Major Branch Point Research Articles

Colon Cancer and Pregnancy

AbstractAs the incidence of colorectal cancer (CRC) in the younger population increases and women start their childbearing journey at later ages, CRC in pregnancy will become an increasingly relevant and decreasingly rare occurrence. Unfortunately, there is a dearth of data on this subject given its historical rarity. CRC is often diagnosed at a late stage in pregnancy because of the conflation of symptoms of pregnancy with symptoms of CRC. A major branch point for the diagnosis and treatment of CRC in pregnancy is whether it is diagnosed early or late in pregnancy. The workup for cancer is similar for pregnant and nonpregnant populations and includes endoscopy and staging computed tomography. Treatment is dependent on the stage of cancer and term of pregnancy. This review summarizes the current evidence for diagnosis, workup, and treatment of CRC during pregnancy and explores issues of fertility after a diagnosis of CRC.

Difficult mask ventilation: An objective criterion

Effective mask ventilation plays a critical role in safe management of airway before intubation or after failed intubation. Mask ventilation (MV) is the primary technique of ventilation before tracheal intubation or insertion of any airway device. Its most unique role however, is as a rescue technique for ventilation should tracheal intubation fail or prove difficult. The ability to establish adequate MV has, therefore, become a major branch point in any difficult airway algorithm. Despite it being an important skill, MV has received little attention in the extensive body of literature and book chapters addressing airway management when compared to laryngoscopy and intubation. At present, there is no standard definition for Difficult Mask Ventilation (DMV) that is based on precise and objective criterion. The authors propose a simple objective criterion to categorize difficult mask ventilation based on the best monitoring parameters.

Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera)

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs markedly from that of opium poppy and other members of the Ranunculales, most notably in an abundance of BIAs possessing the (R)-stereochemical configuration and the absence of reticuline, a major branchpoint intermediate in most BIA producers. Owing to these unique metabolic features and the pharmacological potential of lotus, we set out to elucidate the BIA biosynthesis network in N. nucifera. Here we show that lotus CYP80G (NnCYP80G) and a superior ortholog from Peruvian nutmeg (Laurelia sempervirens; LsCYP80G) stereospecifically convert (R)-N-methylcoclaurine to the proaporphine alkaloid glaziovine, which is subsequently methylated to pronuciferine, the presumed precursor to nuciferine. While sacred lotus employs a dedicated (R)-route to aporphine alkaloids from (R)-norcoclaurine, we implemented an artificial stereochemical inversion approach to flip the stereochemistry of the core BIA pathway. Exploiting the unique substrate specificity of dehydroreticuline synthase from common poppy (Papaver rhoeas) and pairing it with dehydroreticuline reductase enabled de novo synthesis of (R)-N-methylcoclaurine from (S)-norcoclaurine and its subsequent conversion to pronuciferine. We leveraged our stereochemical inversion approach to also elucidate the role of NnCYP80A in sacred lotus metabolism, which we show catalyzes the stereospecific formation of the bis-BIA nelumboferine. Screening our collection of 66 plant O-methyltransferases enabled conversion of nelumboferine to liensinine, a potential anti-cancer bis-BIA from sacred lotus. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.

AGPAT2 interaction with CDP-diacylglycerol synthases promotes the flux of fatty acids through the CDP-diacylglycerol pathway

AGPATs (1-acylglycerol-3-phosphate O-acyltransferases) catalyze the acylation of lysophosphatidic acid to form phosphatidic acid (PA), a key step in the glycerol-3-phosphate pathway for the synthesis of phospholipids and triacylglycerols. AGPAT2 is the only AGPAT isoform whose loss-of-function mutations cause a severe form of human congenital generalized lipodystrophy. Paradoxically, AGPAT2 deficiency is known to dramatically increase the level of its product, PA. Here, we find that AGPAT2 deficiency impairs the biogenesis and growth of lipid droplets. We show that AGPAT2 deficiency compromises the stability of CDP-diacylglycerol (DAG) synthases (CDSs) and decreases CDS activity in both cell lines and mouse liver. Moreover, AGPAT2 and CDS1/2 can directly interact and form functional complexes, which promote the metabolism of PA along the CDP-DAG pathway of phospholipid synthesis. Our results provide key insights into the regulation of metabolic flux during lipid synthesis and suggest substrate channelling at a major branch point of the glycerol-3-phosphate pathway.

Targeted Muscle Reinnervation in the Lower Leg: An Anatomical Study.

Targeted muscle reinnervation reroutes the ends of cut nerves to reinnervate small motor nerves of nearby muscles, with the goal of reducing neuroma pain and/or improving prosthesis function. Anatomical roadmaps for targeted muscle reinnervation have been established in the upper extremity and thigh, but not for the lower leg. The major branch points of motor nerves and the motor entry points to muscles of the leg were dissected in five cadaver specimens. Leg length was defined as distance from the lateral femoral condyle to the lateral malleolus. The distances from the lateral femoral condyle to major branch points and motor entry points were recorded as percentages of leg length to identify targets for targeted muscle reinnervation. The tibialis anterior and extensor digitorum longus were both acceptable targets in the anterior compartment, with an average 4.4 motor entry points within 10 to 80 percent and 3.0 motor entry points within 20 to 80 percent leg length, respectively. The peroneus longus was the best target in the lateral compartment, with an average 5.8 motor entry points within 20 to 70 percent leg length. The gastrocnemius and soleus were both acceptable targets in the superficial posterior compartment, with an average 4.4 motor entry points within 0 to 40 percent and 6.2 motor entry points within 20 to 80 percent leg length, respectively for each muscle. The flexor digitorum longus was the best target in the deep posterior compartment, with an average 6.0 motor entry points within 30 to 90 percent leg length. Targeted muscle reinnervation is technically feasible in the lower leg. This cadaveric study provides a roadmap for incision placement and identification of motor nerve targets.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.

Faecal microbiota of healthy adults in south India: Comparison of a tribal & a rural population.

Background & objectives:The relevance of the gut microbiota to human health is increasingly appreciated. The objective of this study was to compare the gut microbiota of a group of adult tribals with that of healthy adult villagers in Tamil Nadu, India.Methods:Faeces were collected from 10 healthy tribal adults (TAs) in the Jawadhi hills and from 10 healthy villagers [rural adults (RAs)] in Vellore district, Tamil Nadu. DNA was extracted, and 456 bp segments comprising hypervariable regions 3 and 4 of the 16S rRNA gene were amplified, barcoded and 454 sequenced.Results:Totally 227,710 good-quality reads were analyzed. TAs consumed a millets-based diet, ate pork every day, and did not consume milk or milk products. RAs consumed a rice-based diet with meat intake once a week. In both groups, Firmicutes was the most abundant phylum, followed by Proteobacteria, Bacteroidetes and Actinobacteria. The median Firmicutes-to-Bacteroidetes ratio was 34.0 in TA and 92.9 in RA groups. Actinobacteria were significantly low in TA, possibly due to non-consumption of milk. Clostridium constituted the most abundant genus in both groups, but was significantly more abundant in TAs than RAs, while Streptococcus was significantly more abundant in RA (P<0.05). Analyses of genetic distance revealed that the microbiota were distinctly different between TA and RA, and principal component analysis using 550 distinct taxonomically identifiable sequences revealed a clear separation of microbiota composition in the two groups. Phylogenetic analysis of major microbiota indicated clustering of microbial groups at different major branch points for TAs and RAs.Interpretation & conclusions:Phylum Firmicutes and genus Clostridium constituted the bulk of the faecal microbiota, while significant differences in composition between the groups were probably due to differences in diet and lifestyle.

Parental socioeconomic influences on filial educational outcomes in Scotland: patterns of school-level educational performance using administrative data

In Britain there have been manifest changes in the management and organisation of education, but despite these developments there are still persistent inequalities in pupils’ educational outcomes. These inequalities are consequential because school qualifications are known to influence both pupils’ immediate continuation in education, and their later educational and occupational outcomes. The Scottish school system is similar to the system in England and Wales but there are a distinctive set of qualifications. From the mid-1980s until 2013 the final years of compulsory schooling led up to Standard Grade qualifications. Standard Grades were similar to the General Certificates of Secondary Education (GCSEs) and are worthy of detailed sociological examination because they were the first major branching point in the Scottish education system. A specialist dataset using administrative records was constructed for this project. The dataset comprises young people who undertook Standard Grades in Scottish schools between 2007 and 2011, who were members of the Scottish Longitudinal Study (SLS). We analyse pupils’ subject-area outcomes using a latent variable modelling approach, and explore characteristics associated with the membership of latent educational groups. The analyses uncovered four main latent educational groups. One group had very positive outcomes and pupils were generally more socially advantaged; another group had very poor outcomes and were generally more socially disadvantaged. There were two ‘middle’ groups, which both had similar moderate overall Standard Grade outcomes, but notably different subject-area-level outcomes. We conclude that during school hours these pupils are unlikely to be found drinking Iron Brew WKD in their local parks or at home playing on their Xbox; however, they are also unlikely to be filling out university application forms in the next couple of years.

Modifying an Insect Cell N-Glycan Processing Pathway Using CRISPR-Cas Technology.

Fused lobes (FDL) is an enzyme that simultaneously catalyzes a key trimming reaction and antagonizes elongation reactions in the insect N-glycan processing pathway. Accordingly, FDL function accounts, at least in part, for major differences in the N-glycosylation patterns of glycoproteins produced by insect and mammalian cells. In this study, we used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex N-glycans (GlcNAc2Man3GlcNAc2) in S2 cells. As CRISPR-Cas9 has not been widely used to analyze or modify protein glycosylation pathways or edit insect cell genes, these results underscore its broad utility as a tool for these purposes. Our results also confirm the key role of FDL at the major branch point distinguishing insect and mammalian N-glycan processing pathways. Finally, the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures.

Structural Perspectives on the Evolutionary Expansion of Unique Protein-Protein Binding Sites

Structures of protein complexes provide atomistic insights into protein interactions. Human proteins represent a quarter of all structures in the Protein Data Bank; however, available protein complexes cover less than 10% of the human proteome. Although it is theoretically possible to infer interactions in human proteins based on structures of homologous protein complexes, it is still unclear to what extent protein interactions and binding sites are conserved, and whether protein complexes from remotely related species can be used to infer interactions and binding sites. We considered biological units of protein complexes and clustered protein-protein binding sites into similarity groups based on their structure and sequence, which allowed us to identify unique binding sites. We showed that the growth rate of the number of unique binding sites in the Protein Data Bank was much slower than the growth rate of the number of structural complexes. Next, we investigated the evolutionary roots of unique binding sites and identified the major phyletic branches with the largest expansion in the number of novel binding sites. We found that many binding sites could be traced to the universal common ancestor of all cellular organisms, whereas relatively few binding sites emerged at the major evolutionary branching points. We analyzed the physicochemical properties of unique binding sites and found that the most ancient sites were the largest in size, involved many salt bridges, and were the most compact and least planar. In contrast, binding sites that appeared more recently in the evolution of eukaryotes were characterized by a larger fraction of polar and aromatic residues, and were less compact and more planar, possibly due to their more transient nature and roles in signaling processes.

De novo production of the key branch point benzylisoquinoline alkaloid reticuline in yeast

Microbial biosynthesis for plant-based natural products, such as the benzylisoquinoline alkaloids (BIAs), has the potential to address limitations in plant-based supply of established drugs and make new molecules available for drug discovery. While yeast strains have been engineered to produce a variety of downstream BIAs including the opioids, these strains have relied on feeding an early BIA substrate. We describe the de novo synthesis of the major BIA branch point intermediate reticuline via norcoclaurine in Saccharomyces cerevisiae. Modifications were introduced into yeast central metabolism to increase supply of the BIA precursor tyrosine, allowing us to achieve a 60-fold increase in production of the early benzylisoquinoline scaffold from fed dopamine with no supply of exogenous tyrosine. Yeast strains further engineered to express a mammalian tyrosine hydroxylase, four mammalian tetrahydrobiopterin biosynthesis and recycling enzymes, and a bacterial DOPA decarboxylase produced norcoclaurine de novo. We further increased production of early benzylisoquinoline scaffolds by 160-fold through introducing mutant tyrosine hydroxylase enzymes, an optimized plant norcoclaurine synthase variant, and optimizing culture conditions. Finally, we incorporated five additional plant enzymes – three methyltransferases, a cytochrome P450, and its reductase partner – to achieve de novo production of the key branch point molecule reticuline with a titer of 19.2μg/L. These strains and reconstructed pathways will serve as a platform for the biosynthesis of diverse natural and novel BIAs.

Microbes don't play solitaire: how cooperation trumps isolation in the microbial world.

Primitive cells did not carry a stable organismal genealogicaltrace. Primitive cellular evolution is basically communal. Thehigh level of novelty required to evolve cell designs is aproduct of communal invention, of the universal HGT field,not intralineage variation. It is the community as a whole, theecosystem, which evolves.– Woese (2002)

Is there a safe zone to avoid superficial radial nerve injury with Kirschner wire fixation in the treatment of distal radius? A cadaveric study.

To determine the relation of the superficial radial nerve to bony land-marks and to identify a safe zone for K-wire pinning in the distal radius. The superficial radial nerve was dissected in sixteen upper extremities of preserved cadavers. We found that the superficial radial nerve emerged from under brachioradialis at a mean distance of 8.45(±1.22)cm proximal to the radial styloid. The mean distance from the first major branching point of the superficial radial nerve to the radial styloid were 4.8±0.4cm. All branches of the superficial radial nerve were found to lie in the radial half of an isosceles triangle formed by the radial styloid, Lister's tubercle and the exit point of the superficial radial nerve. There is an elliptical area just proximal to the Lister's tubercle. This area is not crossed by any tendons or nerve. It is bounded by the extensor carpiradialis brevis, extensor pollicis longus. Pinning through the radial styloid is unsafe as the branches of the superficial radial nerve passé close to it. The ulnar half of the isosceles triangle is safe regarding the nerve. The elliptical zone just proximal to the Lister's tubercle is safe regarding the tendons and nerve.

Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis.

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

Single-Stage Minimally Invasive Approach for Large Staghorn Renal Calculus: Points of Technique

Introduction: A large complex staghorn renal calculus has been conventionally dealt with anatrophic pyelolithotomy or multiple access percutaneous nephrolithotomy.1 Although anatrophic nephrolithotomy has been attempted through a minimally invasive approach,2 this inflicts ischemic injury to the renal unit with a consequent nephron loss. Multiple access percutaneous nephrolithotomy may be associated with morbidities like blood loss, arteriovenous fistulas, iatrogenic injuries to the colon and incomplete clearance of calculi with a need for ancillary procedures. We demonstrate a minimally invasive approach that ensures a single-stage complete clearance of staghorn renal calculi with minimal morbidity and no ischemic impact on nephrons. Methods: Patients were evaluated in detail, including clinical, blood profile, and imaging (ultrasound, plain radiography, and CT urogram). Patients with a complex staghorn calculus and preserved parenchymal function underwent a laparoscopic approach through the transperitoneal route. An entry was gained into the Gerotas' fascia followed by identification of the renal hilum and the renal pedicle. After delineating the renal pelvis, an inverted U shaped pyelotomy is created. First attempt is made to remove the stone in toto. If nonretrievable, the staghorn stone is fragmented at major branch points using the lithotrite probe (EMS Lithoclast, probe inserted intracorporeally through the working port) and the major fragments are placed in a retrieval bag. Thereafter, the major caliceal extensions are fragmented using rigid ureteroscopy and lithoclast. Flexible ureteroscope is then inserted intracorporeally through the working port, the minor calices inspected for any residual calculi. After confirming complete clearance of calculi, a ureteric stent is inserted antegrade and the pyelotomy is closed. The ureteral stent is removed 6 weeks postprocedure. Imaging is repeated after the stent removal to ensure complete clearance of calculi. Results and Discussion: Between July 2012 and November 2012, three cases underwent a similar approach for the staghorn renal calculus. The mean age was 38.67 years. The mean body mass index was 23.4 kg/m2. All patients were males. The mean longitudinal stone size was 5.8 cm (range 4.8–7 cm). The laparoscopic approach was possible in all cases. The mean operation duration was 252.67 minutes. The mean blood loss was 95.67 mL. All patients recovered uneventfully. Ureteric stents were removed at 6 weeks postprocedure. In one case, a 7-mL lower caliceal fragment was detected on postprocedure imaging that was retrieved using flexible ureteroscopy during ureteric stent removal. In the other two cases, postoperative imaging revealed the complete clearance of calculi. All patients revealed a normal renal profile till last follow-up. Laparoscopic pyelolithotomy with intracorporeal lithotripsy and usage of rigid and flexible ureteroscopy is an effective option for single-stage management of the complex staghorn calculus. Intracorporeal lithotripsy facilitates removal of calculi that are unable to be retrieved in toto due to branching into calices. The procedure is well-tolerated with a negligible impact on nephrons. No competing financial interests exist. Runtime of video: 6 mins 44 secs

Physical Constraints on Sesquiterpene Diversity Arising from Cyclization of the Eudesm-5-yl Carbocation

The biogenic origins of complex cyclic terpenes derive from the interplay of enzymes and the intrinsic reactivity of carbocation species at major branch-points along intramolecular cyclization pathways to ultimately determine the distribution of terpene skeletal types in nature. Solanaceous plants biosynthesize chemical defense compounds, largely derived from the eremophilane and spirovetivane-type sesquiterpenes. These hydrocarbon skeletons share a common biogenic origin, stemming from alternative Wagner-Meerwein rearrangements of the eudesm-5-yl carbocation during the cyclization of farnesyl pyrophosphate (FPP) catalyzed by sesquiterpene synthases. While the spirojatamane skeleton shares the same carbocation intermediate, this class of sesquiterpenes has not been reported in the Solanaceae and is exceedingly rare in nature. To investigate the physical basis for alternative rearrangements of the eudesm-5-yl carbocation, we carried out quantum mechanics (QM) analyses to calculate the allowable conformations, energies, and transition states linking conformers of the eudesm-5-yl carbocation to the eremophilene, spirovetivane, and spirojatamane skeletons. Additionally, we conducted parallel investigations on simplified decalin carbocation systems to examine the contribution of ring substituents to allowable conformations and rearrangement pathways. Our study reveals that ring substituents expand the conformational space accessible to the eudesm-5-yl carbocation while sterically blocking rearrangements in certain contexts. From our analysis, we define a conformational threshold for each possible rearrangement based on dihedral angles describing transition state geometry. Further, our calculations indicate that methylene migration rearrangements leading to spiro compounds are thermodynamically dominant in the eudesm-5-yl and simpler decalin carabocation systems. Interestingly, the theoretical abundance of sesquiterpene skeletal types arising from the intrinsic reactivity of the eudesm-5-yl carbocation stands in sharp contrast to their currently known natural abundance. The implications of these results for the catalytic tragectories catalyzed by sesquiterpene synthases are discussed.

Aneurysm of the distal anterior cerebral artery radiologically mimicking a ventricular mass

The ventricular system is a rare localization for intracranial aneurysms. Most ventricular aneurysms arise from a distal branch of the choroidal arteries and a major branch point of the circle of Willis. A 41-year-old-man suffering from dizziness of 2 weeks' duration was admitted to the clinic. On radiological examination, he had a well-circumscribed mass involving the frontal horn of the right lateral ventricle without radiological evidence of a prior or recent hemorrhage. Localization and radiological appearance were not typical of a ventricular mass and did not allow diagnosis. After cerebral angiography, an aneurysm arising from the distal anterior cerebral artery was incidentally found in an intraventricular location. This unruptured aneurysm was successfully treated via the endovascular route. The authors describe the unusual case of a distal anterior cerebral artery aneurysm with a dome extending into the right lateral ventricle, which appears to be the first such case in the literature. Angiography may be helpful to neurosurgeons in avoiding the disastrous complications of a biopsy procedure in such unusual cases.

Citrate synthase in Corynebacterium glutamicum is encoded by two gltA transcripts which are controlled by RamA, RamB, and GlxR

Citrate synthase (CS) is located at a major branch point in the metabolism and is required for both tricarboxylic acid and glyoxylic acid cycle activity. Here we show that the CS gene gltA of Corynebacterium glutamicum is monocistronic, but that two transcripts are formed with their transcript initiation sites located 121 bp and 357 bp upstream of the translational start codon, respectively. Northern blot analyses revealed that during growth on acetate the short transcript prevails, whereas during growth on glucose the long transcript is dominant. Further Northern blots, reporter gene fusions, and CS activity measurements in mutants devoid of the transcriptional regulators RamA and RamB or with the global regulator GlxR overexpressed revealed a complex involvement of these regulators in gltA transcription. This was confirmed by demonstrating the direct interaction of isolated RamA, RamB and GlxR proteins with specific gltA promoter regions in vitro. The combined analyses point to an elaborate control of gltA transcript formation, which is possibly required as a dedicated mechanism to balance the total CS activity according to the physiological requirements.

Back-propagation of physiological action potential output in dendrites of slender-tufted L5A pyramidal neurons

Pyramidal neurons of layer 5A are a major neocortical output type and clearly distinguished from layer 5B pyramidal neurons with respect to morphology, in vivo firing patterns, and connectivity; yet knowledge of their dendritic properties is scant. We used a combination of whole-cell recordings and Ca2+ imaging techniques in vitro to explore the specific dendritic signaling role of physiological action potential patterns recorded in vivo in layer 5A pyramidal neurons of the whisker-related ‘barrel cortex’. Our data provide evidence that the temporal structure of physiological action potential patterns is crucial for an effective invasion of the main apical dendrites up to the major branch point. Both the critical frequency enabling action potential trains to invade efficiently and the dendritic calcium profile changed during postnatal development. In contrast to the main apical dendrite, the more passive properties of the short basal and apical tuft dendrites prevented an efficient back-propagation. Various Ca2+ channel types contributed to the enhanced calcium signals during high-frequency firing activity, whereas A-type K+ and BKCa channels strongly suppressed it. Our data support models in which the interaction of synaptic input with action potential output is a function of the timing, rate and pattern of action potentials, and dendritic location.

The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids inArabidopsis thaliana

The aromatic amino acids phenylalanine, tyrosine and tryptophan in plants are not only essential components of protein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acid metabolic pathway, with chorismate serving as a major branch point intermediate metabolite. Yet, the regulation of their synthesis is still far from being understood. So far, only three enzymes in this pathway, namely, chorismate mutase of phenylalanine and tyrosine synthesis, tryptophan synthase of tryptophan biosynthesis and arogenate dehydratase of phenylalanine biosynthesis, proved experimentally to be allosterically regulated. The major biosynthesis route of phenylalanine in plants occurs via arogenate. Yet, recent studies suggest that an alternative route of phynylalanine biosynthesis via phenylpyruvate may also exist in plants, similarly to many microorganisms. Several transcription factors regulating the expression of genes encoding enzymes of both the shikimate pathway and aromatic amino acid metabolism have also been recently identified in Arabidopsis and other plant species.