Exome sequencing in 32 patients with anophthalmia/microphthalmia and developmental eye defects.

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome(TM) (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here.

Microbial studies in the Canadian nuclear fuel waste management program.

Atomic Energy of Canada Limited (AECL) has developed a concept for permanent geological disposal of used nuclear fuel in Canada. This concept, based on a multibarrier system, would involve disposal of nuclear fuel waste in titanium or copper containers, surrounded by compacted clay-based buffer and backfill materials, in a vault 500-1000 m deep in granitic rock of the Canadian Shield. Subsurface environments will not be sterile and an experimental program was initiated in 1991 by AECL to address and quantify the potential effects of microbial action on the integrity of the disposal vault. This microbial program focuses on answering specific questions in areas such as the survival of bacteria in compacted clay-based buffer materials under relevant radiation, temperature and desiccation conditions; mobility of microbes in compacted buffer materials; the potential for microbially influenced corrosion of containers; microbial gas production in backfill material; introduction of nutrients as a result of vault excavation and operation; the presence and activity of microbes in deep granitic groundwaters; and the effects of biofilms on radionuclide migration in the geosphere. This paper summarizes the results to date from the research activities at AECL.

Early and late effects of low-dose famotidine, ranitidine or placebo on pentagastrin-stimulated gastric acid secretion in man.

BACKGROUND: There are no published comparative studies on the effect of low-dose H2-antagonists on pentagastrin-stimulated gastric acid secretion. METHODS: Twenty-four healthy subjects were dosed with either famotidine 10 mg, ranitidine 75 mg or placebo in a balanced three-period cross-over design. The subjects were studied in groups of 12, simultaneously, under identical controlled environmental conditions. Gastric juice was aspirated in 15-min aliquots during sub-maximal (0.6 microgram.h/kg) intravenous pentagastrin stimulation in the third and fourth hours (early period) and the eighth and ninth hours (late period) after oral dosing. The hydrogen ion (H+) content of gastric juice was measured ex vivo, by titrating to pH7 known volumes of gastric aspirate against 0.1 M sodium hydroxide, using a versatile microprocessor-controlled auto-titration unit. Gastric acid output during the period of interest was calculated by adding the hydrogen ion content of 15-min aliquots collected during that period. The geometric mean of the cumulative pentagastrin-stimulated gastric acid output during the early and late periods was determined for the subjects dosed with either famotidine, ranitidine or placebo. Comparisons were performed by ANOVA. RESULTS: During the early period (2-4 h post-dose), When the subjects were given placebo, mean gastric acid output was 46.6 mmol, decreasing by 76% to 11.3 mmol (P < 0.001) when treated with famotidine and by 76% to 11.1 mmol (P < 0.001) when treated with ranitidine. During the late period (7-9 h post-dose), when the subjects were dosed with placebo, mean gastric acid output was 41.2 mmol, decreasing by 38% to 25.7 mmol (P < 0.001) when treated with famotidine and by 27% to 30.0 mmol (P = 0.007) when treated with ranitidine. The difference between the inhibitory effects of famotidine and ranitidine on gastric acid output were non-significant during either period. CONCLUSIONS: Low-dose famotidine and ranitidine, intended for over-the-counter use, inhibit stimulated gastric acid secretion profoundly in the third and fourth hours after an oral dose. Modest effects are still detectable up to 9 h after dosing.

Wound tissue oxygen tension predicts the risk of wound infection in surgical patients.

OBJECTIVE: To test the hypothesis that subcutaneous wound oxygen tension (PsqO2) has a predictive relation to the development of wound infection in surgical patients. DESIGN: A noninterventional, prospective study. SETTING: A university department of surgery. PATIENTS: One hundred thirty operative general surgical patients at notable risk of infection as predicted by an anticipated Study on the Effect of Nosocomial Infection Control (SENIC) score of 1 or greater. OUTCOME MEASURES: PsqO2 was measured perioperatively. Its relation to the subsequent incidence of surgical wound infection was then determined and compared with the SENIC score as a criterion standard. RESULTS: Although the SENIC score and PsqO2 are inversely correlated, PsqO2 is the stronger predictor of infection. Low PsqO2 identified patients at risk and concentrated them in a cohort that was about half the size of that identified by the SENIC score. CONCLUSIONS: Subcutaneous perfusion and oxygenation are important components of immunity to wound infections. The SENIC score identifies systemic physiological variables that are important to the development of wound infection. Nevertheless, PsqO2 is the more powerful predictor of wound infection. Moreover, PsqO2 can be manipulated by available clinical means, and thus may direct interventions to prevent infection.

Pulsed microamperage stimulation: a controlled study of healing of surgically induced wounds in Yucatan pigs.

BACKGROUND AND PURPOSE: Microamperage direct current and microamperage electromagnetic stimulation are used to accelerate healing in bone. Although many clinicians are using microamperage stimulation to relieve pain and facilitate wound healing, there is significant question regarding whether this low-intensity direct current significantly accelerates soft tissue wound repair. The purpose of this study was to determine whether low-voltage pulsed microamperage current (100 microA, 60 V, 0.1 Hz) enhances the healing of superficial, full-thickness, or incisional wounds created to simulate acute abrasions, ulcers, and lacerations. SUBJECTS: Ten adult Yucatan mini pigs served as the subjects for this study. METHODS: Variables associated with healing were measured in 60 matched pairs of surgically induced partial-thickness, full-thickness, and incisional wounds after either sham or anodal (positive-polarity) stimulation with pulsed microamperage current (100 microA) was administered for 1 hour per day for 5 days. Sterile, disposable electrodes (2 x 4 cm) were placed over each wound, which was kept wrapped and protected throughout the study. RESULTS: At 7 days postinjury, all wounds were healing well with no signs of infection. There were no differences in tensile strength, collagen density, maturity, or deposition (hydroxyproline), wound size, or visual appearance between the sham treatment and treatment lesions. No changes in local subcutaneous oxygen or temperature were found in the swine during or after microamperage stimulation. CONCLUSION AND DISCUSSION: This study did not provide any evidence to support the use of microamperage stimulation to accelerate wound healing. No negative effects, however, were found. Further research is needed to determine whether there is a critical interaction between the size of the electrode relative to the wound, the density of the current, the duration of the treatment, the polarity of the treatment electrode, and the acuity or chronicity of wounding and the effectiveness of microcurrent stimulation for wound healing.

Cytochrome P-450 in plant/insect interactions: geraniol 10-hydroxylase and the biosynthesis of iridoid monoterpenoids.

The interactions between plant secondary metabolites (particularly monoterpenes) and insects are discussed. Such metabolites are likely to have influenced the evolution of cyt P450-linked detoxification systems in animals, through animal/plant coevolution. The biosynthesis of many classes of plant secondary metabolites involves cyt P450 enzymes. Of these, one of the best characterised is the geraniol/nerol 10-hydroxylase which catalyses a key step in the biosynthesis of the iridoid class of plant terpenes. It would appear that these monoterpenoids are synthesised (via cyt P450 hydroxylation) from different precursors in different plant species, namely geraniol, its isomer nerol, or the related monoterpenoid, citronellol. We show that cyt P450 from the plants Catharanthus roseus and Nepeta racemosa are capable of hydroxylating geraniol, nerol and citronellol, and thus do not impose precursor specificity on iridoid biosynthesis in plants.

Adjuncts to preparing wounds for closure: hyperbaric oxygen, growth factors, skin substitutes, negative pressure wound therapy (vacuum-assisted closure).

Achieving closure in a chronic wound requires provision of adequate oxygen delivery to the tissue, adequate protein and other nutritional factors, a moist environment, an appropriate inflammatory milieu, dèbridement, and correction of contributing medical diagnoses. In some patients, these conditions are achieved easily, whereas in others, greater effort is required. Adjunctive treatments, including HBO2, growth factors, skin substitutes, and negative-pressure wound therapy (e.g., V.A.C.) can provide the proper conditions for healing in appropriately selected patients.

Life with auntie.

Are journalists the enemies of scientists? The media specialize in communication, an area in which scientists are traditionally perceived to be weak. If barriers to understanding could be broken down then there is a real opportunity for mutual benefit. An appreciation of what drives the media can best be achieved by entering their camp.

Alterations in the proportions of skeletal muscle proteins following a unilateral lesion to the substantia nigra pars compacta of rats.

It is well established that mammalian skeletal muscles exhibit a considerable degree of plasticity and one of the main determining factors of this plasticity is the activity pattern and duration of motoneurone discharge. Lesions to the right substantia nigra pars compacta (SNpc) of six adult rats were made to determine whether altered output from the SNpc ultimately leads to a change in the expression of proteins in contralateral skeletal muscles. After 4 months, altered motor performance was identified by the administration of amphetamine. After 7 months, 30-70% of dopaminergic cells in the SNpc had been destroyed. The protein content of muscles was then quantified from densitometric scans of gels, and expressed as a % of the amount of actin (the protein used as a reference in this study). The lesion affected the expression of different protein isoforms in the fast- and slow-twitch muscles. In slow-twitch soleus muscles, the lesion decreased the proportion of alpha-tropomyosin and increased the proportion of beta-tropomyosin. In the fast-twitch extensor digitorum longus muscles, the lesion increased the proportion of the fast isoform of troponin-T1f, and decreased the proportions of the two isoforms of myosin light chain. This study establishes a connection between the chronic effects of a lesion to the SNpc, with a loss of dopaminergic neurones, impaired motor performance, and altered expression of proteins in skeletal muscle. The implication of these results is that the altered motor function observed in Parkinson's disease may be associated with alterations to the expression of skeletal muscle proteins.

Wound healing in the surgical patient: influence of the perioperative stress response on perfusion.

The clinical effect of surgical stress created by pain, cutting, cold, fear, and hypovolemia is peripheral vasoconstriction which limits wound perfusion and oxygenation and impairs wound healing. Correction of all factors as simultaneously as possible is the goal.

Plasticity in the Sclerites of a Gorgonian Coral: Tests of Water Motion, Light Level, and Damage Cues.

The gorgonian coral Briareum asbestinum contains skeletal elements (sclerites) that vary in length and density within and among local populations. Data from previous work suggested that the sclerite compositions of colonies may be altered in response to environmental cues such as predator damage, water motion, and light level. To test these hypotheses, colonies from shallow reefs were transplanted to racks at a single location where the three environmental factors of interest were artificially manipulated. After 9-14 weeks of growth, sclerite morphologies and densities had not changed in response to shading or to water-motion reductions that mimicked deep-water conditions. However, colonies did respond significantly to two types of simulated predator damage. Following tip amputation, sclerites in the regenerated tips of damaged colonies were shorter and more dense than in the controls. In contrast, mid-branch scarring caused colonies to produce longer sclerites at lower densities. Since long sclerites deter feeding by predatory snails, the increase in sclerite length in response to scarring of mid-branch regions may function as an inducible defense.

Contractile activation and the effects of 2,3-butanedione monoxime (BDM) in skinned cardiac preparations from normal and dystrophic mice (129/ReJ).

Small cardiac myofibrillar preparations were obtained from the right ventricle of normal (129/ReJ) and dystrophic (129/ReJ dy/dy) mice and were chemically skinned in a relaxing solution by exposure to Triton X-100 (3% v/v). (2) The isometric force produced in these skinned cardiac preparations at different sarcomere lengths was measured in solutions of different [Ca2+] and ionic strength. The effect of the negative inotropic drug 2,3-butanedione monoxime (BDM), which is known to act at the myofibrillar level was also investigated. (3) The murine cardiac preparation from normal animals was found to develop 50% maximal force at a pCa (= -log10[Ca2+]) of 5.59 +/- 0.08 and 5.94 +/- 0.03 (mean +/- SD) under physiological (ionic equivalents concentration, I = 154 mM; pH 7.10; [Mg2+] 1 mM) and low ionic strength (I = 94 mM; pH 7.10; [Mg2+] 1 mM) conditions respectively. The isometric force curves were significantly shallower at low ionic strength (Hill coefficient, 1.8 +/- 0.1) than at physiological ionic strength (Hill coefficient, 2.6 +/- 0.3) and the sarcomere length effect on the force-pCa relation was markedly reduced at lower ionic strength. (4) Increasing BDM concentrations in solutions up to 100 mM reduced the maximum Ca2+-activated force of cardiac preparations from normal mice to less than 6% of the control values in a dose dependent fashion. BDM also rendered the cardiac preparations less sensitive to Ca2+ by a factor of up to 1.5 in a process which showed saturation at BDM concentrations higher than 15 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ and Sr2+ activation properties of skinned muscle fibres with different regulatory systems from crustacea and rat.

1. The contractile activation properties of long- (sarcomere length (SL) > 6 microns) and short- (SL < 4 microns) sarcomere fibres from the claw muscle of the yabby (freshwater crustacean, Cherax destructor) and the fast- and slow-twitch fibres from the rat have been investigated using single skinned muscle fibres activated in solutions containing Ca2+ or Sr2+ or both Ca2+ and Sr2+. 2. Sr2+ could not fully activate the contractile apparatus of either the long- or the short-sarcomere yabby preparations and the force-pSr curves for both fibre types were biphasic in shape. 3. The long- and short-sarcomere fibres from the yabby differed in their Ca(2+)- and Sr(2+)-activation properties. Thus the long-sarcomere fibres required a significantly lower [Ca2+] to produce 10% maximum force, had Ca(2+)-activation curves which were significantly shallower, and had a significantly higher ratio between maximum Sr(2+)- and maximum Ca(2+)-activated force than the short-sarcomere fibres. 4. Simultaneous activation with Ca2+ and Sr2+ showed a synergistic effect in the rat muscle fibres where Sr2+ could fully replace Ca2+ in activation. In contrast the results with the long- and short-sarcomere fibres from the yabby showed that in some functional states Sr2+ binds but cannot (or can only partially) activate the regulatory system while in others Sr2+ can fully replace Ca2+ in activating contraction. 5. The synergistic effect between Sr2+ and Ca2+ in mammalian muscle could be quantitatively explained if all regulatory sites involved in contractile activation have a similar value for the ratio between their affinity for Ca2+ and Sr2+. 6. Three distinguishable functional states (based on the fibre's ability to be activated by Sr2+ and Ca2+) were identified in the long-sarcomere fibres from the yabby: one where both Ca2+ and Sr2+ were able to activate contraction and had a relatively high sensitivity to Sr2+; one where both Ca2+ and Sr2+ were able to activate contraction but where the sensitivity to Sr2+ was lower and was more sensitive to a decrease in ionic strength; and one where Sr2+ binding to the regulatory system could not activate contraction. Equivalent states of the three described for the long-sarcomere fibres were also found in the short-sarcomere preparations. However, the short-sarcomere fibres had, in addition, a fourth state which was characterized by the ability of Sr2+ to activate contraction at reduced ionic strength but not at standard ionic strength.(ABSTRACT TRUNCATED AT 400 WORDS)

Free amino acids in claw muscle and haemolymph from Australian freshwater crayfish at different stages of the moult cycle.

Amino acids were measured in claw muscle and haemolymph in the freshwater decapod crustacean, Cherax destructor, at different stages of the moult cycle. The total pool of amino acids in muscles from animals in intermoult (97+/-13 mmol kg(-1) muscle), premoult (80+/-20 mmol kg(-1)) and postmoult (97+/-19 mmol kg(-1)) were not significantly different. Despite the relatively stable total pool of amino acids, there were changes in the concentrations of alanine, glutamine and proline over the moult cycle. Compared to intermoult, claw muscles from animals in premoult had a lower concentration of proline, and animals in postmoult had higher concentrations of alanine and glutamine, but lower concentrations of proline. Concentrations of alanine and glutamine in claw muscle of animals in postmoult were higher and proline concentrations lower than in the same animals during the premoult stage. The concentration of proline in haemolymph was lower in animals in premoult and postmoult compared to intermoult. The total amino acid pool in the claw muscle of Cherax destructor did not change significantly over the moult which is distinctly different to the changes in amino acids reported in the claw muscles of marine decapod crustaceans.

Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle.

The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca-Pi precipitation-induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mM-50 mM range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca-Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mM myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 microM s-1 while the amount of Pi loaded appeared to saturate at around 3.5 mM (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca-Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mM) inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mM) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca-Pi solubility product is exceeded in the sarcoplasmic reticulum lumen.

Differences in maximal activation properties of skinned short- and long-sarcomere muscle fibres from the claw of the freshwater crustacean Cherax destructor.

Single fibres of different sarcomere length at rest have been isolated from the claw muscle of the yabby (Cherax destructor), a decapod crustacean. Fibres of either long (SL > 6 microns) or short (SL < 4 microns) sarcomere length have been mechanically skinned and were maximally activated by Ca2+ and Sr2+ under various experimental conditions (ionic strength, in the presence of 2,3 butanedione monoxime (BDM)) to determine differences in their contractile properties. Isometric force was measured simultaneously with either myofibrillar MgATPase or fibre stiffness in both fibre types. The ultrastructure of individual long- and short-sarcomere fibres was also determined by electron microscopy. The long-sarcomere fibres developed greater tension (30.48 +/- 1.72 N cm-2) when maximally activated by Ca2+ compared with the short-sarcomere fibres (18.60 +/- 0.80 N cm-2). The difference in the maximum Ca(2+)-activated force can be explained by the difference in the amount of filament overlap between the two fibre types. The maximum Ca(2+)-activated myofibrillar MgATPase rate in the short-sarcomere fibres (1.60 +/- 0.27 mmol ATP l-1s-1) was higher, but not significantly different from the ATPase rate in fibres with long-sarcomeres (1.09 +/- 0.14 mmol ATP l-1s-1). As the concentration of myosin is estimated to be higher only by a factor of 1.22 in the short-sarcomere preparations there is no evidence to suggest that the myofibrillar MgATPase activity is different in the long- and short-sarcomere preparations. The maximum Ca(2+)-activated force (P0) of both short- and long-sarcomere fibres was quite insensitive to BDM compared with vertebrate muscle. Force decreased to 60.2 +/- 5.3% and 76.1 +/- 2.7% in the short- and long-sarcomere fibres respectively in the presence of 100 mmol l-1 BDM. The difference in the force depression between the long- and short-sarcomere fibres is statistically significant (p < 0.05). Fibre stiffness during maximum Ca(2+)-activation expressed as percentage maximum force per nm per half sarcomere was higher by a factor of 3.5 in short-sarcomere fibres than in long-sarcomere fibres suggesting that the compliance of the filaments in the long-sarcomere fibres is considerably higher than in the short-sarcomere fibres. Sr2+ could not activate the contractile apparatus to the same level as that seen by Ca2+ in either fibre type: the maximum Sr(2+)-activated force was (20 +/- 3%) and (63 +/- 3%) of the maximum Ca(2+)-activated force response in short- and long-sarcomere fibres, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino acids in haemolymph, single fibres and whole muscle from the claw of freshwater crayfish acclimated to different osmotic environments.

The concentrations of free amino acids were measured in whole claw muscle, single fibres and haemolymph of Australian freshwater crayfish, Cherax destructor, during the intermoult stage. The average total pool of amino acids in short-sarcomere fibres (179 mmol kg(-1)) was 60% greater than in long-sarcomere fibres, due to higher concentrations of alanine, cysteine, glutamate, leucine and proline. The two fibre types exhibited differences in the banding pattern of the isoforms of troponin using gel electrophoresis. The average pool of amino acids in haemolymph was 2.7 mmol kg(-1). Cherax has symmetrical claws and the total pool of amino acids from whole muscles (approx. 79 mmol kg(-1)) was similar in left and right claw muscles. In animals acclimated to osmotic environments between 0 and 220 mOsm, the osmotic pressure of the haemolymph increased from 356 to 496 mOsm, but no systematic changes were observed in the amino acid profiles of muscles or haemolymph. The major findings were that (a) concentrations of amino acids differed between the two major fibre types in claw muscle and (b) amino acids in the muscle fibres did not play a major part in intracellular osmoregulation in Cherax, suggesting this species is an anisosmotic regulator.

A minicomputer system for analyzing and reporting pilot plant fermentor data.

In 1979, a minicomputer system was developed for Hoffmann-La Roche by ABEC, Inc. for the purpose of achieving on-line analysis and reporting of data from 16 70-L pilot-plant fermentors (New Brunswick Scientific Co.). The system consists of a PDP 11/60 computer with 96K core capacity, two RL01 disk drives, two RX01 floppy-disk drives, LA-36 DECwriter terminal, Tektronix CRT, and Versatec printer plotter. DEC, PDP, RSX, RL01, RX01, LA-36, and DECwriter are trademarks of Digital Equipment Corporation. The computer software comprises three distinct groups of programs. RSX-11M is a disk-based operating system that allows quick response to realtime events, such as process monitoring and data acquisition, while carrying out less time-dependent activities, such as program development and graphical output. The AIM (Biles, Inc.) system is used to acquire and convert the voltage signals produced by pilot-plant instrumentation into engineering units. Analysis and graphical output are executed by ABEC and Versatec supplied programs. The most beneficial task performed by the computer is the production of graphical output of a variety of measured and analyzed data. This has led to an increase in personnel productivity and design of more meaningful experiments. An ancillary function of the system is to pick up data logged by a PDP 11/03 computer from a remote fermentation production plant by means of a MODEM interfaced communication link. Production data are analyzed and presented in a form identical with pilot-plant data. The experience with the system is discussed in this article.

Characterization of ultrastructural and contractile activation properties of crustacean (Cherax destructor) muscle fibres during claw regeneration and moulting.

Long-(SL > 6 microns) and short-sarcomere (SL < 4 microns) fibres were isolated from the claw muscle of the yabby (Cherax destructor) during limb regeneration and at different stages of the moult cycle. Long-sarcomere fibres were more susceptible to the changes resulting from the moult-induced atrophy compared with the short-sarcomere fibres. Signs of atrophy included fibre erosion, loss of myosin filaments, a reduction in the diameter of myosin filaments and changes associated with the Z line. The intracellular structure of the fibres, however, remained intact in both fibre types. Fibres taken immediately prior to ecdysis could not be fully activated with Ca2+ or Sr2+ without breaking. In contrast fibres taken within 4 h after ecdysis could develop and maintain full force when activated by Ca2+ or Sr2+. The results suggest that loss of myofibrillar proteins via the moult-induced atrophy and/or events associated with fibre elongation may occur in the period just prior to ecdysis and that these changes may be responsible for the fibres inability to function during the premoult stage. Results from this study showed that short-sarcomere fibres add sarcomeres by at least two different mechanisms (1) transverse sarcomere splitting and (2) Z line splitting. Long-sarcomere fibres appear to be elongated by mechanism(s) other than those used by short-sarcomere fibres which possibly involve large electron dense structures which are positioned between the myofibrils and within the A and I bands. Results from the regenerating chelae limb bud showed that sarcomeres form from separate units comprising myosin filaments and actin filaments anchored into Z lines respectively. These sub-sarcomeric units then join together to form sarcomeres. Myofibril formation is aided by electron dense regions which are closely associated with the membrane system. These fibres although short in length and still within the non-functional limb bud could be activated by Ca2+ and Sr2+ suggesting that full fibre function exists before the chelae become functional. Regenerating muscle fibres consisted predominantly of fibres with short-sarcomeres.