Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA.

The calcitonin (CT)/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is subject to alternative tissue-specific processing of its primary transcript. CT mRNA is the predominant mRNA produced in thyroid C cells, whereas CT gene-related peptide I mRNA is the main product in neurons of the central and peripheral nervous systems. The CT-specific exon 4 is surrounded by weak processing sites. In this study we have investigated whether exon 4 sequences are involved in the tissue-specific selection of the exon 4 splice acceptor site. The results indicate that two separate elements, termed A and B, in the 5' part of exon 4 are required for production of CT-specific RNA. These sequences are located between nucleotides 67 and 88 (A) and nucleotides 117 and 146 (B) relative to the 5' end of exon 4. Variation of the distance between these sequence elements and the 3' splice site of exon 4 does not change the processing choice. These sequence elements are functionally equivalent. CT-specific splicing requires the presence of both sequence A and B or duplicates of either sequence element in exon 4. The effect of these sequences on the RNA processing choice is overruled by mutation of the CT-specific uridine branch acceptor nucleotide into a commonly preferred adenosine residue.

The exon 4 poly(A) site of the human calcitonin/CGRP-I pre-mRNA is a weak site in vitro.

The human calcitonin/CGRP-I (CALC-I) pre-mRNA is processed in a tissue-specific alternative way into either calcitonin (CT) or calcitonin gene-related peptide-I (CGRP-I) mRNA. The exons 1 to 3 are common exons. They are spliced to exon 4, which becomes polyadenylated to form CT mRNA, or to exon 5 and the polyadenylated exon 6 to form CGRP-I mRNA. Polyadenylation at exon 4 and splicing of exon 3 to exon 5 are mutually exclusive processing reactions. Only splicing of exon 3 to exon 5 was detected in vitro, with a minigene containing the exon 3 to exon 5 region. No polyadenylation at the exon 4 poly(A) site could be observed. Investigation of the properties of the exon 4 poly(A) site in vitro shows that it is inefficiently used in vitro. Cleavage and polyadenylation of short RNAs containing only the exon 4 poly(A) site is strongly dependent on the 3' length of the RNA. Downstream sequences located within 39 nucleotides from the cleavage site are required for optimal cleavage and polyadenylation. When the exon 4 poly(A) site in the minigene is replaced with the strong adenovirus L3 or rabbit beta-globin poly(A) sites, these sites can be efficiently used in vitro.

Pore REconstruction and Segmentation (PORES) method for improved porosity quantification of nanoporous materials.

Electron tomography is currently a versatile tool to investigate the connection between the structure and properties of nanomaterials. However, a quantitative interpretation of electron tomography results is still far from straightforward. Especially accurate quantification of pore-space is hampered by artifacts introduced in all steps of the processing chain, i.e., acquisition, reconstruction, segmentation and quantification. Furthermore, most common approaches require subjective manual user input. In this paper, the PORES algorithm "POre REconstruction and Segmentation" is introduced; it is a tailor-made, integral approach, for the reconstruction, segmentation, and quantification of porous nanomaterials. The PORES processing chain starts by calculating a reconstruction with a nanoporous-specific reconstruction algorithm: the Simultaneous Update of Pore Pixels by iterative REconstruction and Simple Segmentation algorithm (SUPPRESS). It classifies the interior region to the pores during reconstruction, while reconstructing the remaining region by reducing the error with respect to the acquired electron microscopy data. The SUPPRESS reconstruction can be directly plugged into the remaining processing chain of the PORES algorithm, resulting in accurate individual pore quantification and full sample pore statistics. The proposed approach was extensively validated on both simulated and experimental data, indicating its ability to generate accurate statistics of nanoporous materials.

Physical strain in daily life of wheelchair users with spinal cord injuries.

Forty-three men (age 33 +/- 9 yr) with spinal cord injuries (SCI) were observed during a normal workday while heart rate was recorded continuously. Physical strain was estimated using the heart rate response expressed relative to the individual heart rate reserve (%HRR). The mean physical strain during the day for group I (C4-C8, N = 9), II (T1-T5, N = 6), III (T6-T10, N = 15), and IV (T10-L5, N = 13) was 38 +/- 8, 29 +/- 12, 22 +/- 8, and 23 +/- 5%HRR, respectively. Prolonged periods (> 15 min) of high strain (> 60%HRR) that might maintain or improve physical capacity were not identified during activities of daily life (ADL), but only during sports activities. The analysis of activity-related strain revealed that specific ADL such as making transfers, entering/leaving car, and negotiating environmental barriers, provoked high levels of strain, especially in those with quadriplegia. Periods of peak strain (> 60%HRR, < 3 min) occurred frequently, also predominantly in those with quadriplegia. It was concluded that the physical strain during ADL is related to the level of lesion and is not of a magnitude and duration that would maintain or improve physical capacity. The periods of peak strain might restrict the mobility and independence of persons with SCI, and, therefore, reduce their quality of life.

Changes in physical strain and physical capacity in men with spinal cord injuries.

To determine longitudinal changes in physical capacity and physical strain during activities of daily living (ADL), 37 men with spinal cord injuries (C4/5-L5) performed an exercise test and various ADL on two occasions (T1 and T2; interval 34.5 +/- 1.5 months). Parameters of physical capacity were aerobic power (VO(2peak)) and maximal power output (PO(max)). Physical strain was estimated by the heart rate response relative to the heart rate reserve. VO(2peak) at T2 (1.75 +/- 0.55 1*min(1)) did not significantly differ from that at T1 (1.67 + 0.47 1*min(-1)). Absolute PO max improved (P < 0.05) from 64.9 +/- 25.9 (T1) to 71.7 +/- 27.2 W (T2), whereas relative PO(max) did not change. Activity level, time since injury, change in body mass, and occurrence of rehospitalization were the most important predictors of changes in physical capacity. Changes in relative VO(2peak) were related (P < 0.05) to changes in strain during transfers to the shower wheelchair (r = -0.39) and shower seat (r = -0.46), and during the curb ascent (r = -0.47). In conclusion, the hypothesized decline in physical capacity did not occur over the 3-yr period. Maintenance of physical capacity, which may in part be achieved through sport participation and improved medical care, together with avoidance of excessive body mass, may be useful to prevent high levels of strain during ADL.

Isometric strength, sprint power, and aerobic power in individuals with a spinal cord injury.

This study investigated in rather specific wheelchair tests the relationships among estimates of isometric upper-body strength (Fiso), sprint power (P30), aerobic power (VO2peak), and maximal power output (POaer) in a group of 44 men (age 34 +/- 12 yr) with longstanding spinal cord injuries ranging from C4/C5 to L5. Fiso was defined as the maximum force that could be exerted on the blocked rims of a stationary wheelchair ergometer. The estimation of P30 involved the measurement of the mean power during a 30-s all-out sprint test on the same wheelchair ergometer. VO2peak and POaer were determined as the peak oxygen uptake and highest sustained power output during a discontinuous progressive maximal exercise test on a motorized treadmill, while subjects used their own daily use wheelchair. Fiso ranged from 1.5 N.kg-1 (mean of both arms) in the group with quadriplegia to 3.4 N.kg-1 in the group with lowest-lesions, and P30 ranged from 0.5 to 1.5 W.kg-1 among the subjects. VO2peak ranged from 13.6 ml.kg-1.min-1 in the group with quadriplegia to 31.3 ml.kg-1.min-1 in the group with lowest-lesions, and POaer ranged from 0.4 to 1.1 W.kg-1. Strong positive relationships (r = 0.81-0.92) were demonstrated among all variables. Regression equations among variables were calculated: P30 = 0.51 Fiso - 0.18 (R2 = 0.75); POaer = 0.34 Fiso - 0.02 (R2 = 0.66); POaer = 0.67 P30 + 0.11 (R2 = 0.81); VO2peak = 6.52 Fiso + 4.15 (R2 = 0.76); VO2peak = 12.03 P30 + 7.43 (R2 = 0.77); VO2peak = 16.81 POaer + 6.44 (R2 = 0.84).(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebrovascular reserve capacity is preserved in a population-based sample of patients with type 2 diabetes mellitus.

BACKGROUND AND PURPOSE: Type 2 diabetes mellitus (DM2) is associated with an increased risk of stroke. DM2 is also associated with cognitive impairments. Vascular dysfunction, such as impaired cerebrovascular reserve capacity (CVR), may be a determinant of these changes, but previous studies on CVR in DM2 have provided variable results in selected populations of patients. We aimed to examine CVR in a population-based sample of DM2 patients. METHODS: The CO(2) reactivity of the middle cerebral artery was examined using transcranial Doppler ultrasonography in 81 DM2 patients and 38 controls. In DM2 patients CVR was correlated with diabetic parameters, vascular risk factors and cognitive functioning. RESULTS: CVR was similar in patients and controls (51 vs. 49%). Within the DM2 group, there was no statistically significant relationship between CVR and DM duration, HbA(1c), albuminuria, blood pressure, intima-media thickness and cognition. CVR tended to be lower in diabetic patients with retinopathy [46 vs. 55%, mean difference: -7.9 (confidence interval -18.0, 2.2)]. CONCLUSION: We conclude that CVR is not impaired in unselected patients with DM2 and probably does not, therefore, play a major role in the aetiology of cognitive impairment.

Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins.

The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP.

Reliability of heart rate responses to non-steady-state activities of daily living in men with spinal cord injuries.

The reliability of heart rate responses to non-steady-state tasks among 37 men with spinal cord injuries (lesion level: C4/5-L5) was examined with a simple heart rate recording device (Sport Tester PE3000). Three identical trials of 6 different transfers and an 8-cm curb ascent were performed on one day (Trial 1 and 2; n = 37) and one week later (Trial 3; n = 12). Pearson's r and intraclass correlations for the highest and the mean heart rate provoked during Trial 1 and 2 ranged from 0.73 to 0.97 for the transfers and from 0.92 to 0.97 for the curb ascent. Correlations were somewhat lower for Trial 1 versus Trial 3. A paired t-test revealed lower heart rate responses to Trial 2 and 3, suggesting a moderate learning effect and/or a reduction in psychological stress. It was concluded that heart rate responses to non-steady-state tasks, as recorded by a Sport Tester PE3000, are reproducible in men with spinal cord injuries.

Responses of subjects with spinal cord injuries to maximal wheelchair exercise: comparison of discontinuous and continuous protocols.

Six male subjects with spinal cord injuries (SCI) participated in this investigation to compare peak values of oxygen uptake (VO2), heart rate (fc), ventilation (VE), respiratory exchange ratio (R) and power output (W) obtained using a discontinuous (DP) and a continuous jump max protocol (JMP) in a maximal wheelchair exercise test on a treadmill. The W increments were achieved by imposing an extra mass upon the wheelchair through a pulley system. The DP involved exercise periods of 3 min separated by 2-min intervals at relative rest. Increments in W consisted of 0.10 or 0.15 W.kg-1 total mass. During the rest intervals no mass was imposed on the wheelchair. The JMP involved an increase in W each minute. Increments and velocity in the JMP were the same as during the exercise periods for DP. Mean peak values for W [99.5 (SD 13.6) W], VO2 [2.13 (SD 0.27) l.min-1, standard temperature and pressure, dry], R [1.25 (SD 0.16)] and VE [82.8 (SD 11.2) l.min-1, body temperature and pressure, saturated] in DP were not different from values observed for W [103.5 (SD 13.1)], VO2 [2.18 (SD 0.31) l.min-1], R [1.17 (SD 0.16)] and VE [78.9 (SD 16.0) l.min-1] in the JMP. The only significant difference was observed for fc: 198 (SD 11) beats.min-1 in DP and 187 (SD 11) beats.min-1 in JMP. The higher values for fc elicited using DP have been discussed. It was concluded that both a DP and a JMP seem to be equally appropriate in determining peak VO2 and peak W in SCI persons.(ABSTRACT TRUNCATED AT 250 WORDS)

Coronary heart disease risk indicators, aerobic power, and physical activity in men with spinal cord injuries.

OBJECTIVE: To compare the lipid and (apo-)lipoprotein profile and blood pressure of men with long-standing spinal cord injuries (SCI) to those of an age-matched able-bodied (AB) population, and to assess the most important determinants of this profile and blood pressure. DESIGN: A cross-sectional study of persons with chronic SCI residing in the community. SETTING: Tests were performed in a university research laboratory. SUBJECTS: Thirty-seven men (age 37.4 +/- 12.0 yrs) with longstanding (14.7 +/- 8.6 yrs) SCI ranging from level C4/5 to L5 volunteered to participate. Comparisons were made with published data from 3,498 AB men, age 20 to 59 yrs, from the same country. MAIN OUTCOME MEASURES: Lipid and lipoprotein profile (total cholesterol [TC], high-, low-, and very low-density lipoprotein cholesterol [HDL-C, LDL-C and VLDL-C, respectively], and triglycerides [TG]), as well as aerobic power, activity level, anthropometric variables, and blood pressure. Multiple regression analyses assessed the most important determinants of the lipid and blood pressure profile. RESULTS: None of the lipid variables were related to the lesion level. TC, HDL-C, and TC/HDL-C were not significantly different from the AB population. The most important determinants of TC, LDL-C, and the ratios TC/HDL and HDL-C/LDL-C were age, smoking behavior, and activity level. Aerobic power was not an important determinant of any lipid or (apo-)lipoprotein or blood pressure. CONCLUSION: Men with long-standing SCI do not appear to have an essentially different coronary heart disease risk profile compared with AB persons. Modifiable risk factors such as activity level, smoking, alcohol consumption, body mass index, and adipose tissue were more important than lesion level and aerobic power in the determination of the lipid and lipoprotein profile, suggesting several potential interventions.

Relationship between physical strain during standardised ADL tasks and physical capacity in men with spinal cord injuries.

To describe physical strain during activities of daily living (ADL), 44 men with spinal cord injuries (C4-L5) performed a set of standardised tasks. The physical strain was defined as the highest heart rate response expressed as a percentage of the individual heart rate reserve (%HRR). The physical strain averaged over the subjects who performed all tasks (n = 24) was (mean +/- SD): 20.2 +/- 7.2 %HRR (washing hands), 20.4 +/- 7.3 %HRR (passing a side-hung door), 28.8 +/- 10.8 %HRR (transfer to a toilet), 31.2 +/- 13.1 %HRR (ascending an 8 cm curb). 33.9 +/- 12.0 %HRR (transfer to a shower seat), 35.1 +/- 10.5 %HRR (transfer to bed), 36.4 +/- 13.3 %HRR (preparing lunch), 37.1 +/- 12.0 %HRR (washing up), 38.7 +/- 14.9 %HRR (ascending a ramp), 39.8 +/- 15.6 %HRR (transfer to a shower wheelchair), 41.4 +/- 12.1 %HRR (changing sheets), and 45.9 +/- 10.4 %HRR (entering a car). Physical strain could be notably high, but large variations among subjects were present. During all tasks, subjects with tetraplegia had significantly higher levels of strain than subjects with low (T6-L5) lesions. Physical strain was inversely related to parameters of physical capacity: isometric strength (r: -0.34 to -0.72), sprint power (r: -0.34 to -0.69), peak oxygen uptake (r: -0.41 to -0.81) and maximal power output (r: -0.52 to -0.82). Parameters of physical capacity were better predictors of physical strain than was the lesion level, and explained 37-71% of the variance in strain during ADL. It was also concluded that the method used in this study provides a quantitative and objective estimation of physical strain and may therefore be a useful tool to identify task difficulty during rehabilitation and to evaluate the results of task and physical training on the physical strain during ADL.