Pulsed-field gel analysis of human immunoglobulin heavy-chain constant region gene deletions reveals the extent of unmapped regions within the locus

The human immunoglobulin heavy-chain constant region gene locus is organized in three main gene groups, the physical distances of which are unknown. Different types of gene deletions, originated by unequal crossingover, have been found to encompass one or more genes in the locus. We have analyzed some of these deletions by means of pulsed-field gel electrophoresis, which allows resolution of large DNA fragments. By identifying a fragment containing two of the main gene groups and by observing the size reduction of this fragment in subjects with deletions, we were able to estimate the distance between the two groups and better locate the pseudogene in-between.     

Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies

The structure of the insulin receptor was studied with polyclonal antibodies obtained from rabbits which were immunized with synthetic peptides having a sequence identity to three regions of the alpha-subunit and five regions of the beta-subunit. None of the alpha-subunit antibodies including alpha-Pep8 (residues 40-49 (Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., and Ramachandran, J. (1985) Nature 313, 756-761), alpha-Pep7 (12 amino acid C-terminal extension (Ebina, Y., Ellis, L., Jarnagin, K., Ederly, M., Graf, L., Clauser, E., Ou, J.-H., Masiar, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., and Rutter, W.J. (1985) Cell 313, 747-758], or alpha-Pep6 (residues 1-7, 9) immunoprecipitated the human insulin receptor solubilized from IM-9 lymphocytes; however, alpha-Pep8 immunoprecipitated the dithiothreitol-reduced receptor. Antibodies prepared against the N terminus of the beta-subunit (alpha-Pep5, residues 780-790) and the ATP binding site (alpha-Pep3, residues 1013-1022) did not react with the intact receptor under any conditions; however, antibodies to the C terminus of the beta-subunit (alpha-Pep1, residues 1314-1324) and to the juxta-membrane region (alpha-Pep3, residues 952-962) immunoprecipitated the solubilized receptor in both its phosphorylated and nonphosphorylated forms. In contrast, the antibody reactive with the regulatory region of the beta-subunit which contains the major autophosphorylation sites (alpha-Pep2, residues 1143-1154) only precipitated the phosphorylated form. Thus the conformation of the extracellular domain of the receptor is rigid and stabilized by disulfide bonds, whereas several regions of the intracellular domain are accessible to antibodies and undergo conformational changes during autophosphorylation.     

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.     

The 18 kDa cytosolic acid phosphatase from bovine liver has phosphotyrosine phosphatase activity on the autophosphorylated epidermal growth factor receptor

In this paper we demonstrate that the cytosofic low-M_r acid phosphatase purified from bovine liver has phosphotyrosine protein phosphatase acitivity on ³²P-autophosphorylated epidermal growth factor (EGF) receptor. This activity was significantly inhibited by orthovanadate and p-hydroxymercuribenzoate; the latter result indicates that free sulfhydryl groups are required for phosphotyrosine phosphatase activity. The enzyme was active in a broad pH range, with maximum activity between pH 5.5 and 7.5. The apparent K_m for ³²P-EGF receptor dephosphorylation was 4 nM. The enzyme appeared to be specific for phosphotyrosine in that it dephosphorylated the autophosphorylated EGF receptor and L-phosphotyrosine, but not ³²P-Ser-casein, L-phosphoserine or L-phosphothreonine. These data suggest that the cytosolic low-M_r acid phosphatase might play a regulatory role in EGF receptor-dependent transmembrane signalling.     

Muscle fatigue and metabolic responses following three different antagonist pre-load resistance exercises

PurposePreload of antagonist muscles can be achieved by reciprocal actions (RAs) or by opposing muscle actions. However, evidence concerning neuromuscular and fatigue responses are scarce.ObjectiveTo compare the effects of different knee flexor (KF) preload methods on knee extension (KE) vastus medialis muscle fatigue, based on EMG-spectral index (FI), load range (LR), total work (TW), blood lactate (LAC) and biceps femoris co-activation (BFc) during resistance exercise.MethodsTwenty-four healthy men (23.5 ± 3.6 yrs) performed three antagonist pre-load isokinetic exercises (4 sets, 10 repetitions, 60° s^?1, 1 min rest between sets): RA (KF contraction immediately followed by KE); Superset (SS; one KF set immediately followed by one KE set); Multiple Set (MS; four KF sets followed by four KE sets).ResultsTotal work was significantly greater in RA. There was no significant decrease in LR between sets in RA. The BFc did not differ between protocols (p = 0.063). However, RA presented greater biceps femoriscoactivation. The FI was greater during SS compared to RA and MS (p < 0.05). The SS had greater LAC when compared to MS and RA (p = 0.005 and p = 0.007, respectively).ConclusionIt is suggested that the RA protocol is more neuromuscular and metabolic efficient during the performance of knee extension resistance exercise.     

Relationship between ventilatory threshold and muscle fiber conduction velocity responses in trained cyclists

The relationship between surface electromyography (SEMG) amplitude and the ventilatory threshold has been extensively studied. However, previous studies of muscle fiber conduction velocity (MFCV) are scarce and present insufficient evidence concerning the relationship between MFCV and metabolic responses during cycling. Based on that fact, the purpose of this study is twofold: (1) to investigate the existence of a MFCV threshold (MFCVT) during cycling and (2) to verify if this possible breakpoint is correlated with the ventilatory threshold (VT) and the SEMG threshold (SEMGT). Eight trained male cyclists (age 36.0 ± 9.7 years) performed an incremental cycling test with initial workload of 150 W gradually incremented by 20 W min^?1 until the exhaustion. Gas analyses were conducted using a breath-by-breath open-circuit spirometry and SEMG were registered from vastus lateralis in each pedaling cycle with a linear array of electrodes. A bi-segmental linear regression computer algorithm was used to estimate VT, MFCVT and SEMGT respectively in the carbon dioxide production (VCO₂), MFCV and electromyography root mean square (EMG RMS) curves. The one way ANOVA for repeated measures did not reveal any significant difference among VT (77.1 ± 7.5% of VO₂max), MFCVT (80.3 ± 10.4% of VO₂max) and SEMGT (81.9 ± 11.7% of VO₂max). The Bland and Altman procedure confirmed a good concordance between SEMGT and VT (Bias = 5.5 of %VO₂max) as well as MFCVT and VT (Bias = 5.2 of %VO₂max). The present findings suggest that muscle fiber conduction velocity threshold is a valid and reliable non-invasive tool to obtain information about ventilatory threshold in trained cyclists.     

Salicylic acid-independent plant defence pathways

Salicylic acid is an important signalling molecule involved in both locally and systemically induced disease resistance responses. Recent advances in our understanding of plant defence signalling have revealed that plants employ a network of signal transduction pathways, some of which are independent of salicylic acid. Evidence is emerging that jasmonic acid and ethylene play key roles in these salicylic acid-independent pathways. Cross-talk between the salicylic acid-dependent and the salicylic acid-independent pathways provides great regulatory potential for activating multiple resistance mechanisms in varying combinations.     

Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor

Keratinocyte growth factor (KGF) is an unusual fibroblast growth factor (FGF) family member in that its activity is largely restricted to epithelial cells, and added heparin/heparan sulfate inhibits its activity in most cell types. The effects of heparan sulfate proteoglycan (HSPG) on binding and signaling by acidic FGF (aFGF) and KGF via the KGFR were studied using surface-bound and soluble receptor isoforms expressed in wild type and mutant Chinese hamster ovary (CHO) cells lacking HSPG. Low concentrations of added heparin (1 microgram/mL) enhanced the affinity of ligand binding to surface-bound KGFR in CHO mutants, as well as ligand-stimulated MAP kinase activation and c-fos induction, but had little effect on binding or signaling in wild type CHO cells. Higher heparin concentrations inhibited KGF, but not aFGF, binding and signaling. In addition to the known interaction between HSPG and KGF, we found that the KGFR also bound heparin. The biphasic effect of heparin on KGF, but not aFGF, binding and signaling suggests that occupancy of the HSPG binding site on the KGFR may specifically inhibit KGF signaling. In contrast to events on the cell surface, added heparin was not required for high-affinity soluble KGF-KGFR interaction. These results suggest that high-affinity ligand binding is an intrinsic property of the receptor, and that the difference between the HSPG-dependent ligand binding to receptor on cell surfaces and the HSPG-independent binding to soluble receptor may be due to other molecule(s) present on cell surfaces.     

Androgen receptor status predicts response to chemotherapy, not risk of breast cancer in Indian women

Voltage gated potassium channels have been extensively studied in relation to cancer. In this review, we will focus on the role of two potassium channels, Ether à-go-go (Eag), Human ether à-go-go related gene (HERG), in cancer and their potential therapeutic utility in the treatment of cancer. Eag and HERG are expressed in cancers of various organs and have been implicated in cell cycle progression and proliferation of cancer cells. Inhibition of these channels has been shown to reduce proliferation both in vitro and vivo studies identifying potassium channel modulators as putative inhibitors of tumour progression. Eag channels in view of their restricted expression in normal tissue may emerge as novel tumour biomarkers.