Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility

Originally thought of as a stomach-derived endocrine peptide acting via its receptors in the central nervous system to stimulate food intake and growth hormone expression, ghrelin and its receptor (growth hormone secretagogue receptor (GHS-R)) are widely expressed in a number of organ systems, including cancer cells. However, the direct functional role of ghrelin and its receptor in tumors of central nervous system origin remains to be defined. Here, we demonstrate that the human astrocytoma cell lines U-118, U-87, CCF-STTG1, and SW1088 express 6-, 11-, 15-, and 29-fold higher levels of GHS-R compared with primary normal human astrocytes. The ligation of GHS-R by ghrelin on these cells resulted in an increase in intracellular calcium mobilization, protein kinase C activation, actin polymerization, matrix metalloproteinase-2 activity, and astrocytoma motility. In addition, ghrelin led to actin polymerization and membrane ruffling on cells, with the specific co-localization of the small GTPase Rac1 with GHS-R on the leading edge of the astrocytoma cells and imparting the tumor cells with a motile phenotype. Disruption of the endogenous ghrelin/GHS-R pathway by RNA interference resulted in diminished motility, matrix metalloproteinase activity, and Rac expression, whereas tumor cells stably overexpressing GHS-R exhibited increased cell motility. The relevance of ghrelin and GHS-R expression was verified in clinically relevant tissues from 20 patients with oligodendrogliomas and grade II-IV astrocytomas. Analysis of a central nervous system tumor tissue microarray revealed that strong GHS-R and ghrelin expression was significantly more common in high grade tumors compared with low grade ones. Together, these findings suggest a novel role for the ghrelin/GHS-R axis in astrocytoma cell migration and invasiveness of cancers of central nervous system origin.     

Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.     

Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM

Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to R_work (R_free) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.     

Decline in Clinical Efficacy of Oral Miltefosine in Treatment of Post Kala-azar Dermal Leishmaniasis (PKDL) in India

BackgroundRecent studies have shown significant decline in the final cure rate after miltefosine treatment in visceral leishmaniasis. This study evaluates the efficacy of miltefosine in the treatment of post kala-azar dermal leishmaniasis (PKDL) patients recruited over a period of 5 years with 18 months of follow-up.MethodologyIn this study 86 confirmed cases of PKDL were treated with two different dosage regimens of miltefosine (Regimen I- 50mg twice daily for 90 days and Regimen II- 50 mg thrice for 60 days) and the clinical outcome assessed monthly. Cure/relapse was ascertained by clinical and histopathological examination, and measuring parasite burden by quantitative real-time PCR. In vitro susceptibility of parasites towards miltefosine was estimated at both promastigote and amastigote stages.ResultsSeventy three of eighty six patients completed the treatment and achieved clinical cure. Approximately 4% (3/73) patients relapsed by the end of 12 months follow-up, while a total of 15% (11/73) relapsed by the end of 18 months. Relapse rate was significantly higher in regimen II (31%) compared to regimen I (10.5%)(P<0.005). Parasite load at the pre-treatment stage was significantly higher (P<0.005) in cases that relapsed compared to the cases that remained cured. In vitro susceptibility towards miltefosine of parasites isolated after relapse was significantly lower (>2 fold) in comparison with the pre-treatment isolates (P<0.005).ConclusionRelapse rate in PKDL following miltefosine treatment has increased substantially, indicating the need of introducing alternate drugs/ combination therapy with miltefosine.     

Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling

We have previously demonstrated that glycosphingolipids are internalized from the plasma membrane of human skin fibroblasts by a clathrin-independent, caveolar-related mechanism and are subsequently transported to the Golgi apparatus by a process that is dependent on microtubules, phosphatidylinositol 3-kinase, Rab7, and Rab9. Here we characterized the early steps of intracellular transport of a fluorescent glycosphingolipid analog, BODIPY-lactosylceramide (LacCer), and compared this to fluorescent transferrin (Tfn), a well established marker for the clathrin pathway. Although these two markers were initially internalized into separate vesicles by distinct mechanisms, they became co-localized in early endosomes within 5 min. These results demonstrate that glycosphingolipid-containing vesicles derived from caveolar-related endocytosis fuse with the classical endosomal system. However, in contrast to Tfn, internalization and trafficking of LacCer was independent of Rab5a, a key regulator of transport to early endosomes. By taking advantage of the monomer/excimer properties of the fluorescent lipid analog, we were also able to visualize LacCer segregation into distinct microdomains of high (red emission) and low (green emission) concentrations in the early endosomes of living cells. Interestingly, the high concentration "red" microdomains co-localized with fluorescent Tfn upon exit from early endosomes and passed through Rab11-positive "recycling endosomes" prior to being transported back to the plasma membrane. These results together with our previous studies suggest that glycosphingolipids internalized by caveolar endocytosis are rapidly delivered to early endosomes where they are fractionated into two major pools, one that is transported via late endosomes to the Golgi apparatus and the other that is returned to the plasma membrane via the recycling compartment.     

Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.     

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.     

Selective caveolin-1-dependent endocytosis of glycosphingolipids

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.     

Bioconjugation of InGaP quantum dots for molecular sensing

Fluorescence-based molecular sensing and cellular imaging are commonly carried out with the application of organic dyes. Quantum dots (QDs) are now recognized as better tools because they are brighter, size tunable, and more photostable than dyes. Most of the proposed QD-based biosensing systems involve elements of known toxicity. The present work reports the functionalization of biocompatible InGaP/ZnS core-shell QDs with anti-bovine serum albumin (anti-BSA) to exploit them as fluorescent probes for antigen detection. Successful bioconjugation was characterized with the absorption and emission spectra showing blue shifts of around 40 and 30 nm, respectively. Gel electrophoresis and particle size distribution studies further confirmed the mass increment of QDs after their functionalization with anti-BSA. Surface plasmon resonance spectrometry has been used to study the affinity of QD-(anti-BSA) probes for bovine serum albumin (BSA). Photoluminescence quenching of the developed probe is observed in the presence of BSA.