We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1-2 h) of the DNA precursor analog bromodeoxyuridine (BrdU) in conjunction with MAb 16A11, we observed Hu+/BrdU+ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu+ cells were ambiguously BrdU+ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation.

A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously. The functional role of this new complex is uncertain. However, a role in protein import related to maintenance of mitochondrial structure is suggested as mitofilin helps regulate mitochondrial morphology and at least four of the associated proteins (metaxins 1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11 is a chaperone-like protein that may have a similar role.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by reduced amounts of the mitochondrial protein frataxin. Frataxin levels in research studies are typically measured via Western blot analysis from patient fibroblasts, lymphocytes, or muscle biopsies; none of these is ideal for rapid detection in large scale clinical studies. Recently, a rapid, noninvasive lateral-flow immunoassay was developed to accurately measure picogram levels of frataxin protein and shown to distinguish lymphoblastoid cells from FRDA carriers, patients and controls. We expanded the immunoassay to measure frataxin directly in buccal cells and whole blood from a large cohort of controls, known carriers and patients typical of a clinical trial population. The assay in buccal cells shared a similar degree of variability with previous studies conducted in lymphoblastoid cells (~10% coefficient of variation in controls). Significant differences in frataxin protein quantity were seen between the mean group values of controls, carriers, and patient buccal cells (100, 50.2, and 20.9% of control, respectively) and in protein extracted from whole blood (100, 75.3, and 32.2%, respectively), although there was some overlap between the groups. In addition, frataxin levels were inversely related to GAA repeat length and correlated directly with age of onset. Subjects with one expanded GAA repeat and an identified frataxin point mutation also carried frataxin levels in the disease range. Some patients displaying an FRDA phenotype but carrying only a single identifiable mutation had frataxin levels in the FRDA patient range. One patient from this group has a novel deletion that included exons 2 and 3 of the FXN gene based on multiplex ligation-dependent probe amplification (MLPA) analysis of the FXN gene. The lateral flow immunoassay may be a useful means to non-invasively assess frataxin levels repetitively with minimal discomfort in FRDA patients in specific situations such as clinical trials, and as a complementary diagnostic tool to aid in identification and characterization of atypical patients.

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A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected. Virulence screens of 400 isolates identified four potentially pathogenic strains of Vibrio spp. subsequently identified as V. coralliilyticus. Strains of this species are known coral pathogens; however, cultures were unable to consistently elicit tissue loss, suggesting an opportunistic role. Using an improved immunoassay, the VcpA RapidTest, a toxic zinc-metalloprotease produced by V. coralliilyticus was detected on 22.3% of diseased Montastraea cavernosa (n = 67) and 23.5% of diseased Orbicella faveolata (n = 24). VcpA + corals had significantly higher mortality rates and faster disease progression. For VcpA − fragments, 21.6% and 33.3% of M. cavernosa and O. faveolata, respectively, died within 21 d of observation, while 100% of similarly sized VcpA + fragments of both species died during the same period. Further physiological and genomic analysis found no apparent differences between the Atlantic V. coralliilyticus strains cultured here and pathogens from the Indo-Pacific but highlighted the diversity among strains and their immense genetic potential. In all, V. coralliilyticus may be causing coinfections that exacerbate existing SCTLD lesions, which could contribute to the intraspecific differences observed between colonies. This study describes potential coinfections contributing to SCTLD virulence as well as diagnostic tools capable of tracking the pathogen involved, which are important contributions to the management and understanding of SCTLD.

Although much progress has been made in identifying genetic defects associated with mitochondrial diseases, the protein expression patterns of most disorders are poorly understood. Here we use immunochemical techniques to describe subunit expression patterns of respiratory chain enzyme complexes II (succinate dehydrogenase: SD) and IV (cytochrome c oxidase: COX) in cultured cells lacking mtDNA (Rho0 cells) derived either chemically by exposure of normal cells to ethidium bromide, or genetically in cells derived from a patient with mtDNA depletion syndrome. Both control cells and early passage patient-derived cells express a normal complement of SD and COX subunit proteins. Ethidium bromide treatment of normal cells and in vitro cell proliferation of patient-derived cells caused both populations to acquire identical Rho0 phenotypes. As expected, they lack mtDNA-encoded subunits COX-I and COX-II. In contrast, nDNA-encoded subunits are affected differentially, with some (COX-VIc) lacking and others (COX-IV, COX-Va, SD 30 and SD 70) maintained at somewhat reduced levels. We suggest that the differential stability of nDNA-encoded subunits in the absence of intact enzyme complexes is due to the ability of some, but not all, subunits to associate as partial complexes in the absence of mtDNA-encoded subunits.

Thyroidal trophic effects on slow-twitch skeletal muscle properties were compared in normally innervated and denervated soleus of rats maintained at different thyroid states. Hypothyroidism caused fast to slow changes in fiber type composition (99% decrease in proportion of type II fibers), ATPase activities (down 20-30%), myosin light chain pattern (54% less fast light chains), calcium uptake by SR (down 60%), LDH activity (down 11%), and isozyme pattern (9% decrease in M-subunits). Changes of similar magnitude but opposite in direction were induced by thyrotoxicosis. Denervations reversed, to varying degrees, the fast to slow transformations observed in hypothyroidism. However the slow to fast changes found in hyperthyroidism were facilitated rather than inhibited by denervation. These latter results clearly show that the hormone effect can be elicited in the absence of motor innervation. Furthermore, denervation alone caused slow to fast changes in euthyroid muscles. From these results, it is proposed that denervation and dysthyreosis alter muscle properties by independent mechanisms. Our data favor a direct action of thyroid hormone over a neurally mediated mechanism.