The mouse Snell's waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells.

The mouse represents an excellent model system for the study of genetic deafness in humans. Many mouse deafness mutants have been identified and the anatomy of the mouse and human ear is similar. Here we report the use of a positional cloning approach to identify the gene encoded by the mouse recessive deafness mutation, Snell's waltzer (sv). We show that sv encodes an unconventional myosin heavy chain, myosin VI, which is expressed within the sensory hair cells of the inner ear, and appears to be required for maintaining their structural integrity. The requirement for myosin VI in hearing makes this gene an excellent candidate for a human deafness disorder.

Molecular motors, membrane movements and physiology: emerging roles for myosins.

Myosins are a large family of structurally diverse mechanoenzymes which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force. Consistent with the ubiquitous association of actin with membranes, many of these novel myosins are membrane-associated. In the past two years, evidence has emerged that suggests roles for actin-based molecular motors in a wide range of membrane phenomena such as cell locomotion, phagocytosis, secretion, organelle transport, signal transduction and mechanoregulation of membrane protein function.

Mechanisms of motor protein reversal.

Members of the kinesin superfamily of microtubule-based motors and the myosin superfamily of actin-based motors that move 'backwards' have been identified. As the core catalytic domains of myosins and kinesins are similar in structure, this raises the intriguing questions of how direction reversal is accomplished and whether kinesins and myosins share mechanisms for switching their motors into reverse.

Porcine myosin-VI: characterization of a new mammalian unconventional myosin.

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC-PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.

Unconventional myosins in inner-ear sensory epithelia.

To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Ibeta is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Ibeta is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Ibeta, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Ibeta probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Physicochemical, biological, functional and toxicological characterization of the European follow-on glatiramer acetate product as compared with Copaxone.

For more than 20 years, Copaxone (glatiramer acetate, Teva), a non-biological complex drug, has been a safe and effective treatment option for multiple sclerosis. In 2016, a follow-on glatiramer acetate product (FOGA, Synthon) was approved in the EU. Traditional bulk-based methods and high-resolution assays were employed to evaluate the physicochemical, functional, and bio-recognition attributes, as well as the in vivo toxicity profile of the active substances in Copaxone and Synthon EU FOGA lots. These tests included quality control tests applied routinely in release of Copaxone lots, as well as additional characterization assays, gene expression studies and a rat toxicity study. Even though the Synthon FOGA was designed to copy and compete with Copaxone, the active substances were found to be similar in only 7 of the tested 14 (50%) methods (similar is defined as within approved specifications or within the inherent microheterogeneity range of tested Copaxone batches, or not showing statistically significant differences). With additional methods applied, consistent compositional differences in attributes of surface charge distribution, molecular size, and spatial arrangement were observed. These marked differences were concordantly observed with higher biological activity of some of the Synthon EU FOGA lots compared with Copaxone lots, including potency and cytotoxicity activities as well as gene expression of pathways that regulate apoptosis, IL-2, and inflammation signaling. These observations raise concerns for immunogenicity differences, particularly in (repeated) substitution settings. Another orthogonal finding demonstrated increased frequency of injection-site local toxicity observations for the Synthon EU FOGA in an in vivo daily dosing rat study, thus warranting further qualification of the link between compositional and functional differences in immunogenicity, and potential impact on long-term efficacy and safety.

Molecular motors: sensing a function for myosin-VIIa.

Mutations affecting myosin-VIIa are known to cause deafness and blindness in human Usher syndrome. Mutation of the Dictyostelium myosin-VII gene has now revealed a role for this unconventional myosin in phagocytic events, providing a possible clue to the role of mammalian myosin-VIIa in the inner ear and retina.

Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae.

Clathrin is important but not essential for yeast cell growth and protein secretion. Diploid Saccharomyces cerevisiae cells heterozygous for a clathrin heavy-chain gene (CHC1) disruption give rise to viable, slow-growing, clathrin heavy-chain-deficient meiotic progeny (G. Payne and R. Schekman, Science 230:1009-1014, 1985). The possibility that extragenic suppressors account for growth of clathrin-deficient cells was examined by deletion of CHC1 from haploid cell genomes by single-step gene transplacement and independently by introduction of a centromere plasmid carrying the complete CHC1 gene into diploid cells before eviction of a chromosomal CHC1 locus and subsequent tetrad analysis. Both approaches yielded clathrin-deficient haploid strains. In mutants missing at least 95% of the CHC1 coding domain, transcripts related to CHC1 were not detected. The time course of invertase modification and secretion was measured to assess secretory pathway functions in the viable clathrin-deficient cells. Core-glycosylated invertase was converted to the mature, highly glycosylated form at equivalent rates in mutant and wild-type cells. Export of mature invertase from mutant cells was delayed but not prevented. Abnormal vacuoles, accumulated vesicles, and Golgi body-derived structures were visualized in mutant cells by electron microscopy. We conclude that extragenic suppressors do not account for the viability of clathrin-deficient cells and, furthermore, that many standard laboratory strains can sustain a CHC1 disruption. Clathrin does not appear to mediate protein transfer from the endoplasmic reticulum to the Golgi body but may function at a later stage of the secretory pathway.

The growing family of myosin motors and their role in neurons and sensory cells.

Biochemical and physiological evidence has suggested that myosins, both conventional and unconventional, are critical for neurosensory activities. In the past few years, this premise has been supported by genetic evidence that has shown that unconventional myosins are essential for the proper functioning of neurons, retina and the sensory cells of the inner ear.

An experimental model for infiltration of malignant lymphoma to the eye and brain.

Currently there is no adequate experimental model available whereby the lethal infiltration of malignant lymphoma to the eye and CNS can be studied. Variant S49 mouse lymphoma cells that exhibit cell-cell adhesion properties (named Rev-2-T-6) were inoculated intraperitoneally into Balb/C mice at the ages of 6-60 days postnatal. Mice inoculated between days 6-11 postnatal developed signs of eye and CNS involvement with an apparent peak (58% of mice) at day 7. None of the mice inoculated beyond day 11 exhibited such signs. Histological analysis of these sites revealed tumorous infiltrates into a variety of structures in the orbit, intraocular tissues, along the optic nerve and in the brain. Additional analysis of the histopathological data, based on the structures demonstrating the highest frequency of lymphoma infiltration, suggests preferred routes of lymphoma entry to the brain and eye. Thus, entry to the brain can occur mainly through the choroid plexus and cranial nerves or cranial nerve ganglia. Entry to the eye may occur from the brain (along the optic nerve), and through hematogenous infiltration of orbital structures. No data were found that would support retrograde infiltration of the lymphoma from the eye to the brain. These findings present an experimental model for addressing the molecular mechanisms that govern homing of malignant lymphoma to the eye and brain, as well as the development of experimental therapeutic modalities for malignant lymphoma in these organs.

Hereditary familial vestibular degenerative diseases.

Identification of genes involved in hereditary vestibular disease is growing at a remarkable pace. Mutant mouse technology can be an important tool for understanding the biological mechanism of human vestibular diseases.

Regulation of gonadotropin secretion in the male: effect of an aromatization inhibitor in estradiol-implanted, orchidectomized dogs.

Testosterone is aromatized to estradiol in both peripheral tissues and the central nervous system. Various authors have suggested that this conversion in the male may be prerequisite for the regulation of gonadotropin secretion by testosterone. Previously, it was reported that inhibition of central nervous system aromatase caused a significant increase in plasma LH in the presence of physiologic testosterone levels (Winter et al, 1983). In order to confirm whether aminoglutethimide, the aromatase inhibitor used in our previous study, either blocked aromatization, or the action of estradiol, the following study was conducted. Fifteen male mongrel dogs were equally divided into three groups. Group 1 dogs were implanted with estradiol-filled polydimethylsiloxane capsules only; Group 2 dogs were implanted with empty capsules and treated with 60 mg b.i.d. of aminoglutethimide; and Group 3 dogs were implanted with polydimethylsiloxane capsules filled with estradiol and treated with aminoglutethimide. Blood samples were drawn for 24 days during pretreatment, capsule implantation, castration, aminoglutethimide administration and capsule removal periods. The postcastration response of both plasma LH and FSH in dogs in groups 1 and 3 was suppressed in the presence of elevated estradiol, whereas that of Group 2 dogs was normal in the absence of estradiol. The results suggest that aminoglutethimide neither directly affects the plasma concentration of either LH or FSH nor blocks the effect of estradiol in inhibiting their release following castration. These data, taken together with our previous work, implicate aromatization of testosterone to estradiol in the control of gonadotropin secretion in the male.

MYO1F as a candidate gene for nonsyndromic deafness, DFNB15.

BACKGROUND: Earlier studies have mapped the autosomal recessive nonsyndromic deafness locus, DFNB15, to chromosomes 3q21.3-q25.2 and 19p13.3-13.1, identifying one of these chromosomal regions (or possibly both) as the site of a deafness-causing gene. Mutations in unconventional myosins cause deafness in mice and humans. One unconventional myosin, myosin 1F (MYO1F), is expressed in the cochlea and maps to chromosome 19p13.3-13.2. OBJECTIVE: To evaluate MYO1F as a candidate gene for deafness at the DFNB15 locus by determining its genomic structure and screening each exon for deafness-causing mutations to identify possible allele variants of MYO1F segregating in the DFNB15 family. METHODS: We used radiation hybrid mapping to localize MYO1F on chromosome arm 19p. We next determined its genomic structure using multiple long-range polymerase chain reaction experiments. Using these data, we completed mutation screening using single-stranded conformational polymorphism analysis and direct sequencing of affected and nonaffected persons in the original DFNB15 family. RESULTS: Radiation hybrid mapping placed MYO1F in the DFNB15 interval, establishing it as a positional candidate gene. Its genomic structure consists of 24 coding exons. No mutations or genomic rearrangements were found in the original DFNB15 family, making it unlikely that MYO1F is the disease-causing gene in this kindred. CONCLUSIONS: Although we did not find MYO1F allele variants in one family with autosomal recessive nonsyndromic hearing loss, the gene remains an excellent candidate for hereditary hearing impairment. Given its wide tissue expression, MYO1F might cause syndromic deafness.

Effect of active immunization against LHRH or LH in boars: reproductive consequences and performance traits.

Forty crossbred boars were equally divided into eight groups at birth. Four groups were immunized (200 micrograms/boar) at 12 wk of age against either luteinizing hormone-releasing hormone (LHRH) conjugated to human serum globulin (LHRH-hSG) in complete Freund's adjuvant (CFA), LHRH-hSG in muramyldipeptide adjuvant (PEP), procine luteinizing hormone (LH) conjugated to hSG (pLH-hSG) in CFA or ovine LH (oLH) in CFA. Equal doses of boosters were given in either PEP or incomplete Freund's adjuvant (IFA) at 16 and 18 wk of age. Two groups of boars were immunized with either hSG + CFA or hSG + PEP (adjuvant controls). Two groups were castrated either at the time of weaning (castrate weaning) or at 16 wk when immunized boars were given their first booster injections (castrate booster). All pigs were slaughtered at 24 wk of age. Serum levels of LH and testosterone (T), LHRH or LH antibody titers, as well as testicular and accessory sex gland weights and histology were determined. By wk 16, LHRH antibody titers began to rise in those boars immunized against LHRH-hSG. Luteinizing hormone-releasing hormone antibody titers on wk 18, 20 and 22 were greater than those at wk 16. By 22 wk of age, LHRH-hSG boars had non-detectable plasma LH and T and reduced weights of testes and acessory sex glands. Boars immunized against oLH did not respond to treatment, whereas pLH-hSG boars showed a reduction in serum T levels and accessory sex gland weights. Immunization had no effect on average daily gain, hot carcass weights or loin eye area.(ABSTRACT TRUNCATED AT 250 WORDS)

The dynamic response of human subjects while seated in car seats.

A pendulum impact method was used to establish the dynamic response of the seated subject. Threaded K wires were placed in the L3 spinous process. The gain and phase angle between the platform and the vertebra were established. The response of the subject was observed while seated on a platform and a variety of other seats. The seats were found to be very important in the attenuation of the impulse, leading to a higher transmissibility. Clinical Relevance Skeletal impact through the lower extremity is quite common in many occupations. The importance of posture and seat design in attenuation of impulses has been established.

Adenovirus L1 52- and 55-kilodalton proteins are present within assembling virions and colocalize with nuclear structures distinct from replication centers.

Analysis of a temperature-sensitive mutant, Ad5ts369, had indicated that the adenovirus L1 52- and 55-kDa proteins (52/55-kDa proteins) are required for the assembly of infectious virions. By using monoclonal antibodies directed against bacterially produced L1 52-kDa protein, the L1 52/55-kDa proteins were found to be differentially phosphorylated forms of a single 48-kDa polypeptide. Both phosphoforms were shown to be present within all suspected virus assembly intermediates (empty capsids, 50 to 100 molecules; young virions, 1 to 2 molecules) but not within mature virions. The mobilities of these proteins in polyacrylamide gels were affected by reducing agents, indicating that the 52/55-kDa proteins may exist as homodimers within the cell and within assembling particles. Immunofluorescence analysis revealed that the 52/55-kDa proteins localize to regions within the infected nucleus that are distinct from viral DNA replication centers, indicating that replication and assembly of viral components likely occur in separate nuclear compartments. Immunoelectron microscopic studies determined that the 52/55-kDa proteins are found in close association with structures that appear to contain assembling virions. These results are consistent with an active but transient role for the L1 products in assembly of the adenovirus particle, perhaps as scaffolding proteins.

Multiple unconventional myosin domains of the intestinal brush border cytoskeleton.

Representatives of class V and class VI unconventional myosins are identified as components of the intestinal brush border cytoskeleton. With brush border myosin-I and myosin-II, this brings to four the number of myosin classes associated with this one subcellular domain and represents the first characterization of four classes of myosins expressed in a single metazoan cell type. The distribution and cytoskeletal association of each myosin is distinct as assessed by both biochemical fractionation and immunofluorescence localization. Myosin-VI exists in both the microvillus and terminal web although the terminal web is the predominant site of concentration. Myosin-V is present in the terminal web and, most notably, at the distal ends of the microvilli, thus becoming the first actin-binding protein to be localized to this domain as assessed by both immunohistochemical and biochemical methods. In the undifferentiated enterocytes of the intestinal crypts, myosin-VI is expressed but not yet localized to the brush border, in contrast to myosin-V, which does demonstrate an apical distribution in these cells. An assessment of myosin abundance indicates that while myosin-II is the most abundant in the cell and in the brush border, brush border myosin-I is only slightly less abundant in contrast to myosins-V and -VI, both of which are two orders of magnitude less abundant than the others. Extraction studies indicate that of these four myosins, myosin-V is the most tightly associated with the brush border membrane, as detergent, in addition to ATP, is required for efficient solubilization.

Nucleolar localization of mouse mammary tumor virus proteins in T-cell lymphomas.

To characterize novel proteins expressed in lymphoma cells, monoclonal antibodies (MAbs) were raised against variant S49 mouse lymphoma cells. Immunoperoxidase analysis with a specific MAb, named M-66, revealed nuclear localization with prominent staining in the nucleoli of both tumorigenic (T-63) cells and nontumorigenic, immunogenic (T-25-Adh) cells. Weak signals were also observed in the cytoplasm and plasma membrane of both cells. Western blot analysis with M-66 antibody revealed a 14-kDa protein in nuclear extracts of both T-25-Adh and T-63 cells. An additional nuclear 21-kDa protein was evident only in T-63 cells. M-66 identified several clones from a T-25-Adh cDNA expression library. These clones demonstrated extensive homology (approximately 95% identity throughout their length) to the mouse mammary tumor virus (MMTV) env and LTR regions. Extensive amino acid sequence homology (approximately 90% identity) between the clones and the env protein was observed. M-66 identified the 14-kDa protein in another MMTV bearing T-cell lymphoma, EL-4. Immunoperoxidase analysis of EL-4 cells with M-66 also revealed prominent nucleolar staining. MMTV-negative cells and MMTV-positive cells of nonlymphocytic origin were devoid of both 14- and 21-kDa proteins. Moreover, an anti-MMTV gp52 (env) antibody precipitated the 21-kDa protein in T-63 cells. We thus suggest that MMTV bearing T-cell lymphomas express nucleolar proteins translated from the env region of MMTV.