VEGFA is necessary for chondrocyte survival during bone development

To directly examine the role of vascular endothelial growth factor (VEGFA) in cartilage development, we conditionally knocked out Vegfa in chondrocytes, using the Col2a1 promoter to drive expression of Cre recombinase. Our study of Vegfa conditional knockout (CKO) mice provides new in-vivo evidence for two important functions of VEGFA in bone formation. First, VEGFA plays a significant role in both early and late stages of cartilage vascularization, since Vegfa CKO mice showed delayed invasion of blood vessels into primary ossification centers and delayed removal of terminal hypertrophic chondrocytes. Second, VEGFA is crucial for chondrocyte survival, since massive cell death was seen in joint and epiphyseal regions of Vegfa CKO endochondral bones. Chondrocytes in these regions were found to upregulate expression of Vegfa in wild-type mice at the time when massive cell death occurred in the Vegfa CKO mice. The expression of the VEGFA receptors Npr1 and Npr2 in epiphyseal chondrocytes and lack of blood vessel reduction in the vicinity of the cartilaginous elements in the Vegfa CKO mice raise the possibility that the observed cell death is the result of a direct involvement of VEGFA in chondrocyte survival. Interestingly, the extensive cell death seen in Vegfa CKO null bones had a striking similarity to the cell death phenotype observed when hypoxia-inducible factor 1 alpha (Hif1a) expression was abolished in developing cartilage. This similarity of cell death phenotypes and the deficient VEGFA production in Hif1a null epiphyseal chondrocytes demonstrate that HIF1 alpha and VEGFA are components of a key pathway to support chondrocyte survival during embryonic bone development.     

Regulation of intra-Golgi membrane transport by calcium

Calcium cations play a critical role in regulating vesicular transport between different intracellular membrane-bound compartments. The role of calcium in transport between the Golgi cisternae, however, remains unclear. Using a well characterized cell-free intra-Golgi transport assay, we now show that changes in free Ca(2+) concentration in the physiological range regulate this transport process. The calcium-chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked transport with an IC(50) of approximately 0.8 mm. The effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was reversible by addition of fresh cytosol and was irreversible when performed in the presence of a Ca(2+) ionophore that depletes calcium from lumenal stores. We demonstrate here that intra-Golgi transport is stimulated by low Ca(2+) concentrations (20-100 nm) but is inhibited by higher concentrations (above 100 nm). Further, we show that calmodulin antagonists specifically block intra-Golgi transport, implying a role for calmodulin in mediating the effect of calcium. Our results suggest that Ca(2+) efflux from intracellular pools may play an essential role in regulating intra-Golgi transport.     

Glucocerebroside treatment ameliorates ConA hepatitis by inhibition of NKT lymphocytes

Concanavalin A (ConA) induces natural killer T (NKT) cell-mediated liver damage. Glucocerebroside (GC) is a naturally occurring glycolipid. Our aims were to determine the effect of GC in a murine model of ConA-induced hepatitis. Mice in groups A and B were treated with GC 2 h before and 2 h following administration of ConA, respectively; group C mice were treated with ConA; group D mice was treated with GC; group E mice did not receive any treatment. Liver damage was evaluated by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver histology. The immune effect of GC was determined by fluorescence-activated cell sorter analysis of intrahepatic and intrasplenic NKT lymphocytes, measurement of cytokine levels, and Western blot analysis for STAT 1, 4, 6, and NF-kappaB expression. The effect of GC on NKT cell proliferation was assessed in vitro. Serum AST and ALT levels were markedly reduced in GC-treated group A mice compared with nontreated group C animals, and histological damage was markedly attenuated in group A. The beneficial effect of GC was associated with a 20% decrease of intrahepatic NKT lymphocytes, significant lowering of serum IFN-gamma levels, and decreased STAT1 and STAT6 expression. In vitro administration of GC led to a 42% decrease of NKT cell proliferation in the presence of dendritic cells but not in their absence. Intraperitoneally administered radioactive GC was detected in the liver and bowel. Administration of GC led to amelioration of ConA hepatitis associated with an inhibitory effect on NKT lymphocytes. GC holds promise as a new immune-modulatory agent.     

The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants

Arabidopsis plants possess a family of nine AtAtg8 gene homologues of the yeast autophagy-associated Apg8/Aut7 gene. To gain insight into how these genes function in plants, first, the expression patterns of five AtAtg8 homologues were analysed in young Arabidopsis plants grown under favourable growth conditions or following exposure to prolonged darkness or sugar starvation. Promoters, plus the entire coding regions (exons and introns) of the AtAtg8 genes, were fused to the beta-glucuronidase reporter gene and transformed into Arabidopsis plants. In all plants, grown under favourable growth conditions, beta-glucuronidase staining was much more significant in roots than in shoots. Different genes showed distinct spatial and temporal expression patterns in roots. In some transgenic plants, beta-glucuronidase staining in leaves was induced by prolonged darkness or sugar starvation. Next, Arabidopsis plants were transformed with chimeric gene-encoding Atg8f protein fused to N-terminal green fluorescent protein and C-terminal haemagglutinin epitope tags. Analysis of these plants showed that, under favourable growth conditions, the Atg8f protein is efficiently processed and is localized to autophagosome-resembling structures, both in the cytosol and in the central vacuole, in a similar manner to its processing and localization under starvation stresses. Moreover, treatment with a cocktail of proteasome inhibitors did not prevent the turnover of this protein, implying that its turnover takes place in the vacuoles, as occurs in yeasts. The results suggest that, in plants, the cellular processes involving the Atg8 genes function efficiently in young, non-senescing tissues, both under favourable growth conditions and under starvation stresses.     

Combined Treatments Reduce Chilling Injury and Maintain Fruit Quality in Avocado Fruit during Cold Quarantine

Quarantine treatment enables export of avocado fruit (Persea americana) to parts of the world that enforce quarantine against fruit fly. The recommended cold-based quarantine treatment (storage at 1.1°C for 14 days) was studied with two commercial avocado cultivars ‘Hass’ and ‘Ettinger’ for 2 years. Chilling injuries (CIs) are prevalent in the avocado fruit after cold-quarantine treatment. Hence, we examined the effect of integrating several treatments: modified atmosphere (MA; fruit covered with perforated polyethylene bags), methyl jasmonate (MJ; fruit dipped in 2.5 μM MJ for Hass or 10 μM MJ for Ettinger for 30 s), 1-methylcyclopropene (1-MCP; fruit treated with 300 ppb 1-MCP for 18 h) and low-temperature conditioning (LTC; a gradual decrease in temperature over 3 days) on CI reduction during cold quarantine. Avocado fruit stored at 1°C suffered from severe CI, lipid peroxidation, and increased expression of chilling-responsive genes of fruit peel. The combined therapeutic treatments alleviated CI in cold-quarantined fruit to the level in fruit stored at commercial temperature (5°C). A successful therapeutic treatment was developed to protect ‘Hass’ and ‘Ettinger’ avocado fruit during cold quarantine against fruit fly, while maintaining fruit quality. Subsequently, treated fruit stored at 1°C had a longer shelf life and less decay than the fruit stored at 5°C. This therapeutic treatment could potentially enable the export of avocado fruit to all quarantine-enforcing countries. Similar methods might be applicable to other types of fruit that require cold quarantine.     

Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway

We have modified the flavor and aroma of tomatoes by expressing the Ocimum basilicum geraniol synthase gene under the control of the tomato ripening-specific polygalacturonase promoter. A majority of untrained taste panelists preferred the transgenic fruits over controls. Monoterpene accumulation was at the expense of reduced lycopene accumulation. Similar approaches may be applicable for carotenoid-accumulating fruits and flowers of other species.     

Mutation in TECPR2 Reveals a Role for Autophagy in Hereditary Spastic Paraparesis

We studied five individuals from three Jewish Bukharian families affected by an apparently autosomal-recessive form of hereditary spastic paraparesis accompanied by severe intellectual disability, fluctuating central hypoventilation, gastresophageal reflux disease, wake apnea, areflexia, and unique dysmorphic features. Exome sequencing identified one homozygous variant shared among all affected individuals and absent in controls: a 1 bp frameshift TECPR2 deletion leading to a premature stop codon and predicting significant degradation of the protein. TECPR2 has been reported as a positive regulator of autophagy. We thus examined the autophagy-related fate of two key autophagic proteins, SQSTM1 (p62) and MAP1LC3B (LC3), in skin fibroblasts of an affected individual, as compared to a healthy control, and found that both protein levels were decreased and that there was a more pronounced decrease in the lipidated form of LC3 (LC3II). siRNA knockdown of TECPR2 showed similar changes, consistent with aberrant autophagy. Our results are strengthened by the fact that autophagy dysfunction has been implicated in a number of other neurodegenerative diseases. The discovered TECPR2 mutation implicates autophagy, a central intracellular mechanism, in spastic paraparesis.