Deletion of admB gene encoding a fungal ADAM affects cell wall construction in Aspergillus oryzae

Mammals possess a unique signaling system based on the proteolytic mechanism of a disintegrin and metalloproteinases (ADAMs) on the cell surface. We found two genes encoding ADAMs in Aspergillus oryzae and named them admA and admB. We produced admA and admB deletion strains to elucidate their biological function and clarify whether fungal ADAMs play a similar role as in mammals. The ?admA?admB and ?admB strains were sensitive to cell wall-perturbing agents, congo red, and calcofluor white. Moreover, the two strains showed significantly increased weights of total alkali-soluble fractions from the mycelial cell wall compared to the control strain. Furthermore, ?admB showed MpkA phosphorylation at lower concentration of congo red stimulation than the control strain. However, the MpkA phosphorylation level was not different between ?admB and the control strain without the stimulation. The results indicated that A. oryzae AdmB involved in the cell wall integrity without going through the MpkA pathway.     

Simultaneous measurement of endothelial cell damage,elastase release and chemiluminescence response during interaction between polymorphonuclear leukocytes and endothelial cells

Using cultured human umbilical cord vein endothelial cells and human blood neutrophils, the interaction between neutrophils and endothelial cells, in vitro, was studied. The aim of the study was to examine whether a respiratory burst stimulation by neutrophils would be observed by neutrophil/endothelial cell interaction and whether the respiratory burst stimulation of neutrophils by endothelial cells could be enhanced by lipopolysaccharide stimulation of neutrophils. The second aim was whether such an effect, or secretion of elastase, could cause an endothelial cell damage in vitro. Chemiluminescence as an indicator of oxygen-derived metabolites produced by neutrophils, elastase release by neutrophils, and endothelial cell damage, based on¹¹¹ In-oxine release from labelled endothelial cells, were measured simultaneously. The present investigation demonstrates that neutrophils can be directly stimulated by endothelial cells. A further amplification of this process following lipopolysaccharide priming up to 10 ng/ml blood could be demonstrated. A slight endothelial cell damage occurs following neutrophil stimulation, although elastase secretion does not increase during interaction between neutrophils and endothelial cells. These results raise the possibility that oxygen-derived metabolites rather than elastase contribute to an endothelial cell damage which might occur in conditions such as endotoxin-induced adult respiratory distress syndrome.     

Effect of essential fatty acids on circulating T cell subsets in patients with colorectal cancer

The effect of essential fatty acids (EFA), given orally as dietary supplements, on the responsiveness in vitro of peripheral blood lymphocytes (PBL), to the mitogen concanavalin A have been studied in 10 patients with localized and 14 patients with advanced colorectal cancer. The degree of lymphocyte activation was assessed by measuring the amount of tritiated [³H]thymidine incorporated into newly synthesised lymphocyte DNA. The results were expressed as stimulation indices. T cell responses to concanavalin A stimulation showed a significant reduction of stimulation indices following EFA supplementation, in both the localized (P=0.026) and advanced (P=0.016) tumour groups, when compared with pretreatment activity in vitro. Mixing experiments, using EFA-supplemented and non-EFA-supplemented lymphocytes with concanavalin A, suggest no enhancement of T suppressor cell activity. Cell surface marker analysis (fluorescence-activated cell sorting for CD phenotyping) revealed a reduction of absolute numbers of CD4⁺ and CD8⁺ lymphocytes following EFA supplementation. The stimulation indices returned to presupplementation values 3 months following cessation of EFA intake. There was no significant change of these indices in the control (no EFA supplementation) advanced tumour group tested. This study suggests that EFA supplementation in patients with colorectal cancer selectively reduces circulating PBL. and T cell subset (including suppressor cells) numbers and/or activity. Such effects may have an important outcome in patients with malignant disease.This work was supported by grants from the Grampian Health Broad, the Royal College of Surgeons of Edinburgh, and Scottish Hospital Endownment Research Trust     

Alterations in the Immunogenic Properties of Sheep Erythrocytes by Sonic Disruption

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 10^7-2 × 10⁸ sonicated SRBC at 6 × 10⁴ g for 30 min [Sup-SRBC]), whose ability to inhibit anti-SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC-primed mice. However, Sup-SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup-SRBC involved helper memory for anti-trinitrophenyl group (TNP) response to challenge with TNP-conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup-SRBC-primed mice was also demonstrated by an in vitro culture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup-SRBC. Repeated stimulation with Sup-SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (10⁶) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.     

A model for the mechanism of stimulation of latex flow in Hevea brasiliensis by ethylene

A model is proposed for ethylene stimulation of latex flow from tapping cuts of Hevea brasiliensis, based on the predicted effects of ethylene on the plasticity and structure of cell walls of the latex vessels. Evidence for the model comes from the effects of ethylene on etiolated shoots of Pisum sativum.     

The mechanism for the effect of selenium supplementation on immunity

Lipid peroxide (LPO) in lymphocytes from mice was evaluated by measuring substances reactive to thiobarbituric acid (TBA). The product resulting from the reaction of TBA with lymphocytes was extracted with n-butyl and fluorescence intensity was determined. The degree of lipid peroxidation, expressed as fluorescence intensity f547, was assessed for stimulation of lymphocytes with concanavalin A (Con A), and was related to lymphocyte proliferation in response to Con A if Se was administered. The lymphocyte proliferation was determined by [³H]thymidine incorporation, expressed as cpm. The effect of superoxide dismutase (SOD), added to cell culture on lymphocyte proliferation was also evaluated. It was found that LPO in lymphocytes before Con A stimulation was significantly less than that after stimulation (p<0.001), and that SOD promoted lymphocyte proliferation dose dependently. The addition of Na₂SeO₃ to lymphocyte culture or supplementation in drinking water to mice decreased the produced LPO in lymphocyte in response to Con A. In the presence of Se, there is an inverse correlation between the levels of LPO in lymphocyte and the stimulated proliferation (r=−0.8902,r=−0.9439). In conclusion, active oxygen species scavenging was proposed as one of the mechanisms for Se to promote immunity.     

DNA Synthesis and Ca2+ Metabolism of Isolated Rat Osteoblast-Like Cells Under Direct Current Stimulation

The experimental model of in vitro culture and direct current stimulation of isolated rat osteoblastlike cells was used to study the effects of mechanoelectric environments on osteogenesis and bone metabolism. DNA synthesis and Ca⁺⁺ concentration in the osteoblastlike cells were measured with an Adherent Cell Analysis and Sorting Interactive Laser Cytometer (ACAS-570). The results showed that a suitable direct current stimulation (i.e., 100 µ;A/cm²) is effective in opening the Ca⁺⁺ passages in the osteoblastlike cell membrane, increasing the intracellular concentration of free calcium ions. This effect can be elicited by the application of direct current for as short a time as about 160 sec and can last for about 110 sec after cessation of stimulation. DNA synthesis is mediated by calcium ion influx after DC stimulation. From the results, we concluded that intra-extra-cel-Mar calcium ion metabolism plays a key role in regulating osteogenesis under stimulation by direct current.     

Cellular Stress Causes Accumulation of the Glucose Transporter at the Surface of Cells Independently of their Insulin Sensitivity

The stimulation of glucose transport in response to various types of stress has been studied. There is no relationship between effects of stress-inducing agents on glucose transport and their effects on cellular protein synthesis. Although the effect of stress on glucose transport appears analogous to its stimulation by insulin, cells that are slightly insulin-sensitive in terms of glucose transport (BHK cells) show a similar degree of stimulation as highly insulin-sensitive cells (differentiated 3T3-L1 cells). External labeling of the transporter protein with a photoactivatable derivative of mannose, 2-N-4-(1-azi-2,2,2-trifluoroethyl) benzoyl-1, 3-bis-(D-mannos-4-yloxy)-propylamine, shows that most of the increased glucose transport activity correlates with an increase in the amount of the transporter on the cell surface. Cells subjected to K⁺-depletion, which inhibits endocytosis and results in an accumulation of receptors at the cell surface, show the same increase in glucose transport as cells exposed to stress; stressed cells show no further increase in glucose transport when subjected to K⁺ depletion. These results support the view (Widnell, C.C., Baldwin, S.A., Davies, A., Martin, S., Pasternak, C.A. 1990. FASEB J 4:1634–1637) that cellular stress increases glucose transport by promoting the accumulation of glucose transporter molecules at the cell surface. Received: 20 June 1995/Revised: 29 September 1995     

Active cell aggregation by immature rat Sertoli cells in primary culture: a role for cell surface glycoproteins.

Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10-day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH-stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0- to 18-hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose-specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of ³H-aminoacid incorporation as well as cell aggregation in 24-hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease ¹²⁵I-FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.     

Sulla Terminologia dei Processi Apomittici

Summary

The Author observing the great disagreement of the terminology usually adopted for the apomictic phenomena, examines the whole series of the phenomena noted both in regards of the formation of the gametophyte and of the embryo. The forming of the gametophyte is fixed on the base of nature of its original cell, normally a haploid spore or »haplospore« (phenomenon of the »haplospory« following the regular meiosis), rarely a diploid spore or »diplospore« (phenomenon of the »diplospory« following the irregular meiosis, as for instance in the case of restitution nucleus, etc.). The original cell can be at least represented by the same cell that usually is the spore mother cell, whether for the outward causes, or for inward causes is lacking the usual reductional process. For the failure of the meiotic process, such a cell cannot be called »spore«.

A cell of the somatic tissue of the sporophyte can even become the original cell of the gametophyte always in case of the absence of the meiosis. These two last cases of apospory are respectively called: »somatic apospory« and »gonial apospory« (= germinal apospory) (Chiarugi 1926).

The formation of the embryo is fixed on the base of the nature of the fecundation. The principal phenomena of the fecundation are represented by the following cases: »amphigamy« (or eugamy, or gamy), »semigamy, »pseudogamy«, »apogamy« »parthenogenesis«. The A. uses the term »apogamy« both for the diploid eggs and for the haploid eggs in which the stimulation of the division is produced by the pollen-tube or by male nuclei, in absence of the gamy.

The term »parthenogenesis« is restricted to the division of the haploid or diploid eggs in which the stimulation is not caused by pollen-tube nor even for the great reason, by the male nuclei existing in it.     

Electrophysiology of Raccoon Cuneocerebellar Neurons

(1) Electrophysiological experiments were undertaken in order to locate and functionally characterize cells of the raccoon main cuneate nucleus (MCN) that can be activated by electrical stimulation of the cerebellum. A total of 98 such units were studied in pentobarbital sodium-anesthetized, methoxyflurane-anesthetized, or decerebrate preparations. Aside from a greater likelihood of resting discharge in the decerebrate preparations, no appreciable variability in physiological properties of the neurons could be attributed to differences in the type of preparation. (2) Using constant latency of response and ability to be blocked by collision as principal criteria, both antidromically (n = 31) and synaptically (n = 67) activated neurons of the main cuneate nucleus could be identified. A small number of MCN neurons could be activated by both cerebellar and thalamic stimulation, but no unit was antidromically activated from both locations. (3) MCN neurons projecting to the cerebellum are located primarily in the ventral polymorphic cell region of the nucleus at and rostral to the obex, corresponding to the “medial tongue” region of Johnson et al (1968). In contrast, neurons synaptically activated from the cerebellum are found throughout the dorsoventral extent of the rostral MCN, including the “clusters” region. (4) The majority of antidromically activated units responded to mechanical stimulation of deeper tissues, and most of these were activated by muscle stretch. Only a small portion (13-15%) of either antidromically or synaptically activated units were classed as light touch units with peripheral receptive fields (RFs) restricted to glabrous surfaces of the forepaw. (5) Glabrous skin RFs located on the digital surfaces are smaller than those located on the palm pads. In both cases, RFs are larger than those associated with primary afferent fibers, but toward the low end of the distribution for MCN neurons not activated by cerebellar stimulation. (6) All MCN units activated by cerebellar stimulation, regardless of modality, respond to mechanical stimulation with trains of irregularly spaced single spikes. Glabrous skin cutaneous mechanoreceptive MCN neurons, whether rapidly or slowly adapting, respond to ramp indentations with an instantaneous frequency which may be described as a power function of ramp velocity, with exponents less than one. These values are in the same range as those previously reported for primary afferents of the cuneate fasciculus (Pubols and Pubols, 1973).     

Transcriptional profiling identifies critical steps of cell cycle reprogramming necessary for Plasmodiophora brassicae‐driven gall formation in Arabidopsis

Plasmodiophora brassicae is a soil‐borne biotroph whose life cycle involves reprogramming host developmental processes leading to the formation of galls on its underground parts. Formation of such structures involves modification of the host cell cycle leading initially to hyperplasia, increasing the number of cells to be invaded, followed by overgrowth of cells colonised by the pathogen. Here we show that P. brassicae infection stimulates formation of the E2Fa/RBR1 complex and upregulation of MYB3R1, MYB3R4 and A‐ and B‐type cyclin expression. These factors were previously described as important regulators of the G2?M cell cycle checkpoint. As a consequence of this manipulation, a large population of host hypocotyl cells are delayed in cell cycle exit and maintained in the proliferative state. We also report that, during further maturation of galls, enlargement of host cells invaded by the pathogen involves endoreduplication leading to increased ploidy levels. This study characterises two aspects of the cell cycle reprogramming efforts of P. brassicae: systemic, related to the disturbance of host hypocotyl developmental programs by preventing cell cycle exit; and local, related to the stimulation of cell enlargement via increased endocycle activity.     

Termination of leech swimming activity by a previously identified swim trigger neuron

Cell Tr2 is a neuron in the subesophageal ganglion of the leech that can trigger swim episodes. In this report, we describe the ability of Tr2 to terminate ongoing swim episodes as well as to trigger swimming. Stimulation of Tr2 terminated ongoing swim episodes in nearly every preparation tested, while Tr2 stimulation triggered swim episodes in only a minority of the preparations. We suggest that the primary role of Tr2 is in the termination rather than the initiation of swimming activity.The swim trigger neuron Tr3 and a swim-gating neuron, cell 21, hyperpolarized during Tr2-induced swim termination. Another swim-gating neuron, cell 204 was sometimes slightly excited, but more often, hyperpolarized during Tr2-induced swim termination. In contrast to these cells, Tr2 stimulation excited another swim-gating neuron, cell 61. The responses of the swimgating cells were variable in amplitude and sometimes not evident during Tr2-induced swim termination. Hence, the effects of Tr2 stimulation on swim-gating neurons seem unlikely to be the direct cause of swim termination.Oscillator cells examined during Tr2-induced swim termination include: 27, 28, 33, 60, 115, and 208. The largest effect seen in an oscillator neuron was in cell 208, which was repolarized by up to 10 mV during Tr2 stimulation. Tr2 stimulation did not produce any obvious synaptic effects in motor neurons DI-1, VI-1, and DE-3. Our findings indicate that other, yet undiscovered, connections are likely to be important in Tr2-induced swim termination. Therefore, we propose that cell Tr2 is probably a member of a distributed neural network involved in swim termination.Abbreviations DP dorsal posterior nerve - Mx midbody ganglion x - Rx neuromere x of the subsesophageal (rostral) ganglion - DE dorsal excitatory motor neuron - DI dorsal inhibitory motor neuron - VI ventral inhibitory motor neuron     

Reexamination of Opioid Stimulation of cGMP Formation in Cell Lines of Neuronal Origin

1. The present study reexamines a previous notion on opioid stimulation of cyclic GMP (cGMP) formation and the retraction of the original findings.2. The effect of opioid agonists on cGMP accumulation in two cell lines of neuronal origin was measured. The proportion of cGMP stimulation in NG108-15 neuroblastoma × glioma hybrid cells resembled the proportion of [Ca²⁺]_in elevation by opioids in this culture. The failure of opioids to stimulate cGMP formation in SK-N-SH human neuroblastoma coincided with the lack of cGMP stimulation by other Ca²⁺ mobilizing agents in these cells. The nitric oxide donor nitroprusside elevated cGMP in both cell lines.3. The implication of the opioid-Ca²⁺-NO-cGMP cellular pathway for opioid activity in vivo is discussed.     

Impact of low‐intensity pulsed ultrasound on the growth of Schizochytrium sp. for omega‐3 production

Schizochytrium sp. is a microalga that is known for its high content of oils or lipids. It has a high percentage of polyunsaturated fatty acids in the accumulated oil, especially docosahexaenoic acid (DHA). DHA is an important additive for the human diet. Large‐scale production of Schizochytrium sp. can serve as an alternative source of DHA for humans as well as for fish feed, decreasing the burden on aqua systems. Therefore, research on improving the productivity of Schizochytrium attracts a lot of attention. We studied the potential of using low‐intensity pulsed ultrasound (LIPUS) in the growth cycle of Schizochytrium sp. in shake flasks. Different intensities and treatment durations were tested. A positive effect of LIPUS on biomass accumulation was observed in the Schizochytrium sp. culture. Specifically, LIPUS stimulation at the ultrasound intensity of 400 mW/cm² with 20 min per treatment 10 times a day with equal intervals of 2.4 h between the treatments was found to enhance the growth of Schizochytrium biomass most effectively (by up to 20%). Due to the nature of cell division in Schizochytrium sp. which occurs via zoospore formation, LIPUS stimulation was inefficient if applied continuously during all 5 days of the growth cycle. Using microscopy, we studied the interval between zoospore formation in the culture and selected the optimal LIPUS application days (Days 0–1 and Days 4–5 of the 5‐day growth cycle). Microscopic images have also shown that LIPUS stimulation enhances zoospore formation in Schizochytrium sp., leading to more active cell division in the culture. This study shows that LIPUS can serve as an additional tool for cost‐efficiency improvement in the large‐scale production of Schizochytrium as a sustainable and environmentally friendly source of omega‐3 (DHA).     

Activation of ERK-FAK signaling pathway and enhancement of cell migration involved in the early interaction between oral keratinocytes and Candida albicans

Purposes  To investigate the early molecular events in oral keratinocytes induced by Candida albicans challenge. Methods  The oral keratinocyte cell line, Tca8113, was used to study the molecular events induced by C. albicans challenge in oral keratinocytes. The phosphorylation statuses of extracellular signal-regulated protein kinase (ERK) and focal adhesion kinase (FAK) upon C. albicans challenge were assessed using specific antibodies and western blotting. Specific inhibitors for ERK and FAK were used to validate the involvement of ERK-FAK signaling cascade. A Transwell insert system-based migration study was performed to evaluate the involvement of the C. albicans-dependent ERK-FAK activation with cell migration. Results  Following the stimulation with C. albicans, a transient activation of ERK was observed, which reached a peak at 10 min post stimulation. Similarly, a transient activation of FAK, the downstream substrate of ERK, was also observed upon C. albicans challenges, which reach the maximum at 20 min. Specific inhibitors for ERK and FAK abolished the C. albicans-induced ERK and FAK activations. The elevated migratory ability of oral keratinocyte was observed upon stimulation with C. albicans, and was synchronous with the ERK-FAK activation. Conclusion  ERK-FAK signaling cascades are involved in the early interaction between the oral keratinocytes and C. albicans, which appears to be linked with the enhanced cell migration. Jing Shi and Xin Zeng contributed equally to this work.     

Immunostimulatory effect of<Emphasis Type="Italic">Bacillus firmus</Emphasis> on mouse lymphocytes

Bacillus firmus (a Gram-positive nonpathogenic and harmless bacterium), was shown to be a strong polyclonal activator of mouse B lymphocytes as estimated by ELISA testing of Ig concentrations in culture supernatants after incubation of BALB/c mouse splenocytes with inactivated bacillus. Synthesis of all main Ig classes and all IgG subclasses was stimulatedin vitro, the considerable effect on IgA formation being the most interesting feature. B cell stimulation was T cell dependent, as was demonstrated by the effect ofB. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+mice. The effect ofB. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-γ and IL-10 (detected by ELISA in splenocyte culture supernatants) showed probable stimulation of Th1 and Th2 subpopulations. Considering the stimulatory effect on IgA formation and macrophage stimulation,B. firmus seems to be a prospective mucosal adjuvant and/or probiotic.     

Mauthner cell-initiated abrupt increase of the electric organ discharge in the weakly electric fishGymnotus carapo

Stimulation of the spinal cord of the electric fish Gymnotus carapo, evoked an abrupt increase in the discharge rate of the electric organ. At the maximum of this response, the rate increased an average of 26 ± 11.8%. The duration of the response was 4.9 ± 2.12 s; its latency was 10.4 ± 1.1 ms. Activation of the Mauthner axon played a decisive role in this phenomenon as indicated by the following: (1) recordings from the axon cap of the Mauthner cell demonstrated that the response was evoked if the Mauthner axon was antidromically activated and (2) a response that was similar to that produced by spinal cord stimulation, was elicited by intracellular stimulation of either Mauthner cell. Stimulation of the eighth nerve could also increase the discharge rate of the electric organ. The effect was greater if a Mauthner cell action potential was elicited. The findings described in the present report, indicate the existence of a functional connection between the Mauthner cell and the electromotor system in Gymnotus carapo. This connection may function to enhance the electrolocative sampling of the environment during Mauthner-cell mediated behaviors. This is a novel function for the Mauthner cell.Abbreviations EHP extrinsic hyperpolarizing potential - EOD electric organ discharge - M-AIR Mauthner initiated abrupt increase in rate - M-cell Mauthner cell - M-axon Mauthner axon - PM pacemaker nucleus - PM-cell pacemaker cell - PPn prepacemaker nucleus - SPPn sublemniscal prepacemaker nucleus