Molt cycle regulation of protein synthesis in skeletal muscle of the blackback land crab, Gecarcinus lateralis, and the differential expression of a myostatin-like factor during atrophy induced by molting or unweighting.

In decapod crustaceans, claw muscle undergoes atrophy in response to elevated ecdysteroids while thoracic muscle undergoes atrophy in response to unweighting. The signaling pathways that regulate muscle atrophy in crustaceans are largely unknown. Myostatin is a negative regulator of muscle growth in mammals, and a myostatin-like cDNA is preferentially expressed in muscle of the land crab, Gecarcinus lateralis (Gl-Mstn). Contrary to prediction, levels of Gl-Mstn mRNA decreased dramatically in both the claw closer and weighted thoracic muscles when molting was induced by either eyestalk ablation (ESA) or multiple limb autotomy (MLA). However, the effect of molt induction was greater in the claw muscle. By late premolt, Gl-Mstn mRNA in the claw muscle decreased 81% and 94% in ESA and MLA animals, respectively, and was negatively correlated with ecdysteroids. Gl-Mstn mRNA in thoracic muscle decreased 68% and 82% in ESA and MLA animals, respectively, but was only weakly correlated with ecdysteroid. Claw and thoracic muscles also differed to varying extents in the expression of ecdysteroid receptor (Gl-EcR and Gl-RXR), elongation factor-2 (Gl-EF-2), and calpain T (Gl-CalpT) in response to molt induction, but levels of the four transcripts were not correlated with ecdysteroid. The downregulation of Gl-Mstn expression in premolt claw muscle coincided with 11- and 13-fold increases in protein synthesis in the myofibrillar and soluble protein fractions, respectively. Furthermore, the rate of the increase in the synthesis of soluble proteins was greater than that of myofibrillar proteins during early premolt (1.4:1, soluble:myofibrillar), but the two were equivalent during late premolt. By contrast, Gl-Mstn mRNA increased 3-fold and Gl-CalpT mRNA decreased 40% in unweighted thoracic muscle; there was little or no effect on Gl-EF-2, Gl-EcR, and Gl-RXR mRNA levels. These data indicate that Gl-Mstn expression is negatively regulated by both ecdysteroids and load-bearing contractile activity. The downregulation of Gl-Mstn in claw muscle may induce the elevated protein turnover associated with remodeling of the contractile apparatus during molt-induced atrophy. The upregulation of Gl-Mstn in unweighted thoracic muscle suggests that this factor is also involved in disuse atrophy when hemolymph ecdysteroid levels are low.

Three calpains and ecdysone receptor in the land crab Gecarcinus lateralis: sequences, expression and effects of elevated ecdysteroid induced by eyestalk ablation.

Crustacean muscle has four calpain-like proteinase activities (CDP I, IIa, IIb and III) that are involved in molt-induced claw muscle atrophy, as they degrade myofibrillar proteins in vitro and in situ. Using PCR cloning techniques, three full-length calpain cDNAs (Gl-CalpB, Gl-CalpM and Gl-CalpT) were isolated from limb regenerates of the tropical land crab Gecarcinus lateralis. All three had highly conserved catalytic (dII) and C2-like (dIII) domains. Gl-CalpB was classified as a typical, or EF-hand, calpain, as the deduced amino acid sequence had a calmodulin-like domain IV in the C-terminus and was most similar to Drosophila calpains A and B. Based on its estimated mass (approximately 88.9 kDa) and cross-immunoreactivity with a polyclonal antibody raised against Dm-CalpA, Gl-CalpB may encode CDP IIb, which is a homodimer of a 95-kDa subunit. It was expressed in all tissues examined, including skeletal muscle, heart, integument, gill, digestive gland, hindgut, nerve ganglia, gonads and Y-organ (molting gland). Both Gl-CalpM and Gl-CalpT were classified as atypical, or non-EF-hand, calpains, as they lacked a domain IV sequence. Gl-CalpM was a homolog of Ha-CalpM from lobster, based on similarities in deduced amino acid sequence, estimated mass (approximately 65.2 kDa) and structural organization (both were truncated at the C-terminal end of dIII). It was expressed at varying levels in most tissues, except Y-organ. Gl-CalpT (approximately 74.6 kDa) was similar to TRA-3 in the nematode Caenorhabditis elegans; domain IV was replaced by a unique ;T domain' sequence. It was expressed in most tissues, except eyestalk ganglia and Y-organ. The effects of elevated ecdysteroid, induced by eyestalk ablation, on calpain and ecdysone receptor (Gl-EcR) mRNA levels in skeletal muscles were quantified by real-time PCR. At 1 day after eyestalk ablation, Gl-EcR and Gl-CalpT mRNA levels increased 15- and 19.3-fold, respectively, in claw muscle but not in thoracic muscle. At 3 days after eyestalk ablation, Gl-EcR and Gl-CalpT mRNA levels in claw muscle had decreased to 2.8-fold and 4.3-fold higher than those in intact controls, respectively, suggesting a feedback inhibition by ecdysteroid. There was no significant effect of eyestalk ablation on Gl-CalpB and Gl-CalpM mRNA levels. Gl-CalpT and Gl-EcR mRNA levels were significantly correlated in both claw and thoracic muscles from intact and eyestalk-ablated animals. The data suggest that Gl-CalpT is involved in initiation of claw muscle atrophy by ecdysteroids. Premolt reduction in claw muscle mass and concomitant remodeling of the sarcomere probably result from post-transcriptional regulation of calpains.

Carbohydrate dynamics and the crustacean hyperglycemic hormone (CHH): effects of parasitic infection in Norway lobsters (Nephrops norvegicus).

The effects of a dinoflagellate parasite (Hematodinium sp.) on carbohydrate metabolism were examined in the Norway lobster, Nephrops norvegicus. Five stages of infection were observed. These included uninfected (Stage 0), subpatently infected (SP), and patently infected (Stage 1-4) lobsters. During patent infection, the concentration of glucose in the hemolymph was reduced significantly from its value of 180 microg ml(-1) in uninfected (Stage 0) lobsters to 25.3 microg ml(-1) in Stage 3-4. These changes were accompanied by significantly lower levels of hepatopancreatic glycogen in lobsters at Stage 2 (2.01 mg g(-1)) and Stage 3-4 (0.84 mg g(-1)) of infection than in those at Stage 0 (16.19 mg g(-1)) and Stage 1 (14.71 mg g(-1)). Due to disruption of the normal feedback loops which control the release of crustacean hyperglycemic hormone (CHH), plasma concentrations increased with the severity of infection from 32.2 fmol ml(-1) in Stage 0 to 106.6 fmol ml(-1) in Stage 3-4. The increased CHH concentrations occurred concomitantly with reduced concentrations of plasma glucose and tissue glycogen. A significantly increased hemolymph CHH titer (107.7 fmol ml(-1)) was also observed during SP infection. It is concluded that the parasite places a heavy metabolic load on the host lobster.

Effects of exogenous serotonin on a motor behavior and shelter competition in juvenile lobsters (Homarus americanus).

Three experiments were conducted to determine (1) the pharmacodynamics of 5-hydroxytryptamine in juvenile lobsters; (2) the effects of 5-hydroxytryptamine, using a range of dosages, on a motor behavior used to escape an aversive situation; and (3) the effect of doses that did and did not inhibit this motor behavior on measures of dominance and shelter competition. The fate of 5-hydroxytryptamine in hemolymph over a 60-min post-injection period showed that the concentration fell rapidly to a low plateau that was maintained for at least 1 h. Low doses of 5-hydroxytryptamine did not affect locomotor behavior, but higher doses inhibited it. Dominance and subsequent possession of a shelter were unaffected by a low dose of 5-hydroxytryptamine but a higher dose that inhibited locomotion resulted in lobsters that lost fights and did not secure or retain possession of the shelter. In the context of dominance and shelter competition, we were unable to demonstrate any advantage of the low dose of exogenous 5-hydroxytryptamine and a severe disadvantage with the higher dose. Previous reports of transient increases in aggression in 5-hydroxytryptamine-treated subordinate lobsters did not take into account motor inhibition as a possible critical variable in aggression.

Crustacean hyperglycemic hormone in the lobster nervous system: localization and release from cells in the subesophageal ganglion and thoracic second roots.

Crustacean hyperglycemic hormones (CHHs) are neuropeptides involved in the regulation of hemolymph glucose. The primary source of CHHs has been identified as the neurosecretory neurons of the eyestalk X-organ and its associated neurohemal organ, the sinus gland. We have identified another source of CHH-like peptides in the nervous system. With the use of immunocytochemistry, cells in the second roots of the thoracic ganglia have been observed to stain positively for CHH-reactive material. We also identified a pair of cells in the subesophageal ganglion that contain large amounts of CHH-reactive material. Depolarization of these cells with elevated potassium mediates a calcium-dependent release of CHH-like material from the ganglion as quantified with an enzyme-linked immunosorbent assay (ELISA).

Quantification of crustacean hyperglycemic hormone by ELISA in hemolymph of the lobster, Homarus americanus, following various stresses.

An ELISA was developed for the crustacean hyperglycemic hormone (CHH) from the lobster, Homarus americanus. It is sensitive to as little as 0.2 fmol of peptide. The assay was used to measure CHH in the hemolymph of intact lobsters after various environmental stresses. Increases in CHH were observed following emersion, exposure to high temperatures (23 degrees and 28 degreesC), and salinity stress (50 and 150% seawater). During emersion, concentrations of hemolymph glucose increased concomitantly with increases in CHH. Significant levels of hemolymph CHH were also measured in lobsters that had been eyestalk-ablated. These latter observations indicate that there may be a source of CHH other than the X-organ/sinus gland in the lobster.

The NO/cGMP pathway and the development of neural networks in postembryonic lobsters.

The nitric oxide/cyclic 3',5'-guanosine monophosphate (NO/cGMP) signaling pathway has been implicated in certain forms of developmental and adult neuronal plasticity. Here we use whole-mount immunocytochemistry to identify components of this pathway in the nervous system of postembryonic lobsters as they develop through metamorphosis. We find that the synthetic enzyme for NO (nitric oxide synthase, or NOS) and the receptor for this transmitter (NO-sensitive soluble guanylate cyclase) are broadly distributed in the central nervous system (CNS) at hatching. In the brain, NOS immunoreactivity is intensified during glomerular development in the olfactory and accessory lobes. Whereas only a few neurons express NOS in the CNS, many more neurons synthesize cGMP in the presence of NO. NO-sensitive guanylate cyclase activity is a stable feature of some cells, while in others it is regulated during development. In the stomatogastric nervous system, a subset of neurons become responsive to NO at metamorphosis, a time when larval networks are reorganized into adult motor circuits. cGMP accumulation was occasionally detected in the nucleus of many cells in the CNS, which suggests that cGMP may have a role in transcription. Based on these findings, we conclude that the NO/cGMP signaling pathway may participate in the development of the lobster nervous system. Furthermore, NO may serve as a modulatory neurotransmitter for diverse neurons throughout the CNS.

An ecdysteroid-responsive gene in a lobster - a potential crustacean member of the steroid hormone receptor superfamily.

The role of ecdysteroids in modulating exoskeletal growth during the moult cycle of Crustacea has been well described. However, little is known about the action of ecdysteroids at the level of gene transcription and regulation in Crustacea. This paper reports the cloning of an ecdysteroid responsive gene, HHR3, a potential Manduca sexta MHR3 homologue in the American lobster, Homarus americanus. Levels of HHR3 expression are up-regulated in response to in vivo injections of premoult concentrations (10(-6) M) of 20-hydroxyecdysone in the epidermal and muscle tissue of the lobster after 6 h. Maximal mRNA levels are observed after 21 h before returning to basal levels. In muscle tissue, elevated levels of HHR3 mRNA follow a time course similar to elevated actin mRNA expression in response to hormonal injection. In contrast, in eyestalk tissue, the HHR3 levels decline up to 21 h post-injection before rising to basal levels after 48 h. Eyestalk, epidermal and leg muscle tissue was extracted over the moult cycle to determine the levels of expression. In muscle, HHR3 is high during the premoult period that corresponds to the period of the moult cycle when the ecdysteroid titre is high. In the epidermis, HHR3 levels are also high during the premoult with elevated levels maintained into the postmoult period. In the eyestalk, mRNA levels of HHR3 show an opposite pattern of expression with low levels during premoult and postmoult and high levels found during the intermoult period. Our results provide novel evidence for an ecdysteroid responsive gene in a crustacean that has many similarities to MHR3 in Manduca and DHR3 in Drosophila melanogaster. This raises the question of whether a similar cascade of ecdysteroid responsive genes exist in other members of Arthropoda such as the Crustacea, as has been demonstrated in Drosophila. In addition, we provide further evidence for negative feedback regulation of ecdysteroids at the site of moult-inhibiting hormone (MIH) production in the lobster eyestalk.

Distribution and regulation of esterases that hydrolyze methyl farnesoate in Homarus americanus and other crustaceans.

Ester hydrolysis of methyl farnesoate (MF) by crustacean tissue homogenates was measured using the substrate [3H]MF in a radiochemical partition assay. Tissues were obtained from the lobster Homarus americanus, penaeid shrimp Sicyonia ingentis, thalanassid shrimp Callianassa californiensis, sand crab Emerita analoga, and spider crab Pugettia producta. The greatest specific activities were recovered from the hepatopancreas (239 to 11,500 pmol MF/min-mg total protein). Hepatopancreatic homogenates of C. californiensis were significantly more active than homogenates from the other species. In the lobster, esterases that hydrolyze MF were associated with lipid storage (R) cells of the hepatopancreas. Enzyme activity of lobster larval homogenates increased 1.5-fold during the second stage of development. The rate of MF hydrolysis by esterases extracted from the juvenile lobster hepatopancreas could not be correlated with molt stage or sex and was not significantly influenced by eyestalk ablation, mandibular organ ablation, or MF injection.

Identification and characterization of methyl farnesoate binding proteins from the crab, Cancer magister.

Methyl farnesoate (MF) binding proteins were identified in the hemolymph of male crabs, Cancer magister, using a tritium-labeled photoaffinity analog of MF, farnesyl diazomethyl ketone (FDK). Crab hemolymph was incubated with [3H]FDK in the presence of increasing amounts of unlabeled MF and the proteins were separated using SDS-PAGE. The associated fluorogram revealed the presence of two specific MF binding proteins with apparent molecular masses of 34 and 44 kDa. MF binding proteins were not detected in other tissues including testes, eyestalks, hepatopancreas, heart, muscle, epidermis, and Y-organs. Unlabeled MF and FDK were capable of displacing [3H]FDK from hemolymph MF binding proteins in a dose-dependent way. The apparent dissociation constant (Kd) of each binding protein for MF and FDK was approximately 65 and 100 nM, respectively, as determined by saturation binding studies. A ligand binding assay followed by Scatchard analysis was used to determine a more accurate apparent Kd value of 145 +/- 10 nM. A single MF binding peak was demonstrated when hemolymph samples incubated with [3H]FDK were electrophoresed under nondenaturing conditions.

Limb Regeneration in the Eye Sockets of Crabs.

The eyestalks of crabs were removed and various tissues of the limbs were autotransplanted into the empty eye sockets to study the capacity of the limb tissue to regenerate in a heterotopic site. Autotransplantation of walking leg tissues into the eye sockets was able to regenerate complete walking legs in the new site. Autotransplantation of tissues of claw digit (dactyl and pollex) or more proximal claw segments (ischium and merus/carpus joint) could regenerate complete claws in the eye sockets. If the autotransplant of claw tissue was contralateral, claws could regenerate with host-site handedness. Sham operations or autotransplantation of frozen claw tissue did not induce regeneration in the eye sockets. These results demonstrate that complete crab claws can regenerate from the eye sockets by autotransplantation of live limb tissue and that the regeneration is not due to the traumatic effect of transplantation. The structure of the limbs regenerated in the eye sockets was determined by the source of the transplanted tissue. Complete claws resulted from autotransplantation of the tissues of the most distal claw segments (claw digits), and the most distal claw segments regenerated first, followed by the proximal claw segments in subsequent molts. Thus tissue from distal portions of crab claw can regenerate proximal portions of the claw in the eye sockets. Such a mode of regeneration is not consistent with the distalization rule of the polar coordinate model, which proposes that distal portions of the limb cannot regenerate proximal portions and that the direction of limb regeneration is always from proximal to distal.

Homeotic Transformation of Crab Walking Leg into Claw by Autotransplantation of Claw Tissue.

Homeotic transformation is defined as transformation of one body part into the likeness of something else. By autotransplantation of crab claw tissue into the autotomized stump of the fourth walking leg, the stump can regenerate a complete claw. Frozen claw tissue, sham operation, or walking leg tissue had no such activity. Contralateral autotransplantation of claw tissue into the autotomized stump of the fourth walking leg can induce the regeneration of a claw with normal handedness. Most of the transformed claws combined features of the claw and the walking leg, suggesting that both host and donor tissues play a role in regeneration. Three possible mechanisms that might account for limb transformation are discussed. Simple intercalary regeneration does not explain all of the observations, but some regulatory events might be taking place during regeneration. Two other processes--secretion of some morphogen by the claw tissue and alteration in the expression of Hox genes--offer alternatives that might explain the results of this study.

Expression of HLA-DR antigen in large bowel carcinoma.

One hundred large bowel carcinomas were studied immunohistochemically with regard to expression of HLA-DR antigen (DR). One or two sections from each tumor including surrounding normal mucosa were examined by a semiquantitative counting system for tumor cells and mucosal and stromal infiltrates of lymphocytes and mononuclear cells (MNCs) with DR expression and the results were applied Chi-square test. The rate of presence of DR positive (DR+) lymphocytes in lymphoid nodules and DR+ lymphocytes/ MNC in the adjacent mucosa and stroma in DR+ carcinoma (50%) was higher (P < 0.01) than in DR- carcinoma (21.9%). Thirty-six carcinomas (36%) were DR+. Three (75%) out of 4 DR+ poorly differentiated carcinomas and six (20%) out of 30 DR+ moderately differentiated carcinomas showed homogeneously strong DR+ expression. There was tendency for poorly differentiated carcinoma to be more homogeneous DR+ expression. According to Dukes' stage, four (80%) out of 5 carcinomas in Dukes' stage D were DR-. An increased infiltration of lymphocytes/MNCs into adjacent mucosa and stroma in large bowel carcinomas is possibly related with DR expression by carcinoma. From the results of this study, we postulated as follows: 1) DR+ tumor cells may act as antigen-presenting cells, 2) They may have an inhibitory effect for distant metastasis, 3) Poorly differentiated carcinoma expressed more DR+ homogeneously.

Metastatic malignant melanoma with "rhabdoid" features.

The authors propose the addition of malignant melanoma to the list of extrarenal neoplasms that may be predominantly composed of polygonal cells with the cytologic features of "rhabdoid" tumor. A review of 313 metastatic melanomas disclosed 49 examples with rhabdoid features, from which 31 had sufficient material for further pathologic and immunohistologic characterization. A control group of 46 nonrhabdoid metastatic melanomas was examined in parallel fashion. In 39% of cases, rhabdoid melanomas manifested relative deletion of S100 protein compared with the control tumors. However, there were no differences in staining with HMB-45. Vimentin immunoreactivity was concentrated in the paranuclear cytoplasm of rhabdoid melanoma cells. However, ultrastructural studies of these cases failed to show corresponding whorls of intermediate filaments and instead demonstrated paracrystalline paranuclear inclusions in profiles of rough endoplasmic reticulum. It is concluded that metastatic rhabdoid melanoma exhibits significant morphologic similarity to other rhabdoid tumors at a light-microscopic level. However, it usually retains enough melanocytic attributes to allow for accurate diagnostic recognition. Probably because patients with metastatic melanoma have an extremely poor prognosis overall, no worsening of biologic behavior was associated with rhabdoid cytomorphologic findings in this tumor type when compared with the control cases.

Intraluminal bypass technique using a condom for protection of coloanal anastomosis.

PURPOSE: Most surgeons carry out temporary diverting colostomy in coloanal anastomosis for mid-rectal or lower-rectal carcinomas. It has been reported that proximal fecal diversion provides no guarantee against anastomotic leaks. Some have proposed the use of the intracolonic bypass tube to prevent anastomotic leakage and colostomy, but colonic necrosis has been reported; it is important to use a safe technique that obviates this. METHODS: The rectum is fully mobilized and transected at the level of the levator diaphragm. The mobilized sigmoid and rectum are resected with their mesenteries, and the prepared distal colon is everted 5 cm using Babcock clamps. The ring of a sterilized condom is then sutured to the mucosa and submucosa of the colon with 4/0 chromic catgut sutures. After completion of coloanal anastomosis, the condom is brought to the exterior, and the mid part is transected. RESULTS: We have used a condom for intraluminal bypass procedures in ten rectal carcinoma patients including five preoperative radiation cases. There was no anastomotic dehiscence, leakage, or colonic necrosis because of a condom. CONCLUSION: We believe that the intraluminal bypass technique using a condom is a very safe, cost-effective, and easily available alternative for coloanal anastomosis.

Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro.

Methyl farnesoate (MF) stimulation of ecdysteroid secretion by Y-organs of the crab, Cancer magister, is demonstrated. Isolated Y-organs that were incubated with a mandibular organ (MO) secreted significantly higher levels of ecdysteroids into the medium when compared to those incubated in the absence of a MO. MOs secrete both farnesoic acid (FA) and MF into the medium and are not themselves a source of ecdysteroids. To determine if MF has a role in the regulation of ecdysteroid secretion, Y-organs from C. magister were incubated with various concentrations of MF. Y-organs in the presence of MF secreted significantly more ecdysteroids into the medium after a 24-hr incubation when compared to controls (P < or = 0.05). The magnitude of this stimulation increased with higher concentrations of MF and increasing incubation times. The response was specific to (2E,6E)-MF. The cis,trans isomer of MF ((2Z,6E)-MF), FA, and farnesyl diazomethyl ketone, a photoaffinity analog of MF, did not have any effect on ecdysteroid secretion from Y-organs. The primary ecdysteroid secreted by C. magister Y-organs comigrates with authentic ecdysone and its secretion is stimulated by MF.

Expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases-1 in granuloma annulare and necrobiosis lipoidica diabeticorum.

Granuloma annular (GA) and necrobiosis lipoidica diabeticorum (NLD) are disorders characterized by granulomatous inflammation and degenerative changes in collagen and elastic fibers. Because these disorders have often been described as being associated with altered extracellular matrix deposition, we studied the in situ expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases (TIMP)-1. Twelve lesions each of GA and NLD of different histopathologic types and durations were examined. Interstitial collagenase mRNA was seen in histiocyte-like cells in one-third of the cases of both diseases, typically in younger lesions. In GA, collagenase mRNA was only detected in lesions of the palisading type. Signal for 92-kDa gelatinase mRNA was observed in eosinophils, which were present in low numbers in five of 12 GA and three of 12 NLD samples. The signal for this enzyme and the presence of eosinophils did not correlate with the age of lesion. TIMP-1 mRNA was consistently expressed by histiocyte-like cells in both disorders. In GA, TIMP-1 mRNA was detected at the outer edge of the palisading granulomas, but in NLD, inhibitor expression was seen in the perivascular and periadnexal accumulation of inflammatory cells. Our data indicate that collagenase and TIMP are expressed early in these disorders and that these proteins may contribute to stromal remodeling associated with necrobiotic lesions. Our results further indicate that the localization of TIMP-1 production may provide a distinction between the two disorders, whereas metalloproteinase expression is not sufficiently specific to aid in the differential diagnosis of GA and NLD.