Characterisation of myosin heavy chain gene variants in the fast and slow muscle fibres of gammarid amphipods.

Recent molecular work has revealed a large diversity of myosin heavy chain (MyHC) gene variants in the abdominal musculature of gammarid amphipods. An unusual truncated MyHC transcript from the loop 1 region (Variant A(3)) was consistently observed in multiple species and populations. The current study aimed to determine whether this MyHC variant is specific to a particular muscle fibre type, as a change in net charge to the loop 1 region of Variant A(3) could be functionally significant. The localisation of different fibre types within the abdominal musculature of several gammarid species revealed that the deep flexor and extensor muscles are fast-twitch muscle fibres. The dorsal superficial muscles were identified as slow fibres and the muscles extrinsic to the pleopods were identified as intermediate fibres. Amplification of loop 1 region mRNA from isolated superficial extensor and deep flexor muscles, and subsequent liquid chromatography and sequence analysis revealed that Variant A(3) was the primary MyHC variant in slow muscles, and the conserved A(1) sequence was the primary variant in fast muscles. The specific role of Variant A(3) in the slow muscles remains to be investigated.

Linking functional molecular variation with environmental gradients: myosin gene diversity in a crustacean broadly distributed across variable thermal environments.

To investigate the molecular basis of temperature adaptation in natural populations we used the candidate gene approach, targeting the myosin heavy chain (MyHC) gene. The functional effects of genetic variation in MyHC have been well characterised, and changes in the flexibility of the surface loops 1 and 2, caused by modulations in length, amino acid composition and charge can play an important role in thermal acclimation in fish. However, the extent that MyHC diversity is influenced by natural thermal gradients is largely unknown. Sequence variation in MyHC cDNA was examined in 7 species of gammarid amphipod with broad latitudinal distributions and differing intertidal thermal habitats in the NE Atlantic and Arctic Oceans. A high degree of diversity was detected in the loop 1 nucleotide sequences, although not all are likely to be functional transcripts, and their deduced amino acid sequences indicated no differences in the length and charge of loop 1 and associated binding kinetics. Four isoforms for loop 2 were detected which differed in sequence length and charge distribution, suggesting functional differences in sliding velocities and ATPase activities. While all species, and indeed most individuals, expressed multiple loop 2 isoforms, analysis of the two species with the greatest number of sequenced clones revealed that G. duebeni, a high-shore species with the highest thermal tolerance, expressed a greater diversity of forms than G. oceanicus, a low intertidal species more sensitive to temperature change. Latitude further influenced MyHC loop 2 diversity in G. duebeni, as the number of isoforms increased in the northern populations. Species-specific variations in MyHC diversity were observed, irrespective of phylogenetic associations revealed by analysis of the mitochondrial cytochrome oxidase 1 (CO1) gene. Overall, it appears that the temporal temperature variations associated with higher intertidal habitat may be a greater selective agent for MyHC isoform diversity in gammarid muscles than broad spatial changes with latitude.

Temperature-dependent developmental variation in lobster muscle myosin heavy chain isoforms.

The temperature- and developmental-regulation of myosin heavy chain (MyHC) expression and primary sequence was investigated in the abdominal musculature of developing Homarus gammarus larvae acclimated to 10, 14 and 19+/-1 degrees C. MyHC loop 1 (ATP binding) and loop 2 (actin binding) regions were sequenced and compared. The deduced amino acid sequence of MyHC loop 1 showed a development-related increase in net charge from +1 to +2 between larval stages 1 and 2, which was not temperature-dependent. In post-settled stage 9 larvae, minor shifts in amino acid sequence occurred at 19 degrees C, and corresponded to a significant up-regulation of fast myosin mRNA expression. However, no temperature-specific loop 1 isoforms were detected. The deduced amino acid sequence of MyHC loop 2 was not affected by temperature, and the net charge remained +4 throughout development. These findings contrast to previous studies using the common carp, in which temperature-specific MyHC isoform genes were expressed in response to disparate thermal regimes. This raises the question as to whether arthropods do not express specific temperature isoforms but instead rely on shifts in fibre type to accommodate alterations in thermal environment.

Comparison of the variable loop regions of myosin heavy chain genes from Antarctic and temperate isopods.

The evolutionary adaptations of functional genes to life at low temperatures are not well characterised in marine and fresh water invertebrates. Temperature has been shown to affect the functional characteristics of fish muscles, with changes in the velocity of shortening and ATPase activity being associated with myosin heavy chain (MyHC) isoform composition and the structure of the surface loop regions. Two PCR products spanning loops 1 and 2 of a MyHC gene from an Antarctic isopod (Glyptonotus antarcticus) were sequenced and compared with those of a temperate isopod (Idotea resecata), slow and fast fibres from lobster (Homarus gammarus) and a cold water amphipod (Eulimnogammarus verrucosus), revealing specific differences between the species, possibly related to fibre type and habitat temperature. The loop 2 region from G. antarcticus myosin was cloned and used for Northern analysis of total RNA from the other species. The cloned myosin cDNA hybridised specifically to a 6.6-kb transcript, in G. antarcticus muscle. In contrast, cDNA probes for lobster slow myosin and actin hybridised to muscle RNA from all species, demonstrating that a distinct MyHC isoform is expressed in the Antarctic isopod, as opposed to the temperate species. The inter- and intra-specific sequence differences in loop 2 region suggest that this may be a site for muscle adaptation to enable function at the low temperatures found in the Southern Ocean.

Nicotinic regulation of c-fos and osteopontin expression in human-derived osteoblast-like cells and human trabecular bone organ culture.

Long-term in vivo studies have highlighted smoking as a risk factor in postmenopausal osteoporosis, bone fracture incidence, and increased nonunion rates. In contrast, there are few data postulating the effects of smoking at the cellular level in human skeletal tissue. In this study, we present novel evidence demonstrating that the nicotinic receptor alpha4 subunit is present in human primary bone cells by using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, we demonstrate direct cellular effects of nicotine on primary human bone cells and blockage of these effects with a nicotinic receptor antagonist, D-tubocurarine. Nicotine effects on cell proliferation were biphasic with toxic, antiproliferative effects at high levels of nicotine (>1 mmol/L) and stimulatory effects at very low levels (0.01-10 micromol/L) after 72 h. This nicotine-induced increase in cell proliferation was inhibited in a dose-dependent manner by the addition of D-tubocurarine. In addition, proliferation effects from low-level treatment correlated with an upregulation of expression of the AP-1 transcription factor, c-fos, within 1 h, which was blocked by incubation with D-tubocurarine. To determine in situ bone cell responses within their trabecular matrix, cores of human bone isolated from biopsies were perfused with 0.1 micromol/L nicotine for 24 h. Western analysis of proteins isolated from the cores highlighted an increase in osteopontin, a bone matrix protein implicated in regulating resorption, which was partially inhibited by the addition of D-tubocurarine. To conclude, our results suggest that nicotine has a direct effect on human bone cells in modulating proliferation, upregulation of the c-fos transcription factor, and the synthesis of the bone matrix protein, osteopontin.

Production of cysteinyl-dopamine during intravenous dopamine therapy.

BACKGROUND: Oxidized dopamine rapidly forms thiol-conjugates with --SH groups on cysteine, glutathione, and proteins. We used cysteinyl-dopamine production as an index of thioester production during intravenous dopamine treatment of critically ill patients. METHODS: Cysteinyl-dopamine and catecholamines were measured by high-performance liquid chromatography with electrochemical detection. The production of cysteinyl-dopamine by purified human neutrophils was measured using dopamine (1 micromol/L) and cysteine (1 mmol/L) concentrations similar to those found during dopamine treatment. To examine the impact of endotoxic shock on cysteinyl-dopamine production, anesthetized rats were given dopamine (12 to 15 microg/kg/min intravenously) with or without endotoxin (50 mg/kg intravenously). RESULTS: In vitro, neutrophils converted 26% of dopamine to cysteinyl-dopamine (30 min at 37 degrees C). Activating neutrophils with zymogen increased dopamine consumption from 26 to 68%, but only 36% appeared as cysteinyl-dopamine. The remainder may have been oxidized to other cysteinyl derivatives. Endotoxin increased cysteinyl-dopamine in rat plasma from 2.5 nmol/L (range <0.2 to 11) to 9.7 nmol/L (range <0.3 to 31, P = 0.1). After four hours, with or without endotoxin, cysteinyl-dopamine was <0.3 nmol/L in cerebrospinal fluid. In the plasma of eight patients receiving dopamine (6 to 20 microg/kg/min for 1 to 3 days), dopamine was 0.5 to 9.9 micromol/L, and cysteinyl-dopamine was 48 to 1660 nmol/L. Cysteinyl-dopamine was 4.3 to 22.6% of dopamine and correlated with leukocyte count (r(2) = 0.388, P = 0.099). CONCLUSIONS: A significant fraction of exogenously administered dopamine reacts with -SH groups of cysteine and probably also with -SH groups on peptides and proteins. During brief dopamine treatment of endotoxic shock in rats, neither dopamine nor cysteinyl-dopamine crossed the blood-brain barrier.

Selected contribution: regulatory pathways involved in mechanical induction of c-fos gene expression in bone cells.

The regulatory pathways involved in the rapid response of the AP-1 transcription factor, c-fos, to mechanical load in human primary osteoblast-like (HOB) cells and the human MG-63 bone cell line were investigated using a four-point bending model. HOB and MG-63 cells showed upregulation of c-fos expression on fibronectin and collagen type I substrates; however, MG-63 cells did not respond on laminin YIGSR substrates. Addition of cytochalasin D and Arg-Gly-Asp peptides during loading did not inhibit the response, whereas addition of beta(1)-integrin antibodies inhibited the load response. The role of Ca(2+) signaling has been demonstrated by blocking upregulation with addition of 2 mM EGTA, which chelates extracellular Ca(2+), and gadolinium (10 microM), which inhibits stretch-activated channels. Addition of the Ca(2+) ionophore A-23187 induced upregulation without loading; however, addition of nifedipine (10 microM), the L-type channel blocker, failed to prevent the load response. Inhibitors of downstream pathways indicated the involvement of protein kinase C. Our results demonstrate a key involvement of Ca(2+) signaling pathways and integrin binding in the c-fos response to mechanical strain.

Sequence analysis and transcript expression of the MEN1 gene in sporadic pituitary tumours.

The majority of pituitary tumours are monoclonal in origin and arise sporadically or occasionally as part of multiple endocrine neoplasia type 1 (MEN1). Whilst a multi-step aetiology involving both oncogenes and tumour suppressor genes has been proposed for their development, the target(s) of these changes are less clearly defined. Both familial and sporadic pituitary tumours have been shown to harbour allelic deletion on 11q13, which is the location of the recently cloned MEN1 gene. We investigated 23 sporadic pituitary tumours previously shown to harbour allelic deletion on 11q13 with the marker PYGM centromeric and within 50 kb of the MEN1 locus. In addition, the use of intragenic polymorphisms in exon 9 and at D11S4946, and of telomeric loci at D11S4940 and D11S4936, revealed that five of 20 tumours had loss of heterozygosity (LOH) telomeric to the menin gene. However, the overall pattern of loss in informative cases was indicative of non-contiguous deletion that brackets the menin gene. Sequence analysis of all MEN1 coding exons and flanking intronic sequence, in tumours and matched patient leucocyte DNA, did not reveal mutation(s) in any of the 23 tumours studied. A benign polymorphism in exon 9 was encountered at the expected frequency, and in seven patients heterozygous for the polymorphism the tumour showed retention of both copies of the menin gene. Reverse transcription polymerase chain reaction analysis of ten evaluable tumours and four normal pituitaries revealed the presence of the menin transcript. Whilst these findings suggest that gene silencing is unlikely to be mechanistic in sporadic pituitary tumorigenesis, they do not exclude changes in the level or stability of the transcript or translation to mature protein. Our study would support and extend very recent reports of a limited role for mutations in the MEN1 gene in sporadic pituitary tumours. Alternatively, these findings may point to an, as yet, unidentified tumour suppressor gene in this region.

Uses and misuses of serum hCG--a clinical audit.

A 24-hour laboratory-based service was established in the City Hospital, Birmingham, England, in 1987 for semi-quantitative serum human chorionic gonadotrophin (hCG) assay and specific clinical criteria for requests were agreed. However, a 309% increase in workload between 1988 and 1992 prompted a detailed clinical audit of this service. During a representative month only 35% of 142 requests conformed to the agreed criteria. Those for routine diagnosis of pregnancy, threatened miscarriage, and 'abdominal pain screen' comprised 49% of total requests and seemed inappropriate. No pregnancy was diagnosed by 'abdominal pain screen'. Adhering to agreed clinical criteria, therefore, would not jeopardise patient care and could halve the cost of the service. As a result of this audit the established criteria for serum hCG estimation were reinforced and re-issued to all relevant clinical teams. Additionally, to improve delivery of service and reduce costs a semi-quantitative urine hCG assay was introduced in the Accident and Emergency Department for use by approved staff. Records in this department showed no increase in hCG requests during the six months after the service change, compared with those in the preceding half year. However, theoretical cost savings of 5173 pounds per annum were eroded by an estimated 10% because 208 (26%) tests were unaccounted for. The reasons for this apparent 'wastage' are unclear, and highlight the difficulty in auditing a biochemical test performed outside the laboratory.