Percoll centrifugation eliminates mold contaminants from cell cultures

This work was supported by a grant and research associateship to N.A. from the Medical Research Council of Canada and the National Cancer Institute of Canada.     

Retinol-induced modification of the extracellular matrix of endothelial cells: Its role in growth control

Summary The growth of the endothelial cell (EC) is tightly regulated throughout the body. Many factors have been implicated in modulating EC growth including diffusible compounds, cell-to-cell interactions, and the extracellular matrix (ECM). Retinol, or vitamin A alcohol, has recently been shown to inhibit the growth of bovine capillary ECs, in vitro. Retinoids are known to modify ECM in other cell systems, and pure ECM components have been shown to effect EC growth rates. We, therefore, examined the role of the matrix in the retinol-induced inhibition of ECs. Cell-free matrices from control and vitamin A-treated ECs were prepared by removing cells with EGTA treatment after 7 d of culture. Matrix proteins were analyzed by solubilizing the matrices in 5M quanidine-HCl and performing Western blot analysis using specific antibodies to matrix proteins. In isolating the ECM, we observed that retinol-treated cultures of ECs were resistant to EGTA removal; retinol-treated ECs required twice the exposure time to EGTA to detach from their matrix than did controls cells. Western blot analysis of matrix proteins derived from control and retinol-treated EC cultures demonstrated a 1.6-fold increase in lamininβ chains and a 2.5-fold increase in fibronectin in the ECM of retinol-treated EC compared to control cell matrix. Functional properties of these matrices were assessed by plating control and Day 6 retinol-treated ECs onto the matrices and measuring attachment and growth by determining cell numbers at 24, 72, and 144 h. These studies revealed that control cells attached in greatest numbers to a control matrix whereas retinol-treated ECs preferentially attached to a matrix derived from retinol-treated cells. Furthermore, control ECs which grew rapidly on a control matrix were growth inhibited on a retinol-derived matrix. These data indicate that vitamin A treatment of ECs effects both their phenotype and influences the composition and the functional properties of their underlying ECM. These studies also demonstrate that alterations of the matrix are at least in part responsible for the growth inhibition of EC by retinol.     

Sustained effects of biofeedback-assisted relaxation therapy in essential hypertension

The usefulness of biofeedback-assisted relaxation as an adjunct or substitute for pharmacotherapy in essential hypertension can be enhanced if the effects are shown to persist after formal treatment has ended. Patients with essential hypertension successfully treated with biofeedback-assisted relaxation were recalled for follow-up yearly after the termination of treatment. Twenty-six of 40 patients met the BP criterion for success. At one-, two-, and three-year follow-up, 31%, 38%, and 27% of the successful completers continued to meet the criterion for success. The pretreatment-posttreatment decreases in BP were accompanied by decreases in forehead muscle tension and urinary cortisol. Forehead muscle tension, urinary cortisol, and anxiety levels were significantly lower than pretreatment one year after the end of treatment. Self-report data were used to assess continued relaxation practice. No relationship was found between practice and any other dependent measure. It appears that some patients trained in biofeedback-assisted relaxation can maintain lowered blood pressure, muscle tension, anxiety, and cortisol levels over the long term; however, the role of relaxation practice in maintaining these lowered levels remains unclear.     

A technique for the long-term application of surface electrodes

A technique is reported for the long-term application of surface electrodes for ambulatory electromyographic (EMG) recording. Prior to electrode application the surrounding skin is lightly painted with tincture of benzoin. This treatment improves adherence to the skin of disposable electrodes and electrode attachment collars, reduces skin trauma associated with electrode removal, and minimizes sensitivity to electrode adhesives.This research was supported in part by NIH grant No. NS25114.     

Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.     

Effects of progesterone and estradiol on the proliferation of phytohemagglutinin-stimulated human lymphocytes

In this paper we report on a study to elucidate whether the response of human lymphocytes to mitogenic stimulation was modified by physiological changes which occur during the menstrual cycle. Experiments with untreated cultures showed intra-individual variation to mitogen stimulation in female lymphocyte cultures, but a significant correlation between the menstrual cycle and the proliferation kinetics of lymphocytes was not found. Consequently, we performed experiments in which two of the hormones that regulate the menstrual cycle in women, estradiol and progesterone, were added to cultured human lymphocytes obtained from both men and women. The results indicate that both hormones at physiological concentrations have the capacity to modify the proliferation of PHA-stimulated human lymphocytes. Therefore, both hormones could play a role in the induction of the intra-individual variation observed in the untreated female cultures. However, in vivo other factors could also modify the proliferation kinetics of human lymphocytes preventing the demonstration of the effects of a single factor, such as the hormonal changes occurring during the menstrual cycle.     

The formation of cytoplasmic channels between tapetal cells inZea mays

Summary In this report we show that large cytoplasmic channels form between the tapetal cells ofZea mays (maize) during the period of tapetal cell differentiation. Tapetal cells are connected by plasmodesmata through their cellulosic cell walls prior to the first meiotic division of the meiocytes. As the tapetal cellulose wall is degraded at the onset of meiosis, both plasmodesmata and cytoplasmic channels measuring 50–200 nm are detectable between tapetal cells. By the time the meiotic tetrad is formed, the cytoplasmic channels are well-established and vary in size from 100–400 nm. The channels, with an average diameter of 200–300 nm, persist after the microspores are released from the callose wall and throughout the period of exine development in microsporogenesis. The channels could potentially allow for free exchange of cytoplasm and organelles. As the tapetal cells begin to pull apart and become vacuolate prior to microspore mitosis, the connecting channels are no longer detectable.     

Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.     

Enxymological characterization of a putative canine analogue of primary hyperoxaluria type 1

This paper concerns an enzymological investigation into a putative canine canalogue of the human autosomal recesive disease primary hyperoxaluria type 1 (alanine:glyoxylate / serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and seribe:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephripathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including d-glycerate dehydrogenese / glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate / serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal humanb liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is anlogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.