Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development.

Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or intrinsic "clock." Taken together, these results suggest that, unlike embryonic and neonatal MyHCs, the expression of slow MyHC in vivo at different developmental stages during gestation is not the result of commitment to a distinct myoblast lineage, but is largely determined by the environment.

Plasticity of the differentiated state.

Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.

Angiotensin-converting enzyme-like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone.

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.

Dynamics of in vivo release of molt-inhibiting hormone and crustacean hyperglycemic hormone in the shore crab, Carcinus maenas.

Very little is known regarding the release patterns or circulating titers of neuropeptides in crustaceans, in particular those concerned with regulation of molting hormone (ecdysteroid) synthesis, molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH), which is also an adaptive hormone, centrally important in carbohydrate metabolism. Furthermore, the currently accepted model of molt control is founded on an untested hypothesis suggesting that molting can proceed only after decline in MIH titer. Accordingly, we measured simultaneous circulating neuropeptide profiles for both MIH and CHH by RIA of purified hemolymph during the molt cycle at fine temporal scale during day/night cycles and seasonally. For CHH we additionally determined release patterns after physiologically relevant stress. Results show that both hormones are released exclusively and episodically, rather than continuously, with notably short half-lives in circulation, suggesting dynamic and short-lived variations in levels of both hormones. During the molt cycle, there are no overt changes in MIH titer, except a massive and unprecedented increase in MIH during late premolt, just before ecdysis. The function of this hormone surge is unknown. Treatment with various stressors (hypoxia, temperature shock) showed that CHH release occurs extremely rapidly, within minutes of stress. Release of CHH after stressful episodes during premolt (when gut endocrine cells synthesize large quantities of CHH) is exclusively from the sinus gland: CHH from the gut is never involved in the stress response. The results show a hitherto unsuspected dynamism in release of MIH and CHH and suggest that currently accepted models of molt control must be reconsidered.

Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus; expression of MIH in non-X-organ tissues.

Synthesis of ecdysteroids (molting hormones) by crustacean Y-organs is regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. We report here the molecular cloning of a cDNA encoding MIH of the edible crab, Cancer pagurus. Full-length MIH cDNA was obtained by using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides based upon the amino acid sequence of MIH, in conjunction with 5'- and 3'-RACE. Full-length clones of MIH cDNA were obtained that encoded a 35 amino acid putative signal peptide and the mature 78 amino acid peptide. Of various tissues examined by Northern blot analysis, the X-organ was the sole major site of expression of the MIH gene. However, a nested-PCR approach using non-degenerate MIH-specific primers indicated the presence of MIH transcripts in other tissues. Southern blot analysis indicated a simple gene arrangement with at least two copies of the MIH gene in the genome of C. pagurus. Additional Southern blotting experiments detected MIH-hybridizing bands in another Cancer species, Cancer antennarius and another crab species, Carcinus maenas.

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression.

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia. Previous work has demonstrated the existence of two isoforms of MO-IH, called MO-IH-1 and -2, that differ by a single amino acid in the mature peptide and one in the putative signal peptide. To study the structural organisation of the crab MIH and MO-IH genes, a genomic DNA library was constructed from DNA of an individual female crab and screened with both MO-IH and MIH probes. The results from genomic Southern blot analysis and library screening indicated that the Cancer pagurus genome contains at least two copies of the MIH gene and three copies of the MO-IH genes. Upon screening, two types of overlapping genomic clone were isolated. Each member of one type of genomic clone contains a single copy of each of the convergently transcribed MO-IH-1 and MIH genes clustered within 6.5kb. The other type contains only the MO-IH-2 gene, which is not closely linked to an MIH gene. There are three exons and two introns in all MIH and MO-IH genes analysed. The exon-intron boundary of the crab MIH and MO-IH genes follows Chambon's rule (GT-AG) for the splice donor and acceptor sites. The first intron occurs within the signal peptide region and the second intron occurs in the coding region of the mature peptide. Sequence analysis of upstream regions of MO-IH and MIH genes showed that they contained promoter elements with characteristics similar to other eukaryotic genes. These included sequences with high degrees of similarity to the arthropod initiator, TATA box and cAMP response element binding protein. Additionally, putative CF1/USP and Broad Complex Z2 transcription factor elements were found in the upstream regions of MIH and MO-IH genes respectively. The implications of the presence of the latter two putative transcription factor binding-elements for control of expression of MIH and MO-IH genes is discussed. Phylogenetic analysis and gene organisation show that MO-IH and MIH genes are closely related. Their relationship suggests that they represent an example of evolutionary divergence of crustacean hormones.

Amino acid sequence of putative moult-inhibiting hormone from the crab Carcinus maenas.

Putative moult-inhibiting hormone (MIH) was isolated from sinus glands of the shore crab Carcinus maenas, and its primary structure determined by automated Edman degradation of endoproteinase derived peptide fragments. MIH is a 78 residue neuropeptide (deduced molecular mass 9181 Da) with three disulphide bridges and unblocked N- and C-termini. MIH shows some homology to the crustacean hyperglycemic hormone (CHH) neuropeptide family. However, consideration of the roles of various members of this group, together with sequence information recently reported, strongly suggests that these neuropeptides may be multifunctional.

Involvement of adenosine cyclic-3',5'-monophosphate in the signal transduction pathway of mandibular organ-inhibiting hormone of the edible crab, Cancer pagurus.

The juvenoid, methyl farnesoate (MF), is synthesized in the mandibular organs (MOs) of crustaceans, under the control of mandibular organ-inhibiting hormone (MO-IH). Using an in vitro assay to measure synthesis of MF by MOs, the effect of a variety of agents that affect signal transduction pathways was investigated. Of the compounds tested, only agents which affect cAMP (forskolin and 8-bromoadenosine cyclic-3',5'-monophosphate) levels were found to mimic the inhibitory action of MO-IH on MF synthesis. To further support these findings, the effect of MO-IH-1 on production of cAMP was investigated. The results demonstrated that MO-IH stimulated a dose-dependent increase in cAMP levels. Furthermore, a maximal 2-fold increase in cAMP was detected after a 5-min exposure of MO membranes to 100 nM MO-IH-1, falling to basal levels thereafter. The results presented strongly support a role for cAMP in the signal transduction mechanism of MO-IH that leads to inhibition of MF synthesis in MOs.

Involvement of cAMP and cGMP in the mode of action of molt-inhibiting hormone (MIH) a neuropeptide which inhibits steroidogenesis in a crab.

In crustaceans, production of molting hormones (or ecdysteroids) by the molting glands (Y-organs; YO), is under negative control exerted by a neuropeptide, the molt-inhibiting hormone (MIH). MIH of the crab Carcinus maenas inhibits in vitro steroidogenesis of basal (intermolt crab) or activated (premolt crab) YO. MIH inhibits secretion of the two ecdysteroids synthesized by crab YO, ecdysone (E) secreted throughout the molting cycle, and 25-deoxyecdysone (25dE), secreted during the premolt period. At a MIH concentration of 10(-8) M, E is reduced about 50% and 25dE 94%. Regardless of the molting stage, this inhibition of steroidogenesis is reversible, dose dependent and measurable after 5 min. On intermolt YO, MIH induced cGMP increase and 8BrcGMP mimics the effect of MIH: at this stage cGMP seems to be involved with MIH inhibition of steroidogenesis. On premolt YO MIH induced a transient increase of cAMP (2-fold) and a long-lasting enhancement of cGMP (60-fold). On active YO, we demonstrated that a low concentration (10(-5) M) of dbcAMP, 8BrcAMP, 8BrcGMP, or agents increasing intracellular cAMP, mimic MIH effects and inhibit steroidogenesis. From these observations it is concluded that both cyclic nucleotides are involved in the mode of action of MIH on activated YO. At this premolt period, MIH/cAMP may act cooperatively with MIH/cGMP in the inhibitory control of steroidogenesis by crab YO.

Erythrocyte folate levels in young and old.

Erythrocyte folate levels were compared among 25 young healthy subjects (mean age, 27 years), 29 healthy subjects aged over 75 living in the community, 62 subjects (mean age, 75) admitted to a geriatric assessment ward (acute illnesses), and 32 subjects (aged over 65) in a ward for long-term physical or mental illnesses. Overall, the female/male sex ratio varied from 2:1 to 3:1. For the three elderly groups, the incidence of low erythrocyte folate levels (less than 100 mmicrogram/100 ml) were 24 percent, 16 percent, and 18 percent, respectively. For the young group, the mean value would be over 296 mmicrogram/100 ml. Dietary folate deficiency may often account for low folate blood levels in the elderly, but other factors should also be implicated, e.g., the ability to absorb folate.

Isotopic scanning of bone in the diagnosis of osteomalacia.

Results of a study performed over 18 months in 21 elderly patients showed that isotopic bone scanning is a sensitive technique for the detection of osteomalacia. It can be used not only to indicate the presence of the disease but the response to treatment.

Amino acid sequences of both isoforms of crustacean hyperglycemic hormone (CHH) and corresponding precursor-related peptide in Cancer pagurus.

Both isoforms of the crustacean hyperglycemic hormone (CHH) and corresponding crustacean hyperglycemic hormone precursor-related peptide (CPRP) derived from HPLC-purified sinus gland extracts from the edible crab Cancer pagurus were fully characterised by microsequencing and mass spectrometry. The amino acid sequences of the CHH isoforms were almost identical except that the N-terminus of the minor isoform (CHH-I), was glutamine rather than pyroglutamate in the major isoform (CHH-II). Both CHH isoforms were of similar biological activity, as tested by in vivo hyperglycemia bioassays and in vitro repression of ecdysteroid synthesis. Comparison with other published CHH and CPRP sequences show that for crabs, these peptides form a distinct group, that the presence of CHH isoforms with free and blocked N-termini seems unique to crabs. It is argued that this phenomenon reflects a slow post-translational modification in sinus gland neurosecretory terminals. This study appears to complete the entire sinus gland inventory of functionally and structurally characterised CHH-related peptides in a crab.

Determination of the amino acid sequence of the moult-inhibiting hormone from the edible crab, Cancer pagurus.

Putative moult-inhibiting hormone (MIH) from sinus glands of the edible crab Cancer pagurus was characterized by high-performance liquid chromatography, followed by fractional bioassay (inhibition of ecdysteroid synthesis by Y-organs) and immunoassay (using antisera raised against Carcinus MIH). This peptide was fully sequenced by automated Edman degradation of endoproteinase-derived fragments. C. pagurus MIH is a 78 residue peptide (M(r) 9194), with free N- and C-termini and three intrachain disulphide bridges. Comparison with previously published MIH sequences confirms a high degree of sequence identity (c. 80%), supporting the view that brachyurans (crabs), possess distinct, structurally similar MIH neuropeptides.

Binding sites of crustacean hyperglycemic hormone and its second messengers on gills and hindgut of the green shore crab, Carcinus maenas: a possible osmoregulatory role.

To determine the possible involvement of crustacean hyperglycemic hormone (CHH) in osmoregulation in crustaceans, ligand binding and second messenger assays were performed on gills and hindgut preparations of the green shore crab Carcinus maenas, whilst midgut gland, previously known as one of the target tissues of CHH served as a control tissue. Classical receptor binding analyses using [(125)I]CHH by saturation and displacement experiments from membrane preparations from gills, hindgut, and midgut glands demonstrated that CHH binding characteristics involved one site, highly specific, saturable, and displaceable kinetics: (gills: K(D) 5.87 +/- 2.05 x 10(-10) and B(MAX) 6.50 +/- 1.15 x 10(-10), hindgut: K(D) 3.54 +/- 1.49 x 10(-10) and B(MAX) 2.31 +/- 0.44 x 10(-10), and midgut gland: K(D) 7.28 +/- 0.9 x 10(-10) and B(MAX) 3.28 +/- 0.25 x 10(-10)) all expressed as M/mg protein. No differences, in terms of displacement were observed between the two CHH isoforms (N-terminally blocked pGlu and unblocked Gln) variants. CHH binding sites appeared to be coupled to a second messenger system involving cGMP in all the tissues examined. Exposure of crabs to dilute seawater increased levels of cGMP, glucose in gills and circulating CHH levels. Other crustacean neuropeptides including crustacean cardioactive peptide, molt inhibiting hormone, L-enkephalin, FMRF-amide, proctolin, and crustacean hyperglycemic hormone precursor-related peptide were tested with regard to possible osmoregulatory roles with reference to changes in second messenger (cAMP and cGMP) concentrations in gill, hindgut, and midgut tissues in vitro, following application at 2 x 10(-8) M but all were found to be inactive. Thus, it seems likely that CHH is a pertinent neurohormone involved in osmoregulation, thus expanding its many functions as a pleiotropic hormone in crustaceans.

Identification and developmental expression of mRNAs encoding crustacean cardioactive peptide (CCAP) in decapod crustaceans.

Full-length cDNAs encoding crustacean cardioactive peptide (CCAP) were isolated from several decapod (brachyuran and astacuran) crustaceans: the blue crab Callinectes sapidus, green shore crab Carcinus maenas, European lobster Homarus gamarus and calico crayfish Orconectes immunis. The cDNAs encode open reading frames of 143 (brachyurans) and 139-140 (astacurans) amino acids. Apart from the predicted signal peptides (30-32 amino acids), the conceptually translated precursor codes for a single copy of CCAP and four other peptides that are extremely similar in terms of amino acid sequence within these species, but which clearly show divergence into brachyuran and astacuran groups. Expression patterns of CCAP mRNA and peptide were determined during embryonic development in Carcinus using quantitative RT-PCR and immunohistochemistry with whole-mount confocal microscopy, and showed that significant mRNA expression (at 50% embryonic development) preceded detectable levels of CCAP in the developing central nervous system (CNS; at 70% development). Subsequent CCAP gene expression dramatically increased during the late stages of embryogenesis (80-100%), coincident with developing immunopositive structures. In adult crabs, CCAP gene expression was detected exclusively in the eyestalk, brain and in particular the thoracic ganglia, in accord with the predominance of CCAP-containing cells in this tissue. Measurement of expression patterns of CCAP mRNA in Carcinus and Callinectes thoracic ganglia throughout the moult cycle revealed only modest changes, indicating that previously observed increases in CCAP peptide levels during premoult were not transcriptionally coupled. Severe hypoxic conditions resulted in rapid downregulation of CCAP transcription in the eyestalk, but not the thoracic ganglia in Callinectes, and thermal challenge did not change CCAP mRNA levels. These results offer the first tantalising glimpses of involvement of CCAP in environmental adaptation to extreme, yet biologically relevant stressors, and perhaps suggest that the CCAP-containing neurones in the eyestalk might be involved in adaptation to environmental stressors.

Effects of exogenous ecdysterone upon moulting, proecdysial development, and limb regeneration in the prawn Palaemon elegans.

Injection of small doses of ecdysterone accelerated moulting and proecdysis in the prawn Palaemon elegans. Injection of large doses of ecdysterone (1-10 micrograms) markedly accelerated proecdysis, but death always occurred prior to or during moulting and was accompanied by abnormal setal development and retarded cuticle formation. Dose-response curves were obtained for a range of hormone doses from 10 to 0.01 micrograms by administering ecdysterone during postmoult (stages A-B) and early premoult (stages DE0-DL0). Accelerated proecdysis and viable moulting were more marked in the group injected during early premoult (stages DE0-DL0). The sensitivity threshold for prawns injected during this time was less than 40 ng g-1 but could not be determined more precisely in view of the range of ecdysterone concentrations used. In contrast, the sensitivity threshold for ecdysterone administered during postmoult (stages A-B) was much higher, between 0.4 and 2 micrograms g-1. The possible mechanisms controlling sensitivity thresholds for ecdysterone are discussed. The effect of ecdysterone on limb regeneration was also studied. Within the concentration range used, ecdysterone was found to have no effect on the rate of limb regeneration.

Neurohormonal control of ecdysteroid biosynthesis by Carcinus maenas Y-organs in vitro, and preliminary characterization of the putative molt-inhibiting hormone (MIH).

Using simple culture techniques, the effects of neurosecretory tissue, sinus gland-conditioned media, and sinus gland extracts upon the biosynthesis of ecdysteroids by Carcinus maenas Y-organs in vitro were investigated. The sinus glands were found to be a major source of a factor which profoundly repressed ecdysteroid synthesis and which did not appear to be species-specific within other brachyurans examined (Liocarcinus, Cancer). It is suggested that the inhibitory factor is produced by the neurosecretory tissues of the medulla terminalis. It is argued that the inhibitory factor is the putative molt-inhibiting hormone (MIH). Partial characterization revealed that MIH is a heat-stable, trypsin-sensitive neuropeptide, eluting on a Sephadex G-50 gel in a range of approximately 6-14 kDa. By consideration of the dose-response characteristics, it is estimated that MIH may be active in the subpicomolar range.

Peptidergic Neurons in Barnacles: An Immunohistochemical Study Using Antisera Raised Against Crustacean Neuropeptides.

Antisera raised against neuropeptides from decapod crustaceans were used to investigate whether balanomorph barnacles produce peptides analogous to those identified in some decapods. The distribution and structure of immunoreactive neurons was examined in Balanus balanus, Balanus perforatus, and Chirona (Balanus) hameri by whole-mount immunohistochemistry. In these species, no immunoreactivity was observed to antisera against CHH (crustacean hyperglycemic hormone), MIH (molt-inhibiting hormone), or RPCH (red-pigment-concentrating hormone), but neurons immunoreactive for pigment-dispersing hormone (PDH) and crustacean cardioactive peptide (CCAP) were observed. In all three species, PDH immunoreactivity was primarily associated with a pair of large (30-50 {mu}m diam.) anterio-ventral perikarya in the ventral ganglion, projecting prominent axons along the great splanchnic nerves, which branched extensively in the segmental splanchnic nerves, directing several arborizing dendrites to the somatic extensor muscles. Occasionally, three pairs of anterio-dorsal perikarya were observed, which projected fine ipsilateral and contralateral axons along the great splanchnic nerves. A further 12 pairs of perikarya, apparently segmentally arranged, were observed in the thoracic ganglion. Several PDH-immunoreactive perikarya and associated branching plexus were observed in the supra-esophageal ganglion. CCAP immunoreactivity was mainly restricted to the ventral ganglion, where three pairs of perikarya (ca. 30-50 {mu}m diam.) projected contralateral descending axons to the cirri. Occasionally a single pair of immunoreactive neurons were observed in the supra-esophageal ganglia. Although the anatomy of the CCAP-immunoreactive neurons in the ventral ganglion of barnacles might be homologous to conserved neural architectures in higher crustaceans, the anatomy of the PDH-immunoreactive neurons seems unique, and the morphology of the two large neurons in the ventral ganglion suggests a neuromodulatory role for this peptide, possibly associated with somatic extension.