"Turning up the heat": role of neurotrophic batokines in the postnatal maturation and remodeling of brown adipose tissue in deer mice.

Activation of brown adipose tissue (BAT) thermogenesis impacts energy balance and must be tightly regulated. Several neurotrophic factors, expressed in BAT of adult laboratory rodents, have been implicated in remodeling the sympathetic neural network to enhance thermogenesis [e.g., nerve growth factor (NGF), neuregulin-4 (NRG4), and S100b]. Here, we compare, to our knowledge, for the first time, the relative roles of three neurotrophic "batokines" in establishing/remodeling innervation during postnatal development and adult cold stress. We used laboratory-reared Peromyscus maniculatus, which rely heavily on BAT-based thermogenesis for survival in the wild, beginning between postnatal days (P) 8 and 10. BAT sympathetic innervation was enhanced from P6 to P10, and exogenous NGF, NRG4, and S100b stimulated neurite outgrowth from P6 sympathetic neurons. Endogenous BAT protein stores and/or gene expression of NRG4, S100b, and calsyntenin-3ß (which may regulate S100b secretion) remained high and constant during development. However, endogenous NGF was low and ngf mRNA was undetectable. Conditioned media (CM) from cultured P10 BAT slices stimulated neurite outgrowth from sympathetic neurons in vitro, which was inhibited by antibodies against all three growth factors. P10 CM had significant amounts of secreted NRG4 and S100b protein, but not NGF. By contrast, BAT slices from cold-acclimated adults released significant amounts of all three factors relative to thermoneutral controls. These data suggest that although neurotrophic batokines regulate sympathetic innervation in vivo, their relative contributions differ depending on the life stage. They also provide novel insights into the regulation of BAT remodeling and BAT's secretory role, both of which are critical to our understanding of mammalian energy homeostasis.NEW & NOTEWORTHY In altricial Peromyscus mice, the developmental shift to endothermy accompanies the establishment of the brown adipose tissue sympathetic neural network. Cultured slices of neonatal BAT secreted high quantities of two predicted neurotrophic batokines: S100b and neuregulin-4, but surprisingly low levels of the classic neurotrophic factor, NGF. Despite low NGF, neonatal BAT-conditioned media was highly neurotrophic. Cold-exposed adults use all three factors to dramatically remodel BAT, suggesting that BAT-neuron communication is life-stage dependent.

Myosin heavy chain expression in the red, white, and ventricular muscle of juvenile stages of rainbow trout.

Juvenile stages of rainbow trout, smaller parr and older juveniles, termed smolts, show differences in red muscle contractile properties: parr red muscle has faster kinetics and a faster maximum shortening velocity than smolt red muscle. A developmental reduction in the number of MHC isoforms as detected by SDS-PAGE between parr and smolt has also been observed. To investigate whether this shift in contractile kinetics results from differential gene expression, three different MHC cDNA fragments, one each from red, white, and ventricular muscle, were identified. The red muscle and ventricular forms are novel MHCs, and the white muscle form is identical to a published MHC from adult trout white muscle. Tissue and developmental stage-specific expression patterns of these MHC isoforms were examined using isoform-specific RT-PCR. Ventricular muscle typically showed only the ventricular form; 60% parr and 80% smolts expressed the ventricular form only. Approximately half of the white muscle samples of either parr or smolts, 58% and 50%, respectively, expressed only white muscle MHC. Red muscle samples were the most heterogeneous, with red muscle MHC found in combination with either the white or ventricular form or both. Combining samples from the anterior and posterior, 8% of parr red muscle samples expressed solely the red muscle MHC form, and 30% of smolt red muscle samples expressed the red muscle form alone. Variations in the relative contribution of each MHC to the red muscle of parr and smolt may explain observed differences in protein composition and contractile properties. J. Exp. Zool. 290:751-758, 2001.

Assembly of mammalian voltage-gated potassium channels: evidence for an important role of the first transmembrane segment.

Three different experimental approaches were used to establish that the first transmembrane segment (S1) is important for K+ channel assembly. First, hydrodynamic analyses of in vitro translated Kv1.1 N-terminal domain containing the S1 segment coassembles to form homotetrameric complexes, whereas deletion of the S1 segment abolishes coassembly. Second, coimmunoprecipitation experiments of cotranslated Kv1.1 and Kv1.5 truncated polypeptides show that the S1 segment is essential for coimmunoprecipitation. Third, dominant negative experiments in Xenopus oocytes confirm that over-expression of either the S1 segment or the N-terminal domain is sufficient for abolishing the expression of functional Kv1.1 and Kv1.5 K+ channels. These data indicate that S1 segment plays an important role in the coassembly of homo- and heterotetrameric K+ channels.

Biochemical characterization and epididymal localization of the maturation-dependent ram sperm surface antigen ESA152.

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS-polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N-linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton-X-114, and solubilizes in chloroform-methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm.

Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction.

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.

Normal band 3-cytoskeletal interactions are maintained on tanktreading erythrocytes.

Normal nonnucleated erythrocytes subjected to continuous hydrodynamic shear exhibit membrane deformation or "tanktreading," a process important for reduction of the bulk viscosity of circulating blood. To characterize the effect of this unique process on the erythrocyte membrane we have measured the lateral diffusion of band 3 during tanktreading. Band 3 is normally constrained through interactions with the spectrin-actin cytoskeleton, therefore, any significant disruption of these interactions would result in alterations in band 3 dynamics. Band 3 of human erythrocytes was labeled with dichlorotriazinyl amino fluorescein. After laser photobleaching of an equatorial stripe, fluorescence images were recorded from cells in the presence or absence of shear. The amplitude of induced nonuniformity in the surface distribution of fluorescence was calculated directly from images of unsheared cells. In shear the bleached line rotated with the tanktreading motion of the cells. The surface integral of fluorescence oscillated with this motion. For this case, the amplitude of photobleaching-induced nonuniformity was defined as the amplitude at the fundamental frequency of fast Fourier transforms in time of the oscillations. Shear stress-induced membrane flow did not interrupt the linkage of band 3 with the erythrocyte cytoskeleton. Diffusion coefficient and mobile fraction (1.5 +/- 0.5 x 10(-10) cm2/s and 54 +/- 11%, respectively) were unaffected by shear. The rate of fluorescence recovery of cells in shear was also similar at the centers and at the edges, where in-plane shear forces are maximal.