Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon.

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.     

Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice

Survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA.     

Analysis of calorimetric data for isodesmic self-associating systems.

ATP and respiration (NADH)-driven NAD(P)⁺ transhydrogenase (EC 1.6.1.1) activities are low in membranes from Escherichia coli cultured on yeast extract medium (17 and 21 nmol/min × mg) but high on glucose (82 and 142 nmol/min × mg). The ATPase and respiratory activities in both cases appeared comparable. Growth of the bacteria in yeast extract medium followed by washing and replacement into a glucose medium showed that after 3 h the energy-linked and energy-independent NAD(P)⁺ transhydrogenase (reduction of acetylpyridine NAD⁺ by NADPH) activities had appeared simultaneously. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in these activities indicating that de novo protein synthesis is required for the induction of energy-linked and -independent NAD(P)⁺ transhydrogenase. It was found that the K_m values for acetylpyridine NAD⁺ and NADPH for the energy-independent reaction in membranes from glucose grown cells (143 and 62 μm) were similar to those in membranes from cells grown on glucose-yeast extract (135 and 45 μm), respectively, but the maximum velocity at infinite acetyl pyridine NAD⁺ and NADPH increased from 353 to 2175 nmol/min × mg. Furthermore, the membrane-bound NAD(P)⁺ transhydrogenase in glucose-yeast extract grown cells showed substrate inhibition at high NADPH and low acetyl pyridine NAD⁺ levels. Further kinetic data demonstrate that the mechanism of the energy-independent NAD(P)⁺ transhydrogenase in E. coli is similar to that of the mitochondrial enzyme and exhibits similar responses to competitive inhibitors at the NAD⁺ and NADPH sites.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

Differential scanning calorimetry of asparate transcarbamoylase and its isolate subunits.

The thermal denaturation of aspartate transcarbamoylas of Escherichia coli was investigated by differential scanning calorimetry. Isolated regulatory and catalytic subunits were heat denatured at 55 and 80 degrees C, respectively. In contrast, the intact enzyme was denatured in two steps. A small endotherm near 73 degrees C was assoicated with denaturation of the regulatory subunits and the major endotherm at 82 degrees C with denaturation of the catalytic subunits. Thus regulatory subunits are stabilized against heat denaturation by more than 17 degrees C when incorporated in the enzyme. Similar conclusions were obtained from measurements of the enthalpy of heat denaturation. Regulatory subunits yielded a much lower value of the enthalpy of denaturation, 1.91 cal/g, than that found for the catalytic subunit, 3.94 cal/g, or typical globular proteins (4 to 6 cal/g). When the regulatory subunits were incorporated into aspartate transcarbamoylase their enthalpy of denaturation was increased 125% (to 4.3 cal/g). The enthalpy of the catalytic subunits in the intact enzyme was increased 38% (enthalpy of denaturation of 5.43 cal/g). Stabilization of the isolated catalytic subunit as well as the intact enzyme was achieved by the addition of the bisubstrate analog N-(phosphonacetyl)-L-aspartate. Similarly the allosteric effectors, CTP and ATP, stabilized the isolated regulatory subunits or those subunits within the intact enzyme. However, the addition of the bisubstrate analog caused a decrease in the enthalpy of denaturation of the regulatory subunits within the enzyme. These results are consistent with other studies of the ligand-promoted conformational changes in the native enzyme.     

Clinical Evaluation of a Disposable Artificial Kidney

The in-vivo performance of a new disposable parallel flow countercurrent type of artificial kidney has been compared with that of the modified four-layered Kiil dialyser. The dialysance of urea and endogenous creatinine in the former was significantly better than in the latter for similar blood flow rates, and, moreover, unlike the dialysance with the Kiil dialyser it continued to improve over 12 hours. Among 100 disposable artificial kidneys tested no failure occurred from blood leaks despite the use of high negative pressures in the dialysate compartment. No pyrogen reactions were observed.These new artificial kidneys were ready for use in less than one-seventh of the time taken to prepare the conventional Kiil dialyser. Other advantages are their low priming volumes, the lack of a pump in the blood line, and the reduced risk of infection.