Influence of oilseed supplement ranging in n-6/n-3 ratio on fatty acid composition and Δ5-, Δ6-desaturase protein expression in steer muscles.

This study investigated effects of roasted or extruded oilseed supplementation ranging in n-6/n-3 ratios from 0.3 to 5.0 on the fatty acid composition and expression of delta-5 desaturase (Δ5d) and Δ6-desaturase (Δ6d) protein in commercial steer cheek (m. masseter) and diaphragm (pars costalis diaphragmatis) muscles. In general, the n-6/n-3 ratio of the diet had a subsequent effect on the muscle n-6/n-3 ratio (P < 0.05), with muscle 18:2n-6 and 18:3n-3 content relating to proportion of dietary soya bean and linseed (P < 0.01). Compared with canola, pure linseed and soya bean diets reduced 14:1c-9 and 16:1c-9 (P < 0.05) but increased 18:1t-11 and c-9,t-11 conjugated linoleic acid (CLA) content (P < 0.01). Oilseed processing had a minor influence but extruded oilseeds increase 18:1t-11 and c-9,t-11 CLA compared with roasted (P < 0.05). Polar lipid 18:3n-3 and n-3 long-chain polyunsaturated fatty acid (LC, ⩾20 carbons PUFA) derivative content increased in relation to dietary linseed supplementation in the diaphragm (P < 0.01), whereas only 18:3n-3 was increased in the cheek (P < 0.01). Protein expression did not differ between diets; however, in each muscle the Δ5d protein expression had a stronger association with the desaturase products rather than the precursors. The relationship between Δ5d protein expression and the muscle LC n-6/n-3 ratio was negative in both muscles (P < 0.05). The relationship between Δ6d protein expression and the LC n-6/n-3 ratio was positive in the cheek (P < 0.001) and negative in the diaphragm (P < 0.05). In conclusion, diet n-6/n-3 ratio affected muscle 18:2n-6 and 18:3n-3 deposition, whereas the Δ5d and Δ6d protein expression had some influence on the polar lipid LC-PUFA profile. Results reaffirm that processed oilseeds can be used to increase the proportion of fatty acids potentially beneficial for human health, by influencing the formation of LC-PUFA and reducing the n-6/n-3 ratio.

Dynamin 2 mediates PDGFRα-SHP-2-promoted glioblastoma growth and invasion.

Dynamin 2 (Dyn2), a large GTPase, is involved in receptor tyrosine kinase (RTK)-promoted cell migration. However, the molecular mechanisms by which Dyn2 regulates RTK-induced cell migration have not been established. Recently, we reported that tyrosine-protein phosphatase non-receptor type 11 (SHP-2) and phosphatidylinositol 3-kinase (PI3K) mediate platelet-derived growth factor receptor-α (PDGFRα)-promoted glioma tumor growth and invasion. Here, we show that Dyn2 is an effector downstream of the PDGFRα-PI3K/SHP-2 signaling in glioma cells. Depletion of endogenous Dyn2 by short hairpin RNAs (shRNAs) inhibited PDGFRα-stimulated phosphorylation of Akt, Erk1/2, Rac1 and Cdc42 activities, glioma cell migration and survival in vitro and tumor growth and invasion in the brains of mice. Dyn2 binds to SHP-2 and PI3K and colocalizes with PDGFRα at the invasive fronts in PDGF-A-stimulated glioma cells. Inhibition of SHP-2 by siRNA knockdown abrogated Dyn2 association with activated PDGFRα and PDGFRα activation of Rac1 and Cdc42, and glioma cell migration, thereby establishing a link between SHP-2 interaction with Dyn2 and the PDGFRα signaling. Furthermore, a dominant-negative SHP-2 C459S mutant inhibited PDGF-A-stimulated glioma cell migration, phosphorylation of Dyn2 and concomitantly blocked PDGFRα-induced Src activation. Inhibition of Src by Src inhibitors attenuated PDGF-A-stimulated phosphorylation of Akt and Dyn2 and glioma cell migration. Additionally, mutations of binding sites to PI3K, SHP-2 or Src of PDGFRα impaired PDGFRα-stimulated phosphorylation of Akt and Dyn2, and Dyn2 association with activated PDGFRα. Taken together, this study identifies Dyn2 as an effector that mediates PDGFRα-SHP-2-induced glioma tumor growth and invasion, suggesting that targeting the PDGFRα-SHP-2-Dyn2 pathway may be beneficial to patients with malignant glioblastomas.

Increased expression of the large GTPase dynamin 2 potentiates metastatic migration and invasion of pancreatic ductal carcinoma.

Pancreatic ductal tumors invade local parenchyma and metastasize to distant organs. Src-mediated tyrosine kinase signaling pathways promote pancreatic ductal adenocarcinoma (PDAC) metastasis, though the molecular mechanisms supporting this invasive process are poorly understood and represent important and novel therapeutic targets. The large GTPase Dynamin 2 (Dyn2), a Src-kinase substrate, regulates membrane-cytoskeletal dynamics although it is yet to be defined if it contributes to tumor cell migration and invasion. Therefore, the goal of this study was to test if Dyn2 is upregulated in human pancreatic tumors and to define its role in cell migration and metastatic invasion using in vitro assays and nude mouse models. Histological analysis showed that 81% of 85 patients had elevated Dyn2 in PDAC. To test if Dyn2 overexpression alters metastatic properties of human pancreatic tumor cells, stable clones of BxPC-3 cells overexpressing either wild-type Dyn2 or a phosphorylation-deficient mutant Dyn2Y(231/597)F known to attenuate Dyn2 function, were generated and analyzed. Importantly, tumor cells overexpressing Dyn2 protruded lamellipodia at twice the rate, migrated faster (180%) and farther (2.5-fold greater distance) on glass and through transwell chambers (2-3-fold more cells through the filter) compared with cells expressing Dyn2Y(231/597)F or vector alone. Further, depletion of Dyn2 and dynamin inhibitors Myristyl trimethyl ammonium bromides and Dynasore significantly reduced cell migration, wound healing and invasion in transwell assays compared with controls. To test the metastatic potential conferred by increased Dyn2 expression, the BxPC-3 cell lines were implanted orthotopically into the pancreas of nude mice. Cells expressing Dyn2-green fluorescent protein exhibited a threefold increase in large distal tumors compared with cells expressing Dyn2Y(231/597)F or vector alone. Finally, histological analysis revealed that Dyn2 is upregulated in 60% of human metastatic pancreatic tumors. These findings are the first to implicate dynamin in any neoplastic condition and to directly demonstrate a role for this mechanoenzyme in invasive cell migration.

A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods.

BACKGROUND: Given its prognostic value, there is renewed interest in molecular staging in non-Hodgkin's lymphoma (NHL) using immunoglobulin heavy and light chain (IgH, IgL) gene rearrangements. AIMS: To compare the efficiency of DNA amplification from fresh frozen and formalin-fixed decalcified paraffin-embedded (FFDPE) bone marrow trephines for use in molecular staging using two methods. METHODS: After manually extracting DNA from 13 FFDPE and 14 fresh frozen trephine biopsy specimens, two methods were used to test for amplifiability: use of the amplification control master mix supplied in the In Vivo Scribe immunoglobulin heavy chain (IgH) clonality kit, which creates 5 amplicons between 96-600 base pairs (bp); and real-time amplification of the beta-globin gene. RESULTS: Using the first method, the mean maximum length of amplicons generated from FFDPE trephines was statistically lower at 300 bp compared to fresh frozen samples, all of which generated amplicons up to 600 bp in size (p<0.001). Real-time amplification of the beta-globin gene showed that the mean crossing threshold of fresh frozen samples was statistically lower than that of FFDPE samples (23.48 (95% CI 22.47 to 24.48) vs 33.64 (95% CI 32.15 to 35.12); p<0.001). CONCLUSIONS: Although amplifiable DNA can be extracted from both fresh-frozen and FFDPE trephine samples for IgH/IgL analysis, freshly frozen specimens are superior as a source of template DNA, especially for higher base pair PCR products.

The role of dynamin 3 in the testis.

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.

The cytosolic domain of APP induces the relocalization of dynamin 3 in hippocampal neurons.

Amyloid precursor protein (APP) has been the subject of intense research to uncover its implication in Alzheimer's disease. Its physiological function is, however, still poorly understood. Herein, we investigated its possible influence on the development of cultured hippocampal neurons. A peptide corresponding to the APP intracellular domain linked to a cell-penetrating peptide was used to alter the interactions of APP with its cytosolic partners. This treatment promoted the concentration of the cytosolic GTPase dynamin 3 (Dyn3) in neurite segments when most untreated cells displayed a homogenous punctate distribution of Dyn3. The Dyn3-labelled segments were excluded from those revealed by APP staining after aldehyde fixation. Interestingly, after aldehyde fixation MAP2 also labelled segments excluded from APP-stained segments. Thus APP is also a marker for the spacing pattern of neurites demonstrated by Taylor & Fallon (2006)J. Neurosci., 26, 1154-4463.

Preservation of rainbow trout (Oncorhynchus mykiss) eyed eggs using a perfluorochemical as an oxygen carrier.

The objective of this study was to maintain the viability of chilled rainbow trout (Oncorhynchus mykiss) eyed eggs during storage using oxygenated perfluorochemical (PFC). Three trials were conducted using eggs at 161, 180 or 217 degree days (days from fertilization x incubation temperature in degrees C). A separate trial was conducted for 147 degree day eggs that were not at the eyed stage. For each trial, eggs were stored in a moisture-saturated atmosphere at 1 degrees C in PFC, water, and 1:1 combinations of PFC and PBS, PFC and 0.3 M glucose, PFC and mineral oil, or PFC and water. The PFC was oxygenated before each trial and all media were oxygenated at weekly intervals during the storage period. Eggs from each trial were also incubated without storage to provide Day 0 results. After 3 and 5 weeks of storage, eggs from each medium were incubated at 10 degrees C until hatch. Hatching percentage was expressed as a percentage of Day 0 results. The percentage of normal alevins that hatched was also determined. There were interactions (P < 0.01) between stage of development and treatment for hatching percentage after 3 and 5 weeks of storage. After 3 weeks of storage, eggs stored at 161, 180, or 217 degree days without PFC had hatching rates of 0-14.3% but eggs stored in any medium with PFC had hatching percentages from 75.1 to 106.4% of Day 0 values. After 5 weeks of storage, eggs stored at 161 degree days in PFC plus PBS or PFC plus water, and eggs stored at 217 degree days in PFC or PFC plus water, had higher (P < 0.05) hatching percentages than eggs stored in any of the other media. Eggs stored at 161 degree days for 5 weeks in PFC and water had a higher (P < 0.05) percentage of normal alevins hatching than eggs stored in PFC and PBS. Because of their early developmental stage, eggs stored at 147 degree days had low hatching percentages, except eggs stored for 3 weeks in PFC or PFC plus PBS. Chilling eyed eggs of rainbow trout to 1 degrees C and storing them in water with PFC as an oxygen carrier can preserve their viability for 5 weeks.

Lack of acrosome formation in Hrb-deficient mice.

The sperm acrosome is essential for sperm-egg fusion and is often defective in men with nonobstructive infertility. Here we report that male mice with a null mutation in Hrb are infertile and display round-headed spermatozoa that lack an acrosome. In wild-type spermatids, Hrb is associated with the cytosolic surface of proacrosomic transport vesicles that fuse to create a single large acrosomic vesicle at step 3 of spermiogenesis. Although proacrosomic vesicles form in spermatids that lack Hrb, the vesicles are unable to fuse, blocking acrosome development at step 2. We conclude that Hrb is required for docking and/or fusion of proacrosomic vesicles during acrosome biogenesis.

Alterations in vesicle transport and cell polarity in rat hepatocytes subjected to mechanical or chemical cholestasis.

BACKGROUND & AIMS: The molecular mechanisms that contribute to the cholestatic condition in hepatocytes are poorly defined. It has been postulated that a disruption of normal vesicle-based protein trafficking may lead to alterations in hepatocyte polarity. METHODS: To determine if vesicle motility is reduced by cholestasis, hepatocytes cultured from livers of bile duct ligation (BDL)- or ethinyl estradiol (EE)-injected rats, were viewed and recorded by high-resolution video microscopy. Cholestatic hepatocytes were analyzed by phalloidin staining and electron microscopy. Functional analysis was done by the sodium fluorescein sequestration assay. RESULTS: In cholestatic hepatocytes, there was a significant decrease in the number of motile cytoplasmic vesicles observed compared with control cells. Further examination of cells from BDL- or EE-treated livers revealed the presence of numerous large intracellular lumina. More than 24% of cells in BDL-treated livers and 19% of cells in EE-treated livers displayed these structures, compared with 1.1% found in control hepatocytes. Phalloidin staining of hepatocytes showed a prominent sheath of actin surrounding the lumina, reminiscent of those seen about bile canaliculi. Electron microscopy revealed that these structures were lined by actin-filled microvilli. Further, these pseudocanaliculi perform many of the functions exhibited by bona fide canaliculi, such as sequestering sodium fluorescein. CONCLUSIONS: Both mechanically and chemically induced cholestasis have substantial effects on vesicle-based transport, leading to marked disruption of hepatocellular polarity.

Mammalian dynamin-like protein DLP1 tubulates membranes.

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) but not GTP. EM of the GTPgammaS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPgammaS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

Mitochondrial division: New partners in membrane pinching.

Mitochondrial division is a complex process requiring the synergistic actions of multiple factors, including mechanical enzymes and accessory proteins. Recent studies have identified a number of these factors and started to elucidate how they act together to bring about mitochondrial fission.

Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape.

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes.

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of approximately 25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.

A functional link between dynamin and the actin cytoskeleton at podosomes.

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin.

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

The dynamin family of mechanoenzymes: pinching in new places.

The large GTPase dynamin is a mechanoenzyme that mediates the liberation of nascent clathrin-coated pits from the plasma membrane during endocytosis. Recently, this enzyme has been demonstrated to comprise an extensive family of related proteins that have been implicated in a large variety of vesicle trafficking events during endocytosis, secretion and even maintenance of mitochondrial form. The potential contributions by the dynamin family to these diverse but related functions are discussed.