"The orchids have been a splendid sport"--an alternative look at Charles Darwin's contribution to orchid biology

Charles Darwin's work with orchids and his thoughts about them are of great interest and not a little pride for those who are interested in these plants, but they are generally less well known than some of his other studies and ideas. Much has been published on what led to his other books and views. However, there is a paucity of information in the general literature on how Darwin's orchid book came about. This review will describe how The Various Contrivances by Which Orchids Are Fertilised by Insects came into being and will discuss the taxonomy of the orchids he studied. It also will concentrate on some of the less well-known aspects of Darwin's work and observations on orchids-namely, rostellum, seeds and their germination, pollination effects, and resupination-and their influence on subsequent investigators, plant physiology, and orchid science.     

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.Download video file.^{(145M, mp4)}     

Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection of post-translational modifications

Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c. Comparing our molecular mass data with calculated values for molecular weight, we were able to identify the correct sequences for several of the major apolipoproteins. Analyses were carried out on the apolipoproteins of ultracentrifugally isolated HDL. Prior to analyses by electrospray ionization mass spectrometry (ESI-MS), the apolipoproteins were separated either by size exclusion or reverse phase chromatography. The molecular masses of apoA-I, proapoA-I, apoA-II, proapoA-II, apoC-I and apoC-III were obtained. Comparing the values obtained for the two strains, differences in the molecular masses of apoA-I, apoA-II and apoC-III were observed. In this study, post-translationally modified apolipoproteins, involving loss of amino acids from both the N- and C-termini, oxidation of methionine residues and possible acylation, were noted following reverse-phase separation. Further analyses by tandem mass spectrometry (MSMS) done on the tryptic digests of apolipoproteins separated by reverse phase chromatography enabled us to confirm sequence differences between the two strains, to verify selected apoA-I sequences that had been entered into the GenBank and to identify which methionines in apoA-I, apoC-III and apoE had been converted to methionine sulfoxides.     

Mitochondrial targeting of growth suppressor protein DLC2 through the START domain

Deleted in liver cancer 2 (DLC2) is a candidate tumor suppressor frequently found to be deleted in hepatocellular carcinoma. In this study, we determined the subcellular localization of DLC2. Co-localization and biochemical fractionation studies revealed that DLC2 localized to mitochondria. In addition, the DLC2-containing cytoplasmic speckles were in proximity to lipid droplets. A DLC2 mutant containing the steroidogenic acute regulatory protein-related lipid transfer (START) domain only showed a localization pattern identical to that of DLC2. Taken together, we have provided the first evidence for mitochondrial localization of DLC2 through the START domain. These findings might have implications in liver physiology and carcinogenesis.     

Secreted PDZD2 exerts concentration-dependent effects on the proliferation of INS-1E cells

PDZD2 (PDZ domain containing 2) is a multi-PDZ protein expressed in pancreas and many other tissues. PDZD2 shows extensive homology to pro-interleukin-16 (pro-IL-16) and is localized mainly to the endoplasmic reticulum. We have recently demonstrated that PDZD2, like pro-IL-16, is proteolytically cleaved at its C-terminus to generate a secreted protein, sPDZD2 (for secreted PDZD2). To understand the possible functional role of PDZD2 in pancreas, we investigated the cellular distribution of PDZD2 in adult pancreas using an antiserum that recognizes both the full-length and secreted forms of PDZD2. Immunohistochemical analysis revealed a strong expression of PDZD2 in pancreatic islet beta cells but not alpha cells. Consistent with the beta-cell-enriched expression of PDZD2, immunoblot analysis indicated expression of both full-length PDZD2 and sPDZD2 in the insulinoma cell line INS-1E. A recombinant sPDZD2 protein was synthesized for study of its functional effect on INS-1E cells. In culture media with limiting serum, co-incubation with sPDZD2 stimulated the proliferation of INS-1E cells. The mitogenic effect of sPDZD2 was concentration-dependent, and was associated with a slight inhibition of the insulin promoter activity at high sPDZD2 concentrations. As a potential mitogen of beta-like cells, sPDZD2 may be useful for the optimization of beta-cell growth and differentiation in vitro.     

Aβ40 oligomers identified as a potential biomarker for the diagnosis of Alzheimer's disease

Alzheimer's Disease (AD) is the most prevalent form of dementia worldwide, yet the development of therapeutics has been hampered by the absence of suitable biomarkers to diagnose the disease in its early stages prior to the formation of amyloid plaques and the occurrence of irreversible neuronal damage. Since oligomeric Aβ species have been implicated in the pathophysiology of AD, we reasoned that they may correlate with the onset of disease. As such, we have developed a novel misfolded protein assay for the detection of soluble oligomers composed of Aβ x-40 and x-42 peptide (hereafter Aβ40 and Aβ42) from cerebrospinal fluid (CSF). Preliminary validation of this assay with 36 clinical samples demonstrated the presence of aggregated Aβ40 in the CSF of AD patients. Together with measurements of total Aβ42, diagnostic sensitivity and specificity greater than 95% and 90%, respectively, were achieved. Although larger sample populations will be needed to confirm this diagnostic sensitivity, our studies demonstrate a sensitive method of detecting circulating Aβ40 oligomers from AD CSF and suggest that these oligomers could be a powerful new biomarker for the early detection of AD.     

Characterization of a Carbon-Carbon Hydrolase from Mycobacterium tuberculosis Involved in Cholesterol Metabolism

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5–9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4–33 times higher specificity for DSHA (k_cat/K_m = 3.3 ± 0.3 × 10⁴ m⁻¹ s⁻¹) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K_d of 51 ± 2 μm. The S114A·DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8–1.9 Åindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the α-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.     

Proliferative and migratory aptitude in pterygium

Pterygium is a chronic fibrovascular overgrowth on the corneal surface and is often associated with inflammation, astigmatism and obstructed vision. The common treatment is surgical removal but post-operative recurrences with more aggressive behavior are common. However, there is a controversy in the pathogenesis of primary pterygium between limbal stem cell failure versus proliferation. In this study, we explore the proliferative and migratory aptitude in pterygium by characterizing the growth and migration pattern of pterygial cells in the head (on the cornea), the neck (over the focal limbus), and the body (on the conjunctiva) epithelia of 12 full-length primary pterygia. Immunofluorescence and quantification analyses showed a spatial expression pattern of markers for stem cells, cell growth, and matrix metalloproteinases. Beside the basal epithelia in all three regions, p63α^strong cells were located in suprabasal layers in head, weak in the body and absent in neck. Pertinent cell proliferation in head than body epithelia was revealed by its higher colony-forming efficiency. ATP-binding cassette transporter glycoprotein family member-2 and cytokeratin-15 were found mainly in the body basal epithelia, similar to that in normal conjunctiva. Much fewer proliferating stem-like cells in the neck region supported the limbal failure as a cause of pterygium formation. Pax6, matrix metalloproteinase-2 and -9 were more expressed in the head than in the other two regions. Our results indicate the importance of pterygium head in tissue growth and invasion and its likely involvement in post-operation recurrence.     

Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking.To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer''s clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.Plasmodium falciparum, the most deadly agent of human malaria, caused around 216 million infections and 655,000 deaths in 2010. The complex parasite life cycle involves the development in a mosquito vector of the Anopheles genus and eventual migration to a human host. In this host, asymptomatic multiplication in the liver cells is followed by parasite release into the bloodstream and erythrocyte invasion. Inside the erythrocytes, parasites grow (trophozoite stage) and multiply asexually (schizont stage), developing into highly specialized invasive forms (merozoites). A fraction of parasites differentiate into gametocytes, the gamete precursors necessary to complete the transmission cycle. Parasite blood stages, responsible for malaria pathogenesis and transmission, actively remodel the host erythrocyte, generating novel membrane compartments to sustain the export and sorting of proteins to the host cell cytosol, membrane skeleton, and plasma membrane. The parasitophorous vacuole membrane (PVM),¹ which surrounds the parasite throughout the erythrocytic cycle, is the site where exported proteins are translocated into the erythrocyte cytosol (1, 2). Membrane-bound structures of parasite origin, the so-called Maurer''s clefts (MCs) (3, 4), form functionally independent compartments at the red blood cell (RBC) periphery and mediate the sorting/assembly of virulence factors en route to the host cell surface (5). In addition, populations of different vesicles (25 and 80 nm) were identified in the RBC cytosol, suggesting the presence of vesicular mediated trafficking for the delivery of cargo to different destinations (6).Membranes are important sites for cellular signaling events, and many proteins with therapeutic potential localize in these cellular compartments (7, 8). Membrane microdomains enriched in sphingolipids and cholesterol, also referred to as lipid rafts, have been extensively studied in different cell types and gained particular interest for their roles in infection and pathogenesis (8, 9). These assemblies are small and dynamic and can be stabilized to form larger microdomains implicated in a wide range of fundamental cellular processes, which vary depending on cell type (10). Sphingolipids exhibit strong lateral cohesion, generating tightly packed regions in the membrane bilayer, and cholesterol acts as a spacer present in both membrane leaflets generating stable, liquid-ordered phase domains in the membrane bilayer (11). Distinct biochemical properties render these membrane assemblies insoluble in nonionic detergents at low temperature, allowing for their enrichment as detergent-resistant membranes (DRMs). Proteins with DRM-raft affinity include glycosylphosphatidyl inositol (GPI)-anchored proteins and acylated, myristoylated, and palmitoylated proteins (11). DRM rafts also restrict free diffusion of membrane proteins, thereby directing the trafficking of proteins and lipids to and from cellular compartments. Because of their endocytic and receptor clustering capacity, an increasing number of pathogens, including Plasmodium falciparum, utilize them when interacting with their target cells for invasion (9, 12).Even though cholesterol-rich membrane microdomains are implicated in fundamental processes in the parasite life cycle, Plasmodium is unable to synthesize sterols and depends entirely on hosts for its cholesterol supply. During merozoite invasion, lipid and protein components of the erythrocyte rafts are selectively recruited and incorporated into the nascent PVM (13, 14). Plasmodium liver stages utilize cholesterol internalized by low-density lipoprotein and synthesized by hepatocytes (15).To shed light on the organization and dynamics of these assemblies during parasite development inside the infected cell, we identified and validated the DRM-raft proteome of the P. falciparum trophozoite/early schizont. Detected proteins only partially overlap with DRM components of the P. falciparum late schizonts (16, 17) or the mixed blood stages of the rodent malaria agent P. berghei (18). Immunolocalization of selected DRM-associated proteins indicated that these assemblies may reside in both exported compartments (PVM, MCs) and intracellular membranes/organelles. The analysis of DRM lipids suggested that distinct microdomains exist in the infected erythrocyte that differ in their relative abundance of cholesterol and phospholipids.