Sexually dimorphic expression of myosin heavy chains in the adult mouse masseter.

Little is known regarding the role of androgenic hormones in the maintenance of myosin heavy chain (MHC) composition of rodent masticatory muscles. Because the masseter is the principal jaw closer in rodents, we felt it was important to characterize the influence of androgenic hormones on the MHC composition of the masseter. To determine the extent of sexual dimorphism in the phenotype of masseter muscle fibers of adult (10-mo-old) C57 mice, we stained tissue sections with antibodies specific to type IIa and IIb MHC isoforms. Females contain twice as many fibers containing the IIa MHC as males, and males contain twice as many fibers containing the IIb MHC as females. There is a modest amount of regionalization of MHC phenotypes in the mouse masseter. The rostral portions of the masseter are composed mostly of type IIa fibers, whereas the midsuperficial and caudal regions contain mostly type IIb fibers. Using immunoblots, we showed that castration results in an increase in the expression of type IIa MHC fibers in males. Ovariectomy has no effect on the fiber type composition in females. We conclude that testosterone plays a role in the maintenance of MHC expression in the adult male mouse masseter.     

On the role of the 200-kDa neurofilament protein at the developing neuromuscular junction

To examine whether the 200-kDa neurofilament protein (200K NFP) is involved in mechanically stabilizing axons, we studied the developmental appearance of immunoreactivity to nonphosphorylated and phosphorylated 200K NFP at the neuromuscular junction. Polyinnervated rat muscle fibers become singly innervated during the first 3 weeks of postnatal life through the process of synapse elimination. If production or post-translational modification of the 200K NFP is actively involved in imparting mechanical stability on neuromuscular synapses, then the selective presence of this protein in only one of several axons at each developing end plate region might make that one axon selectively resistant to elimination. The remaining axons would then be eliminated. Immunoreactivity to the 200K NFP is present on Gestational Day 14 and can be seen in more than one preterminal axon in the end plate region of a muscle fiber during the period of synapse elimination. These results suggest that the 200K NFP is present and phosphorylated early in development and, although the 200K NFP may increase the mechanical stability of axons, this increased stability does not determine the final outcome of synapse elimination.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

Two-dimensional infrared correlation spectroscopy as a probe of sequential events in the thermal unfolding of cytochromes c.

The sequential unfolding events of horse, cow, and tuna ferricytochromes c (cyt c) as a function of increasing temperature over the range 25-81 degrees C were investigated by resolution-enhanced two-dimensional infrared (2D IR) correlation spectroscopy. The 2D IR analysis revealed that in the thermal denaturation of the two mammalian cyts, the overall sequence of unfolding is similar, with denaturation of extended-chain and turn structures occurring prior to unfolding of alpha-helices, followed by denaturation of residual stable extended-chain structures. In tuna cyt c, denaturation of all extended-chain structures precedes the unfolding of alpha-helices. Moreover, in cow cyt c, unfolding of all helical components occurs as one cooperative unit, but in horse and tuna cyts c, the helical components behave as subdomains that unfold separately, as proposed recently by Englander and co-workers for horse cyt c [Bai et al. (1995) Science 269, 192-197; Milne et al. (1999) J. Mol. Biol. 290, 811-822]. At higher temperatures, following the loss of secondary structure, protein aggregation occurs in the three cyts c. The data presented here establish that variations in the thermal unfolding of cyts c can be associated with specific sites in the protein that influence local flexibility yet have little affect on global stability. This study demonstrates the power of resolution-enhanced 2D IR correlation spectroscopy in probing unfolding events in homologous proteins.