Polyalcohol ligand in Cu and Fe cluster chemistry: Synthesis, structures and magnetic properties of {Cu12} and {Fe8} aggregates

Structurally unique {Cu₁₂} and {Fe₈} cluster complexes were synthesized using 2,2,6,6-tetrakis (hydroxymethyl)cyclohexanol (thcH₅) ligand. The polyalcohol thcH₅ ligand consists of a six membered ring in a chair confirmation and five pendant alcohol arms providing pentadentate-anchoring points for coordination chemistry. A wide range of reaction conditions was explored with transition metal ions in order to isolate large cluster complexes. Obtained {Cu₁₂} and {Fe₈} complexes exhibit unprecedented core topologies where thcH₅ encapsulate and bridge between metal centers and mediate magnetic interactions via the superexchange pathways. Both complexes exhibit dominant intramolecular antiferromagnetic interactions leading to singlet spin ground state.     

ACQUISITION OF INORGANIC CARBON BY THE MARINE HAPTOPHYTE ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE)1

Inorganic carbon acquisition has been investigated in the marine haptophyte Isochrysis galbana. External carbonic anhydrase (CA) was present in air‐grown (0.034% CO₂) cells but completely repressed in high (3%) CO₂‐grown cells. External CA was not inhibited by 1.0 mM acetazolamide. The capacity of cells to take up bicarbonate was examined by comparing the rate of photosynthetic O₂ evolution with the calculated rate of spontaneous CO₂ supply; at pH 8.2 the rates of O₂ evolution exceeded the CO₂ supply rate 14‐fold, indicating that this alga was able to take up HCO₃ ^? . Monitoring CO₂ concentrations by mass spectrometry showed that suspensions of high CO₂‐grown cells caused a rapid drop in the extracellular CO₂ in the light and addition of bovine CA raised the CO₂ concentration by restoring the HCO₃ ^? ‐CO₂ equilibrium, indicating that cells were maintaining the CO₂ in the medium below its equilibrium value during photosynthesis. A rapid increase in extracellular CO₂ concentration occurred on darkening the cells, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. Active CO₂ uptake was blocked by the photosynthetic electron transport inhibitor 3‐(3′,4′‐dichlorphenyl)‐1,1‐dimethylurea, indicating that CO₂ transport was supported by photosynthetic reactions. These results demonstrate that this species has the capacity to take up HCO₃ ^? and CO₂ actively as sources of substrate for photosynthesis and that inorganic carbon transport is not repressed by growth on high CO₂, although external CA expression is regulated by CO₂ concentration.     

17beta-Estradiol supplementation reduces tubulointerstitial fibrosis by increasing MMP activity in the diabetic kidney

We previously reported that supplementation with 17beta-estradiol (E2) attenuates albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis in diabetic nephropathy. The present study examined the mechanisms by which E2 regulates extracellular matrix (ECM) metabolism, a process that contributes to the development of glomerulosclerosis and tubulointerstitial fibrosis. The study was performed in female nondiabetic (ND), streptozotocin-induced diabetic (D), and diabetic with E2 supplementation (D+E2) Sprague-Dawley rats for 12 wk. Diabetes was associated with an increase in the renal expression of collagen alpha type IV [ND, 0.22 +/- 0.02; D, 0.99 +/- 0.09 relative optical density (ROD); P < 0.05] and fibronectin protein (ND, 0.36 +/- 0.08; D, 1.47 +/- 0.08 ROD; P < 0.05), as measured by Western blot analysis. E2 supplementation partially attenuated this increase in collagen alpha type IV (D+E2, 0.47 +/- 0.10 ROD) and fibronectin (D+E2, 0.71 +/- 0.16 ROD) protein expression associated with D. Diabetes was also associated with a decrease in the expression of matrix metalloproteinase (MMP) isoform MMP-2 (ND, 0.79 +/- 0.01; D, 0.62 +/- 0.06 ROD; P < 0.05) and MMP-9 protein (ND, 0.49 +/- 0.02; D, 0.33 +/- 0.03 ROD; P < 0.05). E2 supplementation restored MMP-2 and MMP-9 protein to levels similar or even greater than in the ND kidneys (MMP-2, 0.75 +/- 0.06; MMP-9, 0.73 +/- 0.01 ROD). The activities of MMP-2 (ND, 7.88 +/- 0.44; D, 5.60 +/- 0.54 ROD; P < 0.05) and MMP-9 (ND, 29.9 +/- 1.8; D, 12.9 +/- 2.3 ROD; P < 0.05), as measured by zymography, were also decreased with D. E2 supplementation restored MMP-2 and MMP-9 activity to levels similar to that in ND kidneys (MMP-2, 7.66 +/- 0.35; MMP-9, 21.4 +/- 2.9 ROD). We conclude that E2 supplementation is renoprotective by attenuating glomerulosclerosis and tubulointerstitial fibrosis by reducing ECM synthesis and increasing ECM degradation.     

Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture

Without a scale-down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high-capacity microscale system that is typically used in fed-batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 10⁶ mg/cell/day and 7 × 10 ⁷ cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.     

Identification of a homozygous splice site mutation in the dynein axonemal light chain 4 gene on 22q13.1 in a large consanguineous family from Pakistan with congenital mirror movement disorder

Mirror movements (MRMV) are involuntary movements on one side of the body that mirror voluntary movements on the opposite side. Congenital mirror movement disorder is a rare, typically autosomal-dominant disorder, although it has been suspected that some sporadic cases may be due to recessive inheritance. Using a linkage analysis and a candidate gene approach, two genes have been implicated in congenital MRMV disorder to date: DCC on 18q21.2 (MRMV1), which encodes a netrin receptor, and RAD51 on 15q15.1 (MRMV2), which is involved in the maintenance of genomic integrity. Here, we describe a large consanguineous Pakistani family with 11 cases of congenital MRMV disorder reported across five generations, with autosomal recessive inheritance likely. Sanger sequencing of DCC and RAD51 did not identify a mutation. We then employed microarray genotyping and autozygosity mapping to identify a shared region of homozygosity-by-descent among the affected individuals. We identified a large autozygous region of ~3.3 Mb on chromosome 22q13.1 (Chr22:36605976?39904648). We used Sanger sequencing to exclude several candidate genes within this region, including DMC1 and NPTXR. Whole exome sequencing was employed, and identified a splice site mutation in the dynein axonemal light chain 4 gene, DNAL4. This splice site change leads to skipping of exon 3, and omission of 28 amino acids from DNAL4 protein. Linkage analysis using Simwalk2 gives a maximum Lod score of 6.197 at this locus. Whether or how DNAL4 function may relate to the function of DCC or RAD51 is not known. Also, there is no suggestion of primary ciliary dyskinesis, situs inversus, or defective sperm in affected family members, which might be anticipated given a putative role for DNAL4 in axonemal-based dynein complexes. We suggest that DNAL4 plays a role in the cytoplasmic dynein complex for netrin-1-directed retrograde transport, and in commissural neurons of the corpus callosum in particular. This, in turn, could lead to faulty cross-brain wiring, resulting in MRMV.     

Proteomic analysis of zebrafish caudal fin regeneration

The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal α1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism.     

Inorganic carbon acquisition in some synurophyte algae

Some characteristics of photosynthesis of three synurophyte algae, Synura petersenii, Synura uvella and Tessellaria volvocina were investigated to determine the mechanism of inorganic carbon (C(i)) uptake. All three species were found to have no external carbonic anhydrase, no capacity for direct bicarbonate uptake and a low whole-cell affinity for C(i). The internal pH of S. petersenii determined using (14)C-benzoic acid and [2-(14)C]-5,5-dimethyloxazolidine-2,4-dione was pH 7.0-7.5, over an external pH range of 5.0-7.5. Thus, the pH difference between the cell interior of S. petersenii and the external medium was large enough, over the alga's growth range, to allow the accumulation of C(i) by the diffusive uptake of CO(2). Monitoring O(2) evolution and CO(2) uptake by suspensions of S. petersenii at pH 7.0 by mass spectrometry did not indicate a rapid uptake of CO(2), and the final CO(2) compensation concentration reached was 24 +/- 0.7 microM. Furthermore, when the cells were darkened, a brief burst of CO(2) occurred before a steady rate of dark respiration was established, suggesting a loss of CO(2) by photorespiration. An examination of the kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in homogenates of cells of S. petersenii, S. uvella and Mallomonas papillosa showed that values of the K(m) (CO(2)) were 28.4, 41.8 and 18.2 microM, respectively. These species lack the characteristics of cells with a CO(2)-concentrating mechanism because the cell affinity for C(i) appears to be determined by the relatively high CO(2) affinity of the Rubisco of these algae.     

Influence of nutrients,disturbances and site conditions on carbon stocks along a boreal forest transect in central Canada

The interacting influence of disturbances and nutrient dynamics on aboveground biomass, forest floor, and mineral soil C stocks was assessed as part of the Boreal Forest Transect Case Study in central Canada. This transect covers a range of forested biomes–-from transitional grasslands (aspen parkland) in the south, through boreal forests, and into the forested subarctic woodland in the north. The dominant forest vegetation species are aspen, jack pine and spruce. Disturbances influence biomass C stocks in boreal forests by determining its age-class structure, altering nutrient dynamics, and changing the total nutrient reserves of the stand. Nitrogen is generally the limiting nutrient in these systems, and N availability determines biomass C stocks by affecting the forest dynamics (growth rates and site carrying capacity) throughout the life cycle of a forest stand. At a given site, total and available soil N are determined both by biotic factors (such as vegetation type and associated detritus pools) and abiotic factors (such as N deposition, soil texture, and drainage). Increasing clay content, lower temperatures and reduced aeration are expected to lead to reduced N mineralization and, ultimately, lower N availability and reduced forest productivity. Forest floor and mineral soil C stocks vary with changing balances between complex sets of organic carbon inputs and outputs. The changes in forest floor and mineral soil C pools at a given site, however, are strongly related to the historical changes in biomass at that site. Changes in N availability alter the processes regulating both inputs and outputs of carbon to soil stocks. N availability in turn is shaped by past disturbance history, litter fall rate, site characteristics and climatic factors. Thus, understanding the life-cycle dynamics of C and N as determined by age-class structure (disturbances) is essential for quantifying past changes in forest level C stocks and for projecting their future change.