Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Contribution of preterm delivery to perinatal mortality.

A detailed retrospective analysis was made of the records of 486 preterm infants, who accounted for 5-1% of all births during 1973 and 1974. Whereas preterm delivery did not contribute to perinatal mortality in terms of stillbirth, it outweighed all other causes in terms of early neonatal deaths. Preterm birth was responsible for 85% of the early neonatal deaths not due to lethal congenital deformities. Early neonatal mortality rates were closely linked both to gestational age and birth weight and to the reason for preterm birth. Early neonatal mortality was high (97 per 1000) when preterm labour was spontaneous, whether or not associated with material or fetal disease or with multiple pregnancy, but low (27 per 1000) when preterm delivery was elective. Preventing spontaneous preterm labour would considerably reduce neonatal mortality in our community.     

An Examination of the Association between FOXA1 Staining Level and Biochemical Recurrence following Salvage Radiation Therapy for Recurrent Prostate Cancer

Background

Standardly collected clinical and pathological patient information has demonstrated only moderate ability to predict risk of biochemical recurrence (BCR) of prostate cancer in men undergoing salvage radiation therapy (SRT) for a rising PSA after radical prostatectomy (RP). Although elevated FOXA1 staining has been associated with poor patient outcomes following RP, it has not been studied in the specific setting of SRT after RP. The aim of this study was to evaluate the association between FOXA1 staining level and BCR after SRT for recurrent prostate cancer.

Methods

A total of 141 men who underwent SRT at our institution were included. FOXA1 staining levels in primary tumor samples were detected using immunohistochemistry. FOXA1 staining percentage and intensity were measured and multiplied together to obtain a FOXA1 H-score (range 0–12) which was our primary staining measure. P-values ≤ 0.0056 were considered as statistically significant after applying a Bonferroni correction for multiple comparisons.

Results

There was not a significant association between FOXA1 H-score and risk of BCR when considering H-score as an ordinal variable or as a categorical variable (all P≥0.090). Similarly, no significant associations with BCR were observed for FOXA1 staining percentage or staining intensity (all P≥0.14).

Conclusions

FOXA1 staining level does not appear to have a major impact on risk of BCR after SRT.     

Distribution of basement membrane pores in bronchus revealed by microscopy following epithelial removal

The basement membrane of the bronchial epithelium separates the epithelial and mesenchymal compartments. Basement membrane pores allow cells to cross this boundary. We present a method for preparation of samples of human basement membrane allowing us easy visualisation and characterisation of the distribution and persistence of these pores. Columnar epithelial cells were removed from airway samples with gentle scraping with a circular glass coverslip. In contrast, the underlying basal cells required incubation once in dithiothreitol and twice in ethylenediaminetetraacetic acid. Scanning electron microscopy (SEM) at each stage of the epithelial stripping process showed the selective removal of epithelial cells with eventual visualisation of the pores. Using confocal microscopy on blocks of viable tissue, pores were shown to persist in culture for at least 5 days, despite the presence of viable cells in the submucosa. The distribution of pores in tissues determined by SEM was compared to simulations of three distribution patterns (random, clumped, and distributed). The pattern of pores in the samples was consistent with a random distribution. We suggest that basement membrane pores can be generated by the passage of infiltrating cells into the epithelium providing a network suitable for intraepithelial surveillance.     

Effect of paracetamol (acetaminophen) and ibuprofen on body temperature in acute ischemic stroke PISA, a phase II double-blind, randomized, placebo-controlled trial [ISRCTN98608690]

Background

Body temperature is a strong predictor of outcome in acute stroke. In a previous randomized trial we observed that treatment with high-dose acetaminophen (paracetamol) led to a reduction of body temperature in patients with acute ischemic stroke, even when they had no fever. The purpose of the present trial was to study whether this effect of acetaminophen could be reproduced, and whether ibuprofen would have a similar, or even stronger effect.

Methods

Seventy-five patients with acute ischemic stroke confined to the anterior circulation were randomized to treatment with either 1000 mg acetaminophen, 400 mg ibuprofen, or placebo, given 6 times daily during 5 days. Treatment was started within 24 hours from the onset of symptoms. Body temperatures were measured at 2-hour intervals during the first 24 hours, and at 6-hour intervals thereafter.

Results

No difference in body temperature at 24 hours was observed between the three treatment groups. However, treatment with high-dose acetaminophen resulted in a 0.3°C larger reduction in body temperature from baseline than placebo treatment (95% CI: 0.0 to 0.6 °C). Acetaminophen had no significant effect on body temperature during the subsequent four days compared to placebo, and ibuprofen had no statistically significant effect on body temperature during the entire study period.

Conclusions

Treatment with a daily dose of 6000 mg acetaminophen results in a small, but potentially worthwhile decrease in body temperature after acute ischemic stroke, even in normothermic and subfebrile patients. Further large randomized clinical trials are needed to study whether early reduction of body temperature leads to improved outcome.     

The organization of aggrecan in human articular cartilage. Evidence for age-related changes in the rate of aggregation of newly synthesized molecules

The effect of age on the incorporation of newly synthesized aggrecan into the extracellular matrix of human articular cartilage was investigated. This property was measured in a pulse-chase explant culture system by determining the distribution of radiolabeled molecules ([(35)S]sulfate-labeled) between a nondissociating extract (phosphate-buffered saline), which extracts mainly nonaggregated macromolecules, and a dissociating extract (4 M GnHCl) containing mainly aggrecan that was complexed in situ with hyaluronan. The rate of incorporation of aggrecan into aggregates was much slower in mature cartilage than in tissue obtained from younger individuals. Furthermore, autoradiography showed that in mature cartilage, newly synthesized aggrecan is not transported from the pericellular environment within the first 18 h of chase culture, whereas in immature cartilage, it moves into the intercellular space during the same period, i.e. aggrecan is processed in the extracellular space very differently in young and adult articular cartilage. Experiments were also performed to show that the interaction of link protein with newly synthesized aggrecan depends on the maturity of the G(1) domain of aggrecan. This investigation has shown that the extracellular aggregation of aggrecan in adult human articular cartilage involves a number of intermediate structures. These have not been identified in the very young cartilage obtained from laboratory animals or in porcine and bovine articular cartilage obtained from the abattoir.