Paramagnetic and fluorescent probes attached to "buried" sulfhydryl groups in human carbonic anhydrases. Application to inhibitor binding, denaturation and refolding.

1. The single -SH groups in the human carbonic anhydrases B and C have been modified under denaturing conditions. The modified enzymes recover catalytic activity after dilution of the denaturing medium with buffer. By this method a spin label and a fluorescent probe were specifically introduced into the molecules. 2. The modified and reactivated enzymes have similar kinetic properties, inhibitor-binding constants, circular dichroism spectra, and stabilities towards guanidine hydrochloride as the native enzymes. However, the esterase activity of the modified C enzyme is reduced to about 50%. 3. The spectra associated with the probes are insensitive to inhibitor binding in case of the B enzyme, whereas changes of electron paramagnetic resonance spectrum and fluorescence intensity respectively, were observed for the probe-containing C enzymes. The cysteines are located in different parts of the tertiary structures of the homologous B and C enzymes, and these observations suggest that small conformational changes accompanying inhibitor binding are localized to regions of the molecules near the active-site cavity. 4. During denaturation of the spin-labeled B enzyme in 1.7 M guanidine hydrochloride a transient mobilization of the probe occurs, but the mobility is ultimately reduced to a low level. This observation supports previous evidence that denaturation under these conditions, in or near the transition region, mainly yields incorrectly folded molecules rather than stable intermediates between native and fully denatured molecules. 5. During refolding of fully denatured, spin-labeled B and C enzymes the mobility of the probe is drastically reduced within less than 0.1 s after dilution. This would reflect a very short lifetime of the randomly coiled state under these conditions.     

Effects of the PAF-analog and -antagonist CV-6209 on cultured human glioma cell lines

Cell lines of human glioma (U-343 MGa and U-251 MG) and human glia (U-533 CG) origin were cultured as monolayers and exposed to CV-6209, an alkyl-phospholipid analog and antagonist of platelet activating factor. This drug had very potent antiproliferative effects on the studied human glioma cell lines; IC50 was 0.9 microM after 48 h treatment and 0.2 microM after 2 weeks treatment. At these doses no growth inhibitory effect was noted on the normal glia cells. The effects on the glioma cells were reversible in the dose intervals, where cell proliferation, 3H-thymidine and 14C-methionine uptakes were greatly inhibited. The simultaneous administration of platelet activating factor [(R)PAF] did not influence the antiproliferative effects of CV-6209 on the cells cultured as monolayers. The structurally similar analog CV-3988 also had antiproliferative effects, although at 10 times higher concentration than CV-6209. Two other, structurally unrelated, PAF-antagonists (WEB-2086 and TCV-309) gave effects only at very high concentrations. The U-343 MGa cell line was also exposed to CV-6209 when growing as multicellular spheroids. The studies on the spheroid cultures also demonstrated good antitumoral effects with decreases of both the volume growth and the thymidine uptake. The simultaneous administration of (R)PAF reversed the inhibitory effect of CV-6209 on thymidine incorporation. This study demonstrates a strong antitumoral effect at low concentrations of CV-6209. The antiproliferative effects were probably primarily related to the ether-lipid structure and not to the PAF-antagonistic properties. The good antitumoral effect of CV-6209 on both monolayer and spheroid cultures and the possible PAF-antagonistic properties are discussed.     

Promoting islet cell function after transplantation

Engraftment (i.e., the adaptation of transplanted pancreatic islets to their new surroundings with regard to revascularization, reinnervation, and reorganization of other stromal compartments) is of crucial importance for the survival and function of the endocrine cells. Previous studies suggest that transplantation induces both vascular and stromal dysfunctions in the implanted islets when compared with endogenous islets. Thus the vascular density and the blood perfusion of islet grafts is decreased and accompanied with a capillary hypertension. This leads to hypoxic conditions, with an associated shift toward anaerobic metabolism in grafted islets. An improved engraftment will prevent or compensate for the vascular/stromal dysfunction seen in transplanted islets and thereby augment survival of the islet implant. By such means the number of islets needed to cure the recipient will be lessened. This will increase the number of patients that can be transplanted with the limited material available.     

Analysis of free inositol trisphosphate in heart tissue by FPLC and isotachophoresis

Recent, increasing interest in inositol tris(1,4,5)phosphate (IP3) turnover and metabolism has led to a need for a fast and quantitative determination of this compound in various tissues. These requirements are fulfilled by separation in different steps (FPLC and Sephadex G-10) culminating in further separation and quantification by isotachophoresis. Isotachophoresis means a migration of ion species of the same sign in an electrical field with all ions moving with the same velocity. The migration takes place when an electrical field is applied to a system of electrolytes of specific design. Detection was carried out by monitoring conductivity changes. The method is highly sensitive and allows measurements of IP3 in the pmolar range. Linearity was demonstrated over a wide range, 50-4500 pmol. The total imprecision was low with a coefficient of variation of 3%. When determining in biological tissue the recovery was estimated to be close to 100%. The mean content of IP3 in 15 rat heart specimens was 44.3 pmol/mg dry weight corresponding to tissue samples in the order of 4 mg wet weight. By noradrenaline stimulation myocardial content of IP3 increased by 50%.     

Myofibrillar M-band proteins in rat skeletal muscles during development.

The distribution of three myofibrillar M-band proteins, myomesin, M-protein and the muscle isoform of creatine kinase, was investigated with immunocytochemical techniques in skeletal muscles of embryonic, fetal, newborn and four-week-old rats. Furthermore, muscles of newborn rats were denervated and examined at four weeks of age. In embryos, myomesin was present in all myotome muscle fibres of the somites, whereas M-protein was detected only in a small proportion of the myotome muscle fibres and muscle creatine kinase was not detected at all. In fetal and newborn muscles, all fibres contained all three M-band proteins. At four weeks of age, when fibre types (type 1 or slow twitch fibres and type 2 or fast twitch fibres) were clearly discernable, the pattern was changed. Myomesin and muscle creatine kinase were still observed in all fibres, whereas M-protein was present only in type 2 fibres. On the other hand, in muscle fibres denervated at birth all three M-band proteins were still detected. Our results suggest 1) that during the initial stages of myofibrillogenesis expression and incorporation of myomesin into the M-band precede that of M-protein and muscle creatine kinase; 2) that expression and incorporation of all three M-band proteins during fetal development is nerve independent and non coordinated to the expression of different forms of myosin heavy chains, and 3) that the suppression of M-protein synthesis during postnatal development is nerve dependent and reflects the maturation of slow twitch motor units.