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PubMed2004

Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol.

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.

Bacterial Proteins↗
PubMed1993

The toxic effects of formate in dissociated primary mouse neural cell cultures.

Primary dissociated mouse cerebrocortical cell cultures containing both neurons and glial cells were used as an experimental model to study the neurotoxic effects of formate, the putative toxic metabolite of methanol. Neural cells were isolated and prepared from the cerebral cortex of fetal CD-1 mice on Gestational Day 15. Mature 7- to 15-day-old monolayer cultures were exposed to formate (0 to 240 mM) for 8 hr at 37 degrees C over a range of extracellular pH (6.0 to 7.6). Cytotoxicity was evaluated by histopathology, changes in membrane integrity (lactate dehydrogenase release, LDH; [14C]adenine nucleotide leakage), and mitochondrial metabolic activity [reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]. Similar quantitative estimates of cell injury were obtained by LDH release or [14C]adenine nucleotide leakage from prelabeled cells. Exposure of neural cells produced time- and concentration-dependent toxic responses. The concentration of formate that resulted in 50% LDH leakage after an 8-hr incubation was estimated to be 45 mM. As determined by light microscopy, formate (20 to 60 mM) was specifically neuronotoxic, primarily affecting large polygonal neurons. Higher concentrations of formate (> or = 120 mM) induced nonspecific cytotoxicity. MTT reduction appeared to be a more sensitive endpoint by showing significant toxic effects at 20 mM (8-hr incubation), while significant leakage of LDH occurred only at formate concentrations > or = 60 mM. Total intracellular ATP concentration was significantly decreased following a 20 or 40 mM formate exposure for 8 hr. These results are consistent with the hypothesis that formate may inhibit mitochondrial function resulting in decreased intracellular ATP and formate-induced neurotoxicity.

Adenine Nucleotides↗
PubMed1996

Formate-induced alterations in retinal function in methanol-intoxicated rats.

Formic acid is the toxic metabolite in methanol poisoning. Permanent visual damage in methanol-intoxicated humans and non-human primates has been associated with prolonged exposures (> 24 hr) to blood formate concentrations in excess of 7 mM; however, little information is available on the toxicity associated with chronic low-level or repeated exposure to methanol. The present studies compared the effects on retinal function and structure of rapidly increasing formate concentrations typical of acute methanol intoxication with low-level plateau formate concentrations more likely to be generated by subacute or chronic methanol exposure. Rats that accumulated formate concentrations of 8-15 mM developed metabolic acidosis, retinal dysfunction, and retinal histopathologic changes. Retinal dysfunction was measured as reductions in the a- and b-waves of the electroretinogram that occurred coincident with blood formate accumulation. Histopathologic studies revealed vacuolation in the retinal pigment epithelium and photoreceptor inner segments. Rats exposed to formate concentrations ranging from 4 to 6 mM for 48 hr showed evidence of retinal dysfunction in the absence of metabolic acidosis and retinal histopathology. These data indicate that formic acid generated from methanol oxidation acts as a direct retinal toxin. Formate-induced retinal dysfunction in methanol-intoxicated rats can be produced by steadily increasing concentrations of formate and importantly can also be produced by prolonged exposure to lower concentrations of formate. Our findings substantiate evidence based on clinical case reports and a small number of epidemiological studies and support the hypothesis that the visual system toxicity produced by acute, subacute, or chronic methanol poisoning share a common mechanism.

Animals↗
PubMed1994

The hydrogenases and formate dehydrogenases of Escherichia coli.

Escherichia coli has the capacity to synthesise three distinct formate dehydrogenase isoenzymes and three hydrogenase isoenzymes. All six are multisubunit, membrane-associated proteins that are functional in the anaerobic metabolism of the organism. One of the formate dehydrogenase isoenzymes is also synthesised in aerobic cells. Two of the formate dehydrogenase enzymes and two hydrogenases have a respiratory function while the formate dehydrogenase and hydrogenase associated with the formate hydrogenlyase pathway are not involved in energy conservation. The three formate dehydrogenases are molybdo-selenoproteins while the three hydrogenases are nickel enzymes; all six enzymes have an abundance of iron-sulfur clusters. These metal requirements alone invoke the necessity for a profusion of ancillary enzymes which are involved in the preparation and incorporation of these cofactors. The characterisation of a large number of pleiotropic mutants unable to synthesise either functionally active formate dehydrogenases or hydrogenases has led to the identification of a number of these enzymes. However, it is apparent that there are many more accessory proteins involved in the biosynthesis of these isoenzymes than originally anticipated. The biochemical function of the vast majority of these enzymes is not understood. Nevertheless, through the construction and study of defined mutants, together with sequence comparisons with homologous proteins from other organisms, it has been possible at least to categorise them with regard to a general requirement for the biosynthesis of all three isoenzymes or whether they have a specific function in the assembly of a particular enzyme. The identification of the structural genes encoding the formate dehydrogenase and hydrogenase isoenzymes has enabled a detailed dissection of how their expression is coordinated to the metabolic requirement for their products. Slowly, a picture is emerging of the extremely complex and involved path of events leading to the regulated synthesis, processing and assembly of catalytically active formate dehydrogenase and hydrogenase isoenzymes. This article aims to review the current state of knowledge regarding the biochemistry, genetics, molecular biology and physiology of these enzymes.

Anaerobiosis↗
PubMed1980

The orientation of the substrate sites of formate dehydrogenase and fumarate reductase in the membrane of Vibrio succinogenes.

Formate dehydrogenase and fumarate reductase are involved in the electron transport phosphorylation system of Vibrio succinogenes. The orientation of the active sites of these enzymes in the cytoplasmic membrane of the bacterium was investigated with the aim of elucidating the mechanism of energy transduction. This was done by measuring the accessiblities of the enzymes to substrates, dyes and inhibitors both in cells and in cell-derived particles obtained with the French press. 1. After treatment of the cells with lysozyme and EDTA, followed by fractionation, both enzymes were found exclusively in the membranous fractions, while the periplasmic as well as the cytoplasmic fractions were devoid of both of the enzymic activities. 2. The sites of dye interaction of fumarate reductase were inaccessible to non-permeant dyes in cells, but were fully accessible in French-press particles. The Km for succinate as measured with the permeant methylene blue as acceptor was increased ten fold on lysis of the cells. The Km measured in the particles was similar to that of lysed cells and was not altered by lysis. 3. The rates of formate oxidation in the presence of non-permeant dyes and the Km for formate were unaffected by cell lysis. On lysis of French-press particles, formate oxidation with both permeant and non-permeant acceptors was increased about three fold. The extent of stimulation was not altered by inhibition of the enzymic activities. 4. Succinate oxidation by particles was fully inhibited by 4-chloromercuriphenyl sulfonate whereas that of cells was fully resistant. Formate dehydrogenase in cells was inhibited by 4-diazophenyl sulfonate when added together with formate. This compound also inhibited the enzyme in the particles when given in the absence of a lytic agent or after its addition. However, most of the enzyme remained active if the inhibitor was added before the lytic agent. 5. Fumarate and succinate were actively taken up by the cells from the medium, while formate did not even penetrate through the membrane of the bacteria. 6. It is concluded that the substrate and the dye-reactive sites of formate dehydrogenase face the outside, while those of fumarate reductase face the inside of the cytoplasmic membrane of cells of V. succinogenes. About 70% of the French-press particles were inverted with respect to the substrate and dye sites of the enzymes. The membrane is impermeable to formate and does not contain a specific transporter. The electrogenic liberation of protons on the outisde and the uptake of protons from the cytoplasm of the bacteria, which accompany electron transport, can be explained on the basis of the orientation of the substrate sites of the enzymes without net transport of protons across the membrane.

Aldehyde Oxidoreductases↗
PubMed1992

Afferent connections of the parvocellular reticular formation: a horseradish peroxidase study in the rat.

The afferent connections of the parvocellular reticular formation were systematically investigated in the rat with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. The results indicate that the parvocellular reticular formation receives its main input from several territories of the cerebral cortex (namely the first motor, primary somatosensory and granular insular areas), districts of the reticular formation (including its contralateral counterpart, the intermediate reticular nucleus, the nucleus of Probst's bundle, the dorsal paragigantocellular nucleus, the alpha part of the gigantocellular reticular nucleus, the dorsal and ventral reticular nuclei of the medulla, and the mesencephalic reticular formation), the supratrigeminal nucleus and the deep cerebellar nuclei. Moderate to substantial input to the parvocellular reticular formation appears to come from the central amygdaloid nucleus, the parvocellular division of the red nucleus, and the orofacial and gustatory sensory cell groups (comprising the mesencephalic, principal and spinal trigeminal nuclei, and the rostral part of the nucleus of the solitary tract), whereas many other structures, including the substantia innominata, the field H2 of Forel, hypothalamic nuclei, the superior colliculus, the substantia nigra pars reticulata, the retrorubral field and the parabrachial complex, seem to represent relatively modest additional input sources. Some of these projections appear to be topographically distributed within the parvocellular reticular formation. From the present results it appears that the parvocellular reticular formation receives afferents from a restricted group of sensory structures. This finding calls into question the traditional characterization of the parvocellular reticular formation as an intermediate link between the sensory nuclei of the cranial nerves and the medial magnocellular reticular districts, identified as the effector components of the reticular apparatus. Some of the possible physiological correlates of the fiber connections of the parvocellular reticular formation in the context of oral motor behaviors, autonomic regulations, respiratory phenomena and sleep-waking mechanisms are briefly discussed.

Afferent Pathways↗
PubMed1994

Distribution of cholinergic, GABAergic and serotonergic neurons in the medial medullary reticular formation and their projections studied by cytotoxic lesions in the cat.

As part of a larger study concerning the role of neurons in the medial medullary reticular formation in sleep-wake states, the distribution and projections of cholinergic, GABAergic and serotonergic neurons were studied within the lower brainstem of the cat. Cells were plotted with the aid of an image analysis system through the medullary reticular formation and raphe in adjacent sections immunostained for choline acetyltransferase, glutamic acid decarboxylase and serotonin. Immunostained fibres and varicosities were examined and quantified by microdensitometry in regions of the medulla, pons and upper spinal cord in normal and quisqualate-injected animals to assess the loss of local and distant projections following cytotoxic destruction of neurons in the medial medullary reticular formation. Choline acetyltransferase-immunoreactive neurons are unevenly and sparsely distributed, though none the less in significant numbers (estimated at approximately 9080 in total), through the medial medullary reticular formation, and are present in all tegmental fields, including the gigantocellular (approximately 3700) and magnocellular (approximately 1760) rostrally and the ventral (approximately 3240) and paramedian (approximately 380) caudally, and are absent in the midline raphe. Glutamic acid decarboxylase-immunoreactive neurons are more evenly and densely distributed in large numbers (estimated at approximately 18,720) through the medial medullary reticular formation, being present in the gigantocellular (approximately 5960), magnocellular (approximately 8260), ventral (approximately 2280) and paramedian (approximately 2220) tegmental fields, and are also numerous within the raphe magnus and pallidus-obscurus nuclei (approximately 3880). Serotonin-immunoreactive cells are sparse in the medial medullary reticular formation (estimated to total approximately 1540), where they are mainly located in the magnocellular tegmental field (approximately 1340), and are concentrated in larger numbers within the raphe nuclei (approximately 8060). Cholinergic varicose fibres were moderately densely distributed through the medial medullary reticular formation, as well as through more distant lateral, rostral and caudal brainstem and upper spinal regions. After cytotoxic lesions focussed in the gigantocellular and magnocellular tegmental fields, a loss of approximately 55% of the cholinergic neurons in the medial medullary reticular formation was associated with a minor decrease (approximately 35% in optical density measures) of local cholinergic fibres. Small and variable reductions in varicose fibres (and their optical density measures) were detected in distant structures (including the pontine lateral, gigantocellular and subcoerular tegmental fields and the caudal spinal trigeminal nucleus), that were none the less correlated with the number of intact medial medullary cholinergic cells, suggesting that these cells may project to distant brainstem targets, in addition to providing a minor proportion of the local cholinergic innervation of the medial medullary reticular formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Real-time monitoring of the oxalate decarboxylase reaction and probing hydron exchange in the product, formate, using fourier transform infrared spectroscopy.

Oxalate decarboxylase converts oxalate to formate and carbon dioxide and uses dioxygen as a cofactor despite the reaction involving no net redox change. We have successfully used Fourier transform infrared spectroscopy to monitor in real time both substrate consumption and product formation for the first time. The assignment of the peaks was confirmed using [(13)C]oxalate as the substrate. The K(m) for oxalate determined using this assay was 3.8-fold lower than that estimated from a stopped assay. The infrared assay was also capable of distinguishing between oxalate decarboxylase and oxalate oxidase activity by the lack of formate being produced by the latter. In D(2)O, the product with oxalate decarboxylase was C-deuterio formate rather than formate, showing that the source of the hydron was solvent as expected. Large solvent deuterium kinetic isotope effects were observed on V(max) (7.1 +/- 0.3), K(m) for oxalate (3.9 +/- 0.9), and k(cat)/K(m) (1.8 +/- 0.4) indicative of a proton transfer event during a rate-limiting step. Semiempirical quantum mechanical calculations on the stability of formate-derived species gave an indication of the stability and nature of a likely enzyme-bound formyl radical catalytic intermediate. The capability of the enzyme to bind formate under conditions in which the enzyme is known to be active was determined by electron paramagnetic resonance. However, no enzyme-catalyzed exchange of the C-hydron of formate was observed using the infrared assay, suggesting that a formyl radical intermediate is not accessible in the reverse reaction. This restricts the formation of potentially harmful radical intermediates to the forward reaction.

Bacillus subtilis↗
PubMed1994

Isolation and characterization of hypophosphite--resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter.

Hypophosphite was used as a toxic analogue to identify genes whose products have a putative function in the transport of formate. Two Tn10-derived insertion mutants were identified that exhibited increased resistance to high concentrations of hypophosphite in the culture medium. The transposon was located in the identical position in the focA (formate channel; previously termed orf) gene of the pfl operon in both mutants. A defined chromosomal focA nonsense mutant, which showed minimal polarity effects on pfl gene expression, had the same phenotype as the insertion mutants. Results obtained using a hycA-lacZ fusion to monitor changes in the intracellular formate concentration in a focA mutant indicated that the level of formate inside the cell was elevated compared with the wild type. Moreover, it could be shown that there was a corresponding reduction of approximately 50% in the amount of formate excreted by a focA mutant into the culture medium. Taken together, these results indicate that formate accumulates in anaerobic cells which do not have a functional focA gene product and that one function of FocA may be to export formate from the cell. A further significant result was that hypophosphite could substitute for formate in activating hycA gene expression. This hypophosphite-dependent activation of hycA gene expression was reduced 10-fold in a focA null mutant, suggesting that hypophosphite must first enter the cell before it can act as a signal to activate hycA expression. By analogy, these data suggest that focA may also be functional in the import of formate into anaerobic Escherichia coli cells. Site-specific mutagenesis identified the translation initiation codon of focA as a GUG. Therefore, the FocA polypeptide has a molecular weight of 30,958. FocA shows significant similarity at both the primary and secondary structural levels with the NirC protein of E. coli and the FdhC protein of Methanobacterium formicicum. All three proteins are predicted to be integral membrane proteins. A detailed in vivo TnphoA mutagenesis study predicted that FocA has six membrane-spanning segments.

Acetyltransferases↗
PubMed1987

Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum.

The catabolism of sodium formate to acetate and carbon dioxide by the anaerobic acetogen Butyribacterium methylotrophicum was analyzed by fermentation time course and 13C nuclear magnetic resonance studies. Significant hydrogen production and consumption fluxes were observed during formate catabolism but not during the catabolism of formate plus CO. In the latter case, formate and CO were simultaneously consumed and label distribution studies with mixtures of 13C-labeled CO and formate demonstrated their preferential incorporation into the acetate carboxyl and methyl groups, respectively. Hydrogen consumption was inhibited by CO when both were present, whereas hydrogen and formate were simultaneously consumed when CO2 was supplied. Carbon dioxide was required for the conversion of CO to acetate, but a similar need was not observed when methanol plus CO or formate plus CO was present. These analyses indicate a bifurcated single-carbon catabolic pathway in which CO2 is the sole single-carbon compound that directly supplies the carbonyl and methyl group synthesis pathways leading to the formation of acetyl coenzyme A, the primary reduced product. We discuss causes for the reported inability of B. methylotrophicum to use formate as a sole substrate.

Acetates↗
PubMed1992

The squirrel monkey as an experimental model for Plasmodium falciparum erythrocyte rosette formation.

Experimental cerebral malaria was recently found to occur in the squirrel monkey Saimiri sciureus when infected with the human malaria parasite Plasmodium falciparum. This report is concerned with the existence of spontaneous rosette formation ex vivo (infected blood samples) and in vitro (cultured parasites) between red blood cells (RBC) infected with squirrel monkey-adapted P. falciparum isolates and normal squirrel monkey RBC. Transfer of P. falciparum with high rosette formation tendencies (90-100 R+) from one donor monkey to several recipients gave rise to parasites that varied extensively in their ex vivo rosette formation capacity (4-96% R+). However, all individual parasites readily form rosettes after 24 hr of in vitro culture (60-95% R+). Host factors may be involved in the modulation of rosette formation, although it is found to occur both in splenectomized and spleen-intact animals. Cross-rosette formation is seen between parasitized human RBC and normal squirrel monkey RBC and vice versa, and rosettes formed by RBC of the two hosts are similarly affected by pH, temperature, EDTA, trypsin, as well as squirrel monkey and African human hyperimmune IgG. These characteristics of rosette formation are preserved after long-term in vitro culture in human RBC. Rosettes formed by some isolates are highly sensitive to heparin while others are not, suggesting at least two distinct mechanisms of rosette formation. This idea is also supported by the observation that specific squirrel monkey antisera to heparin-sensitive strains does not dissociate rosettes formed by a heparin-resistant strain. The results suggest that rosettes and anti-rosette formation antibodies formed by squirrel monkeys and humans exhibited similar characteristics, and that the squirrel monkey is therefore a good experimental model to study erythrocyte rosette formation and cerebral malaria.

Animals↗
PubMed1998

Protein oxidation: examination of potential lipid-independent mechanisms for protein carbonyl formation.

Previous data indicated that diquat-mediated protein oxidation (protein carbonyl formation) occurs through multiple pathways, one of which is lipid dependent, and the other, lipid independent. Studies reported here investigated potential mechanisms of the lipid-independent pathway in greater detail, using bovine serum albumin as the target protein. One hypothesized mechanism of protein carbonyl formation involved diquat-dependent production of H2O2, which would then react with site-specifically bound ferrous iron as proposed by Stadtman and colleagues. This hypothesis was supported by the inhibitory effect of catalase on diquat-mediated protein carbonyl formation. However, exogenous H2O2 alone did not induce protein carbonyl formation. Hydroxyl radical-generating reactions may result from the H2O2-catalyzed oxidation of ferrous iron, which normally is bound to protein in the ferric state. Therefore, the possible reduction of site-specifically bound Fe3+ to Fe2+ by the diquat cation radical (which could then react with H2O2) was also investigated. The combination of H2O2 and an iron reductant, ascorbate, however, also failed to induce significant protein carbonyl formation. In a phospholipid-containing system, an ADP:Fe2+ complex induced both lipid peroxidation and protein carbonyl formation; both indices were largely inhibitable by antioxidants. There was no substantial ADP:Fe(2+)-dependent protein carbonyl formation in the absence of phospholipid under otherwise identical conditions. Based on the lipid requirement and antioxidant sensitivity, these data suggest that ADP:Fe(2+)-dependent protein carbonyl formation occurs through reaction of BSA with aldehydic lipid peroxidation products. The precise mechanism of diquat-mediated protein carbonyl formation remains unclear, but it appears not to be a function of H2O2 generation or diquat cation radical-dependent reduction of bound Fe3+.

Adenosine Diphosphate↗
PubMed1990

Immunohistochemical study of choline acetyltransferase-immunoreactive processes and cells innervating the pontomedullary reticular formation in the rat.

The present study was undertaken to examine the cholinergic innervation of the brainstem reticular formation in an effort to understand the potential role of cholinergic neurons in processes of sensory-motor modulation and state control. The cholinergic cells and processes within the pontomedullary reticular formation were studied in the rat by application of peroxidase-antiperoxidase immunohistochemistry with silver intensification for choline-acetyltransferase (ChAT). ChAT-immunoreactive cells were located in the pontomesencephalic tegmentum within the laterodorsal and pedunculopontine tegmental (LDT and PPT) nuclei, where they numbered approximately 3,000 on each side and were scattered in the midline, medial, and lateral medullary reticular formation, where they numbered approximately 10,000 in total on each side. The cholinergic neurons within the reticular formation were commonly medium in size and gave rise to multiple dendrites that extended for considerable distances within the periventricular gray or the reticular formation, as is typical of other isodendritic reticular neurons. A prominent innervation of the entire pontomedullary reticular formation was evident by varicose ChAT-immunoreactive fibers that often surrounded large noncholinergic reticular neurons in a typical perisomatic pattern of termination, suggesting a potent influence of the cholinergic innervation on pontomedullary reticular neurons. The contribution of the pontomesencephalic cholinergic neurons to the innervation of the medial medullary and lateral pontine reticular formation was studied by retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin (WGA-HRP) in combination with ChAT immunohistochemistry. A proportion of the cholinergic neurons within the laterodorsal tegmental nucleus (pars alpha) and the pedunculopontine tegmental nucleus were retrogradely labelled on the ipsilateral (10-15%) and contralateral (5-10%) sides from the medial medullary reticular formation, indicating a significant contribution to the cholinergic innervation of this region, which, however, also appeared to derive in part from intrinsic medullary cholinergic neurons. The major fiber system by which the medial medullary reticular formation was reached by the pontomesencephalic cholinergic neurons appeared to correspond to the lateral tegmentoreticular tract. Fibers passed from these cholinergic cells ventrally through the lateral pontine tegmentum, in the region of the subcoeruleus, where they also appeared to innervate by fibres en passage the noncholinergic neurons of the region. A significant proportion of the pontomesencephalic cholinergic neurons were retrogradely labelled from the lateral pontine tegmentum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Connections between the retrosplenial cortex and the hippocampal formation in the rat: a review.

The retrosplenial cortex is situated at the crossroads between the hippocampal formation and many areas of the neocortex, but few studies have examined the connections between the hippocampal formation and the retrosplenial cortex in detail. Each subdivision of the retrosplenial cortex projects to a discrete terminal field in the hippocampal formation. The retrosplenial dysgranular cortex (Rdg) projects to the postsubiculum, caudal parts of parasubiculum, caudal and lateral parts of the entorhinal cortex, and the perirhinal cortex. The retrosplenial granular b cortex (Rgb) projects only to the postsubiculum, but the retrosplenial granular a cortex (Rga) projects to the postsubiculu, rostral presubiculum, parasubiculum, and caudal medial entorhinal cortex. Reciprocating projections from the hippocampal formation to Rdg originate in septal parts of CA1, postsubiculum, and caudal parts of the entorhinal cortex, but these are only sparse projections. In contrast, Rgb and Rga receive dense projections from the hippocampal formation. The hippocampal projection to Rgb originates in area CA1, dorsal (septal) subiculum, and post-subiculum. Conversely, Rga is innervated by ventral (temporal) subiculum and postsubiculum. Further, the connections between the retrosplenial cortex and the hippocampal formation are topographically organized. Rostral retrosplenial cortex is connected primarily to the septal (rostrodorsal) hippocampal formation, while caudal parts of the retrosplenial cortex are connected with temporal (caudoventral) areas of the hippocampal formation. Together, the elaborate connections between the retrosplenial cortex and the hippocampal formation suggest that this projection provides an important pathway by which the hippocampus affects learning, memory, and emotional behavior.

Afferent Pathways↗
PubMed1997

Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation.

Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100ng/mal IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 microg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAK1 and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Lymphokine regulation of granuloma formation in murine schistosomiasis mansoni.

The pathology in schistosomiasis mansoni is primarily due to hepatic and intestinal granuloma formation around deposited eggs and subsequent fibrosis. The intensity of the granulomatous response is dependent on the lymphokines produced by T cells in response to soluble egg antigens (SEA). The present study examined the relationship of three inflammatory lymphokines, IL-2, IL-4, and IFN-gamma in granuloma formation during the acute stage of the murine infection. Repeated injections of anti-IL-2 or anti-IL-4 mAbs in vivo significantly diminished pulmonary granuloma formation in infected mice and demonstrated that both IL-2 and IL-4 were involved during the period of peak granuloma formation. Administration of anti-IL-2 mAb decreased IL-2, but not IL-4 production, while anti-IL-4 mAb treatment decreased both IL-2 and IL-4 production by splenic T cells. Administration of anti-IFN-gamma mAb to infected mice significantly increased pulmonary granuloma formation and increased the production of both IL-2 and IL-4 by splenic T cells. Repeated injections of graded doses of recombinant IFN-gamma given to mice between 6 and 8 weeks of infection decreased pulmonary granuloma formation as well as IL-2 and IL-4 production by splenic T cells. These data demonstrate that the regulation of lymphokines, IFN-gamma, IL-2, and IL-4, is important in influencing the intensity of granuloma formation. IL-2 and IL-4 appear to play a proinflammatory role in granuloma formation, while IFN-gamma may downregulate splenic IL-2 and IL-4 production and, more importantly, the corresponding inflammatory granuloma formation.

Animals↗
PubMed1999

Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum.

Metabolic flux analysis was used to investigate the roles of the acid formation pathways in Clostridium acetobutylicum. The acid formation pathways were revealed to serve different roles in wildtype fermentations than previously expected. Specifically, enzymes known to catalyze butyrate formation were found to uptake butyrate without concomitant production of acetone. This role was further corroborated by flux analysis of a recombinant strain overexpressing the butyrate formation enzymes. Analysis of wildtype fermentation data also revealed an important role for the acetate formation enzymes, namely the cycling of carbon between acetate and acetylCoA during the stationary phase. Next, metabolic flux analysis was used to compare the patterns of activity in two butyrate kinase deficient strains of C. acetobutylicum. The strain developed by gene inactivation, PJC4BK, exhibited a shift in acid formation fluxes toward acetate while the strain developed by antisense RNA strategies, 824(pRD4), did not exhibit such a shift. However, both strains exhibited altered solvent formation patterns. PJC4BK exhibited a strong transient enhancement of solvent formation fluxes. In contrast, 824(pRD4) exhibited relatively lower levels of solvent formation fluxes, although fluxes were sustained over a longer period of time.

Acetic Acid↗
PubMed2000

Reovirus mu2 protein determines strain-specific differences in the rate of viral inclusion formation in L929 cells.

Reovirus infection induces the formation of large cytoplasmic inclusions that serve as the major site of viral assembly. Reovirus strains type 3 Dearing (T3D) and type 1 Lang (T1L) differ in the rate of inclusion formation in L929 cells. The median time of inclusion formation is 18 h in cells infected with T3D and 39 h in cells infected with T1L. Using reassortant viruses that contain combinations of gene segments derived from T1L and T3D, we found that the M1 gene, which encodes the mu2 protein, is the primary determinant of the rate of inclusion formation. The S3 gene, which encodes the nonstructural protein sigmaNS, plays a secondary role in this process. The subcellular location of the mu2 protein was determined by confocal laser scanning microscopy using dual-fluorescence labeling of mu2 and the outer-capsid protein mu1/mu1C. In virus-infected cells, mu2 protein colocalized with other viral proteins in inclusions and was also distributed diffusely in the cytoplasm and nucleus. Expression of recombinant T1L and T3D mu2 proteins resulted in the formation of protein complexes resembling inclusions in both the cytoplasm and the nucleus with kinetics that reflected the strain of origin. The median time of mu2 protein complex formation was 22 h in cells transfected with the T3D M1 gene and 43 h in cells transfected with the T1L M1 gene. These findings suggest that the mu2 protein influences the rate of inclusion formation and contributes to inclusion morphogenesis. The requirement of mu2 protein in inclusion formation was tested by determining the subcellular localization of mu2 in cells infected with temperature-sensitive (ts) mutants that are defective in viral assembly. In contrast to infection with wild-type virus, mu2 did not colocalize with mu1/mu1C protein in subcellular structures that formed in cells infected at nonpermissive temperature with ts mutants tsH11.2, tsC447, and tsG453 with mutations in the M1, S2, and S4 genes, respectively. These results suggest that despite the role of the mu2 protein in controlling the rate of inclusion formation, this process is a concerted function of several reovirus proteins.

Animals↗