GOLPH3L antagonizes GOLPH3 to determine Golgi morphology

GOLPH3 is a phosphatidylinositol-4-phosphate (PI4P) effector that plays an important role in maintaining Golgi architecture and anterograde trafficking. GOLPH3 does so through its ability to link trans-Golgi membranes to F-actin via its interaction with myosin 18A (MYO18A). GOLPH3 also is known to be an oncogene commonly amplified in human cancers. GOLPH3L is a GOLPH3 paralogue found in all vertebrate genomes, although previously it was largely uncharacterized. Here we demonstrate that although GOLPH3 is ubiquitously expressed in mammalian cells, GOLPH3L is present in only a subset of tissues and cell types, particularly secretory tissues. We show that, like GOLPH3, GOLPH3L binds to PI4P, localizes to the Golgi as a consequence of its PI4P binding, and is required for efficient anterograde trafficking. Surprisingly, however, we find that perturbations of GOLPH3L expression produce effects on Golgi morphology that are opposite to those of GOLPH3 and MYO18A. GOLPH3L differs critically from GOLPH3 in that it is largely unable to bind to MYO18A. Our data demonstrate that despite their similarities, unexpectedly, GOLPH3L antagonizes GOLPH3/MYO18A at the Golgi.     

Correction to: Linear relation between leaf xylem water potential and transpiration in pearl millet during soil drying

Plant and Soil - In the original version of this article, equations 4 and 9 unfortunately contained errors     

Fluphenazine Reduces Proteotoxicity in C. elegans and Mammalian Models of Alpha-1-Antitrypsin Deficiency

The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. Elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo. Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models.     

Biogeochemical transformations of amino acids in soil assessed by position-specific labelling

Background and aims

Amino acid turnover in soil is an important element of terrestrial carbon and nitrogen cycles. This study accounts for their driver - the microbial metabolism - by tracing them via the unique isotopic approach of position-specific labeling.

Methods

Three ¹⁴C isotopomers of alanine at five concentration levels combined with selective sterilization were used to distinguish sorption mechanisms, exoenzymatic and microbial utilization of amino acids in soil.

Results

Sorption and microbial uptake occurred immediately. Unspecific microbial uptake followed a linear kinetic, whereas energy-dependent uptake followed Michaelis-Menten. Less than 6 % of the initially added alanine was sorbed to soil, but after microbial transformation products were bound to the soil matrix at higher proportions (5–25 %). The carboxyl group (C-1) was rapidly oxidized by microorganisms, whereas C-2 and C-3 positions were preferentially incorporated into microbial biomass. Dependency of C metabolization on amino acid concentration reflected individual alanine transformation pathways for starvation, maintenance and growth conditions.

Conclusions

This study demonstrates that position-specific labeling determines the mechanisms and rates of C cycling from individual functional groups. This approach reflected underlying metabolic pathways and revealed the formation of new organic matter. We therefore conclude that position-specific labeling is a unique tool for detailed insights into submolecular transformation pathways and their regulation factors.     

Target level blocking of T-cell cytotoxicity for human malignant melanoma by monoclonal antibodies

Cytotoxic T lymphocytes (CTL) for autologous malignant melanoma in culture of a patient AV were induced by restimulation of PBL (peripheral blood leukocytes) with AV melanoma cells in vitro and subcultured in interleukin 2 (IL-2) conditioned media. Monoclonal antibodies detecting six antigenic systems on melanoma cell surfaces were tested for blocking activity on the effector function of subcultured cytolytic T lymphocytes for autologous melanoma cells. The monoclonal antibodies R₂₄ (γ3), specific for the G_D3 disialoganglioside on melanoma cell surfaces and I₂₄ (γM), detecting a similar antigenic determinant, blocked autologous T lymphocytotoxicity for malignant melanoma cells on the target level. The effector function of alloantigen activated cytolytic T lymphocytes generated by coculture of allogeneic PBL with Epstein-Barr virus (EBV) transformed AV B lymphocytes, was blocked by monoclonal antibody R₂₄ when tested against AV melanoma targets, but not when tested against AV B lymphocyte targets. It is concluded that blocking by mAb R₂₄ occurs in this system as a nonspecific effect, unrelated to the specific target antigen recognition by cytotoxic T lymphocytes. Steric hindrance or antibody induced membrane changes may account for the blocking effect of monoclonal antibody R₂₄.     

Chronic ethanol feeding enhances miR-21 induction during liver regeneration while inhibiting proliferation in rats

Liver regeneration is an important repair response to liver injury. Chronic ethanol consumption inhibits and delays liver regeneration in experimental animals. We studied the effects of chronic ethanol treatment on messenger RNA (mRNA) and microRNA (miRNA) expression profiles during the first 24 h after two-thirds partial hepatectomy (PHx) and found an increase in hepatic miR-21 expression in both ethanol-fed and pair-fed control rats after PHx. We demonstrate that the increase of miR-21 expression during liver regeneration is more robust in ethanol-fed rats. Peak miR-21 expression occurs at 24 h after PHx in both ethanol-fed and control rats, corresponding to the peak of hepatocyte S phase in control rats, but not in ethanol-exposed livers in which cell cycle is delayed. The induction of miR-21 24 h after PHx in control rats is not greater than the increase in expression of miR-21 due to sham surgery. However, in the ethanol-fed rat, miR-21 is induced to a greater extent by PHx than by sham surgery. To elucidate the implications of increased miR-21 expression during liver regeneration, we employed unbiased global target analysis using gene expression data compiled by our group. Our analyses suggest that miR-21 may play a greater role in regulating gene expression during regeneration in the ethanol-fed rat than in the control rat. Our analysis of potential targets of miR-21 suggests that miR-21 affects a broad range of target processes and may have a widespread regulatory role under conditions of suppressed liver regeneration in ethanol-treated animals.     

Antibodies to cell surface ganglioside GD3 perturb inductive epithelial-mesenchymal interactions

Most epithelial sheets emerge during embryogenesis by a branching and growth of the epithelium. The surrounding mesenchyme is crucial for this process. We report that branching morphogenesis and the formation of a new epithelium from the mesenchyme in the embryonic kidney can be blocked by a monoclonal antibody reacting with a surface glycolipid, disialoganglioside GD3. In contrast, a more than 10-fold excess of antibodies to adhesive glycoproteins (N-CAM, L-CAM, fibronectin) fails to inhibit morphogenesis. Although the anti-GD3 antibody affected epithelial development, the disialoganglioside GD3 was expressed not in the epithelium, but in the mesenchyme surrounding the developing epithelia. The data raise the intriguing possibility that the anti-GD3 antibody inhibits epithelial development by interfering with epithelial-mesenchymal interactions.     

On maintenance and metabolisms in soil microbial communities

Plant and Soil - Biochemistry is an essential yet undervalued aspect of soil ecology, especially when analyzing soil C cycling. We assume, based on tradition, intuition or hope, that the complexity...