Direct evidence that KLK4 is a hydroxyapatite-binding protein

The protease kallikrein 4 (KLK4) plays a pivotal role during dental enamel formation by degrading the major enamel protein, amelogenin, prior to the final steps of enamel hardening. KLK4 dysfunction is known to cause some types of developmental defect in enamel but the mechanisms responsible for transient retention of KLK4 in semi-hardened enamel matrix remain unclear. To address contradictory reports about the affinity of KLK4 for enamel hydroxyapatite-like mineral, we used pure components in quasi-physiological conditions and found that KLK4 binds hydroxyapatite directly. Hypothesising KLK4 self-destructs once amelogenin is degraded, biochemical analyses revealed that KLK4 progressively lost activity, became aggregated, and autofragmented when incubated without substrate in both the presence and absence of reducer. However, with non-ionic detergent present as proxy substrate, KLK4 remained active and intact throughout. These findings prompt a new mechanistic model and line of enquiry into the role of KLK4 in enamel hardening and malformation.     

The biochemical basis of mitochondrial diseases

Disfunctioning of human mitochondria is found in a rapidly increasing number of patients. The mitochondrial system for energy transduction is very vulnerable to damage by genetic and environmental factors. A primary mitochondrial disease is caused by a genetic defect in a mitochondrial enzyme or translocator. More than 60 mitochondrial enzyme deficiencies have been reported. Secondary mitochondrial defects are caused by lack of compounds to enable a proper mitochondrial function or by inhibition of that function. This may result from malnutrition, circulatory or hormonal disturbances, viral infection, poisoning, or an extramitochondrial error of metabolism. Once mitochondrial ATP synthesis decreases, secondary mitochondrial lesions may be generated further, due to changes in synthesis and degradation of mitochondrial phospholipids and proteins, to mitochondrial antibody formation following massive degradation, to accumulation of toxic products as excess acyl-CoA, to the depletion of Krebs cycle intermediates, and to the increase of free radical formation and lipid peroxidation.     

Non-selective cation and dysfunctional chloride cahnnels in the apical membrane of nasal epithelial cells cultured from cystic fibrosis patients

Chloride channels and non-selective cation channels in the apical membranes of cultured nasal epithelial cells from three cystic fibrosis patients were investigated with the patch-clamp techinique. Outwardly rectifying chloride channels were found in 31% of the inside-out patches, but activity of this channel was never observed in cell-attached patches, even after stimulation with adrenaline. In 30% of the patches with chloride channels, activation occurred immediately after excision. Most of the channels, however, activated only after a membrane depolarization of +40 to +120 mV. Once activated, the chloride channels were indistinguishable from thsoe in nasal epithelial cells of control patients. Amiloride-insensitive, calcium- and voltage-dependent, non-selective cation channels were present in 11% of the cell-attached and 43% of the cell-free patches and could not be distinguished from those in controls. The cystic fibrosis chloride channel defect is conserved in cultured nasal epithelial cells, while a non-selective cation channel is apparently not affected.