An mRNA region of the canine distemper virus fusion protein gene lacking AUG codons can promote protein expression.

Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.

Citrate determines calcium oxalate crystallization kinetics and crystal morphology-studies in the presence of Tamm-Horsfall protein of a healthy subject and a severely recurrent calcium stone former.

BACKGROUND: The aim of this study was to measure the effects of normal (nTHP) and abnormal stone former Tamm-Horsfall protein (SF-THP) on calcium oxalate (CaOx) nucleation and aggregation as well as on crystal morphology, in presence or absence of citrate. METHODS: Nucleation and aggregation of CaOx crystals from a supersaturated, stirred solution (200 mM NaCl, 10 mM Na-acetate, pH 5.70, 5 mM Ca and 0.5 mM Ox) were studied by spectrophotometric time-course measurements of OD at 620 nm (OD(620)). Measured parameters were induction time t(I) (time to induce formation of detectable particles), S(N), (slope of increase of OD(620), mainly due to crystal nucleation), and S(A), (slope of decrease of OD(620) after equilibrium has been reached, due to crystal aggregation). Effects of citrate, nTHP and SF-THP on these parameters were measured, and scanning electron microscopy (SEM) was performed. RESULTS: At 1.5, 2.5 and 3.5 mM, citrate increased t(I) and inhibited crystal nucleation (by 78-87%) as well as aggregation (by 63-70%), and smaller CaOx crystals (length/width ratio 1.7+/-0.1) than under standard conditions (length/width 3.9+/-0.5) were visible (P<0.001). Normal THP at 30 and 40 mg/l inhibited crystal nucleation and, more strongly, aggregation (inhibition 76-81%). SEM revealed a decrease in length/width ratio to 2.6+/-0.4 (P=0.051 vs standard conditions) and less aggregation than without nTHP. At all concentrations tested, SF-THP reduced t(I) (P=0.0001 vs standard conditions) and promoted aggregation (inhibition -48 to -33%); crystals were elongated with a length/width ratio of 4.3+/-0.6 (P<0. 05 vs nTHP). When simultaneously present with nTHP, citrate enhanced the inhibitory effects of nTHP, producing the smallest (length/width 1.5+/-0.1) and least aggregated crystals. Finally, 3.5 mM citrate turned promotory SF-THP into a crystallization inhibitor with abundant small and clustered, but not aggregated crystals. CONCLUSION: Citrate appears to be the main determinant of CaOx crystallization rates and crystal morphology in the presence of nTHP as well as SF-THP. Its effects appear to predominate over those of THP, since even promotory SF-THP is turned into a crystallization inhibitor in the presence of citrate. This re-emphasizes at a morphological level what has been concluded from functional as well from clinical studies, namely that citrate is needed in urine at equimolar concentrations to calcium in order to prevent the formation of large crystal aggregates in presence of abnormal THP.

High-calcium intake abolishes hyperoxaluria and reduces urinary crystallization during a 20-fold normal oxalate load in humans.

BACKGROUND: The aim of the study was to test whether increasing dietary calcium intake lowers intestinal oxalate absorption and thereby prevents hyperoxaluria and urinary crystallization during a 20-fold normal oxalate load in healthy subjects. METHODS: Fourteen healthy male volunteers (age 23-44 years, BMI 21.5-27.7 kg/m2) collected 24-h urines while on free-choice diet as well as on two standardized diets. The latter contained 2545 kcal, 2500 ml of mineral water, 102 g of protein, 13.6 g of sodium chloride and 2220 mg of oxalate (approximately 20-fold content of an average diet). Subjects were studied twice while on the standardized diet, once while eating a normal amount of calcium (1211 mg/day, oxalate-rich diet), and once while eating 3858 mg of calcium/day (calcium and oxalate-rich diet). RESULTS: Compared with the free-choice diet (322+/-36 micromol/d), UOx x V increased to 780+/-72 micromol/d on the oxalate-rich diet (P=0.001) and fell again to 326+/-31 micromol/d on calcium and oxalate-rich diet (P=0.001 vs oxalate-rich diet). Urinary glycolate (a metabolic precursor of Ox) always remained below the upper limit of the normal range and did not change between different diets, indicating that changes in UOX x V reflect respective variations in intestinal absorption of Ox. Uca x V was 4.60+/-0.45 mmol/d on the free-choice diet and 3.20+/-0.32 mmol/d on the oxalate-rich diet (P=0.011 vs free-choice diet); it increased to 7.28+/-0.74 mmol/d on the calcium- and oxalate-rich diet (P=0.001 vs free-choice and oxalate-rich diets). As indicated by the AP (CaOx) index (Tiselius), urinary supersaturation did not vary significantly between the three diets. In freshly voided morning urines (studied in 8/14 subjects) on the oxalate-rich diet, CaOx crystals or crystal aggregates of up to 80 microm diameter were found in 5/8 urines, whereas this never occurred on the free-choice diet and only t once on the calcium- and oxalate-rich diet. CONCLUSION: . Increasing calcium intake while eating Ox-rich food prevents dietary hyperoxaluria and reduces CaOx crystallization in healthy subjects. This further illustrates that dietary counseling to idiopathic calcium-stone formers should ensure sufficient calcium intake, especially during oxalate-rich meals.

Simultaneous measurements of calcium oxalate crystal nucleation and aggregation: impact of various modifiers.

Rates of nucleation and aggregation of calcium oxalate crystals were derived from 20-min time course measurements of OD620 after mixing solutions containing CaCl2 and K2C2O4 at 37 degrees C, pH 5.7, ionic strength (IS) 0.21, with constant stirring (500 rpm); final assay concentrations were 4.25 mM calcium and 0.5 mM oxalate, respectively. The maximum increase of OD620 with time, termed SN, mainly reflects maximum rate of formation of new particles and thus crystal nucleation. After equilibrium has been reached, OD620 progressively decreases despite ionized calcium staying constant and no new particles being formed, due to crystal aggregation. Rate of aggregation, SA, is derived from the maximum decrease in OD620 with time. SN and SA are not independent, as indicated by a positive correlation (r = 0.844, P = 0.0001). Among the modifiers studied, citrate at 0.5-2.5 mM lowered both SN and SA in a concentration-dependent manner (P < 0.01 for all comparisons vs control). Chondroitin-6-sulfate at 6.25-25 mg/l moderately lowered SN, whereas it strongly inhibited aggregation (P < 0.01 vs control). At 6.8-20.4 mg/l, albumin did not affect nucleation, whereas it inhibited aggregation in a concentration-dependent manner (P < 0.005 vs control for all comparisons).

Citrate and calcium effects on Tamm-Horsfall glycoprotein as a modifier of calcium oxalate crystal aggregation.

We measured the effects of Tamm-Horsfall glycoprotein (THP) on calcium oxalate monohydrate (COM) crystal aggregation (Ac) in vitro as well as intrinsic viscosities (Vi) of THP at pH 5.7 and 200 mM NaCl and studied the effects of calcium and citrate on these parameters. THP were isolated from 24-h urines of seven male recurrent calcium stone formers (RCSF) and eight age-matched male healthy volunteers (N, normal). At basal conditions, RCSF-THP inhibited Ac by 28 +/- 10% and normal THP by 60 +/- 6% (P = 0.028). In the presence of calcium, increasing THP concentrations from 16 to 28 and 40 mg/l progressively lowered inhibition by RCSF-THP, but not by N-THP. At 40 mg/l, inhibition by N-THP was 27 +/- 9% vs. -43 +/- 8% by RCSF-THP (P = 0.001), i.e., all stone former THP promoted Ac. With an additional 3.5 mM of citrate, inhibition of Ac was 56 +/- 5% by normal and 34 +/- 6% by stone former THP (P = 0.021), and all seven stone former THP again inhibited Ac. Vi of RCSF-THP was higher than that of N at basal conditions (162 +/- 21 vs. 93 +/- 15 ml/g, P = 0.021) and in the presence of 5 mM calcium (352 +/- 54 vs. 118 +/- 17 ml/g, P = 0.001), i.e., RCSF-THP were more self-aggregated, but not when citrate was added (185 +/- 29 vs. 123 +/- 19 ml/g). (ABSTRACT TRUNCATED AT 250 WORDS)