Vasoactive intestinal peptide prevents PKCε-induced intestinal epithelial barrier disruption during EPEC infection.

We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-κB pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKCθ and PKCε during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKCε in EPEC-induced barrier disruption. Furthermore, activation of PKCε and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKCε by VIP pretreatment or the PKCε inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCε inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKCε levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKCε activation, thereby attenuating EPEC-induced barrier disruption.

Activity-dependent activation of presynaptic metabotropic glutamate receptors in locus coeruleus.

Synaptic activation of metabotropic glutamate receptors (mGluRs) in the locus coeruleus (LC) was investigated in adult rat brain slice preparations. Evoked excitatory postsynaptic potentials (EPSPs) resulting from stimulation of LC afferents were measured with current clamp from intracellularly recorded LC neurons. In this preparation, mGluR agonists (+/-)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) activate distinct presynaptic mGluRs, resulting in an inhibition of EPSPs. When two stimuli were applied to afferents at intervals >200 ms, the amplitude of the second [test (T)] EPSP was identical in amplitude to the first [control(C)]. However, when a stimulation volley was delivered before T, the amplitude of the latter EPSP was consistently smaller than C. The activity-dependent depression (ADD) was dependent on the frequency and duration of the train and the interval between the train and T. ADD was potentiated in the presence of an excitatory amino acid (EAA) uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC, 100 microM), changing the T/C ratio from 0.84 +/- 0.05 (mean +/- SE) in control to 0.69 +/- 0.04 in t-PDC (n = 9). In the presence of t-PDC, the depolarizing response of LC neurons to focally applied glutamate was also increased. Together, these results suggest that accumulation of EAA after synaptic stimulation may be responsible for ADD. To test if ADD is a result of the activation of presynaptic mGluRs, the effect of selective mGluR antagonists on ADD was assessed. In the presence of t-PDC, bath applied (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4, 500 microM), a mGluR group III antagonist, significantly reversed the decrease in T/C ratio after a train stimulation [from 0.66 +/- 0.04 to 0.81 +/- 0.02 (mean +/- SE), n = 5]. The T/C ratio in the presence of MAP4 was not different from that measured in the absence of a stimulation volley. Conversely, ethyl glutamic acid (EGLU, 500 microM), a mGluR group II antagonist, failed to alter the T/C ratio. Together, these results suggest that, in LC, group III presynaptic mGluR activation provides a feedback mechanism by which excitatory synaptic transmission can be negatively modulated during high-frequency synaptic activity. Furthermore, this study provides functional differentiation between presynaptic groups II and III mGluR in LC and suggests that the group II mGluR may be involved in functions distinct from those of group III mGluRs.

Cell surface analysis techniques: What do cell preparation protocols do to cell surface properties?

Cell surface analysis often requires manipulation of cells prior to examination. The most commonly employed procedures are centrifugation at different speeds, changes of media during washing or final resuspension, desiccation (either air drying for contact angle measurements or freeze-drying for sensitive spectroscopic analysis, such as X-ray photoelectron spectroscopy), and contact with hydrocarbon (hydrophobicity assays). The effects of these procedures on electrophoretic mobility, adhesion to solid substrata, affinity to a number of Sepharose columns, structural integrity, and cell viability were systematically investigated for a range of model organisms, including carbon- and nitrogen-limited Psychrobacter sp. strain SW8 (glycocalyx-bearing cells), Escherichia coli (gram-negative cells without a glycocalyx), and Staphylococcus epidermidis (gram-positive cells without a glycocalyx). All of the cell manipulation procedures severely modified the physicochemical properties of cells, but with each procedure some organisms were more susceptible than others. Considerable disruption of cell surfaces occurred when organisms were placed in contact with a hydrocarbon (hexadecane). The majority of cells became nonculturable after air drying and freeze-drying. Centrifugation at a high speed (15,000 x g) modified many cell surface parameters significantly, although cell viability was considerably affected only in E. coli. The type of washing or resuspension medium had a strong influence on the values of cell surface parameters, particularly when high-salt solutions were compared with low-salt buffers. The values for parameters obtained with different methods that allegedly measure similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of the in situ microbial cell surface as closely as possible. There is an urgent need for new, reliable, nondestructive, minimally manipulative cell surface analysis techniques that can be used in situ.

Actions of excitatory amino acids on mesencephalic trigeminal neurons.

Mesencephalic trigeminal (MeV) neurons are primary sensory neurons of which the cell soma is located within the brainstem, and is associated with synaptic contacts. In previous studies it has been reported that these cells are resistant to kainic acid excitotoxicity, and have little or no responsiveness to exogenously applied glutamate or selective agonists. In an in vitro slice preparation with intracellular recording, we have found that these cells respond to pressure-applied glutamate, N-methyl-D-aspartic acid (NMDA), kainate (KA), and (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). The kainate and AMPA responses appear to be mediated by different receptors, at least in part, since they exhibit differing sensitivity to an AMPA receptor selective antagonist. The agonists generally evoke larger responses than glutamate and exhibit a long-duration desensitization requiring approximately 10 min for full recovery. Some cross-desensitization between the glutamate agonists is also observed. Mesencephalic trigeminal neurons exhibit high-frequency oscillatory activity during depolarizations that approach threshold potentials, and these could combine with transmitter-induced depolarizations to enhance the excitability of these cells. Previous reports of nonsensitivity to glutamate and to kainate excitotoxicity are attributable to relatively small responses, and to the desensitization expressed by these neurons.

Modeling pH effects on microbial growth: a statistical thermodynamic approach.

This paper applies a statistical thermodynamic approach to the kinetics of microbial growth influenced by pH. A general equation is developed and shown to provide a good theoretical basis for the existing pH models that have been widely used to describe the effects of pH on microbial growth kinetics. Four experimental data sets are used to test the general equation developed. The four data sets exhibited a variety of functional curve shapes, for example, symmetrical and asymmetrical bell-shaped, when the specific growth rate of microorganisms is plotted as a function of pH. All four data sets are found to be well represented by the general equation. The existing pH model was, however, found to represent only one out of four data sets, i.e., the symmetrical case.

P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro.

The neurotransmitter L-glutamate has been associated with a number of developmental events within the central nervous system including synaptogenesis and the refinement of topographically ordered neural maps. As a model for studying such events at the molecular level, we have examined the expression of glutamate and glutamate receptors in neurons that develop from P19 cells in response to retinoids. We report here that many P19-derived neurons do contain glutamate in secretory vesicles and that this glutamate appears to function as a neurotransmitter. The neurotransmitter GABA is also present in these cultures and both glutamate and GABA appeared to co-localize in some neuronal processes. Both neurotransmitters were released from the neurons in response to membrane depolarization. These neurons also express various glutamate receptor subunits including GluR1, GluR4 and NMDAR1 as detected by immunological methods. Using whole-cell patch-clamping, we have recorded spontaneous postsynaptic potentials which increase in both amplitude and frequency with time in culture and which are sensitive to the glutamate antagonist kynurenic acid Thus, P19-derived neurons mature in culture and form electrically active neural networks involving glutamate and glutamate receptors.

Modulation of excitatory synaptic transmission in locus coeruleus by multiple presynaptic metabotropic glutamate receptors.

Metabotropic glutamate receptors have been implicated in modulation of synaptic transmission in many different systems. This study reports the effects of selective activation of metabotropic glutamate receptors on synaptic transmission in intracellularly recorded locus coeruleus neurons in brain slice preparations. Perfusion of either L-2-amino-4-phosphonobutyric acid (L-AP4; 0.1-500 microM) or (+/-)-1-aminocyclopentane-trans-1,3,dicarboxylic acid (t-ACPD; 0.1-500 microM) caused a depression of excitatory postsynaptic potentials in a dose-dependent fashion to about 70% inhibition. Both agonists exerted their effects at relatively low concentrations with estimated EC50s of 2.6 microM and 11.5 microM for L-AP4 and t-ACPD, respectively. This inhibition was not observed with the potent group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG; 100 microM). Conversely, (R)-4-carboxy-3-hydroxyphenyl-glycine (4C-3H-PG), a group I antagonist/group II agonist, and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC), a novel and specific group II agonist, also caused an inhibition of excitatory postsynaptic potentials. Both t-ACPD and L-AP4 produced an increase in paired-pulse facilitation, and failed to change the locus coeruleus response to focally applied glutamate, indicating a presynaptic locus of action. The L-AP4 inhibition was antagonized by (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4: group III antagonist) but not by (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG; mixed antagonist], suggesting that this agonist acts through a type 4 metabotropic glutamate receptor. Conversely, t-ACPD was antagonized by MCPG and by ethyl glutamate (group II antagonist), but not by aminoindan dicarboxylic acid (AIDA; group I antagonist) or MAP4, suggesting that this agonist acts on a type 2 or 3 metabotropic glutamate receptor. Taken together, these results suggest that two pharmacologically distinct presynaptic metabotropic glutamate receptors function in an additive fashion to inhibit excitatory synaptic transmission in locus coeruleus neurons. These receptors may be involved in a feedback mechanism and as such may function as autoreceptors for excitatory amino acids.

Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures.

A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells; a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of fluorescently labelled oligonucleotide probes to rRNA. The method allows monitoring of gene expression and quantification of beta-galactosidase activity in single cells.

Mesencephalic trigeminal neuron responses to gamma-aminobutyric acid.

Mesencephalic trigeminal neurons are primary sensory neurons which have cell somata located within the brain stem. In spite of the presence of synaptic terminals on and around the cell somata, applications of a variety of neurotransmitter substances in earlier studies have failed to demonstrate responses. Using intracellular recording in a brain slice preparation, we have observed prominent depolarizations and decreases in input resistance in response to applications of gamma-aminobutyric acid (GABA) in most recorded mesencephalic trigeminal neurons. Those cells failing to respond were located deeply within the slice, and the low responsiveness was shown to be related to uptake of GABA in the slice. The responses were direct, since they remained during perfusion with a low calcium, high magnesium solution that blocks synaptic transmission. The responses were mimicked by the GABA(A) receptor agonist isoguvacine, and blocked by GABA(A) receptor antagonists. The GABA(B) receptor agonist baclofen evoked no changes in membrane potential or input resistance in neurons exhibiting depolarizations with GABA application. Tests of neuronal excitability during GABA applications indicated that the excitatory effects of the depolarization prevail over the depressant effects of the increase in membrane conductance. In situ hybridization histochemistry indicated that the GABA(A) receptors in Me5 cells are comprised of alpha2, beta2 and gamma2 subunits.

Modeling substrate inhibition of microbial growth.

This article presents a general equation for substrate inhibition of microbial growth using a statistical thermodynamic approach. Existing empirical models adapted from enzyme kinetics, for example, the Haldane-Andrews equation, often criticized for not being physically based for microbial growth, are shown to derive from the general equation in this article, and their empirical parameters are shown to be well defined physically. Three sets of experimental data from the literature are used to test the modeling abilities of the general equation to represent experimental data. The results are compared with those obtained by fitting the same data set to a widely used empirical model existing in the literature. The general equation is found to represent all three experimental data sets better than the alternative model tested. In addition, a graphical method existing in enzyme kinetics is successfully adapted and further developed to determine the number of inhibition sites of a basic functional unit of a bacterial cell.

Construction and use of a new vector/transposon, pLBT::mini-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium.

The previously described pLOFKm transposon delivery plasmid (J.Bacteriol. (1990) 172, 6557-6567) was engineered such that a promotorless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.

Characterisation of carbon dioxide-inducible genes of the marine bacterium, Pseudomonas sp. S91.

Characterisation of two genes in Pseudomonas sp. S91 that are responsive to carbon dioxide is reported. These were identified by random transposon mutagenesis leading to fusion of the Escherichia coli lacZ reporter gene to the genes of interest. Expression of the genes' promoters was quantified by measuring the reporter gene product, beta-galactosidase. beta-Galactosidase synthesis was induced when cells were exposed to 10% CO2 on solid media or during growth in aqueous phase when the culture density was greater than 1 at 610 nm, in either rich or minimal media. Induction of beta-galactosidase synthesis was not due to: increased alkalinity, onset of stationary phase, build up of soluble metabolites in the culture supernatant, or cell density-dependent signalling. The CO2-inducible gene fusions were not induced by other environmental conditions that are known to stimulate global regulators of environmental gene expression. Benzoic acid (2 mM) induced beta-galactosidase synthesis in one of the mutants indicating the Co2 response may involve the intracellular CO2 partial pressure/bicarbonate ion concentration/pH equilibrium.

Neurons derived from P19 embryonal carcinoma cells develop responses to excitatory and inhibitory neurotransmitters.

Cells of the P19 line of embryonal carcinoma cells differentiate into neurons, astrocytes and oligodendrocytes following treatment with retinoic acid. The neurons from these differentiating P19 cultures synthesize a pattern of neurotransmitters that resembles that of neurons of the forebrain. We treated P19 cells with retinoic acid and then implanted them into the striatum of adult rats. After times ranging from 1 to 15 weeks post-implantation, brain slices containing the implanted tissue were prepared and used for intracellular recording of electrical activity and responsiveness to application of neurotransmitters. Within 2 weeks of implantation, the P19-derived neurons had developed responsiveness to the excitatory neurotransmitter glutamate and the inhibitory transmitters gamma-aminobutyric acid and glycine. These neurons also exhibited spontaneous synaptic potentials. The responses to glutamate appear to be mediated by N-methyl-D-aspartic acid as well as non-N-methyl-D-aspartic acid receptor subtypes. Gamma-aminobutyric acid evoked bicuculline-sensitive depolarizing responses in the younger grafts and biphasic depolarizing/hyperpolarizing responses in older ones. Responses to glycine were strychnine sensitive and also showed age-related changes from depolarizing to biphasic character. Synaptic potentials in the younger grafts were exclusively depolarizing, but in older ones both depolarizing and hyperpolarizing events were observed. The synaptic potentials appear to arise from synaptic connections between P19-derived neurons within the grafts. Many of the features of P19-derived neurons are similar to those of neurons in the developing forebrain.

In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line.

The multipotent embryonal carcinoma (EC) P19 cell line differentiates into neurons, glia and smooth muscle following exposure to retinoic acid (RA). RA-induced differentiation is irreversible and the neurons that develop are abundant, post-mitotic, and survive for prolonged periods in culture or when grafted into the CNS of adult rats. Striatal slices containing grafted P19 cells were studied with intracellular recording and labelling techniques to examine the development of electrophysiological and morphological properties of P19-derived neurons over a period of 6 to 120 days after grafting into ibotenic acid lesioned striatum. Cells from 1-week-old grafts had a range of immature electrophysiological characteristics including unstable resting membrane potentials (RMP's) and very high membrane input resistances (Rin's). Many were not able to produce action potentials (AP's). In contrast, the majority of cells recorded from 2- and 3-week-old grafts had stable RMP's, moderate Rin's, and were able to produce regenerative AP's. In grafts over 4 weeks of age, the majority of P19-derived neurons had mature neuronal electrophysiological characteristics including RMP's of -60 mV, Rin's of 100-300 M omega, and overshooting AP's. Morphologically, P19 derived neurons increase in soma size from 12-15 mu in diameter in 7-14-day-old grafts, to 25-35 mu in diameter in grafts 50-120 days old. Developing neurons exhibited a variety of morphotypes with increasingly complex processes and lengths of process extension. Our results demonstrate a developmental progression of the electrophysiology of P19-derived neurons, culminating in mature characteristics closely resembling those of adult rodent hippocampal or cortical pyramidal neurons. The ability to easily alter these cells genetically provides a powerful model for addressing issues specific to neuronal development.

Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.

The effects of surfaces on the physiology of bacteria adhering to surfaces or immobilized within biofilms are receiving more interest. A study of the effects of hydrophobic and hydrophilic substrata on the colonization behavior of a marine bacterium, SW5, revealed major differences in the morphology of SW5 on these surfaces. Using epifluorescence, scanning confocal laser, and on-line visualization (time-lapse video) microscopy, the organisms at hydrophobic surfaces were characterized by the formation of tightly packed biofilms, consisting of single and paired cells, whereas those at hydrophilic surfaces exhibited sparse colonization and the formation of chains more than 100 microns long, anchored at the surface by the terminal (colonizing) cell. The results are discussed in terms of the possible factors inducing the observed morphological differences and the significance of these differences in terms of biofilm structure and plasmid transfer when SW5 is the recipient organism.

Angiotensin II depresses glutamate depolarizations and excitatory postsynaptic potentials in locus coeruleus through angiotensin II subtype 2 receptors.

A previously reported depression of glutamate responses by angiotensin II was investigated to define the nature of this neuromodulatory effect. Studies were carried out in an vitro brain slice preparation containing the locus coeruleus, using intracellular recordings, and iontophoretic, micropressure and bath perfusion methods for application of drugs. The angiotensin action was found to be blocked by a non-peptide antagonist specific for the angiotensin type 2 receptor, and not by an antagonist selective for the type 1 receptor. Excitatory postsynaptic potentials mediated primarily by excitatory amino acids were also depressed by angiotensin II. The angiotensin II depressions of glutamate were shown to be strong and highly specific. The low effectiveness of bath-applied compared with iontophoretically or micropressure-applied angiotensin II was found to be at least partly explained by a rapid degradation by peptidases. Ammonium ions and hydrogen ions were also able to depress glutamate responses, but these effects were not specific for locus coeruleus neurons and were mediated independently of the angiotensin actions. Strong depression by angiotensin II of excitatory postsynaptic potentials as well as exogenously applied glutamate strengthens the strong possibility of a physiological role for this neuromodulatory mechanism. The identification of the type 2 angiotensin receptor subtype as the mediator of this effect indicates a novel functional role for this receptor, since previously recognized functions of angiotensin II in the brain, such as vascular and body fluid regulation, have been associated with the type 1 receptor.

Microbial adhesion in biotechnological processes.

The majority of microorganisms are capable of adhering to surfaces, and we now have a clearer image of the relevance of substratum properties, bacterial surface properties, and molecular conditioning films in adhesion processes. Altered gene expression and increased opportunities for gene transfer are now recognized as consequences of the association of microbes with surfaces. Microbial adhesion leads to biofilm formation; over the past few years, our image of biofilm structure has altered so substantially that a total reassessment of ideas on mass transport of molecules to and from biofilms is essential in environmental biotechnology.

Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum.

P19 embryonal carcinoma cells are pluripotent and can be efficiently induced to differentiate in culture into neurons and astroglia by brief treatment with retinoic acid. Retinoic acid-treated P19 cells survive after grafting into the adult rat striatum and differentiate into neurons and glia within the transplantation site. No tumours develop from the grafted cells which continue to express foreign genes that had been transfected into the parental P19 cells. The neurons in these grafts express a variety of neurotransmitters similar to those formed in retinoic acid-treated P19 cell cultures and they mature to acquire the electrophysiological properties expected of fully developed neurons. These results suggest that P19 cells may be used for studies related to neuronal cell development and maturation and that P19 cells may be considered for cell replacement strategies in neurodegenerative disorders of the central nervous system.

Electrophysiological changes accompanying DSP-4 lesions of rat locus coeruleus neurons.

The electrophysiological characteristics of intracellularly recorded locus coeruleus (LC) neurons in brain stem slices from DSP-4 treated animals have been compared to those from untreated controls. LC neurons from DSP-4 treated animals had action potentials and Ca2+ spikes (elicited in the presence of TTX) of significantly reduced duration compared to controls. These observations suggest that chemical axotomy with DSP-4 reduces Ca2+ conductance in neurons of the locus coeruleus.

Conjugative Plasmid Transfer between Bacteria under Simulated Marine Oligotrophic Conditions.

Marine Vibrio S14 strains and an Escherichia coli strain were starved in artificial seawater (NSS) with no added carbon, nitrogen, or phosphorus. The broad-host-range plasmid RP1 was transferred between the starving S14 strains and also from the E. coli donor to the S14 recipient under oligotrophic conditions, in which mixtures of donor and recipient cells were held on Nuclepore filters either floated on NSS or held such that NSS flowed through the filter. Transconjugants were obtained from S14 donors and recipients starved for at least 15 days before being mixed together for conjugation, whereas transconjugants were recovered from the E. coli donor and S14 recipient for up to 3 days of prestarvation, but not after 5 days. Transconjugants were obtained when there were as few as about 10 and 10 cells of starving S14 donors and recipients, respectively, per ml held on the filters. Starved donor and recipient mixtures incubated at 4 or 26 degrees C, as well as those allowed to mate for 2, 5, or 24 h, all yielded numbers of transconjugants which were not significantly (P > 0.05) different.