Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein

Elongation of very long chain fatty acids 4 (ELOVL4) is a novel member of the ELO family of genes that are involved in fatty acid metabolism. ELOVL4 encodes a putative transmembrane protein of 314 amino acids that carries a possible endoplasmic reticulum (ER) retention/retrieval signal (KXKXX) at the C-terminus. Two distinct mutations, a 5-bp deletion and a complex mutation from the same region in exon 6 of this gene, have been reported so far and are associated with autosomal dominant atrophic macular degeneration (adMD/STGD3). Both of these deletions could result in C-terminal truncation and loss of the ER retention signal in the mutant protein. We expressed the wild-type and mutant proteins in COS-7 and CHO cells to study the intracellular distribution of ELOVL4 and to identify possible implications of the above mutations in its localization. Immunofluorescence analysis of these proteins along with organelle marker antibodies revealed predominant ER localization for wild-type ELOVL4. Targeted deletion of the dilysine motif at the C-terminus of the protein resulted in the loss of ER localization. Immunoelectron microscopy and immunofluorescence analysis revealed a similar ER localization pattern for the protein in human photoreceptors. These data indicate that ELOVL4 is an ER-resident protein, which supports its suggested function in fatty acid elongation. We also demonstrate that the localization of both mutant proteins was dramatically changed from an ER to a Golgi distribution. Our observations suggest that the consequences of defective protein trafficking could underlie the molecular mechanism associated with degeneration of the macula in the patients with adMD/STGD3.     

Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages

Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.     

Physical and functional interaction between Hck tyrosine kinase and guanine nucleotide exchange factor C3G results in apoptosis, which is independent of C3G catalytic domain

The hematopoietic cell kinase Hck is a Src family tyrosine kinase expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells. To study its role in signaling pathways we used the Hck-SH3 domain in protein interaction cloning and identified C3G, the guanine nucleotide exchange factor for Rap1 and R-Ras, as a protein that associated with Hck. This interaction was direct and was mediated partly through the proline-rich region of C3G. C3G could be co-immunoprecipitated with Hck from Cos-1 cells transfected with Hck and C3G. C3G was phosphorylated on tyrosine 504 in cells when coexpressed with Hck but not with a catalytically inactive mutant of Hck. Phosphorylation of endogenous C3G at Tyr-504 was increased by treatment of human myelomonocytic THP-1 cells with mercuric chloride, which is known to activate Hck tyrosine kinase specifically. Coexpression of Hck with C3G induced a high level of apoptosis in many cell lines by 30-42 h of transfection. Induction of apoptosis was not dependent on Tyr-504 phosphorylation or the catalytic domain of C3G but required the catalytic activity of Hck. Using dominant negative constructs of caspases we found that caspase-1, -8, and -9 are involved in this apoptotic pathway. These results suggest that C3G and Hck interact physically and functionally in vivo to activate kinase-dependent and caspase-mediated apoptosis, which is independent of catalytic domain of C3G.     

Cyanobacterial N2 fixation in presence of nitrogen fertilizers

Anabaena oryzae ARM 570 was examined for its growth (chlorophyll and protein), heterocyst frequency, nitrogenase (acetylene reduction) activity, ammonia excretion, and glutamine synthetase and nitrate reductase in response to two levels of urea-N vis-à-vis N2-N. Growth of cyanobacterium increased with duration of incubation. Reduction in heterocyst frequency (40%) was observed at 30 ppm of urea-N, whereas at 60 ppm of urea-N, filaments were completely devoid of heterocysts and no nitrogenase activity was observed. Maximum excretion of ammonia occurred at 30 ppm of urea-N, which was concomitant with minimum glutamine synthetase activity. These results suggested that A. oryzae could be effectively utilized in cyanobacterial biofertilizer programme even in the presence of combined nitrogen, for improving N-budget in rice cultivation.     

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca²⁺ via activation of the steady-state voltage dependent R-type Ca²⁺ channels, endothelin-1 (10^-7 M) and insulin (80 U/ml) were found to induce a sustained increase in cytosolic free Ca²⁺ ([Ca]_i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca²⁺.However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca²⁺ influx and [Ca]_i elevation via activation of the R-type Ca²⁺ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca²⁺ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca²⁺ channel blocker, nifedipine, but were completely reversed by the R-type Ca²⁺ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca²⁺ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).     

Molecular cloning of ELOVL4 gene from cynomolgus monkey (Macaca fascicularis).

ELOVL4, elongation factor of very long chain fatty acids-4, is known to be responsible for autosomal dominant macular degeneration and Stargardt-like macular degeneration. In this study, we cloned the monkey homologue of ELOVL4 and determined the cellular and tissue distribution of the gene product. Sequence analysis of the monkey ELOVL4 gene revealed a high degree of homology between human and monkey. The cloned full-length cDNA of monkey ELOVL4 encoded 314 amino acids, the same length as human and two amino acids longer than mouse. The monkey ELOVL4 conserved the characteristics typical of the super family of ELO enzymes involved in the metabolism of membrane-bound fatty acid elongation. Real-time quantitative PCR demonstrated that the monkey ELOVL4 gene was highly expressed in restricted tissue-specific fashion, not only in the retina but also in the skin (90% of retina) and thymus (111% of retina). Immunohistochemical analysis detected signals predominantly in the photoreceptor layer of the monkey retina.     

Upper airway extraluminal tissue pressure fluctuations during breathing in rabbits.

Transmural pressure at any level in the upper airway is dependent on the difference between intraluminal airway and extraluminal tissue pressure (ETP). We hypothesized that ETP would be influenced by topography, head and neck position, resistive loading, and stimulated breathing. Twenty-eight male, New Zealand White, anesthetized, spontaneously breathing rabbits breathed via a face mask with attached pneumotachograph to measure airflow and pressure transducer to monitor mask pressure. Tidal volume was measured via integration of the airflow signal. ETP was measured with a pressure transducer-tipped catheter inserted in the tissues of the lateral (ETPlat, n = 28) and anterior (ETPant, n = 21) pharyngeal wall. Head position was controlled at 30, 50, or 70 degrees, and the effect of addition of an external resistor, brief occlusion, or stimulated breathing was examined. Mean ETPlat was approximately 0.7 cmH2O greater than mean ETPant when adjusted for degree of head and neck flexion (P < 0.05). Mean, maximum, and minimum ETP values increased significantly by 0.7-0.8 cmH2O/20 degrees of head and neck flexion when adjusted for site of measurement (P < 0.0001). The main effect of resistive loading and occlusion was an increase in the change in ETPlat (maximum - minimum ETPlat) and change in ETPant at all head and neck positions (P < 0.05). Mean ETPlat and ETPant increased with increasing tidal volume at head and neck position of 30 degrees (all P < 0.05). In conclusion, ETP was nonhomogeneously distributed around the upper airway and increased with both increasing head and neck flexion and increasing tidal volume. Brief airway occlusion increased the size of respiratory-related ETP fluctuations in upper airway ETP.     

Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches

Vegetables provide many nutrients in the form of fiber, vitamins, and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement.