Gillian P. Campbell

Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture and in co‐culture with methanogens

When incubated in axenic culture, strains of anaerobic rumen fungi were more active than cellulolytic bacteria in solubilizing barley straw stem fragments 5 to 10 mm in length. Pretreatment with ammonia had little effect on microbial attack. Of three species of methanogens tested, Methanobrevibacter smithii strain PS formed the most stable and reproducible co‐cultures with the fungi and with Ruminococcus albus, and the presence of this organism enhanced the extent of degradation of straw, although this effect was less marked than that previously observed when pure cellulose was used as substrate.

Stimulation of myofibrillar protein degradation and expression of mRNA encoding the ubiquitin‐proteasome system in C2C12 myotubes by dexamethasone: effect of the proteasome inhibitor MG‐132

Addition of the synthetic glucocorticoid, dexamethasone (Dex) to serum‐deprived C2C12 myotubes elicited time‐ and concentration‐dependent changes in Nτ‐methylhistidine (3‐MH), a marker of myofibrillar protein degradation. Within 24 h, 100 nM Dex significantly decreased the cell content of 3‐MH and increased release into the medium. Both of these responses had increased in magnitude by 48 h and then declined toward basal values by 72 h. The increase in the release of 3‐MH closely paralleled its loss from the cell protein. Furthermore, Dex also decreased the 3‐MH:total cell protein ratio, suggesting that myofibrillar proteins were being preferentially degraded. Incubation of myotubes with the peptide aldehyde, MG‐132, an inhibitor of proteolysis by the (ATP)‐ubiquitin (Ub)‐dependent proteasome, prevented both the basal release of 3‐MH (>95%) and the increased release of 3‐MH into the medium in response to Dex (>95%). Northern hybridization studies demonstrated that Dex also elicited similar time‐ and concentration‐dependent increases in the expression of mRNA encoding two components (14 kDa E2 Ub‐conjugating enzyme and Ub) of the ATP‐Ub‐dependent pathway. The data demonstrate that Dex stimulates preferential hydrolysis of myofibrillar proteins in C2C12 myotubes and suggests that the ATP‐Ub‐dependent pathway is involved in this response. J. Cell. Physiol. 181:455–461, 1999.

Stimulation of actin and myosin synthesis in rat gastrocnemius muscle by clenbuterol; evidence for translational control.

1. A transient rise in fractional rates of protein and actomyosin synthesis was observed in gastrocnemius muscles of rats fed clenbuterol for 1-2 days but the muscle RNA:protein ratio was unchanged, therefore protein synthesis per unit RNA (kRNA) also increased. 2. Myosin heavy and light chains and actin showed increased incorporation of [3H]phenylalanine at 2 days; these changes were proportional to increases in total protein synthesis. 3. The ratios actin mRNA:18S RNA and fast myosin heavy chain mRNA:18S RNA were unaffected by clenbuterol. 4. The data suggest that the clenbuterol-induced increase in muscle protein synthesis involves both translational control and increased tissue RNA.

Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C2C12 satellite cells: effect of differentiation and fusion.

In proliferating C2C12 myoblasts, serum and physiological concentrations of insulin and IGF-I stimulated protein synthesis and RNA accretion. After fusion, the multinucleated myotubes remained responsive to serum but not to insulin or IGF-I, even though both insulin and type-I IGF receptor mRNAs increased in abundance. Protein synthetic responses to insulin and IGF-I in myoblasts were not inhibited by dexamethasone, ibuprofen or Ro-31-8220, thus phospholipase A2, cyclo-oxygenase and protein kinase C did not appear to be involved in the signalling mechanisms. Neither apparently were polyphosphoinositide-specific phospholipase C or phospholipase D since neither hormone increased inositol phosphate, phosphatidic acid, choline or phosphatidylbutanol production. Only the phosphatidylinositol-3-kinase inhibitor, wortmannin, and the 70 kDa S6-kinase inhibitor, rapamycin, wholly or partially blocked the effects of insulin and IGF-I on protein synthesis. 2-deoxyglucose uptake remained responsive to insulin and IGF-I after fusion and was also inhibited by wortmannin. The results suggest that the loss of responsiveness after fusion is not due to loss of receptors, but to the uncoupling of a post-receptor pathway, occurring after the divergence of the glucose transport and protein synthesis signalling systems, and that, if wortmannin acts at a single site, this is prior to that point of divergence.

Effects of insulin, pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism.

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 μM) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.

Immunohistochemical evidence for an association of ribosomes with microfilaments in 3T3 fibroblasts.

Ribosome distribution in cultured fibroblasts was investigated immunohistochemically using antibodies which recognize the 60S ribosomal subunit. After treatment of cells with buffer containing 25mM KCl and 0.05% Nonidet-P40 immunostained material was present in punctate patterns and linear arrays consistent with some ribosomes being associated with the cytoskeleton. Treatment of the cells with 130mM KCl caused loss of both the beaded lines of immunostaining and micro-filaments. Double immunostaining showed ribosomes to be closely associated with microfilaments.

Rapid response of protein synthesis to insulin in 3T3 cells: effects of protein kinase C depletion and differences from the response to serum repletion.

Quiescent 3T3 cells grown in media containing 4% foetal calf serum showed different responses to insulin and to serum repletion (to 12%). Insulin stimulated protein synthesis within 1 h and this early response was insensitive to actinomycin D. The later insulin response showed progressive sensitivity to actinomycin D. The serum response was slower, not occurring until 1 h, and was inhibited by actinomycin D. Depletion of cell protein kinase C by pre-treatment with phorbol ester caused a total block of the immediate response to insulin but had little effect on the response to serum or the later response to insulin. Acute phorbol ester treatment stimulated protein synthesis.

Methanogenic bacterial consortia degrading 2-methylbutyric acid

Two co‐cultures, each of a coccobacillus plus a Gram‐negative rod, were isolated from a cattle waste digester. The cultures were distinguished by production of methane from acetate only, or from either acetate or butyrate. 2‐Methylbutyrate, added initially as a growth factor, was degraded to propionate but only during growth on acetate or butyrate. Valerate and 2‐ and 3‐methyl valerate were not degraded.

Development of insulin sensitivity in rat skeletal muscle Studies of glucose transporter and insulin receptor mRNA levels

Expression of GLUT‐4 and insulin receptor mRNAs was investigated in rat skeletal muscle by Northern hybridization. GLUT‐4 mRNA was barely detectable in foetal muscle, was expressed at low levels by 1–8 days and at 2–3‐fold higher levels during and after weaning (18–40 days). In contrast there was little change in insulin receptor mRNA levels prior to weaning and a reduction in mRNA abundance between 18 and 40 days. Weaning rats on to a diet rich in fat prevented the increase in GLUT‐4 abundance seen between 15 and 29 days in animals weaned on a high‐carbohydrate diet.

Increased protein synthesis response to insulin in fibroblasts treated with the diacylglycerol kinase inhibitor R59022

Insulin stimulated protein synthesis in quiescent 3T3 fibroblasts. This effect of the hormone was greater in the presence of the diacylglycerol kinase inhibitor R59022 (10−5M) over a range of insulin concentrations from 1μU to 1 mU/ml; R59022 increased the sensitivity of cells to insulin. The amount of radioactive diacylglycerol recovered from cells prelabelled with [3H]glycerol was increased transiently in response to insulin; the response was larger and prolonged in cells given the kinase inhibitor. The results (i) support the hypothesis that diacylglycerol production is part of the signal pathway by which insulin stimulates protein synthesis and (ii) suggest that inhibition of diacylglycerol breakdown leads to increased sensitivity to the hormone.