Got milk? The secret life of laticifers

Laticifers are specialized cells that occur in over 20 plant families in several unrelated angiosperm orders. Although laticifers are likely to be of polyphyletic origin, their occurrence is considered a morphological indicator of relatedness among species. The classification of laticifers is based on developmental patterns and overall morphology. The cytoplasmic latex exuded in response to damage often includes specialized metabolites, such as cardenolides, alkaloids and natural rubber. Laticifers provide an effective location to store defense metabolites, although not all latex-bearing plants accumulate bioactive natural products. Ecophysiological studies have shown that latex and its associated metabolites are vital for the defense of plants against insects. The anatomy, development and physiology of laticifers are discussed with a focus on evolutionary and ecological perspectives.     

Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses

Recent experimental evidence has demonstrated that bacteria acquire resistance to viruses by incorporation of short transcribed nucleotide sequences into regions of clustered regularly interspaced short palindromic repeats (CRISPR). We have analysed community genomic data from acidophilic microbial biofilms and discovered that evolution of the CRISPR regions in two distinct Leptospirillum group II bacteria occurs fast enough to promote individuality in otherwise nearly clonal populations. Comparative genomics strongly indicates very recent lateral transfer of the CRISPR locus between these populations, followed by significant loss of spacer sequences and locus expansion by unidirectional heterogeneous addition of new spacer sequences. Diversification of the CRISPR region is inferred to be a population-level response to the rapidly changing selective pressure of phage predation. Results reinforce the importance of phage–host interactions in shaping microbial ecology and evolution over very short time scales.     

Deoxygenated Disaccharide Analogs as Specific Inhibitors of ��1�C4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

The disaccharide peracetylated GlcNAcβ1–3Galβ-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acα2–3Galβ1–4(Fucα1–3) GlcNAc; sLe^X) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe^X assembly, and (iii) generation of several glycans related to sLe^X. In this report, we show that GlcNAcβ1–3Galβ-O-naphthalenemethanol binds to the acceptor site of human β1–4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcβ1–2Manβ1–6Man, which is present on N-linked glycans. The 4′-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe^X formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N₃, -NH₂, or -OCH₃ instead of the hydroxyl groups at C-3′- and C-4′-positions of the terminal N-acetylglucosamine residue also blocked sLe^X formation in cells. The reduction of sLe^X by the 4′-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.The sialylated, fucosylated tetrasaccharide, sLe^X,³ is a common carbohydrate determinant present in many O-GalNAc-linked mucins and N-linked glycans that act as selectin ligands (see Ref. 1 and references therein). Expression of sLe^X endows tumor cells with the capacity to bind to platelets and endothelial cells in the vasculature via P- and E-selectins, thus facilitating hematogenous metastasis possibly through protection against innate immune cells and by adhesion to the blood vessel wall. Strategies for blocking selectin-carbohydrate interactions include (i) competition by soluble recombinant forms of selectins, glycoprotein ligands, and glycolipids, (ii) peptides based on the primary sequence of the carbohydrate binding site, (iii) anti-selectin antibodies, (iv) oligosaccharides related to Lewis^X, (v) inositol polyanions and sulfated sugars, (vi) heparin, and (vii) molecular mimics of sLe^X, including oligonucleotides (reviewed in Refs. 2 and 3). Analogs of acceptor substrates of the various glycosyltransferases involved in glycan biosynthesis provide another class of potential inhibitors (reviewed in Refs. 4 and 5). Although many of these analogs are effective in vitro, they generally do not exhibit inhibitory activity in cells due to poor membrane permeability. The large number of polar hydroxyl groups and the lack of membrane transporters for oligosaccharides in most cells presumably prevent their uptake (6).In contrast to many of the inhibitors described above, peracetylated disaccharides (e.g. acetylated Galβ1–4GlcNAcβ-O-naphthalenemethanol (NM), acetylated Galβ1–3GalNAcα-O-NM, and acetylated GlcNAcβ1–3Galβ-O-NM) inhibit sLe^X biosynthesis in cells (6–9). These compounds are taken up by cells by passive diffusion and acted on by cytoplasmic or membrane-associated carboxyesterases, which remove the acetyl groups. The compounds gain access to the biosynthetic enzymes located in the Golgi complex, where they serve as substrates, priming oligosaccharide synthesis and generating products related to O-GalNAc-linked mucin oligosaccharides. Priming in this manner diverts the assembly of the O-linked chains from endogenous glycoproteins, resulting in inhibition of expression of terminal Lewis antigens that are recognized by selectins. Inhibition occurs at a much lower dose than for monosaccharide-based agents, such as GalNAcβ-O-benzyl (∼25 μm versus 1–2 mm, respectively) (10, 11). Furthermore, the disaccharides appear to selectively affect sLe^X formation, since sLe^a expression was unaffected (12). By blocking selectin ligand expression, these compounds block both experimental and spontaneous metastasis (12, 13).In this study, we have examined acetylated disaccharide analogs that have been modified so that after deacetylation their activity as substrates would be altered. Characterization of the 4′-deoxy derivative using β1–4-galactosyltransferase 1 as a model showed that it acts by competitively inhibiting the enzyme. Interestingly, the peracetylated form of this analog maintains the capacity to inhibit sLe^X expression in U937 lymphoma cells and Lewis lung carcinoma (LLC) cells and block tumor formation in vivo. Thus, the deoxy analog presumably inhibits one or more galactosyltransferases in vivo, thereby blocking sLe^X formation and experimental tumor cell metastasis without generation of oligosaccharide products.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Temperature-induced reversible conformational change in the first 100 residues of alpha-synuclein

Natively disordered proteins are a growing class of anomalies to the structure-function paradigm. The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. We noticed a dramatic difference in dilute solution 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type alpha-synuclein and two disease-related mutants (A30P and A53T), with spectra collected at 35 degrees C showing fewer cross-peaks than spectra acquired at 10 degrees C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing alpha-synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H-15N HSQC spectra obtained at 35 degrees C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of alpha-synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions.     

A presenilin-independent aspartyl protease prefers the gamma-42 site cleavage

beta-Amyloid peptides (Abeta40 and Abeta42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Abeta is derived from sequential cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. gamma-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the gamma-site cleavage of APP. Secretion of both Abeta40 and Abeta42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Abeta from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Abeta42 accumulation; thus the intracellular Abeta42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Abeta, especially Abeta42, can be generated in the absence of PS based on an in vitrogamma-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent gamma-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Abeta40 and Abeta42 generation, respectively. These results indicate that these PS-independent gamma-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Abeta42 as the predominant product.     

No unique effect of intergroup competition on cooperation: non-competitive thresholds are as effective as competitions between groups for increasing human cooperative behavior

Explaining cooperation remains a central topic for evolutionary theorists. Many have argued that group selection provides such an explanation: theoretical models show that intergroup competition could have given rise to cooperation that is costly for the individual. Whether group selection actually did play an important role in the evolution of human cooperation, however, is much debated. Recent experiments have shown that intergroup competitions do increase human cooperation, which has been taken as evidence for group selection as a mechanism for the evolution of cooperation. Here we challenge this standard interpretation. Competitions change the payoff structure by creating a threshold effect whereby the group that contributes more earns an additional prize, which creates some incentive for individuals to cooperate. We present four studies that disentangle competition and thresholds, and strongly suggest that it is thresholds – rather than competitions per se – that increase cooperation. Thus, prior intergroup competition experiments provide no evidence of a unique or special role for intergroup competition in promoting human cooperation, and shed no light on whether group selection shaped human evolution.