Gitelman syndrome associated with chondrocalcinosis and severe neuropathy: a novel heterozygous mutation in SLC12A3 gene.

Gitelman syndrome (GS) is an inherited salt-wasting tubulopathy characterized by hypocalciuria, hypokalemia, hypomagnesemia and metabolic alkalosis, due to inactivating mutations in the SLC12A3 gene. Symptoms may be systemic, neurological, cardiovascular, ophthalmological or musculoskeletal. We describe a 70 year-old patient affected by recurrent arthralgias, hypoesthesia and hyposthenia in all 4 limbs and severe hypokalemia, complicated by atrial flutter. Moreover, our patient reported eating large amounts of licorice, and was treated with medium-high dosages of furosemide, thus making diagnosis very challenging. Genetic analysis demonstrated a novel heterozygous mutation in the SLC12A3 gene; therefore, we diagnosed GS and started potassium and magnesium replacement. GS combined with chondrocalcinosis and neurological involvement is quite common, but this is the first case of an EMG-proven severe neuropathy associated with GS. Herein, we underline the close correlation between hypomagnesemia, chondrocalcinosis and neurological involvement. Moreover, we report a new heterozygous mutation in exon 23 (2738G>A), supporting evidence of a large genetic heterogeneity in this late-onset congenital tubulopathy.

[Assessment of urinary cotinine in a group occupationally exposed to nicotine]. / Valutazione della cotinina urinaria in un gruppo di esposti professionalmente alla nicotina.

In a Tobacco processing company, located in central Italy, both air nicotine and, one of its metabolites, the urinary cotinine were assessed, as part of the evaluation of the effectiveness of recently implemented technical preventive controls. Urinary cotinine measurements ranged from 9,63 to 234,09 microg/gr creat. in non-smokers and from 178,79 to 2476,19 microg/gr creat. in smokers. Urinary cotinine proved useful as an exposure biological index especially in terms of population monitoring. On the contrary, the meaning of the measured values were less clear in single workers, because of significant both intra- and inter-individual variability.

Dimerization, stability and electrostatic properties of porcine beta-lactoglobulin.

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico-chemical characterization of porcine beta-lactoglobulin, purified from pooled milk: size-exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine beta-lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer-dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess alpha helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine beta-lactoglobulin in the crystal structure. Interaction studies revealed that porcine beta-lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.

NMR structural determination of viscotoxin A3 from Viscum album L.

The high-resolution three-dimensional structure of the plant toxin viscotoxin A3, from Viscum album L., has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 12 degrees C (the structure has been deposited in the Protein Data Bank under the id. code 1ED0). Experimentally derived restraints including 734 interproton distances from nuclear Overhauser effect measurements, 22 hydrogen bonds, 32 φ angle restraints from J coupling measurements, together with three disulphide bridge constraints were used as input in restrained molecular dynamics, followed by minimization, using DYANA and Discover. Backbone and heavy atom root-mean-square deviations were 0.47+/-0.11 A (1 A=10(-10) m) and 0.85+/-0.13 A respectively. Viscotoxin A3 consists of two alpha-helices connected by a turn and a short stretch of antiparallel beta-sheet. This fold is similar to that found in other thionins, such as crambin, hordothionin-alpha and -beta, phoratoxin A and purothionin-alpha and -beta. The difference in the observed biological activity for thionins of known structure is discussed in terms of the differences in the calculated surface potential distribution, playing an important role in their function through disruption of cell membranes. In addition, the possible role in DNA binding of the helix-turn-helix motif of viscotoxin A3 is discussed.

Simulation of electrostatic effects in Fab-antigen complex formation.

A model based on the Poisson-Boltzmann equation has been used to model electrostatics in Anti-p24 (HIV-1) Fab-antigen association. The ionization state at different pH values has been simulated and the results have been used to estimate the stability at different pH values and to generate electrostatic potential maps at physiological ionic strength. The analysis of the electrostatic potential at the solvent-accessible surface shows that residues involved in binding are mostly found in the highest, but also in lowest potential regions. Brownian dynamics simulations have been used to estimate the enhancement of the association rate due to electrostatics which appears limited (approximately 2 at 150 mM ionic strength and approximately 3 at 15 mM ionic strength). A much more pronounced effect is observed upon increase of the charge of the diffusing particle. These results compare well with results obtained previously in similar studies on different systems and may serve to estimate the expected order of magnitude of electrostatic effects on association rates in antibody-antigen systems.

Electrostatic properties of bovine beta-lactoglobulin.

Bovine beta-Lactoglobulin (BLG) has been studied for many decades, but only recently structural data have been obtained, making it possible to simulate its molecular properties. In the present study, electrostatic properties of BLG are investigated theoretically using Poisson-Boltzmann calculations and experimentally following pH titration via NMR. Electrostatic properties are determined for several structural models, including an ensemble of NMR structures obtained at low pH. The changes in electrostatic forces upon changes in ionic strength, solvent dielectric constant, and pH are calculated and compared with experiments. pK(a)s are computed for all titratable sites and compared with NMR titration data. The analysis of theoretical and experimental results suggests that (1) there may be more than one binding sites for negatively charged ligands; (2) at low pH the core of the molecule is more compact than observed in the structures obtained via restrained molecular dynamics from NMR data, but loop and terminal regions must be disordered.

Intermolecular protein interactions in solutions of bovine lens beta L-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles.

We report the magnetic field dependence of 1/T1 of solvent water protons and deuterons (nuclear magnetic relaxation dispersion, or NMRD, profiles) for solutions of steer lens beta L-crystallin. Such data allow the study of intermolecular protein interactions over a wide concentration range, here 1-34% vol/vol, by providing a measure of the rotational relaxation time of solute macromolecules. We conclude that, for approximately less than 5% protein, the solute particles are noncompact, with a rotationally averaged volume approximately three times that of a compact 60-kD sphere. (Earlier results for alpha-crystallin, approximately 1,000 kD, from optical and osmotic measurements (Vérétout and Tardieu, 1989. J. Mol. Biol. 205:713-728), show a similar, approximately twofold, effect). At intermediate concentrations, to approximately 20% protein, there is evidence for limited association or oligomerization, as found for the structurally related gamma II-crystallin (Koenig et al. 1990. Biophys. J. 57:461-469), to a limiting size about two-thirds that of alpha-crystallin. The difference in NMRD behavior of the three classes of crystallins is consonant with their differing osmotic properties (Vérétout and Tardieu. J. Mol. Biol. 1989, 205:713-728; Kenworthy, McIntosh, and Magid. Biophys. J. 1992. 61:A477; Tardieu et al. 1992. Eur. Biophys. J. 21:1-12). We indicate how the unusual structures and interactions of these three classes of proteins can be combined to optimize transparency and minimize colloid osmotic difficulties in eye lens.

Magnetization transfer in cross-linked bovine serum albumin solutions at 200 MHz: a model for tissue.

We report results for proton 1/T1, 1/T2, and K, the rate of magnetization transfer from solvent to solute, for 5 and 10 wt. % solutions of bovine serum albumin, both native and chemically cross-linked, in undeuterated and approximately 50% deuterated water, at 4.7 T (200.1 MHz) and 19 degrees C. At this field, although K > 1/T1 for the cross-linked samples, magnetization transfer contributes little to 1/T1 directly. Therefore K was measured using off-resonance irradiation of the protein protons. The data for all the samples can be fit using a theoretical model for magnetization transfer, with three parameters: the intrinsic longitudinal relaxation rates of solute and solvent protons, and K. The magnitude of K is so large that the newly-identified, long-lived (approximately 1 microseconds) hydration sites (S.H. Koenig, R.D. Brown III, and R. Ugolini, Magn. Reson. Med., 29, 77 (1993)) must be invoked to account for K, as is necessary to explain the differential effects of cross linking on the magnetic field dependence of 1/T1 of protons and deuterons and the large 1/T1 and 1/T2 values below approximately 20 MHz in immobilized systems. Although these sites are few in number, their long resident lifetime becomes the correlation time for magnetization transfer when protein is immobilized, accounting for the large value of K. Recent data from several laboratories have shown that cross-linked protein, as used here, is a good model for 1/T1 and 1/T2 of tissue, as a function of temperature and magnetic field.

A unified view of relaxation in protein solutions and tissue, including hydration and magnetization transfer.

Protein in water solution increases magnetic relaxation rates of solvent nuclei to an extent that depends on magnetic field strength and molecular weight. Koenig and Schillinger (J. Biol. Chem. 244, 3283 (1969)) showed that a small fraction of the water molecules in the first hydration shell, bound irrotationally with a residence lifetime in the range 0.1 to 10 microseconds, would account for the phenomena. No experiments, as yet, have proven the existence of such long-lived waters, nor yielded a value for their lifetime. Analogous measurements on solutions of both denatured and cross-linked protein give data different from that of native protein, but much like results for tissue. By comparing proton and deuteron relaxation rates in solutions of native and cross-linked protein, it is possible to demonstrate the existence of these relatively long-lived waters; the data indicate that 1% of a monolayer of the waters of hydration of protein have lifetimes that cluster near 1 microsecond and, it is argued, are held in place by multiple hydrogen bonds. Assigning shorter lifetimes for waters held by fewer bonds, it is possible to develop a unified view of relaxation of water nuclei in protein solutions and in tissue, and to relate it to recent crystallographic data on hydrated protein.