Polymorphic Protein Crystal Growth: Influence of Hydration and Ions in Glucose Isomerase.

Crystal polymorphs of glucose isomerase were examined to characterize the properties and to quantify the energetics of protein crystal growth. Transitions of polymorph stability were measured in poly(ethylene glycol)/NaCl solutions, and one transition point was singled out for more detailed quantitative analysis. Single crystal x-ray diffraction was used to confirm space groups and identify complementary crystal structures. Crystal polymorph stability was found to depend on the NaCl concentration, with stability transitions requiring > 1 M NaCl combined with a low concentration of PEG. Both salting-in and salting-out behavior was observed and was found to differ for the two polymorphs. For NaCl concentrations above the observed polymorph transition, the increase in solubility of the less stable polymorph together with an increase in the osmotic second virial coefficient suggests that changes in protein hydration upon addition of salt may explain the experimental trends. A combination of atomistic and continuum models was employed to dissect this behavior. Molecular dynamics simulations of the solvent environment were interpreted using quasi-chemical theory to understand changes in protein hydration as a function of NaCl concentration. The results suggest that protein surface hydration and Na+ binding may introduce steric barriers to contact formation, resulting in polymorph selection.

Applicability of the Ussing chamber technique to permeability determinations in functionally distinct regions of the gastrointestinal tract in the rat.

BACKGROUND: Ussing chambers are commonly utilized for in vitro investigations into gastrointestinal permeability. However, their sensitivity and applicability to the small intestine have not been well characterized. METHODS: In order to investigate the effects of experimentally induced damage and the relative contribution of the mucosa and muscularis externa layers to transmural permeability in the small intestine, stomach and colon, normal rat intestinal tissues were mounted in Ussing chambers with or without removal of the muscularis externa or mucosal layers. Gastric tissues were damaged in vivo by exposure to indomethacin (100 mg kg(-1)), while ileal tissues were damaged in vitro by 0.4 M NaCl. Tissue damage was assessed histologically, while permeability parameters included conductance (G), potential difference (PD) and mucosal to serosal flux of horseradish peroxidase (HRP). RESULTS: Damage localized to the tissue edges (edge damage) accounted for 25%-50% of the exposed epithelial length in the ileum, while less than 20% of stomach and colon epithelium was affected by edge damage. In the damaged stomach, a 20% reduction in epithelialization was accompanied by increases in G (P < 0.001) and HRP (P < 0.01) flux. Removal of the muscularis externa did not affect mucosal viability in the undamaged ileum or colon although HRP flux in the colon, but not ileum, was increased (P < 0.01). Removal of the ileal mucosa produced increases in G and HRP flux, while PD was maintained. CONCLUSION: We conclude that the Ussing chamber technique is suitable for application to studies of gastric and colonic permeability in rats. However, owing to the prevalence and extent of edge damage in the small intestine, we would caution against the use of this technique for permeability studies in this region of the gastrointestinal tract in the rat.

Effects of insulin-like growth factor-I and LR3IGF-I on regional blood flow in normal rats.

Two studies were conducted to investigate the haemodynamic effects of IGF-I and its analogue LR3IGF-I in normal anaesthetised rats. Infusion of IGF-I intravenously, at a dose of 125 micrograms/kg/h, for 20 min in the first study resulted in renal blood flow being significantly elevated by 35% above baseline. Mean arterial blood pressure (MABP) at this IGF-I dose fell by 18% of baseline, with LR3IGF-I also causing a significant decline in MABP (by 15%) at the dose of 125 micrograms/kg/h. In the second study the intravenous administration of IGF-I or LR3IGF-I, at a dose of 125 micrograms/kg/h, over a period of 60 min, resulted in MABP being significantly lowered by 25% of baseline values. Regional blood flow rates were determined using radioactive microspheres, 15 microns in diameter, injected systemically at the end of the peptide infusion period. The gastrocnemius, a representative skeletal muscle, was the only vascular region to show a significant increase in blood flow after IGF-I (by 58%) or LR3IGF-1 (by 308%) infusion. Vascular resistance in the brain was significantly reduced after infusion of IGF-I (by 60%) or LR3IGF-I (by 48%) as compared with vehicle. Skeletal muscle vascular resistance was also reduced by IGF-I (by 41%) and more particularly by LR3IGF-I (by 77%) in comparison to vehicle. These alterations to vascular tone produced by IGF infusion may be related to the central nervous system and systemic cardiovascular side-effects that have been reported during IGF-I administration in humans.

Effects of chronic renal failure on plasma clearance of insulin-like growth factor I, des-(1-3)IGF-I, and LR3IGF-I.

Using a rat model of chronic renal failure (CRF), we examined insulin-like growth factor I (IGF-I) clearance, degradation, organ distribution, and IGF binding profiles in plasma. The effects of IGF-binding proteins (IGFBP) on IGF clearance and degradation in CRF were studied using the IGF-I analogues des-(1-3)IGF-I and LR3IGF-I, which bind poorly to IGFBP. Although total clearance of IGF-I was not significantly altered in CRF, half-life and area under the curve were increased in the rapid distribution phase and were reduced in the slow elimination phase. Total clearance of LR3IGF-I was significantly increased. Reduced binding of IGF-I in the 150-kDa complex and increased binding to smaller-molecular-weight IGFBP were observed in CRF. Increased degradation of both IGF-I and LR3IGF-I was associated with reduced IGF binding in the 150-kDa complex. The results suggest that the accumulation of lower-molecular-weight IGFBP with reduced IGF binding in the 150-kDa complex, associated with increased degradation of peptide, may explain, at least in part, the reduced bioactivity of IGF-I observed in CRF.

Enhanced body growth in uremic rats treated with IGF-I and growth hormone in combination.

The efficacy of seven days treatment with IGF-I (1.7 mg/kg/day), growth hormone (GH) (2 mg/kg/day), IGF-I+GH (1.7 mg/kg/day + 2 mg/kg/day) or vehicle, in promoting growth was investigated in female Sprague-Dawley rats with 5/6 nephrectomies (N = 8 per group). Treatment commenced after chronic renal failure had been present for seven weeks. Significant increases in body weight gain were found in all groups versus control, with IGF-I+GH causing the greatest response, and increased body weight gains correlating with increased nitrogen retention. GH treatment alone significantly stimulated food intake. IGF-I+GH resulted in close to additive increases in food conversion efficiency (18.8%, 21.5% and 39.6% increases with IGF-I, GH and IGF-I+GH, respectively, over control levels) and longitudinal bone growth (39%, 37% and 67% increases with IGF-I, GH and IGF-I+GH, respectively, vs. control). Serum insulin and cholesterol levels significantly decreased with IGF-I and IGF-I+GH treatment. Creatinine clearance did not change, suggesting there were no effects of treatment on kidney function. Although IGF-I at the doses used did not result in a greater anabolic response than GH, IGF-I+GH caused significantly enhanced growth while reducing serum insulin and cholesterol levels.

Ultraviolet sensitivity of three cone types in the aphakic observer determined by chromatic adaptation.

Sensitivities at 5.5 deg off-fovea from the aphakic observer superimposed on orange, purple and blue fields were obtained to estimate short-, middle- and long-wavelength cone spectra respectively. The middle-wavelength mechanism had a visible wavelength maximum resembling a nomogram plus an UV sensitivity fitting a cis-peak. The short (and, to a lesser extent, the long) wavelength cone sensitivities are higher in the UV than expected.

Effects of insulin-like growth factor-I peptides in rats with acute renal failure.

The effect of insulin-like growth factor-I (IGF-I) administration on body weight gain and the rate of recovery of renal function was investigated in rats following an acute episode of renal ischaemia. Since the des(1-3)IGF-I and LR3IGF-I variant forms of IGF-I have been shown to be more potent than IGF-I, their effects were also examined. Acute renal failure was produced in male Sprague-Dawley rats by clamping both renal arteries for 45 min. Treatment was commenced at the time of renal artery occlusion with vehicle (0.1 mol acetic acid/l; control group), IGF-I (2.0 mg/kg per day), des(1-3)IGF-I (2.0 mg/kg per day) or LR3IGF-I (1.5 mg/kg per day) by s.c. osmotic pump, and continued for 7 days, with rats being held in metabolism cages. Glomerular filtration rate (GFR) was estimated by the use of 51Cr-EDTA continuously infused i.p. via osmotic pump. Following the episode of renal ischaemia, body weight gain and nitrogen retention were significantly improved in all three peptide-treated groups, and serum urea concentrations were reduced in the groups treated with IGF-I and des(1-3)IGF-I. However, there was no evidence of the variants having any increased potency over the growth effects of IGF-I itself. GFR was significantly reduced, urine output was increased and urinary concentrating ability was reduced in all groups compared with normal rats, with no significant effect of the IGF peptides being apparent. A closer examination of the acute effects of LR3IGF-I on renal function was undertaken by measuring GFR for 3 days before and 3 days after renal ischaemia in two groups of rats, treated for the latter 3 days with either vehicle (controls) or LR3IGF-I (1.5 mg/kg per day). LR3IGF-I treatment following renal ischaemia resulted in a significantly greater fall in GFR than in controls, urinary osmolality was also significantly reduced, and fractional excretion of sodium was increased. In addition, there was histological evidence of a greater degree of tubular epithelial calcification in the kidneys of the rats treated with LR3IGF-I. This study showed that administration of IGF peptides at doses sufficient to cause significant improvement in anabolic status did not improve renal function in rats following an acute episode of renal ischaemia. Indeed the LR3IGF-I variant of IGF-I had a deleterious effect on renal function in the early stage of the recovery period.

Genetic heterogeneity in X-linked amelogenesis imperfecta.

The AMELX gene located at Xp22.1-p22.3 encodes for the enamel protein amelogenin and has been implicated as the gene responsible for the inherited dental abnormality X-linked amelogenesis imperfecta (XAI). Three families with XAI have been investigated using polymorphic DNA markers flanking the position of AMELX. Using two-point linkage analysis, linkage was established between XAI and several of these markers in two families, with a combined lod score of 6.05 for DXS16 at theta = 0.04. This supports the involvement of AMELX, located close to DXS16, in the XAI disease process (AIH1) in those families. Using multipoint linkage analysis, the combined maximum lod score for these two families was 7.30 for a location of AIH1 at 2 cM distal to DXS16. The support interval around this location extended about 8 cM proximal to DXS92, and the AIH1 location could not be precisely defined by multipoint mapping. Study of recombination events indicated that AIH1 lies in the interval between DXS143 and DXS85. There was significant evidence against linkage to this region in the third family, indicating locus heterogeneity in XAI. Further analysis with markers on the long arm of the X chromosome showed evidence of linkage to DXS144E and F9 with no recombination with either of these markers. Two-point analysis gave a peak lod score at DXS144E with a maximum lod score of 2.83 at theta = 0, with a peak lod score in multipoint linkage analysis of 2.84 at theta = 0. The support interval extended 9 cM proximal to DXS144E and 14 cM distal to F9.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I and its N-terminal modified analogues induce marked gut growth in dexamethasone-treated rats.

The effects of insulin-like growth factor-I (IGF-I) on the gut of 150 g dexamethasone-treated rats were compared with those of two analogues with reduced affinity for IGF-binding proteins, des(1-3)IGF-I and LR3IGF-I, an N-terminal-extended variant. Administration of IGF-I for 7 days to rats made catabolic by co-treatment with dexamethasone induced a dose-dependent increase in total gut weight, with the highest dose of IGF-I (695 micrograms/day) increasing gut weight by up to 60%, and gut weight as a fraction of body weight by up to 32%. Effects were apparent in all regions of the gut examined, including the stomach, small intestine and colon. Histological and biochemical analyses of the intestine showed that cross-sectional mass, rather than gut length, was increased, and proportional increases in wet weight, protein and DNA content per unit length were measured in both the mucosa and muscularis layers. The rate of duodenal protein synthesis measured on day 7 of treatment was not increased by IGF-I treatment. The IGF-I analogues had qualitatively similar effects to IGF-I, but were consistently severalfold more potent, providing evidence that IGF-binding proteins reduce the biological activity of exogenous IGF-I in the gut. The results indicate that the gut is one of the most sensitive IGF-I target tissues, and that potency in vivo correlates with a reduced interaction with IGF-binding proteins.

Strain differences in the opsonisation of Staphylococcus epidermidis.

Ten isolates of coagulase-negative staphylococci, collected from patients receiving treatment with continuous ambulatory peritoneal dialysis (CAPD), exhibited marked differences in the degree of opsonisation when incubated in 10% and 1% pooled human serum, 10% and 1% heat-treated serum, Hanks' Balanced Salt Solution, and timed peritoneal dialysis (PD) effluent. The addition of exogenous IgG to PD effluent results in a greater increase in opsonisation in those fluids with the weakest inherent opsonic activity, but is ineffective against the majority of isolates in the absence of heat-labile opsonic activity. The results of this in vitro study suggest that host resistance to CAPD peritonitis due to coagulase-negative staphylococci may be determined as much by the characteristics of the contaminating strain, as by the opsonising activity of PD effluent.

HLA-A1 and steroid-induced cataracts in renal transplant patients.

Posterior subcapsular (PSC) lens changes were found in 32 of 33 steroid-treated renal transplant patients examined with the slit lamp techniques of direct and retroillumination. Only one of 33 age-matched controls had any PSC changes. Six (18%) of the renal transplant patients were bilaterally surgically aphakic because of PSC cataracts that developed while the patient was receiving steroid therapy after transplant surgery. Steroid-induced PSC cataracts were associated with the presence of human leukocyte antigen (HLA) A1, but not with the patient's age, cause of renal failure, total dose of steroids, or duration of steroid therapy.

Clear air turbulence: detection by infrared observations of water vapor.

"Forward-looking" infrared measurements of water vapor from the C-141A Kuiper Airborne Observatory of the National Aeronautics and Space Administration Ames Research Center show large, distinctly identifiable, signal anomalies from 4 to 10 minutes in advance of subsequent encounters with clear air turbulence (CAT). These anomalies are characteristically different from the signals not followed by CAT encounters. Results of airborne field trials in which the infrared radiometer was used indicate that, out of 51 situations, 80 percent were CAT alerts followed by CAT encounters, 12 percent were "false alarms" (CAT alerts not followed by CAT encounters), and 8 percent were CAT encounters not preceded by an infrared signal anomaly or CAT alert.