Retinoic acid receptor alpha2 is a growth suppressor epigenetically silenced in MCF-7 human breast cancer cells.

Retinoic acid (RA) receptor (RAR) beta2 has been shown to be underexpressed in human breast cancer cells, including MCF-7 cells, and recent reports have suggested that hypermethylation of the RAR beta2 promoter and 5'-UTR is the underlying cause. Here we show that RAR alpha2 is also underexpressed in MCF-7 breast cancer cells, at both the message and the protein level, relative to normal or nontumorigenic breast epithelial cells. Bisulfite sequencing of the CpG island in the RAR alpha2 promoter revealed highly penetrant and uniform cytosine methylation in MCF-7 cells. Pretreatment with the DNA methyltransferase inhibitor, azacytidine, followed by treatment with RA and a histone deacetylase inhibitor, trichostatin A, resulted in partial promoter demethylation and RAR alpha2 induction, which strongly suggested that promoter hypermethylation is responsible for RAR alpha2 underexpression. We compared the outcome of ectopic expression in MCF-7 cells of matched levels of RAR alpha2 and RAR beta2. On the basis of a clonogenic assay, RAR alpha2 displayed ligand-dependent growth-suppressive activity similar to that of RARb eta2; thus, 10 and 20 nM RA inhibited clonogenic growth by 52 and 80%, respectively, in RAR alpha2-transfected cells compared with 75 and 77%, respectively, in RAR beta2-transfected cells. We conclude that the silencing of the RAR alpha2 promoter by hypermethylation may play a contributory role in the dysregulation of RA signaling in mammary tumorigenesis.     

Reaerosolization of Fluidized Spores in Ventilation Systems

This project examined dry, fluidized spore reaerosolization in a heating, ventilating, and air conditioning duct system. Experiments using spores of Bacillus atrophaeus, a nonpathogenic surrogate for Bacillus anthracis, were conducted to delineate the extent of spore reaerosolization behavior under normal indoor airflow conditions. Short-term (five air-volume exchanges), long-term (up to 21,000 air-volume exchanges), and cycled (on-off) reaerosolization tests were conducted using two common duct materials. Spores were released into the test apparatus in turbulent airflow (Reynolds number, 26,000). After the initial pulse of spores (approximately 10¹⁰ to 10¹¹ viable spores) was released, high-efficiency particulate air filters were added to the air intake. Airflow was again used to perturb the spores that had previously deposited onto the duct. Resuspension rates on both steel and plastic duct materials were between 10⁻³ and 10⁻⁵ per second, which decreased to 10 times less than initial rates within 30 min. Pulsed flow caused an initial spike in spore resuspension concentration that rapidly decreased. The resuspension rates were greater than those predicted by resuspension models for contamination in the environment, a result attributed to surface roughness differences. There was no difference between spore reaerosolization from metal and that from plastic duct surfaces over 5 hours of constant airflow. The spores that deposited onto the duct remained a persistent source of contamination over a period of several hours.     

Isolation and characterization of a starchless mutant of Chlorella pyrenoidosa STL-PI with a high growth rate, and high protein and polyunsaturated fatty acid content

The growth characteristics, biochemical composition and ultrastructure of a novel starchless mutant of Chlorella pyrenoidosa, designated as STL‐PI, are compared to the same characteristics of its parental strain, C. pyrenoidosa 82T. The STL‐PI mutant had a 22 ± 5% higher growth rate, and 24.5 ± 4.2% more protein than the parental strain, 82T. Furthermore, the STL‐PI mutant accumulated 20.4% more polyunsaturated fatty acids and 18% less saturated fatty acids than the parental 82T. When the parental 82T was cultured in a nitrogen‐free media, their starch content increased from 6.8 ± 2.8% to 22.5 ± 3.1%. In contrast, the STL‐PI mutant produced no starch, regardless of the growth conditions. Instead, the mutant cells responded to nitrogen limitation by further increasing their lipid content from 25.2 ± 1.2% to 38.0 ± 2.3% per dry weight. Transmission electron micrography revealed that nitrogen limitation typically stimulates the formation of starch granules in the chloroplast of 82T cells. Yet no starch granules were observed in the STL‐PI cells. Instead, only the formation of large lipid globules was observed in the mutant cells. These results demonstrate that the starchless mutant STL‐P1 possesses novel physiological and phytochemical properties distinct from the 82T cells: their cells were deficient in starch synthesis and showed higher growth rates and productivity than 82T cells.     

FURTHER CHARACTERIZATION OF BOVINE KERATOHYALIN

Extraction of serial sections of cattle hoof epidermis with solutions of calcium chloride, magnesium chloride, potassium chloride, sodium chloride, guanidine hydrochloride, ammonium sulfate, and potassium phosphate buffer (pH 7.0) at varying salt concentrations demonstrates that keratohyalin (KH) is extracted by these salts at certain molarities. Under given conditions of time and temperature, each salt has a specific extraction pattern, and similar salts have similar extraction patterns. Dialysis of the salt extracts of hoof epidermis against distilled water results in the macroaggregition of KH, as assayed by histochemical methods. Although the various macroaggregates appear identical at the histochemical level, they display different ultrastructural characteristics. Polyacrylamide gel electrophoresis of the sodium decyl sulfate-solubilized macroaggregates results in the fractionation of a 20 (or more) member homologous series of oligomers. Isolation of the various oligomeric species of bovine keratohyalin and re-electrophoresis indicate that the various KH species can undergo depolymerization. Amino acid analyses of the unfractionated bovine macroaggregates and the various molecular weight species of bovine KH are similar, further demonstrating homology of the oligomers. The molecular weight of the subunit (monomer) of bovine KH is 14,955, estimated from the amino acid analyses.     

Indirect Hemagglutinating Antibody Response to Herpesvirus hominis Types 1 and 2 in Immunized Laboratory Animals and in Natural Infections of Man

Indirect hemagglutinating (IHA) antibody responses to Herpesvirus hominis types 1 and 2 (HVH-1 and HVH-2) were compared to complement-fixing and neutralizing antibody responses in immunized laboratory animals (rabbits, guinea pigs, and hamsters) and in natural infections of man. With the immunized animals, type specificity was seen only in the IHA test and only with antisera produced in hamsters and in the rabbits immunized with HVH-2. In human nongenital infections (considered to be caused predominately by HVH-1), IHA and neutralizing antibodies developed at about the same rate and reached approximately the same levels for HVH-1 and HVH-2. IHA titers tended to be higher than neutralizing antibody titers for both virus types. In genital infections (considered to be caused predominately by HVH-2), there was a rapid IHA antibody response to HVH-2, and the early HVH-2 antibody demonstrable by IHA, but not by neutralization tests, was found to be immunoglobulin M in nature. In genital infections, IHA titers for HVH-2 were markedly higher than neutralization titers, but there was no pronounced difference in neutralizing the IHA antibody titers for HVH-1. Several patients with genital infections fialed to develop IHA antibody for HVH-1. The IHA test possessed no greater sensitivity than did complement fixation or neutralization tests for serodiagnosis of HVH infections. Despite the fact that a number of patients with genital infections produced IHA antibody only for HVH-2, the test was no more effective than the neutralization test in providing a type-specific serodiagnosis of infection, due largely to the fact that the rapid IHA antibody response to HVH-2 prevented demonstration of a further, significant antibody titer increase in a number of cases.     

Kinematics of normal locomotion—A statistical study based on T.V. data

A T.V.-computer system for the measurement and analysis of the kinematics of normal and abnormal locomotion has been developed which has the following characteristics: minimum encumberment to the subject or patient, determination of absolute coordinates of anatomical landmarks, analysis of 3 or 4 strides, and automation of data conversion and analysis. Analysis techniques have been developed which take advantage of the accuracy of the marker coordinates, and with suitable application of noise reduction filtering, permit direct calculation of velocities and accelerations (both linear and angular). Examples of some of these derived parameters vs time are presented. The results of a statistical study on a group of normal subjects walking at slow, comfortable and fast cadences yield the ranges of many velocities and accelerations not previously available.