Expression patterns of <Emphasis Type="Italic">semaphorin7A</Emphasis> and <Emphasis Type="Italic">plexinC1</Emphasis>during rat neural development suggest roles in axon guidance and neuronal migration

Background  

Although originally identified as embryonic axon guidance cues, semaphorins are now known to regulate multiple, distinct, processes crucial for neuronal network formation including axon growth and branching, dendritic morphology, and neuronal migration. Semaphorin7A (Sema7A), the only glycosylphosphatidylinositol-anchored semaphorin, promotes axon growth in vitro and is required for the proper growth of the mouse lateral olfactory tract in vivo. Sema7A has been postulated to signal through two unrelated receptors, an RGD-dependent α1β1-integrin and a member of the plexin family, plexinC1. β1-integrins underlie Sema7A-mediated axon growth and Sema7A function in the immune system. Sema7A-plexinC1 interactions have also been implicated in immune system function, but the neuronal role of this ligand-receptor pair remains to be explored. To gain further insight into the function(s) of Sema7A and plexinC1 during neural development, we present here a detailed analysis of Sema7A and plexinC1 expression in the developing rat nervous system.     

Viable nonsense mutants for the essential gene <Emphasis Type="Italic">SUP45</Emphasis> of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.     

Evidence for a bimodal distribution of <Emphasis Type="Italic">Escherichia coli</Emphasis> doubling times below a threshold initial cell concentration

Background  

In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (τ) only up to a certain threshold cell density. Below this cell concentration (≤ 100 -1,000 CFU mL^-1 ; ≤ 27-270 CFU well^-1) we observed an obvious increase in the τ scatter.     

Assisted Reproductive Technology affects developmental kinetics, <Emphasis Type="Italic">H19</Emphasis> Imprinting Control Region methylation and <Emphasis Type="Italic">H19</Emphasis>gene expression in individual mouse embryos

Background  

In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos.     

Expression of human α<Subscript>1</Subscript>-proteinase inhibitor in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Background  

Human α₁-proteinase inhibitor (α₁-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α₁-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α₁-PI (r-α₁-PI) could provide an attractive alternative. Although r-α₁-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

Modulation of alpha7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells

We showed previously that the expression of ₇-integrin in aortic vascular smooth muscle cells (VSMC) is enhanced in a rat model of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF) on ₇-integrin expression and VSMC adhesion and migration. Expression of the ₇-integrin gene was determined by real-time RT-PCR, whereas protein levels were determined by fluorescence-activated cell sorting analysis. PDGF increased ₇ cell surface protein expression (12 and 24 h: 3.3 ± 0.8- and 3.6 ± 0.4-fold, P < 0.05 vs. control) and mRNA levels (24 h: 3.1-fold, P < 0.05 vs. control) in a time-dependent manner. Actinomycin D and cycloheximide attenuated PDGF-induced increases in ₇-integrin, indicating the involvement of de novo mRNA and protein synthesis. Treatment with the MAPK inhibitors PD-98059, SP-600125, and SB-203580 attenuated PDGF-induced increases in mRNA. In contrast, PD-98059 and SP-600125, but not SB-203580, attenuated PDGF-induced increases in cell surface protein levels. PDGF-treated VSMC adhered to laminin more efficiently (42 ± 6% increase, P < 0.01), and this increase was partially inhibited by anti-₇-integrin function-blocking antibody. However, PDGF did not alter migration on laminin, and there was no effect of the anti-₇-integrin function-blocking antibody on basal or PDGF-stimulated migration. Immunofluorescence imaging revealed an increase in ₇-integrin distribution along the stress fibers. Together, these observations indicate that PDGF enhances ₇-integrin expression in VSMC and promotes ₇-integrin-mediated adhesion to laminin. vascular injury; laminin; mitogen-activated protein kinase     

Differentially expressed genes in embryonic cardiac tissues of mice lacking <Emphasis Type="Italic">Folr1</Emphasis>gene activity

Background  

Heart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs), it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects [1–3]. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRα) gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis.     

A hydrogen-bonding network formed by the B10–E7–E11 residues of a truncated hemoglobin from <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis> is critical for stability of bound oxygen and nitric oxide detoxification

Abstract  

Truncated hemoglobins (trHbs) are distributed from bacteria to unicellular eukaryotes and have roles in oxygen transport and nitric oxide detoxification. It is known that trHbs exist in ciliates of the Tetrahymena group, but trHb structure and function remain poorly understood. To investigate trHb function with respect to stability of bound oxygen and protein structure, we measured the oxygen binding kinetics of Tetrahymena pyriformis trHb, and determined the crystal structure of the protein. The O₂ association and dissociation rate constants of T. pyriformis trHb were 5.5 μM⁻¹ s⁻¹ and 0.18 s⁻¹, respectively. The autooxidation rate constant was 3.8 × 10⁻³ h⁻¹. These values are similar to those of HbN from Mycobacterium tuberculosis. The three-dimensional structure of an Fe(II)–O₂ complex of T. pyriformis trHb was determined at 1.73-? resolution. Tyr25 (B10) and Gln46 (E7) were hydrogen-bonded to a heme-bound O₂ molecule. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom. Furthermore, Tyr25 was hydrogen-bonded to the Gln46 and Gln50 (E11) residues. Mutations at Tyr25, Gln46, and Gln50 increased the O₂ dissociation and autooxidation rate constants. An Fe(III)–H₂O complex of T. pyriformis trHb was formed following reaction of the Fe(II)–O₂ complex of T. pyriformis trHb, in a crystal state, with nitric oxide. This suggests that T. pyriformis trHb functions in nitric oxide detoxification.     

<Emphasis Type="Italic">Iridaea cordata</Emphasis> (Gigartinales,Rhodophyta): responses to artificial UVB radiation

The effects of UVB radiation on the different developmental stages of the carrageenan-producing red alga Iridaea cordata were evaluated considering: (1) carpospore and discoid germling mortality; (2) growth rates and morphology of young tetrasporophytes; and (3) growth rates and pigment content of field-collected plant fragments. Unialgal cultures were submitted to 0.17, 0.5, or 0.83 W m⁻² of UVB radiation for 3 h per day. The general culture conditions were as follows: 12 h light/12 h dark cycles; irradiance of 55 μmol photon^.per square meter per second; temperature of 9 ± 1°C; and seawater enriched with Provasoli solution. All UVB irradiation treatments were harmful to carpospores ( 0.17  \textW \textm ^{- 2} = 40.9 ±6.9% 0.17\;{\text{W}}\,{{\text{m}}^{ - 2}} = 40.9 \pm 6.9\% , 0.5  \textW \textm ^{- 2} = 59.8 ±13.4% 0.5\;{\text{W}}\,{{\text{m}}^{ - 2}} = 59.8 \pm 13.4\% , 0.83  \textW \textm ^{- 2} = 49 ±17.4% 0.83\;{\text{W}}\,{{\text{m}}^{ - 2}} = 49 \pm 17.4\% mortality in 3 days). Even though the mortality of all discoid germlings exposed to UVB radiation was unchanged when compared to the control, those germlings exposed to 0.5 and 0.83 W m⁻² treatments became paler and had smaller diameters than those cultivated under control treatment. Decreases in growth rates were observed in young tetrasporophytes, mainly in 0.5 and 0.83 W m⁻² treatments. Similar effects were only observed in fragments of adult plants cultivated at 0.83 W m⁻². Additionally, UVB radiation caused morphological changes in fragments of adult plants in the first week, while the young individuals only displayed this pattern during the third week. The verified morphological alterations in I. cordata could be interpreted as a defense against UVB by reducing the area exposed to radiation. However, a high level of radiation appears to produce irreparable damage, especially under long-term exposure. Our results suggest that the sensitivity to ultraviolet radiation decreases with increased algal age and that the various developmental stages have different responses when exposed to the same doses of UVB radiation.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Protein,fatty acid,and pigment content of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> under different light regimes

The effects of irradiance and photoperiod on growth rates, chlorophyll a, β-carotene, total protein, and fatty acid content of Chlorella vulgaris were determined. The maximum growth rate (1.13 day⁻¹) was at 100 μmol photons m⁻² s⁻¹ and 16:8-h light/dark photoperiod. Chlorophyll a and β-carotene contents significantly differed under different light regimes with chlorophyll a content lower at high irradiance and longer light duration, while β-carotene showed the inverse trend. The total protein and fatty acid content also significantly differed in different light regimes; the maximum percentage of protein (46%) was at 100 μmol photons m⁻² s⁻¹ and 16:8 h photoperiod, and minimum (33%) was at 37.5 μmol photons m⁻² s⁻¹ and 8:16 h photoperiod; the total saturated fatty acids increased, while monounsaturated and polyunsaturated fatty acids decreased with increasing irradiance and light duration.     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.     

Transcriptional regulation of mouse alpha <Emphasis Type="Italic">A-crystallin</Emphasis> gene in a 148kb <Emphasis Type="Italic">Cryaa</Emphasis> BAC and its derivates

Background  

αA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12–24 hours, compared to the expression of the endogenous Cryaa gene.     

Fed-batch production of a bioflocculant from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The constant-rate fed-batch production of the polygalacturonic acid bioflocculant REA-11 was studied. A controlled sucrose-feeding strategy resulted in a slight improvement in biomass and a 7% reduction in flocculating activity compared with the batch process. When fed with a 3 g l⁻¹ urea solution, the flocculating activity was enhanced to 720 U ml⁻¹ in 36 h. High cell density (2.12 g l⁻¹) and flocculating activity (820 U ml⁻¹) were obtained in a 10-l fermentor by feeding with a sucrose-urea solution, with values of nearly two times and 50% higher than those of the batch process, respectively. Moreover, the residual sucrose declined to 2.4 g l⁻¹, and residual urea decreased to 0.03 g l⁻¹. Even higher flocculating activity of 920 U ml⁻¹ and biomass of 3.26 g l⁻¹ were obtained by feeding with a sucrose-urea solution in a pilot scale fermentation process, indicating the potential industrial utility of this constant-rate feeding strategy in bioflocculant production by Corynebacterium glutamicum.     

Bioreduction of Cu(II) by Cell-Free Copper Reductase from a Copper Resistant <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NA

Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L⁻¹ of Cu(II) from medium amended with 200 mg L⁻¹ of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L⁻¹ after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L⁻¹ of copper, with more then 60 μg L⁻¹ of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.