Molecular and cellular basis of ornithine δ-aminotransferase deficiency caused by the V332M mutation associated with gyrate atrophy of the choroid and retina

Gyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5′-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic–γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT_KO). When overexpressed in HEK-OAT_KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs.     

A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems

Objectives

Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes.

Results

A correlation between evaporation and the concentration of Na⁺ was found (R²?=?0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na⁺ concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7?±?6.7% without volume corrections to 6.9?±?6.5% with volume corrections.

Conclusion

A procedure was established to indirectly measure evaporation through a correlation with the level of Na⁺ ions in solution and deriving a simple formula to account for liquid losses.

     

The metazoan meiobenthos along a depth gradient in the Arctic Laptev Sea with special attention to nematode communities

The meiobenthos along a depth transect of oligotrophic sediments in the Arctic Laptev Sea was studied. The meiobenthos followed the general trends reported from other studies: densities decreased with depth in relation to the more limited supply of degradable organic matter at greater depths. Although the sediments along the transect were poor in organic matter in comparison with the NE Atlantic, the densities fitted well with the meiobenthic densities reported from the latter area. It is suggested that the meiobenthos in the cold polar waters is adapted to this extreme environment by a rapid response to short food pulses to the sediments. Nematodes were identified up to genus level and assigned to trophic groups. A total of 32 families comprising 95 genera were found along the transect. The communities were dominated by deposit feeders whose importance increased with depth. Both TWINSPAN and CCA analyses revealed a community shift along the depth transect: a shelf community dominated by Microlaimus and Chromadora could be distinguished from a slope community dominated by Monhystera and Leptolaimus. Generic diversity decreased with depth. Received: 15 March 1997 / Accepted: 29 June 1997     

Epipregnanolone Acts as a Partial Agonist on a Common Neurosteroid Modulatory Site of the GABAA Receptor Complex in Avian CNS

Neurosteroid modulatory sites present in the GABA_A receptor complex in chick optic lobe were investigated, in order to evaluate whether allopregnanolone and alphaxalone act through a common site of action. Results showed that either allopregnanolone or alphaxalone present a single-component enhancement of [³H]flunitrazepam binding with EC₅₀ of 1.18 ± 0.12 and 6.56 ± 0.86 M and E_max of 82.18 ± 5.80 and 62.98 ± 3.73 %, respectively. Epipregnanolone behaved as a partial agonist of these steroid modulatory sites with EC₅₀ of 0.49 ± 0.15 M and E_max 12.34 ± 1.03%. Moreover, the addition of 16 M epipregnanolone to either allopregnanolone or alphaxalone decreased EC₅₀ values to 0.54 ± 0,09 and 1.24 ± 0.25 M respectively, while E_max values were not significantly affected. On the other hand, additivity experiments disclosed that a maximal concentration (16 M) of alphaxalone in the presence of allopregnanolone failed to enhance [³H]flunitrazepam binding in excess of that produced by allopregnanolone alone. Results indicate that not only allopregnanolone and alphaxalone act through a common site of action, but such site is highly stereospeciflc with regard to the neurosteroid spatial configuration.     

Role of prostaglandin H synthase-2-mediated conversion of arachidonic acid in controlling 3T6 fibroblast growth

The specific role(s) of arachidonic acid (AA) and itsmetabolites in the signaling pathways that regulated fibroblast growth was studied. A Western blot analysis demonstrated that prostaglandin Hsynthase-2 (PGHS-2) was expressed by 3T6 fibroblast cultures in RPMI1640 supplemented with fetal calf serum (10%). Dexamethasone, whichinhibits AA release and PGHS-2 expression, significantly reduced cellproliferation. Ketoprofen, a dual cyclooxygenase inhibitor, andCGP-28238, a specific PGHS-2 inhibitor, reduced fibroblastproliferation in a dose-dependent manner. These drugs also reduced[³H]thymidineincorporation into the DNA of fibroblasts. These effects werecorrelated with a decrease in prostaglandin (PG)E₂ levels in the cell medium.However, piroxicam at doses that selectively inhibit PGHS-1 did nothave a significant effect on fibroblast proliferation. Finally, weshowed that the antiproliferative effect of dexamethasone and PGHS-2inhibitors was significantly antagonized whenPGE₂ was added to the culturemedium. Our results suggest that PGHS-2 and prostaglandins such asPGE₂ might play an important rolein the regulation of 3T6 fibroblast growth stimulated by growth factorsof serum.

     

Inactivation and Degradation of CuZn-SOD by Active Oxygen Species in Wheat Chloroplasts Exposed to Photooxidative Stress

Changes in CuZn-SOD actvity and content in isolated wheat chloroplastsunder the light, and the involvement of protease(s) and/or activeoxygen species in this process were studied. Both SOD activityand content decayed with exposure time to photooxidative stress.Ascorbate, a H₂O₂ scavenger, prevented photooxidation-associatedinactivation of SOD, while benzoate, a OH scavenger, preventedSOD degradation. Wheat chloroplasts incubated in the dark didnot hydrolyze exogenous or endogenous SOD, either H₂O₂-pretreatedor not. Protease inhibitors did not prevent SOD degradationunder photooxidative treatment, suggesting that plastid protease(s)did not participate in this process. Purified chloroplast CuZn-SODwas exposed to H₂O₂ and or OH-generating systems. had no effect on either SOD activity or stability (estimated bynative PAGE). H₂O₂ up to 700µM inhibited SOD in a dose-dependentmanner and induced charge/mass changes as seen by native PAGE.OH also reduced SOD activity by inducing its fragmentation.High levels of active oxygen, as can be generated under strongstress conditions, could directly inactivate and degrade chloroplasticSOD. ₁ Present address: Departamento de Biología Vegetal,Univer-sidad de Alcalá de Henares, Alcalá de Henares,E-28871 Madrid, Spain     

The effect of NaCl stress and relief on gas exchange properties of two olive cultivars differing in tolerance to salinity

Young olive plants (Olea europaea L.) were grown either in hydroponic or soil culture in a glasshouse over two growing seasons. Plants were exposed to NaCl concentrations between 0 and 200 mM for 34–35 days followed by 30–34 days of relief from stress to determine the effect of salinity on gas exchange of two cultivars ('Frantoio' and 'Leccino') differing in salt-exclusion capacity. Salinity stress brought about a reduction in net CO₂ assimilation and stomatal conductance in both cultivars, but the effect was more pronounced in the salt tolerant 'Frantoio' than in the salt-sensitive 'Leccino' cultivar. Therefore, gas exchange parameters may be misleading if used to evaluate the salt tolerance of olive genotypes. Recovery in gas exchange parameters during relief from stress was slower in the salt sensitive cultivar. In general, the decline in assimilation reflected the salt-induced reduction in stomatal conductance, but a marked effect on carboxylation efficiency and CO₂ compensation point was measured in plants treated with 200 mM NaCl for four weeks. The cultivar 'Frantoio' showed a 50% reduction in assimilation and stomatal conductance at 146 and 78 mM leaf Na+ concentration (tissue water molar basis) respectively, whereas the corresponding 50% thresholds for the cultivar 'Leccino' were at 275 and 264 mM, respectively.     

Effect of carbon and nitrogen limitation on lignin peroxidase and manganese peroxidaseproduction by Phanerochaete flavido-alba

The ligninolytic enzymes lignin peroxidase (LiP) and manganese dependent peroxidase(MnP), were detected in extracellular fluids of Phanerochaete flavido-alba FPL 106507cultures under carbon or nitrogen limitation. MnP activities were found to be higher than LiPactivities under all growth conditions tested. Higher titres of both peroxidases were obtainedunder carbon limitation in excess nitrogen. Isoelectric points (pIs) observed after FPLC and IEFof concentrated extracellular fluids revealed more acidic pIs for LiP enzymes obtained innitrogen-limited cultures than those in carbon-limited cultures. However, the change in thelimiting growth factor does not significantly affect MnP pIs.     

DNA aberrations in urinary bladder cancer detected by flow cytometry and FISH: prognostic implications.

We evaluated the genetic changes in bladder cancer biopsy by fluorescence in situ hybridization (FISH) and related them to stage and grade of the tumor, ploidy (FCM) and clinical outcome, to determine a simple method to identify tumors with a poorer prognosis. Using FISH the numerical aberrations of chromosomes 1, 7, 9, 17 in tumor's imprints of 70 patients with transitional cell cancer (TCC) were determined. First of all, the data demonstrated that the sensitivity of FISH in detecting quantitative DNA aberrations exceeds FCM's sensitivity. The frequency of chromosome 1 and 9 aberrations did not show significant differences in diploid and aneuploid tumors in different stage and grade. On the contrary, the chromosome 7 and 17 aneusomy showed greater differences between pT1 and pT2-3 tumors (p<0.032 and p<0.0006, respectively) than between stage pTa and pT1. In our investigation, an increasing number of aberrations was observed in all chromosomes examined in tumors of patients who afterwards underwent cystectomy and/or had recurrent tumors. These results suggest that chromosome 7 and 17 aneusomy could be predictive of adverse outcome in a subgroup of patients with superficial tumors at presentation.     

Saponification of esters of chiral alpha-amino acids anchored through their amine function on solid support.

Anchoring an alpha-amino acid residue by its amine function onto a solid support is an alternative to develop chemistry on its carboxylic function. This strategy can involve the use of amino-acid esters as precursors of the carboxylic function. A complete study on the Wang-resin was performed to determine the non racemizing saponification conditions of anchored alpha-amino esters. The use of LiOH, NaOH, NaOSi(Me)3, various solvents and temperatures were tested for this reaction. After saponification and cleavage from the support, samples were examined through their Marfey's derivatives by reversed phase HPLC to evaluate the percentage of racemization.