Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

Nitric oxide (NO) serves as a retrograde messenger to activate neuronal NO synthase in the spinal cord via NMDA receptors.

We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.     

A multiscale computational fluid dynamics approach to simulate the micro-fluidic environment within a tissue engineering scaffold with highly irregular pore geometry

Mechanical stimulation can regulate cellular behavior, e.g., differentiation, proliferation, matrix production and mineralization. To apply fluid-induced wall shear stress (WSS) on cells, perfusion bioreactors have been commonly used in tissue engineering experiments. The WSS on cells depends on the nature of the micro-fluidic environment within scaffolds under medium perfusion. Simulating the fluidic environment within scaffolds will be important for gaining a better insight into the actual mechanical stimulation on cells in a tissue engineering experiment. However, biomaterial scaffolds used in tissue engineering experiments typically have highly irregular pore geometries. This complexity in scaffold geometry implies high computational costs for simulating the precise fluidic environment within the scaffolds. In this study, we propose a low-computational cost and feasible technique for quantifying the micro-fluidic environment within the scaffolds, which have highly irregular pore geometries. This technique is based on a multiscale computational fluid dynamics approach. It is demonstrated that this approach can capture the WSS distribution in most regions within the scaffold. Importantly, the central process unit time needed to run the model is considerably low.

     

Effect of beta-hydroxybutyrate and acetoacetate on insulin and glucagon secretion from perfused rat pancreas

To elucidate the physiological significance of ketone bodies on insulin and glucagon secretion, the direct effects of beta-hydroxybutyrate (BOHB) and acetoacetate (AcAc) infusion on insulin and glucagon release from perfused rat pancreas were investigated. The BOHB or AcAc was administered at concentrations of 10, 1, or 0.1 mM for 30 min at 4.0 ml/min. High-concentration infusions of BOHB and AcAc (10 mM) produced significant increases in insulin release in the presence of 4.4 mM glucose, but low-concentration infusions of BOHB and AcAc (1 and 0.1 mM) caused no significant changes in insulin secretion from perfused rat pancreas. BOHB (10, 1, and 0.1 mM) and AcAc (10 and 1 mM) infusion significantly inhibited glucagon secretion from perfused rat pancreas. These results suggest that physiological concentrations of ketone bodies have no direct effect on insulin release but have a direct inhibitory effect on glucagon secretion from perfused rat pancreas.     

Nuclear matrix association regions of rat alpha 2-macroglobulin gene

We have identified DNA fragments which bind specifically to the nuclear matrix in vitro, termed matrix association regions (MARs), in the first and fourth introns of rat alpha 2-macroglobulin gene. The MAR in the first intron is enriched with sequences closely related to the cleavage consensus of topoisomerase II, and contains the binding site of nuclear factor-alpha, a sequence-specific DNA binding protein reported previously.     

Stereoselective total synthesis of ceramide Di-, Tri- and tetrahexosides of wheat flour

The first total synthesis of glycosphingolipids isolated from wheat flour has been achieved in a regio- and stereo-controlled manner.Abbreviations THF tetrahydrofuran - DMF dimethylformamide Part 53 in the series Synthetic Studies on Cell Surface Glycans     

Correlative histochemical studies on preneoplastic and neoplastic lesions in the kidney of rats treated with nitrosamines

Renal tubular lesions induced in male rats by two different carcinogens, N-nitrosomorpholine (NNM) and N-ethyl-N-hydroxyethylnitrosamine (EHEN), using a limited exposure "stop" protocol were investigated histochemically to demonstrate phenotypic cellular changes. The parameters measured included basophilia, glycogen content and the activity of the enzymes glucose-6-phosphatase (G6PASE), glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma-glutamyl transpeptidase (gamma-GT). The lesions observed were predominantly of either basophilic or oncocytic types. In each case, tubular lesions (altered tubules) appeared to give rise to epithelial tumors (epitheliomas) with the same cellular phenotype. Basophilic tubules and epitheliomas proved to be strongly positive for GAPDH and G6PDH while demonstrating a reduction or loss of G6PASE, ALP, ACP, gamma-GT, and SDH compared with controls and the surrounding proximal or distal tubules. In addition, large basophilic epitheliomas demonstrated an increase in both SYN and PHO activities. In contrast, most oncocytic tubules and oncocytomas characterized by abundant densely granular cytoplasm showed a reduction in the activity of G6PDH, but were intensely positive for SDH. However, a few oncocytic lesions demonstrated a decrease in both SDH and G6PDH activity. Rarely, decreased SDH and elevated G6PDH activities were observed in altered tubules resembling oncocytic tubules. It remains to be clarified whether these tubules represent a variation of the oncocytic lesions or, perhaps, another type of tubular lesion. The results indicate that basophilic and oncocytic epithelial tumors differ in their cytochemical pattern and histogenesis. In line with earlier suggestions, the basophilic tumors apparently originate from the proximal renal tubules, while the oncocytomas develop from the distal parts of the nephron. The basophilic tumors are characterized by an increased pentose phosphate pathway and glycolysis, with a corresponding reduction in mitochondrial respiration. However, the majority of the oncocytomas show an increased activity of the mitochondrial enzyme SDH, and a marked decrease in the activity of the key enzyme of the pentose phosphate pathway.     

N-terminal amino acid sequence of human urinary prokallikrein

The N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. (Formula; see text) The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-Gln-Ser-Arg(COOH). Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the alpha-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.     

Expression of an Artificial Cl<Superscript>−</Superscript> Channel in Microperfused Renal Proximal Tubules

To better understand the process of fluid movement driven by Cl^– conductance, a Cl^– channel-forming peptide was delivered to the luminal membrane of microperfused rabbit renal proximal tubules. When the peptide (NK₄-M2GlyR) was perfused, a significant new conductance was observed within 3 min and stabilized at 10 min. Alteration of the ion composition revealed it to be a Cl^–-specific conductance. Reabsorption of Cl^– (J _Cl) was increased by NK₄-M2GlyR, but not by a scramble NK₄-M2GlyR sequence, suggesting that the active peptide formed de novo Cl^– channels in the luminal membrane of the perfused tubules. In the presence of the peptide, reabsorption of fluid (J _v) was dramatically increased and J _Na and J _Ca were concomitantly increased. We propose that introduction of the new Cl^– conductance in the luminal membrane leads to a coordinated efflux of water across the membrane and an increase in cation translocation via the paracellular pathway, resulting in an increase in J _v. This novel method could prove useful in characterizing mechanisms of fluid transport driven by Cl^– gradients.     

Pre-junctional inhibitory action of prostaglandin E2 on excitatory neuro-effector transmission in the human bronchus

The effects of prostaglandin E₂ (PGE₂) and indomethacin on excitatory neuro-effector transmission in the human bronchus were investigated by tension recording and microelectrode methods. PGE₂ (10⁻¹⁰–10⁻⁹M) suppressed the amplitude of twitch contractions and excitatory junction potentials (e.j.ps) evoked by field stimulation at a steady level of basal tension obtained by the combined application of indomethacin (10⁻⁵M) and FPL55712 (10⁻⁶M). In doses over 10⁻⁸M, PGE₂ reduced the muscle tone and dose-dependently suppressed the amplitude of twitch contractions. Indomethacin (10⁻⁵ or 5 × 10⁻⁵M) reduced the muscle tone and enhanced the amplitude of twitch contractions and e.j.ps evoked by field stimulation in the presence of FPL55712. PGE₂ (10⁻⁹M) had no effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh) (4 × 10⁻⁷M). However, indomethacin (10⁻⁵M) significantly enhanced the ACh-induced contraction of the human bronchus. These results indicate that PGE₂ in low concentrations has a pre-junctional action to inhibit excitatory neuro-effector transmission in addition to a post-junctional action, presumably by suppressing transmitter release from the vagus nerve terminals in the human bronchial tissues.