Testicular myxosporidiasis in anurans,with a description of Myxobolus fallax n. sp.

During studies of amphibian sperm cryopreservation, a new species of myxosporidean parasite (Myxozoa, Myxosporae) was observed in the testes of the Australian dwarf green tree frog Litoria fallax (Peters). Myxosporidiasis was found to have no affect on L. fallax body condition or sperm numbers. Myxobolus spores from L. fallax are morphologically distinct from Myxobolus hylae spores (infecting the sympatric Litoria aurea Lesson) and the three previously named (exotic to Australia) Myxobolus species found in anurans. Myxobolus fallax n. sp. is characterised by: pseudocyst white, spherical to ovoid, 141 x 74 to 438 x 337 microm in diameter (mature); plasmodium with spores loosely arranged within interior. Spores ovoid 13.4 +/- 0.5 (12.6-14.6) microm length, 9.5 +/- 0.4 (8.3-10.6) microm width, 6.8 +/- 0.4 (6.5-7.6) microm depth, 1.4 +/- 0.1 (1.3-1.6) length/width; polar capsules broadly pyriform and equal in size 4.2 +/- 0.3 (3.3-4.7) microm length, 2.4 +/- 0.2 (2.1-2.8) microm width; filament coils 7-8, wound tightly and perpendicular to the longitudinal axis of the capsule; polar filament 34 +/- 7.0 (18-50) microm length; intercapsular appendix and sutural ridge folds absent; and iodinophilous vacuole and mucous envelope lacking. In addition to this new species, data from archival samples of M. hylae are provided which show two morphologically distinct spore types. Both appeared rarely in the same pseudocysts and we cautiously retain the single species.     

Parvicapsula bicornis n. sp. and P. limandae n. sp. (Myxozoa, Parvicapsulidae) in Pleuronectidae (Teleostei, Heterosomata) from Denmark

Two species of Parvicapsula were found in the kidney tubules and the urinary bladder of 2 pleuronectid fish from the northern Oresund, Denmark. The coelozoic, spherical, disporic trophozoites of both species are 10 to 12 pm in diameter. The myxospores of both species are elongate, asymmetrical and slightly curved, and have spherical polar capsules. Parvicapsula bicornis n. sp. (6-8 x 5-6 microm, polar capsule 2.5 microm in diameter) occurs in Pleuronectes platessa. The polar capsules of P. bicornis are arranged symmetrically on either side of the longitudinal axis and its spores differ from other species of Parvicapsula in having two 2-3 microm long posterior processes of different length. Parvicapsula limandae n. sp. (8-11 x 4-5 pm, polar capsule 1.6 microm in diameter) is found in Limanda limanda. The polar capsules are arranged along the longitudinal axis. It differs from Parvicapsula unicornis Kabata, 1962, recorded from L. limanda, in the arrangement of the polar capsules and in the absence of a posterior horn-like projection. The phylogenetic relationship between P. bicornis n. sp., P. limandae n. sp. and other Parvicapsula spp. was examined with their partial small subunit rDNA (SSU rDNA) sequences. P. limandae n. sp. and P. asymmetrica appear to be closely related, while P. bicornis n. sp. and P. minibicornis are the most divergent members of the genus.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.     

Mucus secretion from single submucosal glands of pig. Stimulation by carbachol and vasoactive intestinal peptide

Secretion rates of >700 individual glands in isolated tracheal mucosa from 56 adult pigs were monitored optically. "Basal" secretion of 0.7 +/- 0.1 nl x min(-1) gland(-1) was observed 1-9 h post-harvest but was near zero on day 2. Secretion to carbachol (10 microm) peaked at 2-3 min and then declined to a sustained phase. Peak secretion was 12.4 +/- 1.1 nl x min(-1) gland(-1); sustained secretion was approximately one-third of peak secretion. Thapsigargin (1 microm) increased secretion from 0.1 +/- 0.05 to 0.7 +/- 0.2 nl x min(-1) gland(-1); thapsigargin did not cause contraction of the trachealis muscles. Isoproterenol and phenylephrine (10 microm each) were ineffective, but vasoactive intestinal peptide (1 microm) and forskolin (10 microm) each produced sustained secretion of 1.0 +/- 0.5 and 1.7 +/- 0.2 nl x min(-1) gland(-1), respectively. The density of actively secreting glands was 1.3/mm(2). Secretion to either carbachol or forskolin was inhibited (approximately 50%) by either bumetanide or HCO(3)(-) removal and inhibited approximately 90% by the combined treatments. Mucus secreted in response to carbachol or forskolin was acidic by approximately 0.2 pH units relative to the bath and remained acidic by approximately 0.1 pH units after bumetanide. The strong secretory response to vasoactive intestinal peptide, the acidity of [cAMP](i)-stimulated mucus, and its inhibition by bumetanide were unexpected.     

Kinetics of interconversion of ferrous enzymes, compound II and compound III, of wild-type synechocystis catalase-peroxidase and Y249F: proposal for the catalatic mechanism

With the exception of catalase-peroxidases, heme peroxidases show no significant ability to oxidize hydrogen peroxide and are trapped and inactivated in the compound III form by H2O2 in the absence of one-electron donors. Interestingly, some KatG variants, which lost the catalatic activity, form compound III easily. Here, we compared the kinetics of interconversion of ferrous enzymes, compound II and compound III of wild-type Synechocystis KatG, the variant Y249F, and horseradish peroxidase (HRP). It is shown that dioxygen binding to ferrous KatG and Y249F is reversible and monophasic with apparent bimolecular rate constants of (1.2 +/- 0.3) x 10(5) M(-1) s(-1) and (1.6 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C), similar to HRP. The dissociation constants (KD) of the ferrous-dioxygen were calculated to be 84 microm (wild-type KatG) and 129 microm (Y249F), higher than that in HRP (1.9 microm). Ferrous Y249F and HRP can also heterolytically cleave hydrogen peroxide, forming water and an oxoferryl-type compound II at similar rates ((2.4 +/- 0.3) x 10(5) M(-1) s(-1) and (1.1 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C)). Significant differences were observed in the H2O2-mediated conversion of compound II to compound III as well as in the spectral features of compound II. When compared with HRP and other heme peroxidases, in Y249F, this reaction is significantly faster ((1.2 +/- 0.2) x 10(4) M(-1) s(-1))). Ferrous wild-type KatG was also rapidly converted by hydrogen peroxide in a two-phasic reaction via compound II to compound III (approximately 2.0 x 10(5) M(-1) s(-1)), the latter being also efficiently transformed to ferric KatG. These findings are discussed with respect to a proposed mechanism for the catalatic activity.     

Nitric oxide from perivascular nerves modulates cerebral arterial pH reactivity

In the isolated rat middle cerebral artery (MCA) we investigated the role of nitric oxide (NO)/cGMP in the vasodilatory response to extraluminal acidosis. Acidosis increased vessel diameter from 140 +/- 27 microm (pH 7.4) to 187 +/- 30 microm (pH 7.0, P < 0.01). NO synthase (NOS) inhibition by N(omega)-nitro-L-arginine (L-NNA, 10 microM) reduced baseline diameter (103 +/- 20 microm, P < 0.01) and attenuated response to acidosis (9 +/- 8 microm). Application of the NO-donors 3-morpholinosydnonimine (1 microM) or S-nitroso-N-acetylpenicillamine (1 microM), or of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 100 microM) reestablished pre-L-NNA diameter at pH 7.4 and reversed L-NNA-induced attenuation of the vessel response to acidosis. Restoration of pre-L-NNA diameter (pH 7.4) by papaverine (20 microM) or nimodipine (30 nM) had no effect on the attenuated response to acidosis. Guanylyl cyclase inhibition with 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (5 microM) or NOS-inhibition with 7-nitroindazole (7-NI, 100 microM) reduced baseline vessel diameter (109 +/- 8 or 127 +/- 11 microm, respectively) and vasodilation to acidosis, and restoration of baseline diameter with 8-BrcGMP (30 microM) completely restored dilation to pH 7.0. Chronic denervation of NOS-containing perivascular nerves in vivo 14 days before artery isolation significantly reduced pH-dependent reactivity in vitro (diameter increase sham: 48 +/- 14 microm, denervated: 14 +/- 8 microm), and 8-BrcGMP (30 microM) restored dilation to pH 7.0 (denervated: 49 +/- 31 microm). Removal of the endothelium did not change vasodilation to acidosis. We conclude that NO, produced by neuronal NOS of perivascular nerves, is a modulator in the pH-dependent vasoreactivity.     

Mechanism of reaction of myeloperoxidase with nitrite

Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.     

Water permeability differs between growing and non-growing barley leaf tissues

A pressure probe technique and an osmotic swelling assay were used to compare water transport properties between growing and non-growing tissues of leaf three of barley. The epidermis was analysed in planta by pressure probe, whereas (predominantly) mesophyll protoplasts were analysed by osmotic swelling. Hydraulic conductivity (Lp) and, by implication, water permeability (Pf) of epidermal cells was 31% higher in the leaf elongation zone (Lp=0.5+/-0.2 microm s-1 MPa-1; Pf=65+/-25 microm s-1; means+/-SD of n=17 cells) than in the, non-growing, emerged leaf zone (Lp=0.4+/-0.1 microm s-1 MPa-1; Pf=50+/-15 microm s-1; n=24; P<0.05). Similarly, water permeability of mesophyll protoplasts was by 55% higher in the elongation compared with emerged leaf zone (Pf=13+/-1 microm s-1 compared with 8+/-1 microm s-1; n=57 and 36 protoplasts, respectively; P<0.01). Within the leaf elongation zone, a small population of larger-sized protoplasts could be distinguished. These protoplasts, which originated most likely from parenchymateous bundle sheath or midrib parenchyma cells, had a three-fold higher water permeability (P<0.001) as mesophyll protoplasts. The effect on Lp and Pf of known aquaporin inhibitors was tested with the pressure probe (Au+, Ag+, Hg2+, phloretin) and the osmotic swelling assay (phloretin). Only phloretin, when applied to protoplasts in the swelling assay caused an average decrease in Pf, but the effect varied between isolations. Technical approaches and cell-type and growth-specific differences in water transport properties are discussed.     

Meiotic competence of in vitro grown goat oocytes

The objective of the present study was to grow meiotically incompetent goat oocytes from early antral follicles in vitro and to render them competent to undergo germinal vesicle breakdown. Cumulus-oocyte complexes with pieces of parietal granulosa cells were isolated from follicles 0.35-0.45 mm in diameter using both mechanical and enzymatic methods. The cumulus-oocyte complexes were divided into two groups according to oocyte diameter (group A: < 95 microm; group B: > 95 microm) and cultured for 8 or 9 days on granulosa cell monolayers. Within 8 days of culture, the mean oocyte diameter increased from 86 +/- 0.4 microm to 95 +/- 0.7 microm in group Aand from 106 +/- 0.2 microm to 109 +/- 0.5 microm in group B. After 9 days of culture, the mean diameter of oocytes from groups A and B were 99 +/- 0.5 microm and 112 +/- 0.4 microm, respectively. The meiotic competence of oocytes grown in vitro was evaluated by in vitro maturation. Within 8 days of culture, only 3% of oocytes from group A and 6% of oocytes from group B acquired the ability to undergo germinal vesicle breakdown. After 9 days of culture, 7% of group A oocytes and 42% of group B oocytes were competent to resume meiosis. The expression of p34(cdc2) in oocytes grown in vitro was analysed by the western blot technique. During 9 days of culture, p34(cdc2) accumulated in both groups of growing oocytes, but its concentration was lower than in fully grown oocytes used as controls. The results showed for the first time that goat oocytes from early antral follicles can grow, accumulate p34(cdc2) and acquire the ability to resume meiosis, when cultured for 9 days on granulosa cell monolayers.     

Spectral and kinetic studies on eosinophil peroxidase compounds I and II and their reaction with ascorbate and tyrosine.

Eosinophil peroxidase, the major granule protein in eosinophils, is the least studied human peroxidase. Here, we have performed spectral and kinetic measurements to study the nature of eosinophil peroxidase intermediates, compounds I and II, and their reduction by the endogenous one-electron donors ascorbate and tyrosine using the sequential-mixing stopped-flow technique. We demonstrate that the peroxidase cycle of eosinophil peroxidase involves a ferryl/porphyrin radical compound I and a ferryl compound II. In the absence of electron donors, compound I is shown to be transformed to a species with a compound II-like spectrum. In the presence of ascorbate or tyrosine compound I is reduced to compound II with a second-order rate constant of (1.0+/-0.2)x10(6) M(-1) s(-1) and (3.5+/-0.2)x10(5) M(-1) s(-1), respectively (pH 7.0, 15 degrees C). Compound II is then reduced by ascorbate and tyrosine to native enzyme with a second-order rate constant of (6.7+/-0.06)x10(3) M(-1) s(-1) and (2.7+/-0.06)x10(4) M(-1) s(-1), respectively. This study revealed that eosinophil peroxidase compounds I and II are able to react with tyrosine and ascorbate via one-electron oxidations and therefore generate monodehydroascorbate and tyrosyl radicals. The relatively fast rates of the compound I reduction demonstrate that these reactions may take place in vivo and are physiologically relevant.     

Myxobolus cuneus n. sp. (Myxosporea) infecting the connective tissue of Piaractus mesopotamicus (Pisces: Characidae) in Brazil: histopathology and ultrastructure

The characteristics of Myxobolus cuneus n. sp. and its relationship to the host Piaractus mesopotamicus are described based on light and electron microscopy and histological observations. Polysporic plasmodia measuring 20 microm to 2.1 mm in size were found in 63.3 % of the P. mesopotamicus examined. The parasite was found in the gall bladder, urinary bladder, gills, spleen, fins, head surface, liver and heart. Generative cells and disporoblastic pansporoblasts occurred along the periphery of the plasmodia, and mature spores were found in the internal region. The mature spores had a pear shaped body in frontal view, with a total length of 10.0 +/- 0.6 microm and a width of 5.1 +/- 0.3 microm (mean +/- SD). The spore wall was smooth with sutural folds. The polar capsules were elongated, were pear shaped, and equal in size (length 5.7 +/- 03 microm; width 1.7 +/- 0.2 microm), with the anterior ends close to each other. The polar filaments were tightly coiled in 8-9 turns perpendicular to the axis of the capsule. The plasmodia were always found in connective tissue (wall of the arterioles of the gill filaments, serous capsule of the gall bladder, middle layer and subepithelial connective tissue of the urinary bladder, connective tissue between the rays of the fins, subcutaneous tissue of the head surface and fibrous capsule spleen). The parasite caused important damage in the gills, where development occurred in the wall of gill filament arterioles; a mild macrophage infiltrate was also observed. In advanced developmental stages, the plasmodia caused deformation of the arteriole structure, with a reduction and, in some cases, obstruction of the lumen. The parasite was found throughout the period studied and its prevalence was unaffected by host size, season or water properties.     

Histophatology and ultrastructure of Henneguya caudalongula sp. n. infecting Prochilodus lineatus (Pisces: Prochilodontidae) cultivated in the state of São Paulo, Brazil

The histological and ultrastructural characteristics of a new species of Henneguya and the host reactions to infection by this species are reported. Henneguya caudalongula sp. n. was found in the inter and intralamellar regions of the gills of Prochilodus lineatus (Valenciennes, 1836) cultivated at Center for the Research and Management of Continental Fishing Resources located in the municipality of Pirassununga, state of S?o Paulo, Brazil. The plasmodia were white and round or ellipsoidal and measured 0.2 to 1 mm in length. The development of the parasite was asynchronous and the mature spores were fusiform, with a total length 71 +/- 1.4 microm, body length of 16.6 +/- 0.54 microm and width 4.6 +/- 0.2 microm. The caudal process was 52.6 +/- 1.5 microm long. The polar capsules were elongate (length 6.1 +/- 0.19 microm, width 1.6 +/- 0.15 microm) and of equal size. The polar filament was coiled in 10-11 turns. The prevalence of the parasite was 48.3% and did not vary significantly with the season or host size.     

Reactions of soybean peroxidase and hydrogen peroxide pH 2.4-12.0, and veratryl alcohol at pH 2.4

Peroxidase from soybean seed coat (SBP) has properties that makes it particularly suited for practical applications. Therefore, it is essential to know its fundamental enzymatic properties. Stopped-flow techniques were used to investigate the pH dependence of the reaction of SBP and hydrogen peroxide. The reaction is linearly dependent on hydrogen peroxide concentration at acidic and neutral pH with the second order rate constant k(1)=2.0x10(7) M(-1) s(-1), pH 4-8. From pH 9.3 to 10.2 the reaction is biphasic, a novel observation for a peroxidase at alkaline pH. A fast reaction has the characteristics of the reaction at neutral pH, and a slow reaction shows hyperbolic dependence on hydrogen peroxide concentration. At pH >10.5 only the slow reaction is seen. The shift in mechanism is coincident with the change in haem iron co-ordination to a six-coordinate low spin hydroxy ligated alkaline form. The pK(a) value for the alkaline transition was observed at 9.7+/-0.1, 9.6+/-0.1 and 9.9+/-0.2 by spectrophotometric titration, the fast phase amplitude, and decrease in the apparent second order rate constant, respectively. An acidic pK(a) at 3.2+/-0.3 was also determined from the apparent second order rate constant. The reactions of soybean peroxidase compounds I and II with veratryl alcohol at pH 2.44 give very similar second order rate constants, k(2)=(2.5+/-0.1)x10(4) M(-1) s(-1) and k(3)=(2.2+/-0.1)x10(4) M(-1) s(-1), respectively, which is unusual. The electronic absorption spectra of compounds I, II and III at pH 7.07 show characteristic bands at 400 and 651 nm (compound I), 416, 527 and 555 nm (compound II), and 414, 541 and 576 nm (compound III). No additional intermediates were observed.     

Cerebral arteriolar structure and function in pinealectomized rats

We examined cerebral arteriolar structure and autoregulation of cerebral blood flow (CBF) in control (n = 8), sham-operated (n = 8), pinealectomized (n = 10), and pinealectomized plus melatonin-treated (0.51 +/- 0.01 mg x kg(-1) x day(-1) in drinking water, n = 9) young Wistar rats. The lower limit of CBF autoregulation (LLCBF) was determined by measurement of CBF (in arbitrary units, laser Doppler) during stepwise hypotensive hemorrhage; the arteriolar internal diameter (ID; in microm, cranial window) was also measured. Measurements of ID were repeated during a second stepwise hypotension after smooth muscle cell deactivation (67 mmol/l EDTA). The cross-sectional area (CSA) was measured by histometry. CSA and EDTA-induced vasodilatation decreased after pinealectomy (517 +/- 21 vs. 819 +/- 40 microm(2) in sham and 829 +/- 55 microm(2) in control, P < 0.05, and 81 +/- 4 vs. 102 +/- 5 microm in sham and 104 +/- 4 microm in control, P < 0.05, respectively) and were restored by melatonin (924 +/- 39 microm(2) and 102 +/- 5 microm, respectively). These results suggest that melatonin deprival makes the arteriolar wall thinner and stiffer. However, these changes had little effect on LLCBF. In conclusion, pinealectomy of young rats induces atrophy and decreases distensibility of the cerebral arteriolar wall; these effects are prevented by melatonin. They do not modify LLCBF.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.     

Light and electronic observations on Henneguya ghaffari (Myxosporea,Bivalvulida) infecting the gills and intestine of Nile perch Lates niloticus (Pisces: Teleostei) from Chad and Senegal

Henneguya ghaffari Ali, 1999, described for the first time in Egypt, has been found on gills and intestine of Nile perch Lates niloticus L. from Chad and Senegal (Africa). It formed plasmodia which induced lesions of infected tissues. In fresh state, the spore body was ovoid and its size was 11.07 +/- 0.7 (range 11 to 13) x 7.7 +/- 0.4 (range 7 to 8) microm. The length of the caudal appendages was 44.2 +/- 1.7 (42 to 48) microm. The polar capsules were pyriform, of equal size, with the polar filament showing 4 coils, and measuring 3.17 +/- 0.1 (range 3 to 4) x 2.2 +/- 0.1 (range 1 to 2) microm. The total length of the spore was 55.73 +/- 1.7 (range 53 to 61) microm. At ultrastructural level, our results confirm that in Henneguya species, the sporoplasm is binucleate and the pansporaoblast is disporous.     

Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD

The goal of this study was to examine the role of endogenous copper/zinc (CuZn)-superoxide dismutase (SOD) on superoxide levels and on responses of cerebral blood vessels to stimuli that are mediated by nitric oxide (acetylcholine) and cyclooxygenase-dependent mechanisms (bradykinin and arachidonic acid). Levels of superoxide in the rabbit basilar artery were measured using lucigenin-enhanced chemiluminescence (5 microM lucigenin). Diethyldithiocarbamate (DDC; 10 mM), an inhibitor of CuZn-SOD, increased superoxide levels by approximately 2.4-fold (P < 0.05) from a baseline value of 1.0 +/- 0.2 relative light units x min(-1) x mm(-2) (means +/- SE). The diameter of cerebral arterioles (baseline diameter, 99 +/- 3 microm) was also measured using a closed cranial window in anesthetized rabbits. Topical application of DDC attenuated responses to acetylcholine, bradykinin, and arachidonate, but not nitroprusside. For example, 10 microM arachidonic acid dilated cerebral arterioles by 40 +/- 5 and 2 +/- 2 microm under control conditions and after DDC, respectively (P < 0.05). These inhibitory effects of DDC were reversed by the superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (10 mM). Arachidonate increased superoxide levels in the basilar artery moderately under normal conditions and this increase was greatly augmented in the presence of DDC. These findings suggest that endogenous CuZn-SOD limits superoxide levels under basal conditions and has a marked influence on increases in superoxide in vessels exposed to arachidonic acid. The results also suggest that nitric oxide- and cyclooxygenase-mediated responses in the cerebral microcirculation are dependent on normal activity of CuZn-SOD.     

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.     

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.     

Locally controlled release of basic fibroblast growth factor from multilayered capsules

Biodegradable multilayered capsules encapsulating basic fibroblast growth factor (bFGF) were developed as a cytokine release carrier for drug delivery systems. The multilayered hollow capsules were fabricated via the layer-by-layer (LbL) assembly of chitosan (CT) and dextran sulfate (Dex). The bFGF was encapsulated into the CT/Dex multilayered capsules by controlling the membrane permeability, and the local and sustained release of bFGF from the capsules was examined. At pH < 8.0, the capsule membrane tightened, and FITC-dextran ( Mw = 4000) could not enter the capsules. However, FITC-dextran ( M w = 250000) easily entered the capsules at pH > 8.0, which can be attributed to the electrostatic repulsion of Dex caused by the deprotonation of the amine group in CT. After treatment with acetic acid buffer (pH 5.6), FITC-dextran or bFGF was successfully encapsulated into the capsules. The amount of encapsulated bFGF was approximately 34 microg/1 mg of capsule. Initially, about 30% of the encapsulated bFGF was released in serum-free medium within a few hours, however, the release was sustained over 70 h. When the bFGF encapsulating capsules were added to cell culture medium (serum-free), the mouse L929 fibroblast cells proliferated well for 2 weeks as compared to cultures, where bFGF was added to the medium or where bFGF and empty hollow capsules were added separately. The proliferation is due to the local and sustained release of bFGF from the adsorbent capsule to the cell surface.