Role of the chain termini for the folding transition state of the cold shock protein.

Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.

Diffusional barrier crossing in a two-state protein folding reaction.

There has been some debate as to whether protein folding involves diffusive chain motions and thus depends on solvent viscosity. The interpretation of folding kinetics in viscous solvents has remained difficult and controversial, in that viscogenic agents affect folding rates not only by increasing solvent viscosity but also by increasing protein stability. By carefully choosing experimental conditions, we can now eliminate the effect on stability and show that the folding dynamics of the cold shock protein CspB are viscosity dependent. Thus Kramers' theory of reaction rates rather than transition state theory should be used to describe this folding reaction.

Microsecond folding of the cold shock protein measured by a pressure-jump technique.

A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding. In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 microseconds and then held constant. Thus, unfolding and refolding reactions in the time range from 70 microseconds to 70 s can be followed with this technique. Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions. We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region. For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains. The Phe27Ala mutation affected the rate of unfolding only; the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding. Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.

Changes in growth, po(2) and ph after exposure to oxamate - studies of 2 human tumor-cell lines growing as multicellular spheroids.

The basic metabolism and pO(2) and pH gradients in spheroids were characterized and, in some cases, changed by the addition of the pyruvate analogue oxamate. Two human tumour spheroid types, colon adenocarcinoma HT29 and malignant glioma U118MG, were applied as models. Microelectrode measurements in HT29 spheroids showed steep pO(2) gradients with large differences between surface and center, Delta pO(2), and low central pO(2) values. The HT29 spheroids had rather flat pH gradients. The U118MG spheroids had less steep pO(2) gradients but steeper pH gradients. Determinations of 1-C-14 and 2-C-14 pyruvate oxidation rates, for characterization of the oxidative glucose breakdown as well as of lactate dehydrogenase kinetics, showed consistent results with the microelectrode measurements in that there was high oxidative metabolism in the HT29 spheroids whereas the U118MG spheroids relied more on glycolysis. Western blot investigations of the LDH isoenzyme composition showed different isoenzyme patterns in the two spheroid types with a lack of LDH1 in U118MG spheroids. Addition of 40 mM of oxamate gave decreased 1-C-14- and 2-C-14-pyruvate oxidation rates in the HT29 cells and inhibition of LDH activity in the U118MG cells. Oxamate increased the central pO(2) values in HT29 spheroids and the central pH values in U118MG spheroids. One example of experimental therapy was applied and oxamate acted as a radiation sensitizer in the U118MG system and as a radioprotective substance in the HT29 system. This has to be analysed in more detail.

Effects of cobalt on haem proteins of erythropoietin-producing HepG2 cells in multicellular spheroid culture.

The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

Depth profiles of pH and PO2 in the isolated brain stem-spinal cord of the neonatal rat.

We have measured depth profiles of extracellular pH (pHECR) and PO2 (PtO2) as well as the kinetics of changes of pHECR in the isolated brain stem-spinal cord preparation of the neonatal rat using pH and PO2 microelectrodes that entered from the ventral surface. When the preparation was superfused with control mock cerebrospinal fluid (Control mock CSF; pH = 7.5, PO2 = 630 Torr, PCO2 = 28 Torr, at 27 degrees C), the pH in the medulla diminished with a nearly constant gradient from the surface to a depth of about 1000 microns, the slope being about 0.1 pH unit per 100 microns. A similar gradient in the 200 to 300 microns of the CSF above the surface suggested existence of unstirred layers despite continuously flowing superfusate. The pH gradient in the spinal cord was somewhat smaller than that in the medulla. The PO2 gradients in both medulla and spinal cord were about 100 Torr per 100 microns from 200 microns above to 100 to 200 microns below the surface; PO2 reached zero at about 450 (medulla) to 600 microns (spinal cord). Although the preparation was anoxic and acidic except for a small layer below the surface, respiratory activity was recorded for several hours in C4 phrenic roots. The kinetics of changes in pHECF were recorded at 100 and 200 microns depth while rapidly replacing the control mock CSF by more acidic CSF, either with increased PCO2 ("Respiratory acidosis") or by adding fixed acid ("Metabolic acidosis"). The changes in pHECF were smaller than those in pHCSF, particularly during respiratory acidosis, as a result of the buffering of the brain tissue. Our results show the importance of superficial layers of the ventral medulla in producing respiratory rhythmicity; they further suggest that somewhat alkaline CSF (pH about 7.8) should be used in this preparation to ensure physiologic surface pH values despite unstirred surface layers.

Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2).

Erythropoietin (Epo)-producing hepatoma cells (HepG2) reveal, in addition to the cytochromes of the respiratory chain, a photometrically measurable haem signal with absorbance maxima at 559 nm and 427 nm, suggesting the presence of a b-type cytochrome. This activity exhibited a low midpoint potential, CO-binding spectra and reduction which was insensitive to both cyanide and antimycin. This haem possessed a 22 kDa subunit and might be part of an electron transfer chain similar to the NADPH oxidase, since the NADPH oxidase cytosolic activating factor (p47) could be identified by Western blot analysis. H2O2, which was detected inside the cells by confocal microscopy, might therefore be produced by the suggested electron transfer chain. This cyanide- and antimycin-insensitive but hypoxia-sensitive cytochrome b would be an attractive candidate for controlled Epo production in response to pO2.

The meaning of H2O2 generation in carotid body cells for PO2 chemoreception.

The rat carotid body is able to generate H2O2 in type-I cells with the aid of an electron-transferring chain with cytochrome b as the major component as it can be detected by spectrophotometry as well as confocal laser-microscopy. This cytochrome b is reducible by hypoxia, but not by cyanide, indicating that it does not participate in the energy production by the respiratory chain. The carotid body possesses a glutathione peroxidase (GPO) which scavenges H2O2 and other organic hydroperoxides. The nervous chemoreceptor discharge can be inhibited by external application of hydroperoxides with a similar half maximal value (60-80 microM) as used to stimulate GPO. A hypothetical signal chain is described which suggests the involvement of cytochrome b as an O2 sensor in PO2 chemoreception of the carotid body and the degradation of H2O2 by glutathione to control the K(+)-conductivity of carotid body cells.

Influence of glucose on metabolism and growth of rat glioma cells (C6) in multicellular spheroid culture.

The metabolism and growth of rat glioma C6 cells in multicellular spheroid culture depended strongly on the glucose supply. A low glucose level (0.1 g/l) in the culture medium reduced lactate production, increased oxygen consumption and diminished hydrogen ion production under normoxia as well as hypoxia. A high glucose level (10 g/l glucose) increased lactate production, had no significant influence on oxygen consumption and increased the hydrogen ion production under hypoxia. Hydrogen ion release from cells under normoxic and hypoxic conditions could be significantly diminished by amiloride (l mM), indicating the involvement of the Na+/H+ exchanger. The growth of the C6 spheroids was enhanced under low glucose conditions, possibly due to the more physiological extracellular pH in the deeper regions of the spheroids. The growth was inhibited under high glucose conditions, which seemed to be toxic due to a massive hydrogen production giving acidosis. The glucose supply strongly influenced the local hydrogen ion production inside the C6 spheroids and this might in turn lead to changes in the response to different therapeutic modalities.

Effects of amiloride treatment on U-118 MG and U-251 MG human glioma and HT-29 human colon carcinoma cells.

Human glioma (U-118 MG, U-251 MG) and human colon carcinoma (HT-29) spheroids and monolayers were continuously exposed to amiloride under physiological Na+ and HCO3- conditions. Amiloride in concentrations of 0.1-0.2 mM inhibited growth, while 0.5 mM or higher induced disintegration of the glioma spheroids within 4-6 days. Growth retardation of the HT-29 spheroids was achieved at concentrations of 0.4-0.5 mM and total growth inhibition and disintegration were achieved at 1.0 mM. Monolayer cultures of glioma cells were also more sensitive to amiloride than those of colon carcinoma cells. The higher amiloride concentrations induced pyknotic nuclei mainly in the central areas of the spheroids where the extracellular pH (pHe) was low. The amiloride-sensitive glioma spheroids had lower pHe than the colon carcinoma spheroids. The intracellular pH (pHi), measured in monolayers, was higher (7.11-7.18) in glioma cells than in colon carcinoma cells (6.94). High concentrations of amiloride, 1.0 mM for 1 h in combination with low Na+ concentrations, caused a strong pHi decrease in glioma cells but only a slight decrease in the colon carcinoma cells. The pHi measurements in glioma monolayers were carried out after 2-6 days of continuous exposure to 0.1 mM amiloride at physiological levels of Na+ and HCO3- to simulate the conditions during growth inhibition. After several days this caused, when growth already was inhibited, an acidification of pHi. Parallel measurements with X-ray microanalysis showed an increase of intracellular sodium and a decrease of intracellular potassium in the gliomas, while no such changes were seen in the colon carcinoma cells under identical conditions. It is concluded that the two glioma cell lines were more sensitive to amiloride, both as monolayers and spheroids, than the corresponding cultures of the colon carcinoma cell line. The inhibition of proliferation by amiloride seemed not to have a clear connection to pHi regulation.

Oxygen tensions in two human tumor cell lines grown and irradiated as multicellular spheroids.

Cells from two human cell lines were irradiated both as multicellular tumor spheroids (MTS) and in monolayer culture. Radiation response of MTS was quantified in terms of specific growth delay and proportion cured, and as clonogenic cell survival for monolayer cells. Radiation was applied either as a single or as a split dose with time intervals of 1, 2, and 4 h to determine the rate of sublethal damage repair. Using as endpoint the fraction of MTS cured at an iso-effect level, in MTS of NB-100 neuroblastoma cells repair of sublethal damage was complete within 1 h, whereas in MTS of HN-1 squamous cell carcinoma cells there was still some unrepaired damage left. At a larger dose for NB-100 MTS the repair curve showed a similar shape as for HN-1 spheroids. Using as endpoint specific growth delay, no difference in repair between the various time intervals was observed. In monolayer cells from both cell lines sublethal damage was not fully repaired in the time intervals used. Polarographic microelectrode measurements of oxygen tension inside MTS showed a marked difference in steepness of oxygen tension profiles between MTS from both cell lines. In HN-1 squamous cell carcinoma MTS with diameters up to 500 microns the central pO2 amounted to about 100 Torr, whereas in NB-100 neuroblastoma MTS with the same diameters central pO2-values lower than 30 Torr were observed. NB-100 MTS were irradiated with doses of 5 and 10 Gy gamma rays and subsequently the oxygen tension was measured 1 and 5 h after irradiation. A reoxygenation effect could not be observed, either after single dose or after split dose irradiation. If spheroids may be regarded as a suitable model for tumor responses in vivo, the results from these experiments indicate that reoxygenation is a process eluding polarographic measurements, or that no dramatic changes in oxygen tension are to be expected shortly after high single doses or early in a fractionation scheme.

Influence of glucose and buffer capacity in the culture medium on growth and pH in spheroids of human thyroid carcinoma and human glioma origin.

pH gradients were measured with microelectrodes in cellular spheroids of human glioma (U-118 MG) and thyroid carcinoma (HTh7) origin. pH decreased outside the spheroids and then continuously decreased when the electrode was moved through the spheroid towards the center. The lowest pH values inside the spheroids were in the range 6.7-6.8 when grown under standard conditions with F10 medium. When medium with stronger buffer capacity (Dulbecco's minimum essential medium or Locke's) was used, the gradients in both types of spheroids were less steep. Less steep pH gradients were also obtained in both types of spheroids when the concentration of glucose was lowered to 0.1 g/liter in the medium. In the case of HTh7 spheroids the low pH inside the spheroids under standard culture conditions seemed toxic because the growth rate increased when the spheroids were cultured under conditions giving higher central pH values (high buffer capacity or low glucose concentration). No such growth-stimulating effects could be seen for the U-118 MG spheroids. The growth rate of both types of spheroids was retarded when they were grown in medium with very high glucose concentration (10 g/liter). The thickness of the viable cell layer increased for HTh7 spheroids when the concentration of glucose was lowered to 0.1 g/liter. A decrease in the thickness of the viable layer of U-118 MG spheroids was observed when they were grown at a high glucose concentration (10 g/liter).

Methodological aspects of microelectrode measurements in cellular spheroids.

Different types of oxygen microelectrodes have been tested in measurements on cellular spheroids. The shape of the oxygen gradients varied strongly depending on size and type of the spheroids. No significant differences in the results were obtained when different types of electrodes were applied. All measurements were made in a perfusion chamber. The shape of the gradients did not vary with time in the perfusion chamber. The reproducibility was found good in repeated measurements using the same spheroid. No mechanical or chemical disturbances were seen during the penetration of the spheroids. Changes in the medium flow rate through the chamber did not drastically change the shape of the oxygen gradients. Almost no convection could be seen at the bottom of the chamber close to the spheroids. The composition of the medium was found to be of importance. Lock's solution containing glucose was found to be satisfactory. The potential signals in the double barrel electrodes allowed an accurate determination of the position when the electrode hit the spheroid surface. The information gained from microelectrode measurements in spheroids might be valuable for the understanding of effects of new tumor treatment modalities in which hypoxic cell sensitizers or high LET radiation are utilized.