Continuous neurogenesis in the olfactory brain of adult shore crabs, Carcinus maenas

To scrutinize the common belief that the number of neurons in the CNS of adult decapod crustaceans stays constant, in spite of their dramatic postlarval increase in size, I counted olfactory projection neurons (OPNs) in the brains of differently-sized postlarval shore crabs, Carcinus maenas, and performed in vivo labeling of proliferating cells with 5-bromo-2'-deoxyuridine (BrdU) on brains of adults. The number of OPNs increases continuously throughout the postlarval life of shore crabs and approximately doubles from the very young to the oldest animals. Brain sections from adult crabs labeled with BrdU revealed ongoing proliferation of cells in the lateral soma cluster, which consists of OPN cell bodies, and in the cluster of somata of hemiellipsoid body local interneurons, which are the targets of the OPNs. Post-injection survival times from 5.5 to 120 h revealed a small but relatively constant number of labeled nuclei with neuronal morphology in both soma clusters of all specimens (31.3 +/- 9.5 S.D. nuclei per lateral cluster, n = 29; 20.1 +/- 4.5 S.D. nuclei per hemiellipsoid body cluster, n = 10). The labeled nuclei were located in a distinct proliferative zone in each cluster. There were significantly more labeled nuclei in both soma clusters after a prolonged post-injection survival time of 1 month (71.3 +/- 7.8 S.D. nuclei per lateral cluster, n = 4; 38.2 +/- 7.1 nuclei per hemiellipsoid body cluster, n = 6). In both soma clusters the labeled nuclei formed a compact group that was dislocated from the proliferation zone towards the outer edge of the cluster. In the proliferation zone of the lateral cluster histological stainings revealed cell bodies of typical neuronal shape that are slightly smaller and more intensely stained than the surrounding OPN somata. Some of these cell bodies were captured in various stages of mitosis. Collectively, these data indicate that continuous neurogenesis occurs in the central olfactory pathway of the brain of shore crabs throughout their entire adult life. This unexpected structural plasticity may enable long-lived decapod crustaceans to adapt to ever-changing olfactory environments.     

The binding of Cd(II) to the hemocyanin of the Mediterranean crab Carcinus maenas

In idiopathic dilated cardiomyopathy (IDC), the relation between the signal-averaged electrocardiogram and ventricular tachycardia (VT) remains unclear. In this study, conventional time domain and frequency domain analyses (2-dimensional, spectral temporal mapping and spectral turbulence analysis) of the signal-averaged electrocardiogram were performed in 64 patients with IDC. Eight patients had a history of symptomatic sustained VT and an additional 24 had nonsustained VT recorded during ambulatory electrocardiography. Conventional time domain analysis, using the 25 and 40 Hz filter, and spectral temporal mapping, detected late potentials within the terminal QRS in 8 (13%), 14 (22%) and 18 (28%) patients, respectively. Late potentials were seen more often in patients with than without VT, and in patients with sustained versus nonsustained VT, but these differences were not significant. The predictive accuracy of these techniques in detecting either form of VT were: sensitivity, 22, 25 and 31%; specificity, 97, 81 and 75%; and overall predictive value, 59, 53 and 50%, respectively. Two-dimensional frequency domain analysis of the signal-averaged electrocardiogram revealed a higher energy and area ratio in patients with than without VT (entire QRS), and in patients with sustained versus nonsustained VT (entire QRS and terminal QRS). Spectral turbulence analysis was abnormal in 24 patients (39%), but no differences were observed between patients with and without VT. During follow-up (mean duration 18 +/- 14 months), 5 patients had arrhythmic events (3 died suddenly, 1 had aborted sudden death and 1 developed sustained VT).(ABSTRACT TRUNCATED AT 250 WORDS)     

CYP330A1 and CYP4C39 enzymes in the shore crab Carcinus maenas: sequence and expression regulation by ecdysteroids and xenobiotics

Cytochrome P450 enzymes (CYP enzymes) catalyse important metabolic reactions of exogenous and endogenous substrates, including steroid hormones. Here, we report the first two CYP sequences from the shore crab, Carcinus maenas. Two complete cDNAs isolated from crab hepatopancreas encode CYP enzymes named CYP330A1, the first member of a new family, and CYP4C39. CYP330A1 is closest related to members of the CYP2 family (37.3% identical to mouse CYP2J6) and CYP4C39 is most identical to crayfish CYP4C15 (59.5%). CYP330A1 gene expression was induced in hepatopancreas of male green intermoult crabs by ecdysone and ponasterone A, but also by benzo(a)pyrene and phenobarbital. CYP330A1 induction was not observed in red crabs. The present results indicate that the CYP330A1 enzyme may be involved in ecdysteroid metabolism, presumably catabolism, and in the detoxification of environmental pollutants. Ecdysteroids or xenobiotics did not affect CYP4C39 gene expression. The fact that both ecdysteroids and xenobiotics affect CYP330A1 gene expression indicates that mutual interactions between chemical exposures and endocrine functions may exist in the shore crab.     

Experimental coupling of crab (Carcinus maenas) second maxilla neural motor to an alternating current

Movements of the crab gill bailer were entrained to an alternating current applied to the thoracic ganglion. There was little distortion of the muscle recruitment cycle, both absolute and relative coordination were observed, and the phase of the driven system in the driving cycle was a function of the difference between free running and driving frequencies.     

Infestation of an introduced host, the European green crab, Carcinus maenas, by a symbiotic nemertean egg predator, Carcinonemertes epialti

The recent introduction of the European green crab, Carcinus maenas, to the west coast of the U.S. has provided an opportunity for host transfer of the symbiotic nemertean egg predator, Carcinonemertes epialti, from its native shore crab host, Hemigrapsus oregonensis to the exotic C. maenas. Two surveys of C. maenas in Bodega Harbor, California, revealed that, in March 1995 prevalence of C. epialti on C. maenas was significantly lower than on H. oregonensis (11% versus 74%), but in November 1995 there was no significant difference between the 2 species (79% versus 98%). Only juvenile C. epialti were recovered from C. maenas in March 1995. However, in November 1995, ovigerous C. maenas were harboring actively feeding adult worms. Prevalence in both crab species significantly differed from March to November. Laboratory studies revealed that C. epialti fed and reproduced on eggs of C. maenas. The feeding rate of C. epialti on C. maenas eggs (2.5 eggs/trial) was not significantly different from that on H. oregonensis eggs (3.6 eggs/trial). Our findings suggest that this nemertean may have less host specificity than was previously thought. If C. epialti causes brood mortality of C. maenas in nature, it could potentially impact populations of this exotic crab.     

Cryptic invasions of the crab Carcinus detected by molecular phylogeography

Coastal marine ecosystems world-wide are threatened by invasions of nonindigenous species. The ubiquity of marine sibling species identifiable only by genetic analysis suggests that many invasions are cryptic and therefore undetected, causing an underestimation of the actual number and impacts of invading species. We test this hypothesis with European crabs in the genus Carcinus that have invaded five regions globally. Partial 16S ribosomal RNA gene sequences confirm sibling species status of morphologically similar Atlantic C. maenas and Mediterranean C. aestuarii. Based on 16S rRNA haplotypes, crabs from California, New England and Tasmania were all C. maenas. However, we report the cryptic multiple invasion of both species in Japan and South Africa, where only C. aestuarii and C. maenas, respectively, were previously recognized.     

In vitro production of plasminogen activator by human granulosa cells

Plasminogen activator (PA) production by granulosa cells has been demonstrated in several species. In the human ovary, tissue-type PA and urokinase-type PA antigens have been found in the follicular fluids, but neither PA activity nor mRNA for both enzymes was found in granulosa cells of preovulatory follicles. All of these studies were performed on granulosa cells collected from follicles immediately before ovulation, when the cells were already in the luteal phase. In the attempt to better characterize the PA/plasminogen system in the human ovary, we examined PA and PA inhibitor (PAI) production in cultures of granulosa cells obtained from normally cycling untreated women at different stages of the cycle. In addition, we analyzed granulosa-luteal cells obtained from hormonally stimulated women undergoing gamete intrafallopian tube transfer, as a model of late phase follicular development. Zymographic analysis as well as immunoprecipitation with specific antisera revealed that granulosa cells from follicles at early phases of antral stages secreted high levels of PA of the urokinase type in the medium. No free tissue-type PA activity was found in any of the examined samples. On the contrary, free PAI was undetectable in medium obtained from granulosa cell cultures, and it was abundant in granulosa-luteal cell cultures, where it was found in two forms. These data show that in the human ovary as in that of the rat, PAs and PAIs are tightly time regulated. The timing of PA production in human granulosa cells suggests a role for PA activity at early stages of follicular maturation.     

Angiotensin II induces superoxide anion production by mesangial cells

BACKGROUND: The recognized role of angiotensin II (Ang II) in the pathogenesis of the progression of renal disease cannot be solely attributed to Ang II's hemodynamic effects. Indeed, growth stimulating signals driven by Ang II promote mesangial cell (MC) hypertrophy and extracellular matrix production, prominent features of progressive glomerular injury. Superoxide anion (O2-) avidly interacts with nitric oxide, an endogenous vasodilator that inhibits growth factor stimulated MC growth and matrix production. In addition, O2- acting as an intracellular signal is linked to growth related responses such as activation of mitogen activated protein (MAP) kinases. The studies reported herein were designed to investigate: (a) whether Ang II induces MC O2-production and (b) if increased O2- production elicits growth responses in MC. METHODS: MC were exposed to Ang II for 24 or 48 hours. In some experiments, in addition to Ang II, MC were exposed to: diphenylenieodonium (DPI), an inhibitor of the flavin containing NADH/NADPH oxidase; losartan (LOS), an Ang II type 1 (AT1) receptor blocker; PD 98059, a MAP kinases inhibitor; the protein kinase C inhibitors Calphostin C or H-7; and the tyrosine kinase inhibitors, herbymycin A or genistein. RESULTS: Ang II (10(-5) M to 10(-8) M) dose dependently increased MC O2- production up to 125% above control (ED 50 5 x 10(-7) M). LOS as well as DPI, and the PKC inhibitors blocked Ang II stimulated MC O2- production. Ang II dose dependently increased MC 3H-leucine incorporation, and MC protein content, two markers of MC hypertrophy, as well as 3H-thymidine incorporation, a marker of MC hyperplasia. PD98059, a specific inhibitor of MAP kinases prevented Ang II induced MC hypertrophy. Moreover, LOS, DPI, and the PKC inhibitors each independently inhibited MC 3H-leucine incorporation, thereby establishing the specificity of Ang II induced O2- in driving MC hypertrophy. CONCLUSIONS: The current studies demonstrate a previously unrecognized link between Ang II and MC O2- production that may participate in the pathophysiology of progressive renal disease by concomitantly affecting the hemodynamics of the glomerular microcirculation as well as growth related responses of MC to injury.     

An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas

Appropriate low dosages of vincristine stimulate megakaryocytopoiesis and produce thrombocytosis. In this study of the thrombocytotic action of vincristine, administration of a single dose of 0.1 mg/kg to rats produced an increase in megakaryocyte concentration, diameter and 24 h [3H]thymidine labelling index. Transfusion of one body equivalent of platelets from normal donors prevented stimulation of megakaryocytopoiesis by vincristine whereas platelets from vincristine-treated donors did not. These results suggest that vincristine stimulates megakaryocytopoiesis by altering the functional role of circulating platelets in the regulation of thrombopoiesis.     

In vitro hydrogen production by glucose dehydrogenase and hydrogenase

A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity.     

In vitro inhibition of microbial methane production by 9,10-anthraquinone

Monensin, 2,2-dichloroacetamide, and 9,10-anthraquinone were incubated for 24 h in ruminal fluid and buffer with 100:0, 50:50, and 10:90 forage-concentrate diets. Monensin (.5 ppm of the fluid) increased (P < .05) the molar proportion of propionate in the 50 and 100% forage diets but not in the high concentrate diet. At the same level of addition, 2,2-dichloroacetamide increased (P < .05) the molar proportion of propionate only in the 50:50 forage-concentrate diet. Relative to control cultures, monensin and 2,2-dichloroacetamide numerically decreased methane production in the 10 and 100% forage diets and decreased (P < .05) methane in the 50% forage diet. Hydrogen production was unaffected by treatment. Lack of an effect on fermentation end products in the high concentrate diet was probably a result of the low dose levels. In general, increasing levels of 9,10-anthraquinone (.5, 1.0, and 5.0 ppm) reduced total VFA concentration and the molar proportion of acetate, and increased propionate, butyrate and valerate. Increasing levels of 9,10-anthraquinone caused linear and quadratic decreases (P < .05) in methane production, and increases (P < .05) in hydrogen. There were no consistent effects on ammonia concentration in culture fluid from any of the compounds. In continuous culture of a 10:90 forage-concentrate diet, addition of 9,10-anthraquinone (10 ppm of the fluid/12 h) caused changes similar to those observed in batch culture with the exception of a decreased (P < .05) molar percentage of propionate, which may have been due to the high dose. The data are interpreted to indicate that 9,10-anthraquinone has the ability to alter in vitro microbial fermentation.     

Chitooligosaccharides stimulate Atlantic salmon, Salmo salar L., head kidney leukocytes to enhanced superoxide anion production in vitro

Chitosans and chitooligosaccharides stimulated Atlantic salmon, Salmo salar L., head kidney leukocytes in vitro to produce elevated levels of superoxide anion. Both soluble and insoluble chitooligosaccharides were stimulatory 2 and 7 days after addition. Protein-chitooligosaccharide conjugates were also stimulatory in vitro both at 2 and 7 days after addition. Deacetylation seemed to be of little importance for the stimulatory capacity. High concentrations of the 80% deacetylated chitosan/chitooligosaccharides were toxic to the leukocytes as judged by reduced reduction of nitroblue tetrazolium and morphology.     

水稻硫转运蛋白基因OsST的亚细胞定位

[目的]构建水稻硫酸根转运基因OsST与YFP黄色荧光蛋白融合表达载体,对OsST蛋白进行亚细胞定位.[方法]从水稻叶片的cDNA中克隆OsST基因ORF全长,测序验证后连入pA7-YFP表达载体,通过基因枪将融合载体转入洋葱上表皮细胞,在激光共聚焦显微镜下观察细胞中荧光发光部位.[结果]OSST蛋白定位于细胞膜和核膜上.[结论]为进一步研究硫转运蛋白的功能及硫酸根运输的机理奠定了基础.     

In vitro infection of smooth muscle cells by Chlamydia pneumoniae

Recent observations have shown that both Chlamydia pneumoniae antigens and DNA may be found within atherosclerotic lesions. In this study, we evaluated the ability of C. pneumoniae to infect cells that make up atherosclerotic lesions, including endothelial cells, smooth muscle cells, and cholesterol-loaded smooth muscle cells. The organism readily infected rabbit, bovine, and human aortic smooth muscle cells. Cholesterol-loaded smooth muscle cells were even more susceptible to C. pneumoniae infection. Chlamydia trachomatis inefficiently infected smooth muscle cells, demonstrating that this is not a characteristic of all members of the genus Chlamydia. C. pneumoniae infected bovine endothelial cells poorly. This study demonstrates that C. pneumoniae readily infects one of the important types of cells found within atherosclerotic lesions, i.e., smooth muscle cells with and without cholesterol loading.     

A genetic system involving superoxide causes F1 necrosis in wheat (T. aestivum L.)

A genetic system in wheat is described in which F1 produced by crossing a drought tolerant cultivar C306 and high yielding cultivar WL711 exhibits leaf necrosis leading to the death of the plant. The mechanism underlying hybrid necrosis is not yet known. The hybrid exhibited a higher level of superoxide anion compared to the healthy leaves of parents at similar developmental stages. This increase in superoxide generation preceded necrotic lesion formation and displayed a gradient from the leaf tip to base. The leaf tip where necrotic lesions make their first appearance exhibited a higher level of superoxide compared to the base. Superoxide anion thus appears to play a vital role in necrosis of leaves in F1 hybrid. This genetic system can be a model system for understanding cell death in higher plants.     

In vitro production of parathyroid hormone-related protein by cholesteatoma and normal skin

Severe inflammation and infection of the middle ear occasionally results in clinical evidence of bone resorption but with the addition of the cholesteatoma epithelium it becomes inevitable. This study examined production by the cholesteatoma epithelium of a bone resorbing factor, namely parathyroid hormone-related protein (PTH-rP), which would not be expected in inflammatory states alone. PTH-rP was detected in the conditioned medium of primary and secondary cell cultures derived from 12 cholesteatoma biopsies. The levels of PTH-rP were significantly greater than in control cultures of cells derived from normal scalp tissue. Production by the cholesteatoma epithelium may explain the increased incidence of bone resorption in this disease, particularly in cases where inflammation is minimal.     

In vitro immune modulation by antibodies coupled to tumour cells

Modification of autologous tumour cells to express the immune costimulator B7.1 is a potential strategy for immunotherapy of cancer. Previously, this has involved introduction of genetic material into cells, in vitro culture, and confirmation of the protein product on the cell surface. This is possible only if sufficient tumour is obtainable and efficiently modified in a short time. Whilst progress has been made on ex vivo tumour cell culture and transfection/infection procedures there are still tumour types for which the present means of gene transfer are not efficient enough. We describe a highly efficient in vitro procedure for the modification of over 99% of the cells in a population, allowing the expression of cell surface proteins with potential immune modulatory activities. This procedure, which can be completed in as little as 24 h with no upper limit on cell number, utilizes succinimide esters to label cell surface proteins with biotin covalently. Biotinylated cell membrane proteins then anchor an avidin bridge for immobilizing protein G'-biotin. This can serve to bind immunoglobulin (Ig) molecules via their Fc region such that the variable region of the antibody is freely and functionally available. In the present study the binding of a stimulatory mouse anti-human CD28 monoclonal antibody to the surface of tumour cells is used to show that the modified cells are capable of co-stimulating T cells in vitro. The simplicity of the method, and the use of common reagents, represents a further step towards a realistic, truly 'off-the-shelf', nongene immunotherapy protocol.     

In vitro modulation of L-arginine transport in trophoblast cells by glucose

BACKGROUND: The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. METHODS: This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. RESULTS: At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. CONCLUSION: These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.     

Ultraviolet radiation-induced production of superoxide radicals by selected antibiotics

The ability of certain drugs and chemicals to induce cutaneous phototoxicity and DNA damage has been attributed to free radical formation during photolysis. In this context we have observed that the synergistic action of commonly used antibiotics and ultraviolet radiation (UVR) exhibited strong superoxide radical (O2-) generation potential in the following order: benzylpenicillin > amphotericin > ampicillin > nystatin > spectinomycin > gentamicin. Commercially available penicillin, nystatin, ampicillin and gentamicin also generated O2- under similar conditions. The results suggest that due precaution are necessary to avoid UVR after the intake of photoreactive drugs.     

Effects of liposome-encapsulated hemoglobin on phorbol ester-induced superoxide production and expression of costimulatory molecules by monocytes in vitro

The effects of Neo Red Cell (NRC), a liposome-encapsulated hemoglobin (LEH), on the phorbol ester-induced superoxide production and the expression of costimulatory molecules by human peripheral monocytes were investigated. The treatment of human mononuclear cells with NRC caused the potentiation of superoxide production in response to PMA. The longer incubation (20 h) resulted in a decrease in the PMA-induced superoxide production, which was in parallel to a decrease in the viability of the monocytes. A flow cytometric analysis showed that a slight expression of CD80 (B7-1) on monocytes was induced by NRC treatment, whereas the constitutive expressions of CD86 (B7-2) and CD54 (ICAM-1) were unchanged. The activation of monocytes with interferon-gamma (IFN-gamma) induced the expressions of CD80, CD86, and CD54 under all conditions tested, but NRC treatment tended to decrease the IFN-gamma-induced expression of CD54 on monocytes. These results suggest that the administration of LEH may modify the functions of human monocytes.