Biomedical subjects

A J el Haj

Publications and source records attributed to A J el Haj.

16 recordsLinked to original sources

PubMed1999

Mechanotransduction pathways in bone: calcium fluxes and the role of voltage-operated calcium channels.

Changes in strain distribution across the vertebrate skeleton induce modelling and remodelling of bone structure. This relationship, like many in biomedical science, has been recognised since the 1800s, but it is only the recent development of in vivo and in vitro models that is allowing detailed investigation of the cellular mechanisms involved. A number of secondary messenger pathways have been implicated in load transduction by bone cells, and many of these pathways are similar to those proposed for other load-responsive cell types. It appears that load transduction involves interaction between several messenger pathways, rather than one specific switch. Interaction between these pathways may result in a cascade of responses that promote and maintain bone cell activity in remodelling of bone. The paper outlines research on the early rapid signals for load transduction and, in particular, activation of membrane channels in osteoblasts. The involvement of calcium channels in the immediate load response and the modulation of intracellular calcium as an early signal are discussed. These membrane channels present a possible target for manipulation in the engineering of bone tissue repair.

Biomechanical Phenomena↗

PubMed1999

Osteoblasts derived from load-bearing bones of the rat express both L- and T-like voltage-operated calcium channels and mRNA for alpha 1C, alpha 1D and alpha 1G subunits.

Voltage operated calcium channels (VOCCs) are implicated in osteoblastic mechano- and hormonal transduction. Very little, however, is known about the expression of VOCCs in osteoblasts of load-bearing bones. Here we describe two types of whole-cell calcium current in rat femoral explant-derived osteoblasts. The first is high-voltage activated and sensitive to nifedipine, Bay K8644 and FPL 64176. The second is low-voltage activated and is sensitive to micromolar concentrations of Ni2+. The properties of these two currents are consistent with those of L-type and T-type calcium currents respectively. T-type currents were detected in most cells on the day of passage, the level of expression being significantly lower on subsequent days. L-type currents were also most common on the day of passage but were detected consistently throughout the 4-day period of study. The reverse transcription polymerase chain reaction with non-specific primers directed against all L-type VOCC alpha 1 subunits and then with specific primers directed against sequences from rat brain alpha 1C (L-type), alpha 1D (L-type) and alpha 1G (T-type) VOCC subunits detected transcripts of appropriate size in all four cases. Products from the three sets of specific primer pairs (alpha 1C, alpha 1D, alpha 1G) were sequenced and were identical to their respective rat brain templates.

Animals↗

PubMed1996

Bone marrow stromal cells are load responsive in vitro.

Mechanical load-related effects on bone marrow stromal cells in vitro have been investigated. A dose response of a cyclical load of 1 Hz between 350 ustrain and 2500 ustrain applied to 10-day-old cultures resulted in elevated alkaline phosphatase levels and the number of cells expressing this protein after 2 days. No significant changes in the number of cells expressing or the production of collagen type 1 was observed. A critical stage of development of the cultures must be reached before load-related elevation in alkaline phosphatase expression could be measured independent of the stage at which loading was applied. Using a prostaglandin inhibitor at concentrations previously used in vivo, the load response was abolished. We have demonstrated that bone marrow stromal cells are load responsive in culture and have made preliminary studies into determining the involvement of prostaglandins in this process.

Alkaline Phosphatase↗

PubMed1996

Expression of voltage-operated Ca2+ channels in rat bone marrow stromal cells in vitro.

The expression of voltage-operated Ca2+ currents (VOCCs) in bone marrow stromal cells cultured for 3-30 days has been studied by the use of the whole-cell patch-clamp technique. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) VOCCs were recorded. LVA currents were first detectable after 6-7 days in culture and reached a peak of expression at 8 days, after which both the amplitude and frequency of expression of the current fell rapidly. The current was virtually undetectable in cells cultured for more than 15 days. The HVA current was detectable after 3 days in culture and reached a peak of both amplitude and frequency of expression after 1-2 weeks. This current was expressed consistently throughout the remaining culture period. In cultures treated with dexamethasone (10(-8) mol/L) peak expression of LVA currents still occurred at 7-8 days, but currents were enhanced approximately threefold. Expression of LVA currents was maintained to the end of the culture period. Expression of HVA currents was not significantly modified by treatment of cultures with dexamethasone. Examination of the biophysical and pharmacological (blockade by Ni2+ and diphenylhydantoin) properties of the LVA current in these cells suggests that they may have similarities with the LVA T currents of neuronal cells.

Animals↗

PubMed1996

A comparison of the metabolic cost of protein synthesis in stenothermal and eurythermal isopod crustaceans.

To examine the presence of metabolic cold adaptation in Antarctic isopods, whole animal rates of oxygen uptake (MVo2) and protein synthesis were measured in Glyptonotus antarcticus at 0 degree C and compared with the temperature isopod Idotea rescata at 4 and 14 degrees C. The specific relationship between rates of metabolism and protein synthesis was investigated by injecting animals with cycloheximide, a protein synthesis inhibitor. In G. antarcticus, routine MVo2 was 11.10 +/- 0.89 mumol.kg-1.min-1 (n = 19 animals), and ks was 0.24 +/- 0.04% protein synthesized/day (n = 8 animals). Comparison with I. rescata showed that standardized whole animal MVo2 decreased with temperature (temperature quotient = 1.99), but whole animal ks was considerably lower in the Antarctic isopod; 66 and 22% of total MVo2 was attributable to protein synthesis in G. antarcticus at 0 degree C and I. rescata at 4 degrees C, respectively. The energetic cost of protein synthesis was four times higher in G. antarcticus at 885 +/- 141 mmol ATP/g protein (n = 5 animals) compared with 237 +/- 76 mmol ATP/g protein (n = 6) in I. rescata. G. antarcticus does not show metabolic rate compensation and maintains extremely low ks levels because of the relatively high energetic cost of protein synthesis.

Acclimatization↗

PubMed1995

Development of metabolic enzyme activity in locomotor and cardiac muscles of the migratory barnacle goose.

Preflight development of the goslings was typified by rapid increases in the mitochondrial enzymes of the semimembranosus and heart ventricular muscles resulting in near-adult values by 3 wk of age. In contrast, aerobic capacity of the pectoralis muscle initially developed slowly but showed a rapid increase between 5 and 7 wk of age, in preparation for becoming airborne. Activities of glycolytic enzymes in the pectoralis muscle showed similar patterns of development as those found for the aerobic enzymes, except for hexokinase, which was low at all ages, indicating an adaptation for catabolism of both intracellular glycogen and plasma fatty acids in preference to plasma glucose. Muscle mass specific activity of citrate synthase in the pectoralis increased by only 33% from goslings during the first few days of flight, compared with premigratory geese. Activities of anaerobic glycolytic enzymes in the ventricles were low, but values for hexokinase, which is involved in the phosphorylation of plasma glucose, developed rapidly. Values for lactate dehydrogenase were also high, reflecting the capacity of the heart to catabolize plasma lactate. Substrate flux supplied by carnitine palmitoyltransferase and oxoglutarate dehydrogenase (OGD), in the pectoralis muscles of the premigratory geese, appears to have the smallest excess capacities to meet the requirements of sustained aerobic flight. The average maximum oxygen uptake for premigratory geese during flight, as indicated by values for OGD, is calculated to be 484 ml O2/min (or 208 ml O2.min-1.kg-1).

Aging↗

PubMed1994

Induction of a low voltage-activated, fast-inactivating Ca2+ channel in cultured bone marrow stromal cells by dexamethasone.

The production of biochemical markers associated with the osteoblastic phenotype, and accompanying changes in the expression of voltage-operated Ca2+ channels, have been examined in rat bone marrow stromal cell cultures treated with dexamethasone (10(-8) M). Whole cell clamp analysis of voltage-operated Ca2+ channels in control cultures (using Ba2+ as the charge carrier) revealed primarily a high voltage-activated (HVA), slowly inactivating current, which was enhanced two- to threefold by treatment of the cells with Bay K 8644 (300 nM) and inhibited by nifedipine (4 microM). In dexamethasone-treated cultures, the I-V relationship for inward current was shifted to more positive potentials in comparison with control cells. Most cells in these cultures possessed both the HVA current and also a faster inactivating, low-voltage-activated (LVA), nifedipine-resistant current. These two currents could be separated both by nifedipine and by the use of steady state inactivation of the LVA current. The two components of the Ba2+ current varied widely in their relative size. The combination of LVA and HVA currents seen in dex-induced stromal cells resembles records of voltage-operated Ca2+ channels from cultures of calvarial osteoblasts.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1994

Novel culture procedure permitting the synthesis of proteins by rat calvarial cells cultured on hydroxyapatite particles to be quantified.

A simple culture procedure and assay conditions are described which have permitted us to quantify the synthesis of proteins which are associated with an osteoblastic phenotype, by rat calvarial periosteal cells grown on particulate materials. The main feature of the method is the use of an adhesive which does not permit cells to attach to itself but allows attachment and growth of cells on material particles embedded in it on glass coverslips. Cells were cultured for 27 d on hydroxyapatite particle-coated coverslips. Alkaline phosphatase, osteopontin and collagen type I were monitored in cell lysates from d 10 to d 20. After Western blotting, osteopontin and collagen type I were quantified using specific antisera and enhanced chemiluminescence. Maximum levels coincided with peak alkaline phosphatase activity, after 10 and 17 d. The procedures described will be generally applicable to the comparison of cell behaviour on particulate substrata.

Alkaline Phosphatase↗

PubMed1994

Actin mRNA levels and myofibrillar growth in leg muscles of the European lobster (Homarus gammarus) in response to passive stretch.

An actin cDNA clone from the European lobster, Homarus gamarus, has been generated by RT-PCR and used as a probe to quantify the relative abundance of actin mRNA in lobster leg muscles following imposition of passive stretch by leg flexion. The sarcomere lengths of a population of fibers in the same muscles were measured to provide an indirect marker of myofibrillar growth. Stretch resulted in a 70% increase in actin mRNA levels compared with unstretched controls animals between weeks 1 and 2 after flexion of the legs. Sarcomere lengths increased by 23% immediately after imposition of the stretch. During the same period of observed increase in actin mRNA, the sarcomere lengths returned to their initial values, indicating that longitudinal growth of the myofibrils had occurred. Results are discussed in relation to the role of stretch in crustacean muscle growth during the moult.

Actins↗

PubMed1992

A novel microcarrier bead model to investigate bone cell responses to mechanical compression in vitro.

Periosteal cells grown in macroporous microcarrier bead columns were cyclically loaded with compressive force using a newly developed model. In response to 1/2 h of cyclic compression, RNA synthesis increased significantly by twofold, from 113.4 +/- 19.6 to 260.7 +/- 363 (p < 0.01) after 16 h, whereas DNA synthesis did not increase significantly after 24 h. The microcarrier bead model was calibrated using a linear volume displacement transducer with a range of strain magnitudes applied across the column of 1000-6000 microstrain.

Animals↗

PubMed1992

Regulation of muscle gene expression in Crustacea over the moult cycle.

Muscle growth in Crustacea may occur during specific stages of the moult cycle, focused around ecdysis when the old cuticle is shed and the new cuticle expands. In order to determine the moult stages in which sarcomeric proteins are synthesized and the regulatory factors involved, actin mRNA levels have been measured in the muscles of two crustaceans. These levels have been followed throughout the moult cycle and in response to passive stretch of walking leg muscle in vivo and to exogenous ecydsteroids applied to muscle preparations in vitro. Actin mRNA levels in both claw and leg muscles were elevated during the pre and postmoult stages of the moult cycle. However, varying patterns of expression are found in claw and leg muscle at specific stages of pre and postmoult. There was no increase in actin mRNA expression in extensor leg muscles in vitro after 6 hours exposure to elevated premoult levels of ecdysteroids. Immobilization of intemoult walking legs to maintain the extensor muscle in continuous passive stretch did not result in increased levels of actin mRNA after 5 days. These results are discussed in relation to the regulation of muscle growth over the moult cycle and to the molecular processes which may be responsible for controlling muscle protein synthesis.

Actins↗

PubMed1990

Cellular responses to mechanical loading in vitro.

A technique has been established in which cancellous bone biopsies may be simultaneously perfused and subjected to mechanical load bearing. Assessments of cell viability over a period of 24 h were based on the cAMP response to parathyroid hormone, intracellular lactate dehydrogenase activity, and electron micrograph morphology. Two cellular responses to mechanical loading were demonstrated similar to those that follow "osteogenic" loading in vivo, as reported previously. These were (1) a rise in intracellular G6PD in lining cells immediately after loading, and (2) an increase in RNA synthesis measured in osteocytes 6 h after loading. In vivo the osteogenic response to loading was modulated by indomethacin. In these in vitro experiments, addition of indomethacin inhibited both the loading-related G6PD and the RNA responses.

Animals↗

PubMed1990

Load-induced proteoglycan orientation in bone tissue in vivo and in vitro.

Previous studies of Alcian blue-induced birefringence in adult avian cortical bone showed that a short period of intermittent loading rapidly produces an increased level of orientation of proteoglycans within the bone tissue. In the absence of further loading, this persists for over 24 hours. We have proposed that this phenomenon could provide a means for "capturing" the effects of transient strains, and so provide a persistent, constantly updated strain-related influence on osteocyte populations related to the bones' averaged recent strain history, in effect, a "strain memory" in bone tissue. In our present study, we use the Alcian blue-induced birefringence technique to demonstrate that proteoglycan orientation also occurs after intermittent loading of both cortical and cancellous mammalian bone in vivo and in vitro. We also show that the change in birefringence is proportional to the magnitude of the applied strain, and that the reorientation occurs rapidly, reaching a maximal value after only 50 loading cycles. Examination of electron micrographs of bone tissue after staining with cupromeronic blue allows direct visualization and quantification of the change in proteoglycan orientation produced by loading. This shows that intermittent loading is associated with a realignment of the proteoglycan protein cores, bringing them some 5 degrees closer to the direction of collagen fibrils in the bone matrix.

Alcian Blue↗

PubMed1988

Maternal diabetes in rats. I. Effects on placental growth and protein turnover.

The developmental growth of the rat placenta was investigated between days 14 and 21 of gestation in normal control, gestational-diabetic, established-diabetic, and insulin-maintained-diabetic mothers. While established-diabetic mothers were hyperglycemic for 2 wk before and throughout the pregnancy, gestational-diabetic mothers were only hyperglycemic for the second half of pregnancy. Daily insulin replacements successfully restored normoglycemia. The wet weight and protein content of control placentas increased linearly between days 14 and 21. Although placentas from diabetic animals were initially smaller, placentomegaly was found at full term. Placental glycogen concentrations were also markedly increased in all diabetic animals. These changes were largely prevented by insulin replacement. The changes in placental size during normal development and in association with the diabetic state were explained by measuring placental rates of protein turnover (in vivo). In normal placentas, protein synthetic and degradative rates progressively declined over the last week of gestation. Because synthesis rates were unchanged in placentas of diabetic mothers, it appears that the differences in placental size primarily arise from alterations in protein degradation.

Animals↗

PubMed1987

The influence of chronic dietary intervention on protein turnover and growth of the diaphragm and extensor digitorum longus muscles of the rat.

Changes in weight, protein, RNA and DNA contents of the E.D.L. and diaphragm muscles were studied in conjunction with aging and chronic dietary restriction. Between weaning and senescence both muscles exhibited progressive decreases in their fractional rates of growth, protein synthesis and protein breakdown; these rates being age for age higher in the diaphragm. Dietary restriction (50% of ad libitum food intake) from weaning onwards retarded muscle growth, particularly at the early stages (i.e. 4 weeks) after its implementation. Here the suppression of protein synthesis was due to the combined effects of piece meal feeding and long term reductions in food intake. Later, muscle sizes and total, but not fractional, synthetic rates were consistently decreased by chronic dietary intervention. The onset of the ageing atrophy may also be delayed by underfeeding. The changes in these 2 muscles have been compared to those in the whole animal and other striated muscles, as previously reported by the authors.

Animals↗

PubMed1986

The effect of chronic and acute dietary restriction on the growth and protein turnover of fast and slow types of rat skeletal muscle.

Changes in the growth and protein turnover of the anterior tibialis and soleus muscles were studied in response to acute and chronic dietary restriction (50% of ad libitum intake) between 3 and 149 weeks post partum. The effect of long-term dietary restriction from weaning to senescence was to retard the growth and normal developmental of the two types of skeletal muscle. This was evident from measurements of various parameters of growth, i.e. total protein, RNA and DNA and protein/DNA-P, which were reduced by approximately 50% when compared with age-matched controls. These decreases, however, were not accompanied by a decline in the fractional rate of synthesis (%/day) or ribosomal activity (mg protein/day per mg RNAP). The slowing down of the age-related decline in muscle growth has been attributed to a reduction in RNA capacity (RNA/protein), with similar responses in the fast- and slow-twitch skeletal muscles. The initial effects of piecemeal feeding of this restricted diet on the two types of muscle were also monitored. Short term starvation effects, i.e. 24 hr after feeding a reduced ration, were measured on the protein content and RNA/protein of both the anterior tibialis and soleus muscles; both parameters were unchanged within 24 hr. In contrast, a rapid and significant decline in the ribosomal synthetic activity (mg/d per mg RNAP), and a corresponding fall in the fractional rate of synthesis, occurred within 24 hr of feeding.

Aging↗