Nonlinear decision-making with enzymatic neural networks.

Artificial neural networks have revolutionized electronic computing. Similarly, molecular networks with neuromorphic architectures may enable molecular decision-making on a level comparable to gene regulatory networks1,2. Non-enzymatic networks could in principle support neuromorphic architectures, and seminal proofs-of-principle have been reported3,4. However, leakages (that is, the unwanted release of species), as well as issues with sensitivity, speed, preparation and the lack of strong nonlinear responses, make the composition of layers delicate, and molecular classifications equivalent to a multilayer neural network remain elusive (for example, the partitioning of a concentration space into regions that cannot be linearly separated). Here we introduce DNA-encoded enzymatic neurons with tuneable weights and biases, and which are assembled in multilayer architectures to classify nonlinearly separable regions. We first leverage the sharp decision margin of a neuron to compute various majority functions on 10 bits. We then compose neurons into a two-layer network and synthetize a parametric family of rectangular functions on a microRNA input. Finally, we connect neural and logical computations into a hybrid circuit that recursively partitions a concentration plane according to a decision tree in cell-sized droplets. This computational power and extreme miniaturization open avenues to query and manage molecular systems with complex contents, such as liquid biopsies or DNA databases.

Impact of cirrhosis in patients undergoing laparoscopic liver resection in a nationwide multicentre survey.

BACKGROUND: The aim was to analyse the impact of cirrhosis on short-term outcomes after laparoscopic liver resection (LLR) in a multicentre national cohort study. METHODS: This retrospective study included all patients undergoing LLR in 27 centres between 2000 and 2017. Cirrhosis was defined as F4 fibrosis on pathological examination. Short-term outcomes of patients with and without liver cirrhosis were compared after propensity score matching by centre volume, demographic and tumour characteristics, and extent of resection. RESULTS: Among 3150 patients included, LLR was performed in 774 patients with (24·6 per cent) and 2376 (75·4 per cent) without cirrhosis. Severe complication and mortality rates in patients with cirrhosis were 10·6 and 2·6 per cent respectively. Posthepatectomy liver failure (PHLF) developed in 3·6 per cent of patients with cirrhosis and was the major cause of death (11 of 20 patients). After matching, patients with cirrhosis tended to have higher rates of severe complications (odds ratio (OR) 1·74, 95 per cent c.i. 0·92 to 3·41; P = 0·096) and PHLF (OR 7·13, 0·91 to 323·10; P = 0·068) than those without cirrhosis. They also had a higher risk of death (OR 5·13, 1·08 to 48·61; P = 0·039). Rates of cardiorespiratory complications (P = 0·338), bile leakage (P = 0·286) and reoperation (P = 0·352) were similar in the two groups. Patients with cirrhosis had a longer hospital stay than those without (11 versus 8 days; P = 0·018). Centre expertise was an independent protective factor against PHLF in patients with cirrhosis (OR 0·33, 0·14 to 0·76; P = 0·010). CONCLUSION: Underlying cirrhosis remains an independent risk factor for impaired outcomes in patients undergoing LLR, even in expert centres.

ANTECEDENTES: El objetivo de este estudio fue analizar el impacto de la cirrosis en los resultados a corto plazo después de la resección hepática laparoscópica (laparoscopic liver resection, LLR) en un estudio de cohortes multicéntrico nacional. MÉTODOS: Este estudio retrospectivo incluyó todos los pacientes sometidos a LLR en 27 centros entre 2000 y 2017. La cirrosis se definió como fibrosis F4 en el examen histopatológico. Los resultados a corto plazo de los pacientes con hígado cirrótico (cirrhotic liver CL) (pacientes CL) y los pacientes con hígado no cirrótico (non-cirrhotic liver, NCL) (pacientes NCL) se compararon después de realizar un emparejamiento por puntaje de propension del volumen del centro, las características demográficas y del tumor, y la extensión de la resección. RESULTADOS: Del total de 3.150 pacientes incluidos, se realizó LLR en 774 (24,6%) pacientes CL y en 2.376 (75,4%) pacientes NCL. Las tasas de complicaciones graves y mortalidad en el grupo de pacientes CL fueron del 10,6% y 2,6%, respectivamente. La insuficiencia hepática posterior a la hepatectomía (post-hepatectomy liver failure, PHLF) fue la principal causa de mortalidad (55% de los casos) y se produjo en el 3,6% de los casos en pacientes CL. Después del emparejamiento, los pacientes CL tendieron a tener tasas más altas de complicaciones graves (razón de oportunidades, odds ratio, OR 1,74; i.c. del 95% 0,92-0,41; P = 0,096) y de PHLF (OR 7,13; i.c. del 95% 0,91-323,10; P = 0,068) en comparación con los pacientes NCL. Los pacientes CL estuvieron expuestos a un mayor riesgo de mortalidad (OR 5,13; i.c. del 95% 1,08-48,6; P = 0,039) en comparación con los pacientes NCL. Los pacientes CL presentaron tasas similares de complicaciones cardiorrespiratorias graves (P = 0,338), de fuga biliar (P = 0,286) y de reintervenciones (P = 0,352) que los pacientes NCL. Los pacientes CL tuvieron una estancia hospitalaria más larga (11 versus 8 días; P = 0,018) que los pacientes NCL. La experiencia del centro fue un factor protector independiente de PHLF (OR 0,33; i.c. del 95% 0,14-0,76; P = 0,010) pacientes CL. CONCLUSIÓN: La presencia de cirrosis subyacente sigue siendo un factor de riesgo independiente de peores resultados en pacientes sometidos a resección hepática laparoscópica, incluso en centros con experiencia.

Effects of the masticatory demand on the rat mandibular development.

The influence of masticatory loading stimulus on mandibular development is not fully clear. In this paper, experimental alterations in the daily muscle use, caused by a changed diet consistency, were continuously monitored, while adaptations in bone and cartilage were examined. It is hypothesised that decreased muscular loading will result in a decrease in the growth factor expression and mandible growth. Fourteen 21-day-old Wistar strain male rats were randomly divided into two groups and fed on either a hard or soft diet for 14 weeks. An implanted radio-telemetric device recorded continuously muscle activity of the superficial masseter muscle. Chondroblast proliferation in the condylar cartilage was identified by insulin-like growth factor-1 receptor (IGF-1r) immunostaining. Furthermore, an X-ray was taken for cephalometric analysis. In the soft-diet group, the duty time of the superficial masseter muscle at higher activity levels was significantly lower than that in the hard-diet group. This decrease in muscular loading of the jaw system was accompanied by: a significant reduction in (i) articular cartilage thickness, (ii) expression of IGF-1r immunopositive cells and (iii) mandible ramus height. In conclusion, a decrease in masticatory demand during the growth period leads to insufficient mandibular development.

Solid tumor size on high-resolution computed tomography and maximum standardized uptake on positron emission tomography for new clinical T descriptors with T1 lung adenocarcinoma.

BACKGROUND: To better describe clinical T descriptors using solid tumor size (the maximum dimension of the solid component of the tumor) on high-resolution computed tomography (HRCT) and maximum standardized uptake value (SUVmax) on F-18-fluorodeoxyglucose positron emission tomography/CT (FDG-PET/CT). PATIENTS AND METHODS: We examined 610 consecutive patients with clinical stage IA lung adenocarcinoma who underwent complete resection. Recurrence-free survival (RFS) was assessed on the basis of whole tumor size (maximum dimension of the tumor), solid tumor size, or a combination of solid tumor size and SUVmax. RESULTS: RFS based on whole tumor size was not significantly different between patients with tumors measuring ≤2 cm and 2-3 cm (P = 0.089), whereas RFS based on solid tumor size was significantly different (P < 0.0001). We divided patients into four groups on the basis of solid tumor size and SUVmax: group 1: solid tumor size ≤2 cm, SUVmax ≤1.8; group 2: solid tumor size ≤2 cm, SUVmax >1.8; group 3: solid tumor size 2-3 cm, SUVmax ≤3.6; and group 4: solid tumor size 2-3 cm, SUVmax >3.6. Groups 2 and 3 were combined because they showed similar RFS each other. RFS was significantly different among these groups: group 1 versus groups 2 + 3, P < 0.0001; groups 2 + 3 versus group 4, P = 0.019. CONCLUSIONS: Both solid tumor size on HRCT and SUVmax on FDG-PET/CT reflect prognosis well in patients with clinical stage IA lung adenocarcinoma and may support new clinical T descriptors.

A cross-sectional study of sarcopenia in Japanese men and women: reference values and association with cardiovascular risk factors.

In this study of Japanese men and women, we determine reference values for sarcopenia and test the hypothesis that sarcopenia is associated with risk factors for cardiovascular disease, independent of waist circumference. A total of 1,488 Japanese men and women aged 18-85 years participated in this study. Appendicular muscle mass (AMM) was measured by dual-energy X-ray absorptiometry. Reference values for classes 1 and 2 sarcopenia (skeletal muscle index: AMM/height2, kg m-2) in each sex were defined as values one and two standard deviations below the sex-specific means of reference values obtained in this study from young adults aged 18-40 years. The reference values for class 1 and class 2 sarcopenia were 7.77 and 6.87 kg m-2 in men and 6.12 and 5.46 kg m-2 in women. In subjects both with class 1 and class 2 sarcopenia, body mass index and % body fat were significantly lower than in normal subjects. Despite whole-blood glycohaemoglobin A1c in men with class 1 sarcopenia was significantly higher than in normal subjects, and brachial-ankle pulse wave velocity in women both with class 1 and class 2 sarcopenia were significantly higher than in normal subjects, using one-way ANCOVA with adjustment for the covariate of waist circumference. Although sarcopenia is associated with thin body mass, it is associated with more glycation of serum proteins in men and with greater arterial stiffness in women, independent of waist circumference.

FcepsilonRI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells.

A significant increase of mRNA expression of thymic stromal lymphopoietin (TSLP) has been reported in the bronchial mast cells (MCs) of asthmatic subjects; however, the mechanism underlying the upregulation of TSLP mRNA and protein remains unknown. FcepsilonRI-mediated activation of human MCs upregulated TSLP mRNA expression by 5.2+/-2.9-fold, while activation of the MCs using lipopolysaccharide and polyriboinosinic:polyribocytidylic acid failed to upregulate TSLP. Stimulation of MCs with interleukin (IL)-4 alone did not affect the TSLP mRNA expression, while pre-incubation of MCs with IL-4 for 48 h significantly enhanced the FcepsilonRI-mediated TSLP mRNA expression (by 53.7+/-15.9-fold; p<0.05) and the amount of TSLP in the cell pellets increased significantly from 23.4+/-4.3 pg mL(-1) to 121.5+/-3.7 pg mL(-1) (p<0.0001). However, the released TSLP was rapidly degraded by proteases that were released by MCs. We identified the population of cells expressing TSLP in the lungs of 16 asthmatic and 11 control subjects by immunohistochemistry. The percentage of TSLP-positive MCs in the total population of MCs was significantly increased in asthmatic airways (p<0.0001). Thus, MCs are able to store TSLP intracellularly and to produce TSLP following aggregation of FcepsilonRI in the presence of IL-4.

Advancement of magma fragmentation by inhomogeneous bubble distribution.

Decompression times reported in previous studies suggest that thoroughly brittle fragmentation is unlikely in actual explosive volcanic eruptions. What occurs in practice is brittle-like fragmentation, which is defined as the solid-like fracture of a material whose bulk rheological properties are close to those of a fluid. Through laboratory experiments and numerical simulation, the link between the inhomogeneous structure of bubbles and the development of cracks that may lead to brittle-like fragmentation was clearly demonstrated here. A rapid decompression test was conducted to simulate the fragmentation of a specimen whose pore morphology was revealed by X-ray microtomography. The dynamic response during decompression was observed by high-speed photography. Large variation was observed in the responses of the specimens even among specimens with equal bulk rheological properties. The stress fields of the specimens under decompression computed by finite element analysis shows that the presence of satellite bubbles beneath a large bubble induced the stress concentration. On the basis of the obtained results, a new mechanism for brittle-like fragmentation is proposed. In the proposed scenario, the second nucleation of bubbles near the fragmentation surface is an essential process for the advancement of fragmentation in an upward magma flow in a volcanic conduit.

Case Report of Extensive Isolated Spontaneous Celiac Trunk Dissection After Liver Transplantation.

Arterial dissection is a rare complication after liver transplantation (LT). We report a case of extensive isolated spontaneous celiac trunk dissection (ISCTD) up to the proper hepatic artery, left gastric artery, and splenic artery after living donor liver transplantation. A 48-year-old woman with cryptogenic liver cirrhosis underwent living donor liver transplantation. Intraoperative and postoperative Doppler ultrasound revealed sufficient flow in the hepatic artery, portal vein, and hepatic vein. On postoperative day (POD) 10, Doppler ultrasound showed reduction of hepatic arterial flow. On POD 16, a contrast-enhanced computed tomography scan showed that the ISCTD extended to the proper hepatic artery, left gastric artery, and splenic artery with an entry tear on the proximal side of the celiac trunk. Although the computed tomography scan showed ischemia of a small part of the liver, blood flow to the liver was kept to some extent. Because all false lumens were occluded by thrombi and the liver enzyme levels normalized, we chose conservative therapy with antiplatelet agents. The patient was discharged on POD 53. She remains well without any liver dysfunction after 18 months with reduction in all false lumens and a patent hepatic artery. Several cases of ISCTD have been reported apart from LT, most of which were treated with conservative therapy. We conclude that conservative therapy could be the first choice in ISCTD even after LT.

Coordination of microtubules and the actin cytoskeleton is important in osteoclast function, but calcitonin disrupts sealing zones without affecting microtubule networks.

Bone-resorbing osteoclasts form sealing zones and ruffled borders toward the bone surface. The sealing zone consists of a ring-like alignment of F-actin dots and surrounds the ruffled border, from which protons are secreted into the bone surface. Vacuolar-type proton ATPase (V-ATPase) in osteoclasts is a ruffled border-associated enzyme responsible for the proton secretion. We studied the interaction between microtubules and the actin cytoskeleton in osteoclasts. Confocal microscopic observation revealed that osteoclasts on glass coverslips, dentine slices and Osteologictrade mark discs formed the ring-like structure of F-actin dots, and microtubules overlapped the top of the F-actin dots. Osteoclasts cultured on dentine formed resorption pits within 48 h. The treatment of osteoclasts with cytochalasin D, an F-actin-depolymerizing reagent, induced perturbation of the microtubules in osteoclasts on glass and inhibited their pit-forming activity on dentine in a dose-dependent and reversible manner. Conversely, nocodazole, a microtubule-depolymerizing reagent, disrupted sealing zones and inhibited pit-forming activity of osteoclasts in a dose-dependent and reversible manner. V-ATPase showed a tendency to be localized inside sealing zones in osteoclasts. Treatment of osteoclasts with calcitonin induced both disruption of sealing zones and dispersion of V-ATPase to the whole area of the cytoplasm within 60 min. The microtubule networks in osteoclasts remained unchanged for 60 min even in the presence of calcitonin. These results suggest that coordination of the actin cytoskeleton and microtubules is important in the function of osteoclasts, but calcitonin selectively affects the actin cytoskeleton and induces the dispersion of V-ATPase without causing significant changes in the microtubules.

Effects of calcitonin on the function of human osteoclast-like cells formed from CD14-positive monocytes.

Calcitonin inhibits bone-resorbing activity of osteoclasts. Expression of mRNA of calcitonin receptor (CTR) and its related proteins was examined in human osteoclasts and their progenitors. CD14-positive (CD14 + macrophages) in the monocytes prepared from human peripheral blood cells differentiated into macrophages (CD14 +) presence of macrophage colony-stimulating factor (M-CSF) or into osteoclast-like cells (OCLs) in the presence of M-CSF plus receptor activator of NFkappaB ligand. CD14 macrophages expressed mRNA of CTR-like receptor (CRLR), receptor activity modifying protein (RAMP) 1, RAMP2, and RAMP3, but not CTR. In contrast, OCLs expressed mRNA of CTR but not CRLR or RAMPs. Human OCLs cultured on dentine slices formed actin rings (corresponding to clear zones) and resorption pits on the slices. Calcitonin disrupted actin rings and inhibited the pit-forming activity of OCLs. CTR is known to couple to cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The effect of calcitonin on actin ring disruption was partially blocked by adding H-7, an inhibitor of both PKA and PKC. Both forskolin, an activator of PKA, and phorbol myristate, an activator of PKC, disrupted actin rings in OCLs. These results suggest that both PKA- and PKC-mediated signals are involved in calcitonin-induced inhibition of human OCL function.

Prognostic value of genetically diagnosed lymph node micrometastasis in non-small cell lung carcinoma cases.

The predictive value of lymph node micrometastasis, detected by immunohistochemical or genetic methods, is well appreciated in terms of prognosis. However, a major problem is high false-positive rates, because most methods focus on cytokeratin, which is a component not only of carcinoma but also normal epithelial and nonepithelial cells. Mutant allele-specific amplification (MASA) can detect DNAs derived from cancer cells itself, reportedly with high sensitivity. It was, therefore, used with nested-PCR using p53 or K-ras mutation for analysis of lymph node micrometastasis in non-small cell lung carcinoma (NSCLC) patients in the present study, in comparison with the immunohistochemical method using an anti-cytokeratin reagent for the same samples. Lymph nodes from 31 NSCLC patients with p53 and K-ras mutated tumors (30 and 1, respectively) staged as pathological (p)-T1-4 N0-1 and M0 were examined. Genetic and immunohistochemical methods demonstrated positive reactions in 34 (15%) and 61 (27%) of 229 lymph nodes, respectively (9 cases, 29%, and 24 cases, 77%). The concordance with the two methods was 77%, but 13 (39%) of 34 genetically positive lymph nodes could not be detected by immunohistochemistry (IHC). Of 22 cases with p-N0 disease, 6 (27%) were genetically positive in hilar and/or mediastinal lymph nodes, and 4 (67%) of them died after cancer relapse. In contrast, none of the patients without micrometastasis died of cancer (P < 0.001, log rank analysis). Of the same p-N0 patients, 17 (77%) were positive by IHC, and 4 (24%) of them died of cancer, whereas 5 negative patients did not suffer cancer relapse. Survival did not significantly differ between cases positive and negative (P = 0.246) by IHC. According to the g-N (N factor restaged by a genetic method), patients with g-N1 and g-N2 disease had a shorter survival than those with g-N0 disease (P = 0.042 and P < 0.001, respectively). However, no significant difference was observed with grading by IHC. Thus, detection of micrometastasis in regional lymph nodes with the MASA method, in other words with a carcinoma-specific marker, is of greater prognostic significance for early stage NSCLC patients than immunohistochemical results. This approach should facilitate selection of patients for whom postoperative adjuvant chemotherapy should be performed.

Synthesis of various kinds of esters by four microbial lipases.

Ester synthesis by microbial lipases, using homogeneous enzyme preparations, were investigated. The amount of synthesized ester was estimated by alkalimetry, and products were identified by thin-layer chromatography and infrared spectroscopy. Lipases from Aspergillus niger, Rhizopus delemar, Geotrichum candidum and Penicillium cyclopium synthesized esters from oleic acid and various primary alcohols. Only Geotrichum candidum lipase synthesized esters of secondary alcohols. Esters of tertiary alcohols, phenols or sugar alcohols were not synthesized by any lipase. Rather high concentrations of alcohol were required to synthesize the esters of ethylene glycol, propylene glycol or trimethylene glycol. Lipases from Aspergillus niger and Rhizopus delemar synthesized oleyl esters of various fatty acids and some dibasic acids. In contrast, lipases from Geotrichum candidum and Penicillium cyclopium synthesized oleyl esters only from medium or long chain fatty acids.

Glyceride synthesis by four kinds of microbial lipase.

Apart from their usual mechanism of action, lipases from Aspergillus niger and Rhizopus delemar also catalyzed the synthesis of glycerides from oleic acid and glycerol. Lipases from Geotrichum candidum and Penicillium cyclopium were inactivated by oleic acid, but were stable in the presence of casein, albumin or buffer of appropriate pH. Lipases from Aspergillus niger and Rhizopus delemar synthesized glycerides from, not only fatty acid, but dibasic acids and aromatic acids, making ester bonds only at position 1 and 3 of glycerol. In contrast, lipases from Geotricum candidum and Penicillium cyclopium synthesized glycerides only from long chain fatty acids, and made ester bonds at all three available positions of the glycerol molecule.

Coordinated Regulation of the Genes Participating in Starch Biosynthesis by the Rice Floury-2 Locus.

The recessive floury-2 (flo-2) locus of rice (Oryza sativa L.), which is located on chromosome 4, causes a strong reduction in expression of the gene encoding an isoform of branching enzyme RBE1 in immature seeds 10 d after flowering. Mapping of the RBE1 gene demonstrated the localization on rice chromosome 6, suggesting that the wild-type Floury-2 (Flo-2) gene regulates RBE1 gene expression in trans. However, reduced expression of the genes encoding some other starch-synthesizing enzymes, including another isoform of branching enzyme RBE3 and granule-bound starch synthase, was also found in the flo-2 seeds. In spite of the low level of RBE1 gene expression in the immature seeds of the flo-2 mutants, the RBE1 gene was equally expressed in the leaves of the wild type and flo-2 mutants. Thus, these results imply that the Flo-2 gene may co-regulate expression of some of the genes participating in starch synthesis possibly in a developing seed-specific manner.

The Tokyo subway sarin attack--lessons learned.

The sarin gas attack in the Tokyo subway system is reviewed from a clinical toxicology perspective. Based on the lessons learned from this attack, the following areas should be addressed on a global scale. First, an adequate supply of protective equipment is required, including level B protective equipment with a pressure demand breathing apparatus. In addition, a system should be established that enables a possible cause to be determined based on symptoms, physical findings, general laboratory tests, and a simple qualitative analysis for poisonous substances. If an antidote is needed, the system should enable it to be administered to the victims as quickly as possible. Preparation for a large-scale chemical attack by terrorists requires the prior establishment of a detailed decontamination plan that utilizes not only mass decontamination facilities but also public facilities in the area. A system should be established for summarizing, evaluating, and disseminating information on poisonous substances. Finally, a large-scale scientific investigation of the Tokyo sarin attack should be conducted to examine its long-term and subclinical effects and the effects of exposure to asymptomatic low levels of sarin.

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.

Phosphate transporter gene family of Arabidopsis thaliana.

Using a high-affinity phosphate transporter gene of Arabidopsis thaliana, PHT1, as a probe, three Arabidopsis homologs were isolated, named PHT4, PHT5 and PHT6, in addition to the previously isolated PHT2 and PHT3. The amino acid sequences deduced from the three nucleotides were 32-42% homologous with microbial phosphate transporters of Saccharomyces cerevisiae (PHO84), Neurospora crassa (PHO-5) and Glomus versiforme (GvPT). PHT1, PHT2, PHT3 and PHT6 were clustered in a 25-kbp region of chromosome V. PHT1 and PHT4 transcripts were detected in roots. Interestingly, suspension-cultured cells expressed only PHT4. PHT4 and PHT5 located within a genetic distance of 6.4 cM on chromosome II, and were close to a phosphate accumulation mutant pho2. Genomic sequencing revealed no difference in the sequences of the two genes in both pho2 and wild-type. The PHT4 transcript was expressed at similar levels in the mutant and wild-type. These results demonstrate that neither PHT4 nor PHT5 is allelic to PHO2.

A fine physical map of Arabidopsis thaliana chromosome 5: construction of a sequence-ready contig map.

A fine physical map of Arabidopsis thaliana chromosome 5 was constructed by ordering the clones from YAC, P1, TAC and BAC libraries of the genome using the sequences of a variety of genetic and EST markers and terminal sequences of clones. The markers used were 88 genetic markers, 13 EST markers, 87 YAC end probes, 100 YAC subclone end probes, and 390 end probes of P1, TAC and BAC clones. The entire genome of chromosome 5, except for the centromeric and telomeric regions, was covered by two large contigs 11.6 Mb and 14.2 Mb long separated by the centromeric region. The minimum tiling path of the chromosome was constituted by a total of 430 P1, TAC and BAC clones. The map information is available at the Web site http://www.kazusa.or.jp/arabi/.

Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions.

The sequence determination of the entire genome of the Synechocystis sp. strain PCC6803 was completed. The total length of the genome finally confirmed was 3,573,470 bp, including the previously reported sequence of 1,003,450 bp from map position 64% to 92% of the genome. The entire sequence was assembled from the sequences of the physical map-based contigs of cosmid clones and of lambda clones and long PCR products which were used for gap-filling. The accuracy of the sequence was guaranteed by analysis of both strands of DNA through the entire genome. The authenticity of the assembled sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA using the assembled sequence data. To predict the potential protein-coding regions, analysis of open reading frames (ORFs), analysis by the GeneMark program and similarity search to databases were performed. As a result, a total of 3,168 potential protein genes were assigned on the genome, in which 145 (4.6%) were identical to reported genes and 1,257 (39.6%) and 340 (10.8%) showed similarity to reported and hypothetical genes, respectively. The remaining 1,426 (45.0%) had no apparent similarity to any genes in databases. Among the potential protein genes assigned, 128 were related to the genes participating in photosynthetic reactions. The sum of the sequences coding for potential protein genes occupies 87% of the genome length. By adding rRNA and tRNA genes, therefore, the genome has a very compact arrangement of protein- and RNA-coding regions. A notable feature on the gene organization of the genome was that 99 ORFs, which showed similarity to transposase genes and could be classified into 6 groups, were found spread all over the genome, and at least 26 of them appeared to remain intact. The result implies that rearrangement of the genome occurred frequently during and after establishment of this species.

A Ca(2+)-dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize.

In C4 plants the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is regulated by phosphorylation/dephosphorylation which is mediated by light/dark signals. The study using protein kinase inhibitors showed that the inhibition pattern of maize PEPC-protein kinase (PEPC-PK) is similar to that of myosin light chain kinase, a Ca(2+)-calmodulin-dependent PK. The kinase activity was also inhibited by EGTA and the inhibition was relieved by Ca2+. These results suggest that PEPC-PK is Ca(2+)-dependent in contrast with previous observations by other research groups.