IN VIVO MEASUREMENT OF ACTIVE OXYGEN PRODUCTION IN THE BROWN ALGA FUCUS EVANESCENS USING 2′,7′-DICHLOROHYDROFLUORESCEIN DIACETATE1

Intracellular production of active oxygen in the brown alga Fucus evanescens C. Ag. was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA) to the fluorescent dye 2′,7′-dichlorofluorescein (DCF), both in emersed and immersed seaweeds. Algae were incubated in seawater containing DCFH-DA under a range of conditions, and it was also possible to load algae with DCFH-DA and then follow subsequent DCF production in emersed tissue. DCF formation was linear for at least 2 h in both darkness and light, with the rate of formation increasing with the light level. DCF formation was temperature dependent. It also increased when algae were treated with H₂O₂ or methyl viologen (paraquat), which disrupts photosystem 1 electron transport and increases O^?₂ production. Exogenous catalase reduced in vivo DCF production, presumably by lowering cellular concentrations of H₂O₂. Hydrogen peroxide was released into the seawater by illuminated algae resulting in external dye conversion to DCF. However, this does not interfere with in vivo measurement of DCF by loaded, washed algae because DCF leakage appeared to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untreated controls. Overall, our data suggest that DCFH-DA is a potentially very useful probe for studying active oxygen metabolism in seaweeds subjected to environmental stresses.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

Renal cell culture using autopsy material from children with cystinosis

Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations.     

Ultrastructure of spermatids and spermatozoa in the cyclostomatous bryozoan Tubulipora (Bryozoa,Cyclostomata)

Summary Differentiation of spermatids to mature spermatozoa in the bryozoan Tubulipora liliacea was studied by transmission electron microscopy. The spermatozoon of Tubulipora is of a filiform, modified type, and has evolved from the primitive type as an adaptation to a specialized biology of fertilization. The head of the spermatozoon consists of a small, conical acrosome capping an elongated, cylindrical, anteriorly tapering nucleus. A basal invagination in the nucleus contains the proximal portion of the axoneme and a dense attachment matrix. The flagellar axoneme has the typical 9+2 structure. Four elongated rodshaped mitochondria with typical cristae surround the axoneme in the cylindrical middle piece. Granular electron-dense material is accumulated in the form of four columns alternating with four long cylindrical mitochondria. The mitochondrial middle piece is separated externally from the tail region by an involution of the plasma membrane. The tail region contains a cytoplasmic sheath with accessory fibers surrounding the axoneme. Nine outer, coarse fibers extend posteriorly paralleling the nine doublets of the axoneme. The coarse fibers develop from electron-dense plate-like structures associated with the doublets of the axoneme. A characteristic feature in spermiogenesis is that spermatozoa develop in tetrads. There seem to be significant differences in spermatozoan ultrastructure between the three bryozoan classes Stenolaemata, Gymnolaemata, and Phylactolaemata. The differences indicate different lines of evolution of fertilization biology in these groups.Abbreviations used in the figures a acrosome - av acrosomal vesicles - ax axoneme - c coarse fiber - d electron dense rod - m mitochondrion - mp middle piece - Scale bars=0.5 m - mt microtubule - n nucleus - ne nuclear envelope - p nuclear protrusion - pm plasma membrane - t tail     

Dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia

Transmissible murine colonic hyperplasia, cuased by a variant of Citrobacter freundii (4280). was shown to be modified by diet and by host strain and species. Four different diets fed to mice inoculated with C frundii 4280 were found to have a significant but varying influence on the severity of hyperplasia. Diet also influenced the colonic crypt height of uninoculated, control mice. F344 rats, Syrian hamsters, and NIH Swiss [N:(S)], C57BL/6J, C3H/HeJ, and DBA/2J mice were inoculated with C freundii 4280. Marked strain differences were noted in the mice in mortality and severity of the colonic hyperplasia. The NIH Swiss mice had the greatest and the C57BL/6J mice had the least mucosal hyperplasia. The rats and hamsters did not develop disease or maintain infection after inoculation with the organism. Twenty isolates of Citrobacter from a range of biologic sources were inoculated into susceptible mice, but only mice inoculated with C freundii 4280 developed the disease.     

A highly repetitive, mariner-like element in the genome of Hyalophora cecropia.

A highly repetitive DNA element, homologous to the mariner transposon of Drosophila mauritiana was found in the intron of the gene for cecropin A, an antibacterial peptide from the Cecropia moth. The mariner-like elements (MLE) represent a homogeneous population with a copy number of about 1000/genome. Sequencing analysis showed it to be 1255 base pairs long, including 38-base pair terminal inverted repeats. The MLE contains a defective reading frame. Nevertheless, the putative product is clearly homologous to the predicted translation product encoded by mariner. In consonance is also the fact that the inverted repeats are highly conserved between the two elements and that the overall DNA homology is 48%. Since the mariner element is present in several Drosophila species closely related to Drosophila melanogaster and since MLE is present in the lepidopteran Cecropia, a route of horizontal transfer is indicated rather than vertical transmission from a common ancestor. This suggests the possible use of mariner for the construction of an interspecies vector.     

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.     

Photo-CIDNP study of the interaction between the glucocorticoid receptor DNA-binding domain and glucocorticoid response elements

Summary Photo-CIDNP studies were performed on two protein fragments that both contain the double zinc-finger DNA-binding domain of the glucocorticoid receptor. In the absence of DNA, Tyr⁴⁵² and Tyr⁴⁷⁴ are polarised in both fragments while Tyr⁴⁹⁷ is not. Addition of a palindromic glucocorticoid response element (GRE) results in the suppression of Tyr⁴⁷⁴ polarization while the polarization of Tyr⁴⁵² is unaffected. The same result is observed upon adding a half GRE to the protein fragment indicating that the suppression of Tyr⁴⁷⁴ polarization is not due to protein-protein contacts but to interaction with DNA.     

Vegetation types and patterns in Senegal based on multivariate analysis of field and NOAA-AVHRR satellite data

Abstract. Woody vegetation was studied in 44 sites in Senegal. Species composition and density were analysed with TWINSPAN and Canonical Correspondence Analysis; 16 vegetation types were defined. A supervised, multispectral and multitemporal classification of day and night, dry season NOAA-AVHRR imagery was used to identify their distribution with a classification accuracy ranging from 60–100%.