Validity of one-repetition maximum predictive equations in men with spinal cord injury.

STUDY DESIGN: Cross-sectional study. OBJECTIVES: The study aimed (a) to test the cross-validation of current one-repetition maximum (1RM) predictive equations in men with spinal cord injury (SCI); (b) to compare the current 1RM predictive equations to a newly developed equation based on the 4- to 12-repetition maximum test (4-12RM). SETTING: SARAH Rehabilitation Hospital Network, Brasilia, Brazil. METHODS: Forty-five men aged 28.0 years with SCI between C6 and L2 causing complete motor impairment were enrolled in the study. Volunteers were tested, in a random order, in 1RM test or 4-12RM with 2-3 interval days. Multiple regression analysis was used to generate an equation for predicting 1RM. RESULTS: There were no significant differences between 1RM test and the current predictive equations. ICC values were significant and were classified as excellent for all current predictive equations. The predictive equation of Lombardi presented the best Bland-Altman results (0.5 kg and 12.8 kg for mean difference and interval range around the differences, respectively). The two created equation models for 1RM demonstrated the same and a high adjusted R2 (0.971, P<0.01), but different SEE of measured 1RM (2.88 kg or 5.4% and 2.90 kg or 5.5%). CONCLUSION: All 1RM predictive equations are accurate to assess individuals with SCI at the bench press exercise. However, the predictive equation of Lombardi presented the best associated cross-validity results. A specific 1RM prediction equation was also elaborated for individuals with SCI. The created equation should be tested in order to verify whether it presents better accuracy than the current ones.

Mitogenic and oncogenic properties of the small G protein Rap1b.

It has been widely reported that the small GTP-binding protein Rap1 has an anti-Ras and anti-mitogenic activity. Thus, it is generally accepted that a normal physiological role of Rap1 proteins is to antagonize Ras mitogenic signals, presumably by forming nonproductive complexes with proteins that are typically effectors or modulators of Ras. Rap1 is activated by signals that raise intracellular levels of cAMP, a molecule that has long been known to exert both inhibitory and stimulatory effects on cell growth. We have now tested the intriguing hypothesis that Rap1 could have mitogenic effects in systems in which cAMP stimulates cell proliferation. The result of experiments addressing this possibility revealed that Rap1 has full oncogenic potential. Expression of Rap1 in these cells results in a decreased doubling time, an increased saturation density, and an unusual anchorage-dependent morphological transformation. Most significantly, however, Rap1-expressing cells formed tumors when injected into nude mice. Thus, we propose that the view that holds Rap1 as an antimitogenic protein should be restricted and conclude that Rap1 is a conditional oncoprotein.

Identification of GTP-binding proteins in turtle urinary bladder epithelial cells.

Water and electrolyte transport in turtle urinary bladder closely resembles that present in the mammalian collecting tubule. Although cAMP is known to participate in the control of mucosal transport processes, the GTP-binding inhibitory Gi and stimulatory Gs proteins which link receptors on the cell surface to the adenylate cyclase system remain to be identified in this urinary epithelium. To this end, individual cells harvested from the mucosal surface of the turtle bladder were isolated using a discontinuous density Ficoll gradient. Examination by electron microscopy of the material from the different layers of the Ficoll gradient confirmed that bands II and III contained carbonic anhydrase-rich cells and granular cells, respectively. Identification of Gi and Gs in carbonic anhydrase-rich and granular cells was accomplished using pertussis (PT) and cholera toxins to promote [32P] ADP ribosylation of the proteins. Separation of Gi and Gs from other cell proteins was accomplished using polyacrylamide gel electrophoresis and autoradiography. Pretreatment of cells with 0.2% triton X-100 substantially magnified the ADP-ribosylation of Gi by PT. A doublet form of Gi was present in the 40-kD region and indicated heterogeneity of the PT substrate in granular and carbonic anhydrase-rich cells. Gs was observed as a single polypeptide at the 42-kD region in both cell types. A distinct 45-kD peptide not present in mammalian collecting tubule was identified by both toxins in granular cells and by cholera toxin in carbonic anhydrase-rich cells. In summary, this investigation identified and characterized Gi and Gs proteins in carbonic anhydrase-rich and granular cells from the mucosa of turtle urinary bladder.(ABSTRACT TRUNCATED AT 250 WORDS)

Pertussis toxin induces structural changes in G alpha proteins independently of ADP-ribosylation.

Pertussis toxin catalyzes ADP-ribosylation of a family of GTP-binding proteins (G alpha proteins) involved in signal transduction. It is thought that this activity is responsible for the attenuating effects of the toxin on the actions of a number of hormones and neurotransmitters. By utilizing specific antisera for detecting on electrophoretic transfer blots (Western blots) alpha proteins that are subject to ADP-ribosylation, it was found that treatment of these proteins with pertussis toxin resulted in shifts in their electrophoretic mobility and marked enhancement of their immunoreactivity compared to untreated proteins. No changes in mobility or immunoreactivity with specific antisera were observed with beta subunits of G proteins. Both effects on alpha proteins required the same ingredients, including detergents, ATP, and sulfhydryl reducing agents, that other studies have shown are required for activation of the ADP-ribosylating activity of pertussis toxin. However, NAD+, the substrate for ADP-ribosylating activity, was not required. Moreover, inhibition of the ADP-ribosylating activity by 50 mM nicotinamide failed to block the NAD-independent effects of the toxin. These findings indicate that the toxin induces structural changes in alpha proteins independently of its ADP-ribosylating activity and raise the possibility that these structural changes are primary to ADP-ribosylation and causative of many of the biological effects of pertussis toxin.

Prevention of alpha 2-adrenergic inhibition on ADH action by pertussis toxin in rabbit CCT.

The present studies were performed to investigate the mechanism whereby alpha 2-adrenergic receptor occupancy inhibits the hydrosmotic action of antidiuretic hormone (ADH) in isolated cortical collecting tubules (CCT). The ADH-ribosyltransferase activity of pertussis toxin (PT) was used to promote covalent modification in CCT Ni, the inhibitory regulatory protein of adenylate cyclase, which presumably mediates the alpha 2-adrenergic inhibition of water flow. Tubules preincubated with PT were studied after the addition of ADH and then after the superimposition of clonidine. In these studies, the inhibition of Jv (water absorption, nl X mm-1 X min-1) and Pf (water permeability coefficient, cm/s), by the addition of 10(-4) M clonidine to the bath, was attenuated by PT in a concentration-dependent manner. Reversal of the inhibitory action of clonidine was accomplished with a concentration of 1.0 micrograms/ml PT. To further elucidate the molecular basis of Ni-mediated transduction of the alpha 2-adrenergic signal, ADP-ribosylation studies were undertaken in membrane preparations of dissected CCT segments. PT ADP ribosylated a 40,000 Mr peptide which was proportional to the amount of membrane protein added. Furthermore, pretreatment of CCT during dissection with 0.5 micrograms/ml PT dramatically decreased the susceptibility of the subunit of Ni (alpha i) to be subsequently ADP ribosylated by PT, when compared with CCT preparations not previously treated with PT. Cholera toxin ADP ribosylated a 42,000 Mr peptide from CCT membranes and PT pretreatment did not interfere with the reaction. We conclude that CCT segments have both the pertussis and cholera toxin substrates and the effect of clonidine to attenuate ADH action is mediated through Ni.

Adenosine diphosphate ribosylation of G proteins by pertussis and cholera toxin in isolated membranes. Different requirements for and effects of guanine nucleotides and Mg2+.

ADP ribosylation of membranes by pertussis toxin (PT) and cholera toxin (CT) was studied as a function of addition of ATP, various guanine nucleotides, Mg2+, and inorganic phosphate (Pi). ADP ribosylation of a 40 kilodalton (kDa) band by PT is markedly enhanced by ATP and GTP and is strongly inhibited by Pi or Mg2+. GTP analogs (GTP gamma S and GMP-adenyl-5'-yl imidodiphosphate) were less effective. In contrast, ADP ribosylation of two substrates for CT (of 42 and 50 kDa) is stimulated by Pi, Mg2+, and GTP or GTP analogs such as GTP gamma S, but is unaffected by ATP. These stimulatory conditions correlate well with GTP-mediated activation of stimulated nucleotide-binding regulatory component of adenyl cyclase. Optimal conditions for ADP ribosylation by PT do not correlate simply with conditions thought to lead to stabilization of an inactive form of inhibitory nucleotide-binding regulatory component of adenyl cyclase (Gi) or Gi-like protein; rather, the data suggest the involvement of both a stimulatory nucleotide site on PT (positively affected by either ATP or GTP) and a stabilizing site on the PT substrate (affected by GDP, GDP beta S, or GTP). Treatment of membranes with Lubrol PX increased ADP ribosylation by PT by as much as 25- to 30-fold, but inhibited the action of CT. Using defined conditions for ADP ribosylation by PT and CT, distinct labeling patterns were observed in thyroid, brain, corpus luteum, liver, heart, and erythrocytes membranes. All membranes were more intensely labeled by PT rather than CT.

Incubation of bovine thyroid slices with thyrotropin is associated with a decrease in the ability of pertussis toxin to adenosine diphosphate-ribosylate guanine nucleotide regulatory component(s).

Pretreatment of bovine thyroid slices with TSH resulted in desensitization of TSH-sensitive adenylyl cyclase activity but no change in stimulatory nucleotide binding regulatory component of adenylyl cyclase (Gs) activity assessed by reconstitution of the Gs-defective cyc-S49 adenylyl cyclase system. Possible changes in substrates for pertussis toxin (PT)-induced ADP ribosylation due to TSH treatment and/or in endogenous ADP ribosylation of membrane proteins were explored. Using 10 microM [32P]NAD+ as substrate, endogenous ADP ribosylation was not observed in membranes from control or TSH-treated slices. ADP ribosylation of alpha-subunits of Gs by cholera toxin was also unaffected by incubation of thyroid slices with TSH. In contrast, ADP ribosylation of 40 kilodalton (kDa) substrates for PT was decreased between 40% and 60% by TSH treatment. This effect of TSH was dependent on its concentration and the time of incubation of the slices and was specific for labeling of the 40 kDa PT substrate. Prostaglandin E1 treatment of thyroid slices, which results in a much smaller homologous desensitizing effect, did not result in changes in ADP ribosylation by PT. The effect of incubation of slices with TSH was abolished by pretreatment of the membranes with 0.3-1.0% Lubrol PX, which increased the labeling of the 40 kDa polypeptides. The data suggests that TSH induces in thyroid tissue a redistribution of 40 kDa polypeptides changing their availability to PT.