Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

Counterpoint. Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

Anti-idiotype (Id) vaccine therapy has been tested and shown to be effective, in several animal models, for triggering the immune system to induce specific and protective immunity against bacterial, viral and parasitic infections. The administration of anti-Id antibodies as surrogate tumor-associated antigens (TAA) also represents another potential application of the concept of the Id network. Limited experience in human trials using anti-Id to stimulate immunity against tumors has shown promising results. In this “counterpoint” article, we discuss our own findings showing the potential of anti-Id antibody vaccines to be novel therapeutic approaches to various human cancers and also discuss where anti-Id vaccines may perform better than traditional multiple-epitope antigen vaccines. Received: 27 December 1999 / Accepted: 27 January 2000     

Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.     

Functions of heat shock proteins in the system of adaptive immunity. Construction of vaccines

One of the most important aspects of heat shock proteins (HSP) functioning, namely their role in reactions of innate and adaptive immunity, was reviewed in the article. Mechanisms of involvement of HSP in processing and presentation of antigens to T-lymphocytes were described. Principles of construction of prophylactic and therapeutic vaccines on the basis of HSP were set out. Assessment of range of indications for their use and possible risks related with inductive action of vaccines on the development of immunopathologic processes was a specially discussed topic.     

Hippocalcin: a new solution to an old puzzle

In hippocampal pyramidal neurons, calcium entry following an action potential burst results in a slow afterhyperpolarization (sAHP) that critically regulates subsequent excitability. Although this potassium current was described two decades ago, the mechanism whereby the rise in intracellular calcium generates the sAHP was, until now, not known. In this issue of Neuron, Tzingounis et al. now show that calcium binding to hippocalcin, a member of the NCS family, is one of the necessary steps involved in production of the sAHP.     

Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: Lessons learned from HIV‐1 protease inhibition

Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV‐1 protease inhibition and the molecular designs targeted a panel of wild‐type and drug‐resistant mutant HIV‐1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders. Proteins 2015; 83:351–372. © 2014 Wiley Periodicals, Inc.     

Mutations in Either TUBB or MAPRE2 Cause Circumferential Skin Creases Kunze Type

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a β-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.     

Multiple costimulatory modalities enhance CTL avidity

Recent studies in both animal models and clinical trials have demonstrated that the avidity of T cells is a major determinant of antitumor and antiviral immunity. In this study, we evaluated several different vaccine strategies for their ability to enhance both the quantity and avidity of CTL responses. CD8(+) T cell quantity was measured by tetramer binding precursor frequency, and avidity was measured by both tetramer dissociation and quantitative cytolytic function. We have evaluated a peptide, a viral vector expressing the Ag transgene alone, with one costimulatory molecule (B7-1), and with three costimulatory molecules (B7-1, ICAM-1, and LFA-3), with anti-CTLA-4 mAb, with GM-CSF, and combinations of the above. We have evaluated these strategies in both a foreign Ag model using beta-galactosidase as immunogen, and in a "self" Ag model, using carcinoembryonic Ag as immunogen in carcinoembryonic Ag transgenic mice. The combined use of several of these strategies was shown to enhance not only the quantity, but, to a greater magnitude, the avidity of T cells generated; a combination strategy is also shown to enhance antitumor effects. The results reported in this study thus demonstrate multiple strategies that can be used in both antitumor and antiviral vaccine settings to generate higher avidity host T cell responses.     

The cockroach Leucophaea maderae needs more than juvenile hormone, vitellogenin and reserves to make a yolky egg

For the cockroach Leucophaea maderae the developmental profile of lipophorin (Lp) concentrations in the hemolymph was determined through the entire vitellogenic period. At mid-vitellogenesis the concentrations of Lp had risen to 6 times the level at emergence and then declined to 2/3 of such high values at ovulation. The racemic 10R,10S-JH-III bound to Lp with an affinity of K(d) = 5.76 nM and the natural enantiomer 10R-JH-III with a K(d) = 1.60 nM. Injections of anti-Lp into mated females caused a significantly reduced rate of oocyte growth and a substantial degree of oosorption. Injections of gamma-globulin did not significantly reduce oocyte growth and caused only a small number of oocytes to resorb. Starvation after mating had similar effects as treatment with anti-Lp. Because of the high affinity of JH to Lp and since Lp occurs in micromolar concentrations during vitellogenesis one can assume that practically all JH is bound and not available for hydrolysis by the JH esterases. Lp appears to function as an inhibitor of JH metabolism by the JHEs through substrate depletion. One may conclude that a normal rate of egg growth is only achieved when titers of Lp exceed those of JH and remove major portions of this substrate from degradation by the JHEs.