Calcineurin A‐β is required for hypertrophy but not matrix expansion in the diabetic kidney

Calcineurin is an important signalling protein that regulates a number of molecular and cellular processes. Previously, we found that inhibition of calcineurin with cyclosporine reduced renal hypertrophy and blocked glomerular matrix expansion in the diabetic kidney. Isoforms of the catalytic subunit of calcineurin are reported to have tissue specific expression and functions. In particular, the β isoform has been implicated in cardiac and skeletal muscle hypertrophy. Therefore, we examined the role of calcineurin β in diabetic renal hypertrophy and glomerular matrix expansion. Type I diabetes was induced in wild-type and β^−/− mice and then renal function, extracellular matrix expansion and hypertrophy were evaluated. The absence of β produced a significant decrease in total calcineurin activity in the inner medulla (IM) and reduced nuclear factor of activated T-cells (NFATc) activity. Loss of β did not alter diabetic renal dysfunction assessed by glomerular filtration rate, urine albumin excretion and blood urea nitrogen. Similarly, matrix expansion in the whole kidney and glomerulus was not different between diabetic wild-type and β^−/− mice. In contrast, whole kidney and glomerular hypertrophy were significantly reduced in diabetic β^−/− mice. Moreover, β^−/− renal fibroblasts demonstrated impaired phosphorylation of Erk1/Erk2, c-Jun N-terminal kinases (JNK) and mammalian target of rapamycin (mTOR) following stimulation with transforming growth factor-β and did not undergo hypertrophy with 48 hrs culture in high glucose. In conclusion, loss of the β isoform of calcineurin is sufficient to reproduce beneficial aspects of cyclosporine on diabetic renal hypertrophy but not matrix expansion. Therefore, while multiple signals appear to regulate matrix, calcineurin β appears to be a central mechanism involved in organ hypertrophy.     

Effect of surgery to implant motion and force sensors on vertical ground reaction forces in the ovine model

Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruciate ligament (ACL). While calibration problems preclude direct measurement in patients, ACL forces can conceivably be measured in animals after technical challenges are overcome. For example, motion and force sensors can be implanted in the animal but investigators must determine the extent to which these sensors and surgery affect normal gait. Our objectives in this study were to determine (1) if surgically implanting knee motion sensors and an ACL force sensor significantly alter normal ovine gait and (2) how increasing gait speed and grade on a treadmill affect ovine gait before and after surgery. Ten skeletally mature, female sheep were used to test four hypotheses: (1) surgical implantation of sensors would significantly decrease average and peak vertical ground reaction forces (VGRFs) in the operated limb, (2) surgical implantation would significantly decrease single limb stance duration for the operated limb, (3) increasing treadmill speed would increase VGRFs pre- and post operatively, and (4) increasing treadmill grade would increase the hind limb VGRFs pre- and post operatively. An instrumented treadmill with two force plates was used to record fore and hind limb VGRFs during four combinations of two speeds (1.0 m/s and 1.3 m/s) and two grades (0 deg and 6 deg). Sensor implantation decreased average and peak VGRFs less than 10% and 20%, respectively, across all combinations of speed and grade. Sensor implantation significantly decreased the single limb stance duration in the operated hind limb during inclined walking at 1.3 m/s but had no effect on single limb stance duration in the operated limb during other activities. Increasing treadmill speed increased hind limb peak (but not average) VGRFs before surgery and peak VGRF only in the unoperated hind limb during level walking after surgery. Increasing treadmill grade (at 1 m/s) significantly increased hind limb average and peak VGRFs before surgery but increasing treadmill grade post op did not significantly affect any response measure. Since VGRF values exceeded 80% of presurgery levels, we conclude that animal gait post op is near normal. Thus, we can assume normal gait when conducting experiments following sensor implantation. Ultimately, we seek to measure ACL forces for ADLs to provide design criteria and evaluation benchmarks for traditional and tissue engineered ACL repairs and reconstructions.     

Protandim attenuates intimal hyperplasia in human saphenous veins cultured ex vivo via a catalase-dependent pathway

Human saphenous veins (HSVs) are widely used for bypass grafts despite their relatively low long-term patency. To evaluate the role of reactive oxygen species (ROS) signaling in intima hyperplasia (IH), an early stage pathology of vein-graft disease, and to explore the potential therapeutic effects of up-regulating endogenous antioxidant enzymes, we studied segments of HSV cultured ex vivo in an established ex vivo model of HSV IH. Results showed that HSV cultured ex vivo exhibit an ~ 3-fold increase in proliferation and ~ 3.6-fold increase in intimal area relative to freshly isolated HSV. Treatment of HSV during culture with Protandim, a nutritional supplement known to activate Nrf2 and increase the expression of antioxidant enzymes in several in vitro and in vivo models, blocks IH and reduces cellular proliferation to that of freshly isolated HSV. Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide (O₂^??) and the lipid peroxidation product 4-HNE. Blocking catalase activity by cotreating with 3-amino-1,2,4-triazole abrogated the protective effect of Protandim on IH and proliferation. In conclusion, these results suggest that ROS-sensitive signaling mediates the observed IH in cultured HSV and that up-regulation of endogenous antioxidant enzymes can have a protective effect.     

IGF-I and mechanical environment interact to modulate engineered cartilage development

Bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid scaffolds and cultured for four weeks using in vitro systems providing different mechanical environments (static and mixed Petri dishes, static and mixed flasks, and rotating vessels) and different biochemical environments (medium with and without supplemental insulin-like growth factor I, IGF-I). Under all conditions, the resulting engineered tissue histologically resembled cartilage and contained its major constituents: glycosaminoglycans, collagen, and cells. The mechanical environment and supplemental IGF-I (a) independently modulated tissue morphology, growth, biochemical composition, and mechanical properties (equilibrium modulus) of engineered cartilage as previously reported; (b) interacted additively or in some cases nonadditively producing results not suggested by the independent responses, and (c) in combination produced tissue superior to that obtained by modifying these factors individually.     

Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.     

Precision of the Gonyaulax circadian clock

Under constant conditions, the circadian bioluminescent glow rhythm in populations (10(5) cells) of Gonyaulax polyedra is accurate to within 2 min/day. On successive days following the transfer to constant conditions, however, the glow exhibits a progressively broader waveform, implying that individual clocks in the population are drifting out of synchrony. Analysis of the glow waveform suggests that the standard deviation in circadian period among individual clocks is about 18 min and that the period of a given clock varies by less than this from one day to the next.     

Crystallization of the complex of actin with gelsolin segment 1.

Crystals of a 1:1 complex between human gelsolin segment 1 and actin have been grown from solutions containing polyethylene glycol 6000. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 57.4 A, b = 70.4 A, c = 184.5 A. They are moderately stable to X-rays and diffract to beyond 2.5 A. There is one molecule of complex in the asymmetric unit.     

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products

Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar.