SUMMARYGhost crabs possess rapid running capabilities, which make them good candidates for comparing invertebrate exercise physiology with that of more extensively studied vertebrates. While a number of studies have examined various aspects of running physiology and biomechanics in terrestrial crabs, none to date have defined the basic skeletal muscle fiber types that power locomotion. In the current study, we investigated skeletal muscle fiber types comprising the extensor and flexor carpopodite muscles in relation to running performance in the ghost crab. We used kinematic analyses to determine stride frequency and muscle shortening velocity and found that both parameters are similar to those of comparably sized mammals but slower than those observed in running lizards. Using several complementary methods, we found that the muscles are divided into two primary fiber types: those of the proximal and distal regions possess long sarcomeres (6.2±2.3 μm) observed in crustacean slow fibers and have characteristics of aerobic fibers whereas those of the muscle mid-region have short sarcomeres (3.5±0.4 μm) characteristic of fast fibers and appear to be glycolytic. Each fiber type is characterized by several different myofibrillar protein isoforms including multiple isoforms of myosin heavy chain (MHC), troponin I (TnI), troponin T (TnT) and a crustacean fast muscle protein, P75. Three different isoforms of MHC are differentially expressed in the muscles, with fibers of the mid-region always coexpressing two isoforms at a 1:1 ratio within single fibers. Based on our analyses, we propose that these muscles are functionally divided into a two-geared system, with the aerobic fibers used for slow sustained activities and the glycolytic mid-region fibers being reserved for explosive sprints. Finally, we identified subtle differences in myofibrillar isoform expression correlated with crab body size, which changes by several orders of magnitude during an animalʼs lifetime.

Infections caused by enterotoxigenic Escherichia coli (ETEC)are the leading cause of traveler's diarrhea and the major cause of diarrheal disease in underdeveloped nations, especially among children. ETEC, which is usually transmitted by food or water contaminated with animal or human feces, is estimated to be responsible annually for more than 650 million cases of enteric infections and nearly 800,000 deaths (29). Infection begins with ingestion of bacteria, followed by elaboration of enterotoxin and bacterial colonization of the gut, and presents as a profuse watery diarrhea which disseminates the bacteria back into the environment (10).ETEC strains are lactose-fermenting E. coli strains that produce a heat-labile enterotoxin (LT, hereafter referred to as LT-I), heat-stable enterotoxins (ST), or both and colonization factors which enable ETEC to colonize the small intestine (22). The pathogenesis of ETEC is dependent on the strains' capacity to produce LT-I and/or ST (10, 29). LT-I is closely related functionally, antigenically, and structurally to cholera toxin (CT), the heat-labile enterotoxin produced by Vibrio cholerae. Antiserum against CT neutralizes the toxicity of LT-I, and antiserum against LT-I neutralizes the toxicity of CT (15). Structurally, LT-I and CT are oligomeric proteins composed of an A polypeptide which is noncovalently coupled to a pentameric array of B polypeptides (15). The A polypeptide of LT-I and CT is enzymatically active and catalyzes an ADP-ribosylation of the G s ␣ regulatory protein in the intoxicated cell. Ribosylation of this regulatory protein constitutively activates adenylate cyclase, the enzyme which catalyzes production of cyclic AMP (cAMP) (3,20). Accumulation of cAMP induces the intoxicated cell to secrete electrolytes and chloride ions, thus generating the watery diarrhea, which is symptomatic of intoxication. Intracellular accumulation of cAMP modulates other cellular processes such as protein kinase activity, activation of calcium channels, etc. (15). Binding of LT-I and CT to ganglioside receptors is mediated by the B polypeptides. Gangliosides are members of a heterogeneous family of sialylated glycosphingolipids expressed on the surface of eukaryotic cells (9). Based on these characteristics, LT-I and CT have been designated as members of the large family of toxins known as the A 1 B 5 ADP-ribosylating heat-labile enterotoxins (HLTs).LT-IIa and LT-IIb, two new members of the A 1 B 5 family of HLTs produced by E. coli, were recently described (11,12,27). While it is clear that LT-IIa and LT-IIb are evolutionarily related to LT-I and CT, there are major differences between the two groups of enterotoxins. LT-IIa and LT-IIb are antigenically distinguishable from LT-I and CT and from each other (12). These antigenic differences are reflected in the low amino acid sequence similarity of the A polypeptides and the virtual absence of amino acid sequence homology of the B polypeptides between the two groups (LT-I and CT versus LT-IIa and LT-IIb) (35). To distinguish betwee...

Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense.

Vaccinations are extremely effective at combating infectious diseases. Many conserved antigen (Ag) targets, however, are poorly immunogenic. Protein subunit vaccines frequently elicit only humoral immune responses and fail to confer protection against serious intracellular pathogens. These barriers to vaccine development are often overcome by the use of appropriate adjuvants. Heat-labile enterotoxins (HLT) produced by enterotoxigenic strains of Escherichia coli are potent adjuvants when administered by mucosal or systemic routes. The efficacy of the type II HLT, however, has not been well-defined when administered by the intradermal (ID) route. Using a murine ID immunization model, the adjuvant properties of LT-IIb and LT-IIc, two type II HLTs, were compared with those of LT-I, a prototypical type I HLT. While all three HLT adjuvants enhanced Ag-specific humoral responses to similar levels, LT-IIb and LT-IIc, in contrast to LT-I, induced a more vigorous Ag-specific CD8+ T cell response and proffered faster clearance of Listeria monocytogenes in a challenge model. Additionally, LT-IIb and LT-IIc induced distinct differences in the profiles of the Ag-specific CD8+ T cell responses. While LT-IIc stimulated a robust and rapid primary CD8+ T cell response, LT-IIb exhibited slower CD8+ T cell expansion and contraction kinetics with the formation of higher percentages of effector memory cells. In comparison to LT-I and LT-IIc, LT-IIb evoked better long-term protection after immunization. Furthermore, LT-IIb and LT-IIc enhanced the total number of dendritic cells (DC) in the draining lymph node (DLN) and expression of costimulatory molecules CD80, CD86, and CD40 on DCs. In contrast to LT-I, LT-IIb and LT-IIc induced less edema, cellular infiltrates, and general inflammation at the site of ID injection. Thus, LT-IIb and LT-IIc are attractive comprehensive ID adjuvants with unique characteristic that enhance humoral and cellular immunity to a co-administered protein Ag.

We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of ␣ENaC using the serine protease subtilisin. Trimeric channels formed with ␣FM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ϳ10-fold decrease in amiloride-sensitive whole cell conductance as compared with ␣WT (2.2 versus 21.2 microsiemens ( S)). Both ␣WT and ␣FM were activated to similar levels by subtilisin cleavage. Channels formed with ␣FD, a construct that deleted the segment between the two furin sites (⌬175-204), exhibited an intermediate conductance of 13.2 S. More importantly, ␣FD retained the ability to be activated by subtilisin to 108.8 ؎ 20.9 S, a level not significantly different from that of subtilisin activated ␣WT (125.6 ؎ 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of ␣ was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with ␣LD, a construct that extended the deleted sequence of ␣FD by 17 amino acids (⌬175-221). Channels with ␣LD were uncleaved, exhibited low baseline activity (4.1 S), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.It is well established that serine proteases activate the epithelial sodium channel (ENaC). 2 Activation occurs by direct mechanisms that induce channel subunit cleavage (1, 2) as well as those that are cleavage-independent but may involve cleavage of protease-activated membrane receptors (3). Channel cleavage studies have established that cellular proteases such as furin endogenously cleave the channel ␣ and ␥ subunits. Mutation of identified endogenous cleavage sites on both of these subunits diminished baseline activity, demonstrating a role for cleavage in ENaC activation.The acute effects of ENaC cleavage have largely relied on examining the effects of the protease trypsin on the ␣ and ␥ subunits. These studies have examined the effects of cleavage on wild type and furin cleavage-deficient ENaC in oocytes and epithelial cells (1,4,5). Although these have markedly improved our understanding of channel activation by serine proteases, they suffer from the main shortcoming that trypsin is a non-selective serine protease that can cleave after a single arginine residue (6, 7), and therefore, it only offers a limited tool for examining the mechanisms of cleavage at specific sites. Consistent with the reduced specificity for trypsin is the observation that ENaC retains its cleavage by this protease after mutation of consensus cleavage sites for furin (1).Despite their limitations, these studies have ind...