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PubMed1999

Pathways bring dramatic clinical results.

Although physicians at Good Samaritan Health System in Kearney, NE, first offered stiff resistance to case managers' efforts to implement a clinical pathway program, efforts to educate the physicians about the benefits of pathways have led to a turn-around in which physicians now are asking the director of case management to create even more pathways. One key step in promoting the pathways was to create a reporting profile capable of evaluating clinical pathways. In addition, the hospital produced a video for each pathway that describes the patient's care from preadmission through discharge. Most significantly, case managers at Good Samaritan avoided creating an adversarial relationship with physicians in the system. They dodged charges of pathways being "cookbook medicine" by establishing a system in which pathways are not automatically applied; physicians have to write an order for a patient to be placed on a pathway.

Attitude of Health Personnel↗

PubMed1992

Convergence of reflex pathways excited by distension and mechanical stimulation of the mucosa onto the same myenteric neurons of the guinea pig small intestine.

The effects on morphologically and electrophysiologically characterized myenteric neurons of activation of intestinal reflex pathways were examined in vitro. Opened segments of guinea pig small intestine were pinned serosa down in an organ bath that had two balloons set into its base. A 5-10-mm-wide strip of myenteric plexus between the balloons was exposed from the mucosal side, and neurons were impaled with microelectrodes. Reflex pathways were stimulated by inflation of the balloons to distend the intestinal wall, and by deforming the exposed mucosal villi with a brush. Impaled neurons were classified electrophysiologically as AH-neurons or S-neurons (Hirst et al., 1974) and were injected with biocytin to determine their shapes and projections. None of the 58 AH-neurons responded to distension. In contrast, 63 of 131 S-neurons responded to distension with a burst of fast EPSPs; about one-third of the responding S-neurons received input from ascending reflex pathways, one-third received input from descending reflex pathways, and one-third received input from both ascending and descending pathways. Most neurons in this last group supplied extensive varicose branches to the tertiary plexus and were probably longitudinal muscle motor neurons. Neurons receiving input from only one pathway usually projected in the direction of that pathway; many of these were circular muscle motor neurons. Almost all neurons responding to distension were also excited by deforming the villi. Responses evoked by distension or deforming the mucosa declined when stimuli were repeated at intervals less than 10 sec. This was seen in ascending and descending pathways but was more prominent in the former. Deforming the mucosa evoked a normal response even when the response to repeated distensions had disappeared. It is concluded that distension and deforming the mucosa excite separate populations of sensory neurons to activate reflex pathways that converge onto common motor neurons and probably onto common interneurons.

Animals↗

PubMed2004

A model for the role of the proline-linked pentose-phosphate pathway in phenolic phytochemical bio-synthesis and mechanism of action for human health and environmental applications.

The combination of immunodeficiency, inflammatory process and nutritional status that is characteristic of infective and food-borne illness is more evident in chronic diet- and environment-influenced chronic diseases such as diabetes, obesity, cardiovascular disease, cancer, arthritis and neuro-degeneration diseases. These chronic diseases tend to be oxidation-linked and may manifest in communities around the world, irrespective of income. In addressing the challenges of the above diseases, a significant role for dietary phytochemicals is emerging. Phytochemicals are required from a spectrum of food for at least their antioxidant role, if not for other properties, to protect tissues from activities that manifest themselves into what we call chronic disease. Among the diverse groups of phytochemicals, phenolic antioxidants and antimicrobials from food plants are being targeted for designed dietary intervention to manage major oxidation-linked diseases such as diabetes, cardiovascular diseases, arthritis, cognition diseases and cancer. Foods containing phenolic phytochemicals are also being targeted to manage bacterial infections associated with chronic diseases such as peptic ulcer, urinary tract infections, dental caries and food-borne bacterial infections. Plants produce phenolic metabolites as a part of growth, developmental and stress adaptation response. These stress and developmental responses are being harnessed to design consistent phytochemical profiles for safety and clinical relevancy using novel tissue culture and bioprocessing technologies. The biochemical strategy for harnessing phenolic phytochemicals for human health and wellness is based on the hypothesis that phenolic metabolites in plants are efficiently produced through an alternative mode of metabolism linking proline synthesis with pentose-phosphate pathway. In this model, stress-induced proline biosynthesis is coupled to pentose-phosphate pathway, driving the synthesis of NADPH(2) and sugar phosphates for anabolic pathways, including phenolic and antioxidant response pathways, while simultaneously providing reducing equivalents needed for mitochondrial oxidative phosphorylation in the form of proline as an alternative to NADH from Krebs/TCA cycle. Based on this model, tissue culture techniques and elicitation concepts have been used to stimulate phenolic metabolites with an antioxidant response in germinating seeds, sprouts and clonal lines of dietary plants. From our initial investigations, a model has been proposed in which the proline-linked pentose-phosphate pathway is suggested to be critical for modulating protective antioxidant response pathways in diverse biological systems, including biochemical and cellular pathways important for human health. The proposed proline-linked pentose-phosphate pathway model provides a mechanism for understanding the mode of action of phenolic phytochemicals in modulating antioxidant pathways and provides avenues by which dietary approaches may manage oxidation-linked chronic and infectious diseases. The model also has implications for the development of antimicrobial phenolic phytochemicals against bacterial pathogens in an era of increasing antibiotic resistance. Further, this model also has relevance for improving fungal and yeast-based food bioprocessing for designing functional foods and for environmental bioremediation using plant and microbial systems, as well as for improving agricultural and food systems in harsh environments.

Antioxidants↗

PubMed1988

CR2 is a complement activator and the covalent binding site for C3 during alternative pathway activation by Raji cells.

Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.

Burkitt Lymphoma↗

PubMed1995

Eosinophil granule cationic proteins regulate the classical pathway of complement.

Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by interfering with C1 and may exert this activity in vivo.

Blood Proteins↗

PubMed1993

Motor axons preferentially reinnervate motor pathways.

Motor axons regenerating after transection of mixed nerve preferentially reinnervate distal motor branches and/or muscle, a process termed "preferential motor reinnervation." Collaterals of a single motor axon often enter both sensory and motor Schwann cell tubes of the distal stump; specificity is generated by pruning collaterals from sensory pathways while maintaining those in motor pathways. Previous experiments in the rat femoral nerve model evaluated reinnervation of the femoral motor branch and quadriceps muscle as a unit. In this study, pathway contributions are analyzed separately by denying muscle contact, or by reinnervating muscle through inappropriate, formerly sensory pathways. Motor axons preferentially reinnervate motor pathways, even when these pathways end blindly in a silicon tube. If the femoral nerve is removed as a graft and reinserted with correct or reversed alignment of the sensory and motor branches, more motoneurons reinnervate muscle through correct motor than through incorrect sensory pathways. Motor pathways thus differ from sensory pathways in ways that survive Wallerian degeneration and transplantation as a graft, and that can be used by regenerating motor axons as a basis for collateral pruning and specificity generation.

Animals↗

PubMed1999

Divergence of smooth muscle target and sympathetic pathway cell phenotypes in the orbit of the developing rat.

The periorbital sheath serves as a major pathway for sympathetic nerves traveling to distal orbital targets in the rat. This tissue accommodates sympathetic fiber sprouting in the neonate but becomes impassable by postnatal day 30 (PND 30). In contrast, smooth muscle target remains receptive to sympathetic ingrowth. To determine the attributes of receptive and nonreceptive tissues, we compared periorbital pathway and target tissue phenotypes prior to (PND 5 and PND 15) and after (PND 30 and PND 60) the period when pathway receptivity is lost. Both pathway cells and superior tarsal smooth muscle cells expressed alpha-smooth muscle actin and smooth muscle myosin heavy chain throughout development. At PND 5-15, both tissues also expressed vimentin, collagen IV, laminin 1 and laminin beta2, whereas fibronectin was detected only in pathway tissue. At PND 30, vimentin, collagen IV, and fibronectin were absent in tarsal muscle but were robust in pathway tissue. Laminin 1 and laminin beta2 expression was maintained in muscle; however, in pathway cells, laminin 1 declined modestly, and laminin beta2 decreased precipitously to barely detectable levels. Quantitative competitive polymerase chain reaction showed that nerve growth factor mRNA was present in the pathway throughout development at levels that were greater than both surrounding connective tissue and tarsal muscle. We conclude that the loss of pathway receptivity to sympathetic nerve ingrowth is associated with a transition from a phenotype similar to fetal smooth muscle cells to one that is more consistent with myofibroblast-like cells.

Animals↗

PubMed1988

Specific configurations of fibronectin-containing particles correlate with pathways taken by neural crest cells at two axial levels.

Although neural crest (NC) cells can potentially enter a number of intertissue spaces, they select a particular pathway that varies depending on the axial level. In the cranial region, NC cells enter the dorsal-lateral pathway (i.e., immediately subjacent to the ectoderm) and avoid the ventral pathway (i.e., pathway between the mesoderm and neural tube and within the mesodermal cell population), whereas in the trunk region, the majority of the NC cells enter the ventral pathway (i.e., between the somite and neural tube) and not the dorsal-lateral pathway. Our working hypothesis is that one determining factor in directing NC cell migration is the composition and/or intermolecular associations of the extracellular matrix (ECM) in these pathways. Histochemical staining, immunostaining, and lectin-binding studies on cryofixed and conventionally fixed tissue were conducted to initially characterize the ECM found in potential NC cell pathways prior to and during initial NC cell migration at two different axial levels. We found that, regardless of the axial level, the pathways into which NC cells eventually enter possessed a characteristic ECM arrangement. This arrangement included: 1) the presence of multicomponent, glycoprotein-containing spherical particles (0.1-0.5 micron in diameter); and 2) a low-sulfated ECM content. Although all particles contained fibronectin, only those in specific regions were able to bind to a monoclonal antibody directed to the cell-binding domain of fibronectin, suggesting that the conformation of fibronectin may be important in the expression of any in situ function of the molecule.

Animals↗

PubMed1981

Demonstration of multiple antegrade and retrograde atrioventricular pathways.

A patient with paroxysmal supraventricular tachycardia had discontinuous antegrade (1a-A2, H1-H2) and retrograde (V1-V2, A1-A2) conduction curves suggesting dual A-V nodal pathways in both directions. Atrial echoes occurred with premature atrial pacing only at short A1-A2 coupling intervals after long antegrade (A2-H2) and retrograde (H2-A3) conduction intervals. Premature ventricular stimulation revealed ventricular echoes simultaneously with a sudden increase in the V2-A2 interval. The echo zone coincided with the slow pathway curve. Following atropine the echo zone was extended over the slow and fast pathway curves. Slow pathway conduction was observed at long and sort V1-V2 coupling intervals. Following isoproterenol ventricular stimulation initiated two cycles of ventricular echoes with relatively long retrograde (V2-A2, Ve-Ae) and short antegrade (A2-He, Ae-He) conduction times, the earliest atrial activation being observed in the low right atrium before the left atrium and the high right atrium. Antegrade fast and slow pathways as well as retrograde fast pathway conduction appeared to be confined to the A-V node. Retrograde slow pathway conduction may progress through a slow or fast A-V nodal pathway slowed by antegrade concealed conduction. However, an accessory pathway with long conduction times located near the septum cannot be ruled out entirely.

Atrioventricular Node↗

PubMed1995

The folding mechanism of barstar: evidence for multiple pathways and multiple intermediates.

The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25 degrees C has been characterized using rapid-mixing techniques. Earlier studies had established the validity of the three-state US <--> UF <--> N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where US and UF are the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, IS1 and IS2 as well as a late native-like intermediate, IN, are present on the folding pathways of US, and an early intermediate IF1 on the folding pathway of UF, when barstar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of IN, and the rates of formation of N at three different concentrations of GdnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, IN is not fully populated on the US-->IS1-->IN-->N pathway because the rate of its formation is so slow that the US <--> UF <--> N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the US-->IS1-->IN transition is so fast that IN is fully populated. In 0.6 M GdnHCl, IN appears not to be fully populated because an alternative folding pathway, US-->IS2-->N, becomes available for the folding of US, in addition to the US-->IS1-->IN-->N pathway. Measurement of the binding of the hydrophobic dye 1-anilino-8-naphthalenesulphonate (ANS) during folding indicates that ANS binds to two distinct intermediates, IM1 and IM2, that form within 2 ms on the US-->IM1-->IS1-->IN-->N and US-->IM2-->IS2-->N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of UF.

Anilino Naphthalenesulfonates↗

PubMed2010

MAP Kinase: SUMO pathway interactions.

The convergence and coordinated cross talk of different signalling pathways forms a regulatory network which determines the biological outcome to environmental cues. The MAPK pathways are one of the important routes by which extracellular signals are transduced into intracellular responses. Through protein phosphorylation mechanisms, they can play a pivotal role in regulating other posttranslational modifications such as protein acetylation and ubiquitination. In addition, protein sumoylation has emerged as an important pathway which also functions through post-translational modification. The SUMO pathway modulates a diverse range of cellular processes including signal transduction, chromosome integrity, and transcription. Interestingly, recent studies have provided links between the SUMO and MAPK signalling pathways which converge to modulate transcription factor activity. This was first demonstrated by the observation that the activation of the ERK pathway caused de-sumoylation of the transcription factor, Elk-1. Furthermore, a growing number of links are now being made between the MAPK pathway and protein sumoylation. Given the nature of protein sumoylation in diverse biological functions, it is not surprising that the effect of MAPK pathways on sumoylation varies between different proteins. Here, we describe protocols that can be used in studying the cross talk between the MAPK and SUMO pathways, particularly at the level of gene regulation.

Amino Acid Sequence↗

PubMed1986

Separate, Ca2+-activated K+ and Cl- transport pathways in Ehrlich ascites tumor cells.

The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl- transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl- transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl- conductance is increased 14- and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl- conductance is rate limiting. An A23187-induced activation of 42K and 36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: inhibited by quinine which blocks the Ca2+-activated K+ channel, unaffected by substitution of NO-3 or SCN- for Cl-, and inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl- conductance. The Cl- conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl- transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl- transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl- transport pathway. The activated anion transport pathway is unselective, carrying both Cl-, Br-, NO-3, and SCN-. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl- transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by 42K flux experiments and indirectly in media where the dominating anion (SCN-) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from 42K flux measurements at high external K+, and from net K+ flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.

Animals↗

PubMed1989

Uptake of lysine and proline via separate alpha-neutral amino acid transport pathways in Mytilus gill brush border membranes.

Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel, Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na+ and alpha-neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar alpha-neutral amino acids and cationic amino acids, but was not affected by beta-neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with a Jmax of 550 pmol/mg-min and a Kt of 5 microM. The AK pathway did not have a strict requirement for Na+, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li+ and K+, as well as Na+. Harmaline inhibited the transport of lysine in solutions containing either Na+ or K+. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar alpha-neutral amino acids, proline, and alpha-(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with a Jmax of 180 pmol/mg-min and Kt of 4 microM. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na+. Na+-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na+ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.

Alanine↗

PubMed2002

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria.

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).

Cysteine↗

PubMed2001

Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus.

The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by 13C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments. H. saccharolyticus grown on complex media containing glucose or fructose specifically 13C-labeled at C1 and C3, formed acetate and small amounts of lactate. The 13C-labeling patterns, analyzed by 1H- and 13C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea. Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed. After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased. Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose. Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses. The data indicate that, in the archaeon H. saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.

Biodegradation, Environmental↗

PubMed1997

Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase.

Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the 13C label in alanine synthesized by cells grown with [1-13C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD+ and NADP+ as cosubstrates, showing a 100-fold higher affinity for NADP+. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP+. A striking feature was the influence of NADP+ and NAD+ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases.

Amino Acid Sequence↗

PubMed2004

Immunohistochemical distribution of proteins belonging to the receptor-mediated and the mitochondrial apoptotic pathways in human placenta during gestation.

The balance between cell death and cell proliferation and its regulation are essential features of many physiological processes and are particularly important in fetal morphogenesis and adult tissue homeostasis. Apoptosis is a type of cell suicide that is activated in two main ways: through a receptor-mediated pathway or through a mitochondrial pathway. We have investigated the immunohistochemical distribution of proteins belonging to these two pathways in human placenta during gestation by comparing their expression levels between the first and third trimester of gestation. In the first trimester, the receptor-mediated pathway prevails over the mitochondrial pathway with a moderate/intense expression of its three components, viz., Fas ligand (FasL), Fas, and caspase-8, and weak positivity of anti-apoptotic FLIP, these proteins being mainly localized in the cytotrophoblast compartment. In the third trimester of gestation, there is an increased expression of mitochondrial pathway proteins, viz., Apaf-1 and caspase-9. We have also investigated the expression level of caspase-3, the primary effector caspase of both pathways, and have observed that it is moderately expressed during gestation, being mainly localized in the cytotrophoblast during the first trimester and in both placental compartments during the third trimester of gestation. Thus, both pathways actively function in human placenta to execute cell death. By means of immunoelectron microscopy, we have further shown that, in human placenta, the two proteins of the mitochondrial pathway together with caspase-3 are localized both in the cytoplasm and in the nucleus. In particular, Apaf-1 and caspase-9 are distributed near to the nuclear envelope suggesting an important role for these two proteins in disrupting the nuclear-cytoplasmic barrier.

Adult↗

PubMed2001

Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana.

Serine biosynthesis in plants proceeds by two pathways; the glycolate pathway which is associated with photorespiration and the pathway from 3-phosphoglycerate which is presumed to take place in the plastids. The 3-phosphoglycerate pathway (phosphorylated pathway) involves three enzymes catalyzing three sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP). cDNA and genomic clones encoding these three enzymes from spinach and Arabidopsis thaliana were isolated by means of heterologous probe screening, homologous EST clones and genetic complementation in an Escherichia coli mutant. The identity of the isolated cDNAs was confirmed by functional complementation of serine auxotrophy in E. coli mutants and/or the detection of catalytic activity in the recombinant enzymes produced in E. coli. Northern blot analyses indicated the most preferential expression of these three genes in light-grown roots. In contrast, the mRNAs of two proteins involved in the glycolate pathway (H-protein of glycine decarboxylase multienzyme complex and serine hydroxymethyltransferase) accumulated to high levels in light-grown shoots. Environmental stresses, such as high salinity, flooding and low temperature, induced changes in mRNA levels of enzymes in the plastidic phosphorylated serine biosynthetic pathway but not in that of the glycolate pathway. These results indicate that the plastidic 3-phosphoglycerate pathway plays an important role in supplying serine in non-photosynthetic tissues in plants and under environmental stresses.

Arabidopsis↗