Proteoglycan-targeted antibodies as markers on non-Hodgkin lymphoma xenografts

Summary A family of mono- and polyclonal antibodies raised against proteoglycans or their subcomponents served as novel markers to characterize the phenotypes of three non-Hodgkin lymphoma xenograft lines (HT 58 lymphoblastic, HT 117 centroblastic, HT 130 centrocytic) together with normal, human peripheral blood B lymphocytes. These xenografted NHL lines, maintained by serial transplantations on artificially immunosuppressed mice, expressed very similar B-cell-related antigens and differences on the cell surface (HT 58 > HT 117 > HT 130 > B cells) when they were exposed to monoclonal antibodies (mAb) to cartilage proteoglycans. Anti-proteoglycan antibodies used in this study recognize complex epitopes of core protein segment associated with carbohydrate, shared by human cartilage proteoglycans and certain lymphoma cells. The binding of these antibodies was independent of cell-cycle phase. The results suggest that the anti-proteoglycan mAbs could be used as new phenotypic markers to individualize non-Hodgkin lymphomas.     

Focus on Weed Control: Indaziflam Herbicidal Action: A Potent Cellulose Biosynthesis Inhibitor

Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) have a potentially broad-acting herbicidal mode of action and are also useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam as a CBI and provide insight into its inhibitory mechanism. Indaziflam-treated seedlings exhibited the CBI-like symptomologies of radial swelling and ectopic lignification. Furthermore, indaziflam inhibited the production of cellulose within <1 h of treatment and in a dose-dependent manner. Unlike the CBI isoxaben, indaziflam had strong CBI activity in both a monocotylonous plant (Poa annua) and a dicotyledonous plant (Arabidopsis [Arabidopsis thaliana]). Arabidopsis mutants resistant to known CBIs isoxaben or quinoxyphen were not cross resistant to indaziflam, suggesting a different molecular target for indaziflam. To explore this further, we monitored the distribution and mobility of fluorescently labeled CELLULOSE SYNTHASE A (CESA) proteins in living cells of Arabidopsis during indaziflam exposure. Indaziflam caused a reduction in the velocity of YELLOW FLUORESCENT PROTEIN:CESA6 particles at the plasma membrane focal plane compared with controls. Microtubule morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of plasma membrane-localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting1-independent reduction in the normal coincidence rate between microtubules and CESA particles. As a CBI, for which there is little evidence of evolved weed resistance, indaziflam represents an important addition to the action mechanisms available for weed management.Cellulose is a composite polymer of β-1,4-linked glucan chains and is the main load-bearing structure of plant cell walls (Jarvis, 2013). Although cellulose is a relatively simple polysaccharide molecule, its synthesis is quite complex. The principle catalytic unit is a plasma membrane (PM)-localized protein complex referred to as the cellulose synthase complex (CSC; Davis, 2012). In plants, the CSC, visualized with freeze fracture microscopy, is a solitary, hexagonal rosette-shaped complex (Herth and Weber, 1984; Delmer, 1999) and at least three of the catalytic CELLULOSE SYNTHASE A (CESA) proteins are required in each CSC for the production of cellulose (Desprez et al., 2007; Persson et al., 2007). In addition to CESAs, several accessory proteins have been discovered to be necessary for the production and deposition of cellulose, such as KORRIGAN (Lane et al., 2001), COBRA (Roudier et al., 2005) and CELLULOSE SYNTHASE INTERACTING1 (CSI1; Gu et al., 2010), as well as several others that are yet to be identified. The loss of function in any of the aforementioned proteins causes complete or partial loss of anisotropic growth in cells undergoing expansion, resulting in radial swelling. Severe radial swelling in rapidly expanding tissue is also a common symptomology observed in seedlings treated with cellulose biosynthesis inhibitors (CBIs). Therefore, numerous potential herbicidal targets exist (mechanisms of action) for the broad group of known CBIs.Classification of an herbicide to the CBI designation was traditionally achieved by short-term [¹⁴C]radioisotope tracer studies focused on the incorporation of Glc into cellulose (Heim et al., 1990; Sabba and Vaughn, 1999). More recently, time-lapse confocal microscopy of reporter-tagged CESA proteins (Paredez et al., 2006) has been used to further classify CBIs. CBIs can be classified into at least three primary groups based on how treatment disrupts the normal tracking and localization of fluorescently labeled CESAs (for review, see Brabham and DeBolt, 2012). The disruption is, it can be assumed, the result of the inhibitory mechanism of the CBI. In the first group, isoxaben and numerous other compounds cause YELLOW FLUORESCENT PROTEIN YFP):CESAs to be depleted from the PM and concomitantly accumulate in cytosolic vesicles (called small CESA compartments or microtubule-associated cellulose synthase compartments; Paredez et al., 2006; Crowell et al., 2009; Gutierrez et al., 2009) The second group, consisting only of dichlobenil (DCB), causes YFP:CESAs to become immobilized and hyperaccumulated at distinct foci in the PM (Herth, 1987; DeBolt et al., 2007b). The third group influences CSC-microtubule (MT)-associated functions resulting in errant movement and localization of YFP:CESAs (DeBolt et al., 2007a; Yoneda et al., 2007). These different disruption processes suggest that each CBI group targets a different aspect of the complex cellulose biosynthetic process.A lack of evolved weed resistance in the field suggests that CBIs are potentially underutilized tools for weed control (Sabba and Vaughn, 1999; Heap, 2014). CBIs have also been useful research tools in decoding fundamental aspects of cellulose biosynthesis. An exogenous application of a CBI provides spatial and temporal inhibition of cellulose. Resistance screens to CBIs have uncovered key genes in cellulose biosynthesis (Scheible et al., 2001; Desprez et al., 2002). Furthermore, CBIs such as isoxaben have also been effective in linking accessory proteins with CESAs in the CSC (Robert et al., 2005; Gu et al., 2010). Therefore, it is important to extend our range of CBI compounds. Indaziflam (Fig. 1A), an herbicide introduced by Bayer Crop Science, was recently proposed to be a CBI and was reported to have a photosystem II inhibition value of 9.4 (Meyer et al., 2009; Dietrich and Laber, 2012). Indaziflam is labeled for use in turf, for perennial crops, and for nonagricultural situations for preemergent control of grasses and broadleaf weeds (Meyer et al., 2009; Brosnan et al., 2011). The aim herein was to investigate indaziflam as a CBI and to characterize its inhibitory effect on cellulose biosynthesis.Open in a separate windowFigure 1.Indaziflam is a fluoroalkytriazine-containing compound that inhibits elongation in seedlings of P. annua and Arabidopsis. A, Chemical structure of indaziflam. B to D, Images of 7-d-old seedlings treated with increasing concentrations of indaziflam. B shows light-grown P. annua seedlings (indaziflam concentrations from left to right are 0, 100, 250, 500, 1,000, 5,000, and 10,000 pm). C and D show light-grown and dark-grown Arabidopsis seedlings, respectively (indaziflam concentrations from left to right are 0, 100, 250, 500, 1,000, and 2,500 pm). Indaziflam treatment induced swollen cells. E, Representative images of the primary root of P. annua grown in plates for 4 d with and without 10 nm indaziflam. F, Transgenic Arabidopsis seedlings expressing GFP:PIP2 were examined by laser scanning confocal microscopy and images represent visualization of the primary root grown vertically for 7-d plates without and with 250 pm indaziflam. PIP2, Plasma membrane intrinsic protein2. Bar = 10 mm in B, 5 mm in C and D, 2 mm in E, and 50 μm in F.     

Characterization of the Histoplasma capsulatum-induced granuloma

Rising rates of Histoplasma capsulatum infection are an emerging problem among the rapidly growing population of immune-compromised individuals. Although there is a growing understanding of systemic immunity against Histoplasma, little is known about the local granulomatous response, which is an important component in the control of infection. The focus of this article is the characterization of Histoplasma-induced granulomas. Five days after i.p. infection, infected macrophage appear in the liver and lung; however, no granulomas are apparent. Two days later, well-formed sarcoid granulomas are abundant in the lung and liver of infected mice, which contain all visible Histoplasma. Granulomas are dominated by macrophage and lymphocytes. Most of the Histoplasma and most of the apoptotic cells are found in the center of the lesions. We isolated liver granulomas at multiple time points after infection and analyzed the cellular composition, TCR gene usage, and cytokine production of granuloma-infiltrating cells. The lesions contain both CD4+ and CD8+ T cell subsets, and T cells are the primary source of IFN-gamma and IL-17. The main source of local TNF-alpha is macrophage. Chemokines are produced by both infiltrating macrophage and lymphocytes. Dendritic cells are present in granulomas; however, T cell expansion seems to occur systemically because TCR usage is very heterogeneous even at the level of individual lesions. This study is the first direct examination of host cellular responses in the Histoplasma-induced granuloma representing the specific interface between host and pathogen. Our studies will allow further analysis of key elements of host Histoplasma interactions at the site of chronic infection.     

Dendritic cells amplify T cell-mediated immune responses in the central nervous system

Neuroinflammation often starts with the invasion of T lymphocytes into the CNS leading to recruitment of macrophages and amplification of inflammation. In this study, we show that dendritic cells (DCs) facilitate T-T cell help in the CNS and contribute to the amplification of local neuroinflammation. We adoptively transferred defined amounts of naive TCR-transgenic (TCR) recombination-activating gene-1-deficient T cells into another TCR-transgenic mouse strain expressing different Ag specificity. Following adoptive transfers, we coinjected DCs that presented one or multiple Ags into the brain and followed the activation of T cells with defined specificities simultaneously. Injection of DCs presenting both Ags simultaneously led to significantly higher infiltration of T cells into the brain compared with injection of a mixture of DCs pulsed with two Ags separately. DCs mediated either cooperative or competitive interactions between T cell populations with different specificities depending upon their MHC-restricting element usage. These results suggest that DC-mediated cooperation between brain-infiltrating T cells of different Ag specificities in the CNS plays an important role in regulation of neuroinflammation. This work also implies that blocking Ag-specific responses may block not only the targeted specificities, but may also effectively block their cooperative assistance to other T cells. Therefore, these data justify more attention to Ag-specific therapeutic approaches for neuroinflammation.     

Rate remapping: when the code goes beyond space

Rate remapping is a conjunctive code that potentially enables hippocampal place cells to jointly represent spatial and nonspatial information. In this issue of Neuron, Rennó-Costa et al. introduce a theoretical model wherein the convergence of the medial and lateral entorhinal excitatory inputs, combined with local inhibition, explains hippocampal rate remapping.     

Structure of Penaeus stylirostris Densovirus,a Shrimp Pathogen

Penaeus stylirostris densovirus (PstDNV), a pathogen of penaeid shrimp, causes significant damage to farmed and wild shrimp populations. In contrast to other parvoviruses, PstDNV probably has only one type of capsid protein that lacks the phospholipase A2 activity that has been implicated as a requirement during parvoviral host cell infection. The structure of recombinant virus-like particles, composed of 60 copies of the 37.5-kDa coat protein, the smallest parvoviral capsid protein reported thus far, was determined to 2.5-Å resolution by X-ray crystallography. The structure represents the first near-atomic resolution structure within the genus Brevidensovirus. The capsid protein has a β-barrel “jelly roll” motif similar to that found in many icosahedral viruses, including other parvoviruses. The N-terminal portion of the PstDNV coat protein adopts a “domain-swapped” conformation relative to its twofold-related neighbor similar to the insect parvovirus Galleria mellonella densovirus (GmDNV) but in stark contrast to vertebrate parvoviruses. However, most of the surface loops have little structural resemblance to any of the known parvoviral capsid proteins.The Parvoviridae family is a family of small DNA viruses that is divided into two subfamilies, the Parvovirinae that infect vertebrates and the Densovirinae that infect invertebrates. Penaeus stylirostris densovirus (PstDNV), also known as infectious hypodermal and hematopoietic necrosis virus (IHHNV), belongs to the Densovirinae subfamily and was first reported as a highly lethal disease of juvenile shrimp in 1983 (22). The virus has significant commercial impact on the shrimp farming industry, causing mass mortality and severe deformations in penaeid shrimp during catastrophic epidemics in marine aquaculture facilities worldwide (14). PstDNV is closely related to the mosquito brevidensoviruses (35), which have the potential to be used as biological control agents of mosquito-borne diseases, such as malaria (30), dengue, chikungunya, and yellow fever (8).The single-stranded DNA genome of parvoviruses is encapsidated within a nonenveloped, icosahedral protein shell of less than 280 Å in external diameter. The capsid consists of 60 structurally equivalent subunits that are composed of the major viral coat protein and a few copies of N-terminally extended variants of the major capsid protein. A phospholipase A2 (PLA2) activity in the unique N-terminal extension of the largest minor capsid protein plays a crucial role during parvoviral host cell infection (7, 12, 13, 20, 46). The structures of the major capsid protein of several vertebrate parvoviruses have previously been determined to near-atomic resolution (1, 18, 23, 37, 41, 43, 44). However, the only high-resolution structure available for the invertebrate subfamily is that of the insect parvovirus Galleria mellonella densovirus (GmDNV) (36). The central motif of parvoviral capsid proteins is an eight-stranded, antiparallel β-barrel “jelly roll” fold. The surface of the virion, however, is formed by large insertions connecting the strands of the β-barrel, thereby creating features that govern antigenicity, receptor binding, and most intersubunit contacts. Surface characteristics common to most parvoviruses are protrusions at or around the icosahedral threefold axes, depressions on the twofold axes, and canyons surrounding the fivefold axes. At each fivefold apex, a cylindrical pore connects the interior of the virus particle with its exterior surroundings. In full virions, these pores are occupied by a glycine-rich motif in the N-terminal region of the major capsid protein, presumably positioning the N-terminal peptide for externalization. The general surface topology of GmDNV is smoother, probably due to smaller loop insertions. The structure of some of these insertions has diverged from vertebrate parvoviruses beyond recognition (4, 36). The N-terminal portions of twofold-related subunits in GmDNV have swapped their positions relative to those of the vertebrate parvoviruses. A cryo-electron microscopy (cryo-EM) study of Aedes albopictus densovirus, a brevidensovirus, has shown that its surface features are different from GmDNV and the mammalian parvoviruses, in particular in having prominent protrusions at the fivefold axes (9).Although it has been reported that PstDNV contains four structural proteins, as determined by SDS-polyacrylamide gel electrophoresis (3), these data do not fit the coding sequence (35). The 4.1-kb DNA genome of PstDNV (3) encodes in the 3′ half of the plus strand just one structural protein of 329 amino acids, as of now the smallest reported parvoviral capsid protein, and in the 5′ half of the plus strand two nonstructural proteins (666 and 363 amino acids) (35). Having only a single type of capsid protein is an unusual feature for viruses in the Parvoviridae family, where capsids are generally reported to contain two or more coat protein variants. A stretch of 11 amino acids in the N-terminal region of the capsid protein (17-DAHNEDEEHAE-27) is reminiscent of the PLA2 catalytic site (35), but it lacks important conserved motifs of PLA2s. Consequently, but curiously, PstDNV does not have the enzymatic activity that has previously been described as a requirement for parvoviral infectivity.We report here the three-dimensional (3D) crystal structure of recombinant, empty virus-like particles (VLPs) of the shrimp parvovirus PstDNV at 2.5-Å resolution. The loops connecting the strands of the structurally conserved jelly roll motif differ considerably in structure and length from other parvoviruses. The near-atomic resolution structure might provide the basis for the design of capsid binding antiviral compounds that may protect shrimp against parvoviral infection (16, 32, 42). Furthermore, the structure might aid the targeting of monoclonal antibodies to gain functional data about the role of the Brevidensovirus capsid protein during the infection cycle. Such information in turn may permit the design of densovirus-based delivery systems for drugs or pest control agents in aquacultural facilities. The small dimensions of PstDNV VLPs can be advantageous for their possible use as nanoparticles for antigen presentation and transport of immune stimulatory substances or interfering RNAs (21, 26). Additionally, the small size of the PstDNV capsid protein makes the system attractive as a model for studying assembly mechanisms of icosahedral virus capsids.     

Mitochondrial dysfunction in amyotrophic lateral sclerosis

The etiology of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains to be better understood. Based on the studies from ALS patients and transgenic animal models, it is believed that ALS is likely to be a multifactorial and multisystem disease. Many mechanisms have been postulated to be involved in the pathology of ALS, such as oxidative stress, glutamate excitotoxicity, mitochondrial damage, defective axonal transport, glia cell pathology and aberrant RNA metabolism. Mitochondria, which play crucial roles in excitotoxicity, apoptosis and cell survival, have shown to be an early target in ALS pathogenesis and contribute to the disease progression. Morphological and functional defects in mitochondria were found in both human patients and ALS mice overexpressing mutant SOD1. Mutant SOD1 was found to be preferentially associated with mitochondria and subsequently impair mitochondrial function. Recent studies suggest that axonal transport of mitochondria along microtubules and mitochondrial dynamics may also be disrupted in ALS. These results also illustrate the critical importance of maintaining proper mitochondrial function in axons and neuromuscular junctions, supporting the emerging “dying-back” axonopathy model of ALS. In this review, we will discuss how mitochondrial dysfunction has been linked to the ALS variants of SOD1 and the mechanisms by which mitochondrial damage contributes to the disease etiology.     

Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper–zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein–dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.