Coupling between substrate binding and allosteric regulation in ribozyme catalysis.

The contribution of substrate binding to allosteric regulation in the ribozyme catalysis has been investigated using allosteric ribozymes consisting of the hammerhead ribozyme and a flavin mononucleotide (FMN) aptamer. Kinetic parameters were measured for various lengths of the substrates with a wide range of binding energy. The maximum cleavage rate of each ribozyme was retained with the long substrates. However, the cleavage rates largely decreased by the truncation of the substrates according to loss in the free energy of substrate binding. The high sensitivity to the substrate lengths is attributed to the increase in the energetic requirement for the catalytic core folding, which is caused by the incorporation of the aptamer region. One role of FMN binding is assisting the promotion of the core folding through the stabilization of the aptamer domain. The allosteric effect is significantly expressed only when the substrate binding energy is insufficient for the core folding of the ribozyme-substrate complex. This type of allosteric interaction dominates the substrate dependency of another type of regulation. These results demonstrate that an adequate correlation between the type of regulation and the substrate binding is responsible for the effective allosteric interaction in the kinetic process.

Protein kinase C activator inhibits progression of osteoarthritis induced in rabbit knee joints.

To study the role of protein kinase C (PKC) in cartilage tissue in osteoarthritis, experimental osteoarthritis was induced in the knee joints of rabbits by resection of the anterior cruciate ligament (ACL). At 4 weeks after the operation, osteoarthritic changes varying from surface irregularities and cleft formation to loss of the tangential layer were observed, and cloning or hypocellularity of the chondrocytes was observed mainly in the transitional and radial layers. The PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or non-PKC-activating phorbol ester 4 alpha-phorbol-12,13-didecanate (PDD) was administered intraarticularly once a week from the day of the operation for 3 weeks. Histologic evaluation with a rating scale was carried out. In the TPA-administered group, cartilage structures were preserved almost completely, and score of the cartilage lesion was significantly less than that in animals administered PDD or in nonadministered controls. A chondroprotective role of PKC under mechanical stress was suggested.

Alpha and epsilon isozymes of protein kinase C in the chondrocytes in normal and early osteoarthritic articular cartilage.

Protein kinase C (PKC) isozymes (alpha, beta I, beta II, gamma and, epsilon-PKC) were examined immunocytochemically in control and mechanically induced osteoarthritic knee joints in rats. beta I, beta II, and gamma PKC-positive cells were not observed in sham-operated or osteoarthritic knee joints. alpha-PKC, which was observed only in the cells in the subchondral bony layer in the controls, appeared in the chondrocytes in the superficial and columnar layers of the osteoarthritic knees. epsilon-PKC was observed in the control chondrocytes in the superficial portion of the columnar layers. In early osteoarthritic joints, however, epsilon-PKC-positive chondrocytes disappeared from the superficial portion of columnar layer and increased in number in the middle columnar layers. The appearance and changes in distribution of these PKC-positive chondrocytes, either under normal or osteoarthritic conditions, have not been reported previously. The role of the redistribution of PKC isozymes is still unclear as to whether they are involved in initiating destructive processes, reflect attempted cell repair mechanisms, or are simply a consequence of the cellular changes.

Production of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry.

Polyclonal subtype-specific antibodies were developed against three subtypes of GABA transporters (GAT1, GAT2 and GAT3). By immunoblot analysis, each antibody detected a single band that could be blocked by absorption of the antibody with the respective antigen. GAT2 was found in various tissues, while GAT1 and GAT3 were detected only in the brain. GAT1 was distributed throughout the brain with the highest amount in the olfactory bulb, CA3 region of the hippocampus, layer I of the cerebral cortex, piriform cortex, superior colliculus, interpeduncular nucleus and nucleus spinal tract of the trigeminal nerve, while the GAT3 was densely found in the olfactory bulb, thalamus, hypothalamus, pons and medulla, globus pallidus, central gray, substantia nigra, deep cerebellar nuclei and nucleus spinal tract of the trigeminal nerve but not in the hippocampus, cerebral cortex, caudate-putamen and cerebellar cortex. GAT2 immunoreactivity was faint throughout the brain but was concentrated in the arachnoid and ependymal cells. Both GAT1 and GAT3 were found in the neuropil but not in the cell bodies nor in the white matter. These results suggest that GAT1, GAT2 and GAT3 are expressed in different cells and that GAT1 and GAT3 are involved in distinct GABAergic transmission while GAT2 may be related to non-neuronal function.