Kinesin was first identified biochemically as a microtubule-dependent motor protein responsible for transport of membranous organelles in the axon. It is now recognized that microtubule-dependent motor proteins form a large gene family, kinesin superfamily proteins (KIFs). The human genome contains 45 KIF genes. KIFs have high homology at the so-called "motor domain", which is a globular domain responsible for moving along microtubules by hydrolysis of adenosine triphosphate (ATP). Outside the motor domain, the sequence is unique to each member. The motors bind to the "cargoes", the molecule to be transported, at this domain. KIFs transport many different types of cargoes including membranous organelles, protein complexes, and messenger RNAs (mRNAs). They play important roles in a wide variety of intracellular transport, such as transport from Golgi to plasma membrane pathways involved in exocytosis and endocytosis, axonal transport, transport in dendrites, and special transport called intraflagellar transport. In addition, recent molecular genetic experiments have uncovered unexpected roles for KIFs in the regulation of such physiologic processes as higher brain function, tumor suppression, and developmental patterning, through modulating signal transduction. They also play important roles in mitosis. Dysfunctions of KIF motors underlie some human diseases, including Schizophrenia, epilepsy, neuropathy, diabetes, and cancer. The molecular mechanisms by which different KIFs recognize and bind to specific cargoes, and how their binding is regulated, have been identified for some KIFs.

Microtubules are formed by polymerization of α- and β-tubulin. They are intrinsically in a dynamic turnover state, subject to treadmilling and dynamic instability. Microtubule-associated proteins (MAPs) are a heterogeneous group of proteins that have microtubule-binding domains. MAPs are abundantly expressed in the brain, and historically MAPs (e.g., MAP1, MAP2, and tau) derived from the brain have been studied extensively. MAP4 is ubiquitously expressed. These classical MAPs are filamentous proteins of various lengths, ranging from 50 to 185 nm. They have a microtubule-binding domain and a projection domain that extends as a filamentous structure. MAPs stabilize microtubules by binding along the side of microtubules. In addition, by extending the projection domain from the microtubule surface, MAPs can produce microtubule bundles of various densities. Tau and MAP2C predominantly expressed in the axon produce microtubule bundles of ~20 nm spacing between microtubules. MAP2 predominantly expressed in the dendrite produces microtubule bundles with ~65 nm spacing. The microtubule bundles produced by tau/MAP2C and MAP2 resemble microtubule domains in axons and dendrites, respectively. These microtubule bundles are extended from the cell as axon- or dendrite-like processes. Therefore, MAPs serve as determinants of microtubule organization within the cell, particularly in neurons. There are also newly identified MAPs, some of which destabilize microtubules. Binding of MAPs to microtubules is regulated by phosphorylation. In some neurodegenerative diseases including Alzheimer׳s disease, hyperphosphorylated tau precipitates as filaments on its own and may be one of the important factors to determine the progress of the disease.

Although bacterial infection is a major cause of death even after reduced-intensity conditioning (RIC) for allogeneic stem cell transplantation (SCT), little is known about the epidemiology and risk factors. The incidence of bacterial infection in 43 patients who received allogeneic bone marrow transplantation (BMT) using a RIC regimen was compared with that in 68 patients who received BMT using a myeloablative conditioning regimen, and risk factors for bacterial infection were identified. Before engraftment, incidences of febrile neutropenia (FN) and documented infections (DI) were significantly decreased in RIC patients (FN: 59.5% vs. 89.6%, P<0.01, DI: 4.8% vs. 17.9%, P<0.01). However, incidence of bacterial infection was significantly increased in RIC patients in the post-engraftment phase (53.8% vs. 11.1%, log-rank, P<0.01). Blood stream was the most frequent focus of infection in both groups. In multivariate analysis, RIC and acute graft-versus-host disease were revealed to be significant risk factors for bacterial infection in this phase. In summary, risk of bacterial infection after engraftment was significantly higher in RIC patients, although infection was decreased before engraftment, and we need to develop a RIC-specific strategy against bacterial infection after RIC SCT.

Molecularly targeted therapy has become widely used in clinical settings over the last decade. Although it was initially expected that molecularly targeted drugs have fewer side effects, it is becoming increasingly apparent that molecularly targeted drugs have an unanticipated repertoire of side effects. Some side effects are serious, some are manageable, some are rare, and some are frequent. Some may affect individuals with predisposing factors. In this minireview, we briefly summarize how the side effects of molecularly targeted drugs were discovered when new classes of drugs were introduced. We also summarize the clinical characteristics of these side effects focusing on progressive multifocal leukoencephalopathy (PML) associated with the use of an integrin antagonist, cardiotoxicity associated with the use of some tyrosine kinase inhibitors, and hypertension associated with the use of angiogenesis inhibitors. We also review the molecular mechanisms underlying these side effects. Research on the mechanisms underlying these side effects has revealed previously unknown physiological roles of targeted molecules. Awareness and understanding of the side effects of molecularly targeted drugs is important for those working in clinical practice and conducting basic research.

Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

Intracellular transport is fundamental for neuronal morphogenesis, function and survival. Many proteins are selectively transported to either axons or dendrites. In addition, some specific mRNAs are transported to dendrites for local translation. Proteins of the kinesin superfamily participate in selective transport by using adaptor or scaffolding proteins to recognize and bind cargoes. The molecular components of RNA-transporting granules have been identified, and it is becoming clear how cargoes are directed to axons and dendrites by kinesin superfamily proteins. Here we discuss the molecular mechanisms of directional axonal and dendritic transport with specific emphasis on the role of motor proteins and their mechanisms of cargo recognition.

Microtubules are formed by polymerization of α - and β -tubulin. They are intrinsically in dynamic turnover state, subject to treadmilling and dynamic instability. Microtubule-associated proteins (MAPs) are a heterogeneous group of proteins that have microtubule-binding domains. MAPs are abundantly expressed in the brain, and historically MAPs (e.g., MAP1, MAP2, and tau) derived from the brain have been studied extensively. MAP4 is ubiquitously expressed. These classical MAPs are filamentous proteins of various lengths, ranging from 50 to 185 nm. They have microtubule-binding domain and the projection domain that extends as filamentous structure. By binding along the side of microtubules, they stabilize microtubules. In addition, by extending the projection domain from the microtubule surface, they produce microtubule bundles of various densities. Tau and MAP2C predominantly expressed in the axon produce microtubule bundles of ~20 nm distances. MAP2 predominantly expressed in the dendrite produces microtubule bundles of ~65 nm distances. The microtubule bundles produced by tau/MAP2C and MAP2 resemble microtubule domains in axons and dendrites, respectively. These microtubule bundles are extended from the cell as axon- or dendrite-like processes. Therefore, MAPs serve as determinants of microtubule organization within the cell, particularly in neurons. There are some other groups of newly identified MAPs, and some of them destabilize microtubules. Binding of MAPs to microtubules is regulated by phosphorylation. In some neurodegenerative diseases including Alzheimer's disease, hyperphosphorylated tau precipitates as filaments on its own and may be one of the important factors to determine the progress of the disease.

Kinesin superfamily proteins (KIFs) are motor proteins that transport membranous organelles and macromolecules fundamental for cellular functions along microtubules. Their roles in transport in axons and dendrites have been studied extensively, but KIFs are also used in intracellular transport in general. Recent findings have revealed that in many cases, the specific interaction of cargoes and motors is mediated via adaptor/scaffolding proteins. Cargoes are sorted to precise destinations, such as axons or dendrites. KIFs also participate in polarized transport in epithelial cells as shown in the apical transport of annexin XIIIb-containing vesicles by KIFC3. KIFs play important roles in higher order neuronal activity; transgenic mice overexpressing KIF17, which transports N-methyl-d-asp (NMDA) receptors to dendrites, show enhanced memory and learning. KIFs also play significant roles in neuronal development and brain wiring: KIF2A suppresses elongation of axon collaterals by its unique microtubule-depolymerizing activity. X-ray crystallography has revealed the structural uniqueness of KIF2 underlying the microtubule-depolymerizing activity. In addition, single molecule biophysics and optical trapping have shown that the motility of monomeric KIF1A is caused by biased Brownian movement, and X-ray crystallography has shown how the conformational changes occur for KIF1A to move during ATP hydrolysis. These multiple approaches in analyzing KIF functions will illuminate many basic mechanisms underlying intracellular events and will be a very promising and fruitful area for future studies.

Molecular motors such as kinesin superfamily proteins (KIFs), dynein superfamily proteins and myosin superfamily proteins have diverse and fundamental roles in many cellular processes, including neuronal development and the pathogenesis of neuronal diseases. During neuronal development, KIFs take significant roles in the regulation of axon-collateral branch extension, which is essential for brain wiring. Cytoplasmic dynein together with LIS1 takes pivotal roles in neocortical layer formation. In axons, anterograde transport is mediated by KIFs, whereas retrograde transport is mediated mainly by cytoplasmic dynein, and dysfunction of motors results in neurodegenerative diseases. In dendrites, the transport of NMDA and AMPA receptors is mediated by KIFs, and the motor has been shown to play a significant part in establishing learning and memory.

Kinesin was first identified biochemically as a microtubule-dependent motor protein responsible for transport of membranous organelles in the axon. It is now recognized that microtubule-dependent motor proteins form a large gene family, kinesin superfamily proteins (KIFs). The human genome contains 45 KIF genes. KIFs have high homology at the so-called 'motor domain', which is a globular domain responsible for moving along microtubules by hydrolysis of adenosine triphosphate (ATP). Outside the motor domain, the sequence is unique to each member. The motors bind to the 'cargoes', the molecule to be transported, at this domain. KIFs transport many different types of cargoes including membranous organelles, protein complexes, and messenger RNAs (mRNAs). They play important roles in a wide variety of intracellular transport, such as transport from Golgi to plasma membrane pathways involved in exocytosis and endocytosis, axonal transport, transport in dendrites, and special transport called intraflagellar transport. In addition, recent molecular genetic experiments have uncovered unexpected roles for KIFs in the regulation of such physiologic processes as higher brain function, tumour suppression, and developmental patterning. They also play important roles in mitosis. Functions and/or dysfunctions of KIF motors underlie some diseases. The molecular mechanisms by which different KIFs recognize and bind to specific cargoes, and how their binding is regulated, have been identified for some KIFs.

Recent studies have revealed that kinesin, dynein and myosin each form large superfamilies and participate in many different intracellular transport systems. Importantly, these motor proteins play significant roles in the pathogenesis of a variety of diseases. Studies using knockout mice for kinesin KIF1B have led to the identification of the cause of a human hereditary neuropathy, Charcot-Marie-Tooth disease type 2A. The function of members of the dynein superfamily whose existence has previously only been confirmed through genome databases, has been revealed by studies of immotile cilia syndrome. Unconventional myosins have been shown to function in the inner-ear cells by examination of hereditary human hearing impairment and studies using mouse models. In addition, some diseases are caused by mutations, not in the motor itself, but in the proteins associated with the motor proteins. Here, we discuss the relationship of these motor proteins and how they contribute to disease in molecular terms.

Mouse brain expresses multiple kinesin superfamily proteins (KIFs), which are involved in vesicle transport. The expression of KIFs is developmentally regulated, and both the mRNA and proteins of KIF2 and KIF4 are expressed abundantly in the juvenile brain. To elucidate the role of individual kinesin superfamily motor proteins during regenerative outgrowth of axons, we examined the mRNA expression of KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, and KIF5 in adult mouse dorsal root ganglion cells after sciatic nerve crush. Seven to fourteen days after the nerve crush, the mRNA expression pattern of neurofilament and beta-tubulin isotypes suggested that the regenerative outgrowth of axons was active. At these stages, levels of mRNA for KIF1A, KIF1B, KIF2, KIF3A, KIF3B, KIF4, and KIF5 were 50-80% of control. The levels of mRNA for KIF4, which are detected in juvenile brain but not in the adult, were under the detection limit in both control and regenerating dorsal root ganglion cells. Because mRNA of neither KIF2 nor KIF4 increased significantly, the results suggest that the gene expression of KIFs during regeneration does not recapitulate the embryonic development and support the hypothesis that different series of events take place during the regenerative and embryonic outgrowths of axons. In contrast, mRNA for cytoplasmic dynein was slightly increased, up to 140%. This is consistent with the hypothesis that retrograde transport plays critical roles in regeneration such as the transport of neurotrophic factors.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.

To understand the mechanisms of transport for organelles in the axon, we isolated and sequenced the cDNA encoding KIF4 from murine brain, and characterized the molecule biochemically and immunocytochemically. Complete amino acid sequence analysis of KIF4 and ultrastructural studies of KIF4 molecules expressed in Sf9 cells revealed that the protein contains 1,231 amino acid residues (M(r) 139,550) and that the molecule (116-nm rod with globular heads and tail) consists of three domains: an NH2-terminal globular motor domain, a central alpha-helical stalk domain and a COOH-terminal tail domain. KIF4 protein has the property of nucleotide-dependent binding to microtubules, microtubule-activated ATPase activity, and microtubule plus-end-directed motility. Northern blot analysis and in situ hybridization demonstrated that KIF4 is strongly expressed in juvenile tissues including differentiated young neurons, while its expression is decreased considerably in adult mice except in spleen. Immunocytochemical studies revealed that KIF4 colocalized with membranous organelles both in growth cones of differentiated neurons and in the cytoplasm of cultured fibroblasts. During mitotic phase of cell cycle, KIF4 appears to colocalize with membranous organelles in the mitotic spindle. Hence we conclude that KIF4 is a novel microtubule-associated anterograde motor protein for membranous organelles, the expression of which is regulated developmentally.

To further elucidate the mechanism of organelle transport, we cloned a novel member of the mouse kinesin superfamily, KIF1B. This N-terminal-type motor protein is expressed ubiquitously in various kinds of tissues. In situ hybridization revealed that KIF1B is expressed abundantly in differentiated nerve cells. Interestingly, KIF1B works as a monomer, having a microtubule plus end-directed motility. Our rotary shadowing electron microscopy revealed mostly single globular structures. Immunocytochemically, KIF1B was colocalized with mitochondria in vivo. Furthermore, a subcellular fractionation study showed that KIF1B was concentrated in the mitochondrial fraction, and purified KIF1B could transport mitochondria along microtubules in vitro. These data strongly suggested that KIF1B works as a monomeric motor for anterograde transport of mitochondria.

Dynamin is a member of a new GTPase family, which includes the mouse Mx protein, the yeast VPS1 and the Drosophila shibire gene product. A high homology with the shibire product suggests a role for dynamin in the endocytotic process, but it is expressed only in mature neurons. We identified two additional dynamin-like proteins in rats, by using the polymerase chain reaction with degenerate primers corresponding to the GTP-binding areas conserved between dynamin and VPS1. The full coding sequence of one of them, dynamin-2, revealed that it has 848 amino acids and has great similarity with brain dynamin and the shibire product. Northern blot analysis and in situ hybridization revealed its expression to be specific to the seminiferous tubules in the testis. Dynamin-2 (testis type dynamin) was expressed in germ-cell-depleted testis as well, indicating its expression in Sertoli cells. Our data imply that a number of dynamin family proteins, which are products of distinct genes, may play different roles specific to each cell type in the same rat.

A mouse gene (referred to as Emb) encoding a novel class of POU domain is described. The Emb POU domain shares only 40-50% homology to that of any other POU proteins. Nonetheless, the Emb POU domain can bind to the octamer sequence like other POU domains. Emb is a single-copy gene, located on the distal region of mouse chromosome 15. It is expressed in embryo throughout post-implantation stages, where the most prominent expression is seen in developing central nervous system. In the adult, it is highly expressed in brain, whereas weaker expression can be detected in other organs such as testis, skeletal muscle, and kidney. The expression in adult brain is most evident in neurons of hypocampus formation. Two types of Emb mRNA are expressed in brain; one type encodes a protein of 301 amino acids residues, whereas the other codes for a protein with two extra amino acids residues added at the amino-terminal end of POU domain. These two mRNA species are generated by alternative splicing by utilizing an unusual splice acceptor site: CCTCCCTCTG/. Emb mRNA expressed in testis, on the other hand, encodes a smaller protein lacking most of the amino-terminal region.