Influence of Hydrocortisone on Chick Embryo Retina Development

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.     

Glomerular podocytes in cultured rat kidney slices

Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO₂/95% O₂ environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 m of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P < 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.

Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.     

Effects of prolonged sodium restriction on the morphology and function of rat adrenocortical autotransplants

Summary Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions. The hypothesis is advanced that the lack of splanchnic nerve supply and chromaffin medullary tissue in regenerated nodules may be the cause of such an impairment.     

Diurnal beta-endorphin changes in human cerebrospinal fluid

Plasma and cerebrospinal fluid (CSF) beta-endorphin levels were determined by a RIA method in seven hydrocephalic male patients. The samples were simultaneously collected every two hours from 8 AM to 12 midnight and every hour from 1 AM to 7 AM. In both plasma and CSF beta-endorphin levels showed significant time-related variations during the 24 hour period. These results suggest the existence of diurnal CSF beta-endorphin variations analogous to those observed in plasma.     

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.     

Summer biomass of a population of Iridaea cordata (Gigartinaceae,Rhodophyta) from Antarctica

Results of a seasonal study on biomass in an infralittoral population of Iridaea cordata from Terra Nova Bay (Ross Sea, Antarctica) are reported. Thalli were collected during the IX Italian Antarctic Expedition (austral summer 1993–94). The population studied is that living at depths of 4 to 6 m, where the highest density of plants occurred. The highest value of biomass (wet weight 3440 g m^–2) was found at the beginning of summer. In that period 72.5% of biomass was from 128 specimens belonging to weight classes 8 (>16 to 32 g) to 10 (>64 g), corresponding to 13.4% of the population in numbers. Small (<1 g) and medium (1 to 8 g) specimens provided the remaining biomass of 5% and 22.5%, respectively. During the month of January, the number of heavy specimens decreased. At the end of that month biomass reached a minimum of 2225 g m^–2. In February the biomass increased to 3169 g m ^–2, 72% of which was from 120 specimens belonging to weight classes 7 (>8 to 16 g) to 9 (>32 to 64 g), which numerically represented 18.5% of the population. Data showed that biomass depended mainly on the presence of large heavy specimens, even though they were always few in number. Moreover, the occurrence of such large thalli at the beginning of summer suggests that Iridaea cordata continues to grow during the long antarctic winter.