagens, and beef bouillon cubes which contain beef extract about 0.1 part per million. Since such mutagens may be potentially carcionogenic and are formed during ordinary cooking procedures, their occurrence raises questions about possible risks to human health.

Interferon-gamma (IFN-gamma) treatment increases osteoclastic bone resorption in vivo in patients with malignant osteopetrosis (OP). The treatment effect was studied in vitro in osteoclasts generated by culturing peripheral white blood cells (PWBC) from OP patients and normal human control subjects. Osteoclasts were treated with or without IFN-gamma prior to the end of the culture period. Osteoclasts from normal subjects were large in size (161 +/- 18 microm in diameter) with >10 nuclei per osteoclast. These cells showed intense staining for tartrate-resistant acid phosphatase (TRAP), expressed abundant calcitonin receptors (CTR), and formed numerous resorption pits on bovine bone slices, indicative of authentic osteoclasts. In contrast, similarly cultured osteoclasts from OP patients were smaller in size (18 +/- 3 microm in diameter), with 2-3 nuclei per osteoclast, and stained lightly for TRAP. However, IFN-gamma treatment of osteoclasts from OP patients resulted in the formation of larger osteoclasts (171 +/- 33 microm in diameter) with >10 nuclei per cell, similar in appearance to osteoclasts from normal subjects. IFN-gamma stimulation increased the intensity of TRAP staining (p < 0.0001) to levels near that of the normal osteoclasts. Unstimulated osteoclasts from 6 OP patients had a significantly lower baseline level of superoxide production, as measured by nitroblue tetrazolium reduction (p < 0.0001), compared with normal osteoclasts. IFN-gamma markedly increased (p < 0.0001) superoxide production. Whereas there was a 3-fold increase in superoxide generation in OP patients' osteoclasts, osteoclasts from control subjects had only a small and insignificant increase in superoxide production after IFN-gamma treatment.

1. R-(+)-Pulegone was administered orally to rats and the urinary metabolites were investigated. Six metabolites were isolated and purified using column and thin layer chromatographic techniques. Metabolites were identified by i.r., n.m.r. and mass spectral analyses. 2. The neutral metabolites isolated from urine of rats treated with pulegone (I) were: pulegone (II), 2-hydroxy-2(1'-hydroxy-1'-methylethyl)-5-methylcyclohexanone (III), 3,6-dimethyl-7a-hydroxy-5,6,7,7a-tetrahydro-2(4H)-benzofuranone (V) and menthofuran (VII). Metabolites II and III were also excreted in conjugated form. 3. Acidic metabolites isolated from urine of rats treated with pulegone (I) were: 5-methyl-2(1'-methyl-1'-carboxyethylidene)cyclohexanone (IV) and 5-methyl-5-hydroxy-2(1'hydroxy-1'-carboxyethyl)cyclohexanone (VI).

Newborn rats were segregated into litters of 6 and 22 pups. Undernourished groups were maintained for 21 and 53 days at which time a portion of each group was sacrificed and the remainder refed until 121 days. The amount of myelin isolated from the central nervous system was decreased in nutritionally deprived animals but there was little change in its chemical composition or intereference in the normal chemical maturation of the membrane. The only consistent change was a reduction in phosphatidyl ethanolamine plasmalogen. This deficit tended to be corrected by nutritional rehabilitation and was most complete in those animals refed ad lib. diets for the longest time periods prior to sacrifice. The relatively normal chemical composition of myelin obtained from undernourished animals differs from changes described in myelin membranes isolated from patients with destructive or degenerative diseases of the nervous system. This seems to be further evidence that the reduction in myelination in undernutrition is a result of decreased synthesis of the membrane.

No abstract

An unselected population of newborn infants at a tertiary perinatal center was the subject of an investigation of isoimmune neonatal neutropenia (INN) to determine the incidence of the disorder and further characterize its clinical and immunologic aspects. We screened 1465 consecutively born infants for neutropenia on the first day of life, and evaluated those with neutropenia for the presence of antineutrophil antibodies utilizing EDTA-microagglutination and indirect immunofluorescence. Of the 16 infants with persistent neutropenia, 3 were confirmed to have INN, representing 2/1000 live births and 1.5% of special care nursery admissions during the period. INN is not a rare disorder and should be considered in the evaluation of all infants with neutropenia, with or without infection. Our rapidly expanding knowledge of the neutrophil-specific antigen system is refining our ability to diagnose and treat immune neutropenias.

A bioassay capable of detecting carcinogenic substances that are associated with the elevated incidence of cancer in the urban environment would be important for epidemiologic and environmental analyses. The feasibility of using the Salmonella mutagenesis system developed by Ames for this purpose has been tested by analyzing Chicago air particulate samples. Active material, as evidenced by enhanced rates of mutation, both in the presence of microsomes and in their absence, is readily extractable from samples of air particulates. Dose-response curves have been obtained from such extracts of 15 successive samples, taken at intervals during 1975 from a sampling site in South Chicago. A method for analyzing such data in order to evaluate the relative mutagenic activity of different samples is described. The presence of a number of mutagenic constituents has been demonstrated by means of thin-layer chromatography of particularly active samples, in which the active material is located by mutagenic analysis of successive chromatographic zones. Mass spectrometer analysis of material isolated from an original sample in this way indicates that benzo[a]pyrene and benzo[e]pyrene, which are known to be mutagenic and carcinogenic, are present. It is concluded that within certain constraints, which are described, the methodology can serve the purpose of an environmental bioassay for organic carcinogens.