Versatile steroid molecules at the end of the aldosterone pathway

18-hydroxycorticosterone converts spontaneously and reversibly to a variety of less polar forms and derivatives, some of which are precursors to aldosterone. In particular, 21-hydroxy-11 beta, 18-oxido-4-pregnene-3,20-dione (18-DAL) is hydroxylated to aldosterone with high yields in the presence of malate and NADP+, at pH 4.8. 18-DAL also behaves as a metabolic intermediate between 18-OH-B and aldosterone according to time-course and trapping experiments. Consequently, the final steps of the aldosterone pathway at pH 4.8 could be identified as 18-OH-B, 18-DAL and aldosterone, in this sequence. The submitochondrial distribution of aldosterone biosynthesis is compatible with this postulate. The work also shows that some forms of 18-OH-B are promoters of hydrogen transport in renal tubuli and that this regulation may be independent of sodium reabsorption. These results suggest a regulatory model, new in steroid biology, according to which steroid molecules bearing an oxidized angular C18-methyl may undergo structural changes between precursor ("P") and hormonal ("H") forms in response to homeostatic requirements.     

Eighteen-deoxyaldosterone and other less polar forms of 18-hydroxycorticosterone as aldosterone precursors in rat adrenals

Samples containing as precursors either 18-hydroxycorticosterone (18-OH-B) in its M form, or this converted to less polar forms at pH 2 (ACM), or M or ACM enclosed in liposomes from adrenal lipids were incubated at pH 7.4, 4.8 or 3.3 in the presence or absence of quartered rat adrenals for 1 and 2 h. Optimal (10%) yields of aldosterone were obtained when (a) ACM was incubated at pH 4.8 and (b) M enclosed in liposomes was suspended in buffer and shaken without enzyme at pH 3.3. When conditions (a) were supplemented with malate and NADP, 16% of ACM was converted to aldosterone. ACM contained 80% of a fraction which, according to 13C NMR spectroscopy, was identical to 18-deoxyaldosterone (18-DAL). Experiments in which radioactivity from corticosterone (B) or M was trapped by radioinert M or 18-DAL disclosed a pathway comprising sequentially B, 18-OH-B, 18-DAL and aldosterone, and the combined evidence of this work, an enzymatic hydroxylation of 18-DAL to aldosterone.     

pH-dependent changes of ganglioside biosynthesis in neuronal cell culture

Ganglioside biosynthesis was studied in primary cultured murine cerebellar cells after labeling with [14C]galactose. A shift in biosynthesis from "a"-series to "b"-series gangliosides was observed after lowering the pH of the culture medium from 7.4 to 6.2; this effect was fully reversible on changing back to pH 7.4. The observed regulatory effect of pH is in accordance with a recent model of ganglioside biosynthesis. Sialyltransferase II (ST II), the first enzyme for biosynthesis of "b"-series gangliosides, is more active at pH 6.2 than Gal-NAc-transferase, the first enzyme for synthesis of "a"-series gangliosides, which is more active than sialyltransferase II at pH 7.4.     

The development and application of a radioimmunoassay for 18-hydroxy-corticosterone.

A sensitive and specific radioimmunoassay has been developed for 18-hydroxy-corticosterone (18-OH-B) and applied to the measurement of this steroid in peripheral plasma. High specific activity label (3H-18-OH-B) was prepared using the incubation of 3H-corticosterone with duck adrenal mitochondria. Antisera were produced by immunisation with 18-OH-B gamma-lactone 3-oxime conjugated to bovine serum albumin. The antibodies examined showed 100% cross-reactivity with 18-hydroxy-deoxycorticosterone gamma-lactone (18-OH-DOC gamma-lactone), but minimal cross-reactivity with other steroids. Paper chromatography was used to separate 18-OH-DOC gamma-lactone from 18-OH-B gamma-lactone. The interassay precision was 7.6% and the intra-assay precision 11.0%. The accuracy of the method was confirmed by showing a linear relationship between amounts of 18-OH-B added and amounts of 18-OH-B gamma-lactone measured (y = 0.854 X +15.1, r = 0.9. p less than 0.001). The mean plasma level in normal subjects on an ad libitum sodium intake was 225 +/- 92.7 (SD) pg/ml (n = 17) when standing, and 99 +/- 38.3 (SD) pg/ml (n = 6) after lying down for 30 minutes.     

Cholesterol precursors stabilize ordinary and ceramide-rich ordered lipid domains (lipid rafts) to different degrees. Implications for the Bloch hypothesis and sterol biosynthesis disorders

Genetic disorders of cholesterol biosynthesis result in accumulation of cholesterol precursors and cause severe disease. We examined whether cholesterol precursors alter the stability and properties of ordered lipid domains (rafts). Tempo quenching of a raft-binding fluorophore was used to measure raft stability in vesicles containing sterol, dioleoylphosphatidylcholine, and one of the following ordered domain-forming lipids/lipid mixtures: dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), a SM/cerebroside mixture or a SM/ceramide (cer) mixture. Relative to cholesterol, early cholesterol precursors containing an 8-9 double bond (lanosterol, dihydrolanosterol, zymosterol, and zymostenol) only weakly stabilized raft formation by SM or DPPC. Desmosterol, a late precursor containing the same 5-6 double bond as cholesterol, but with an additional 24-25 double bond, also stabilized domain formation weakly. In contrast, two late precursors containing 7-8 double bonds (lathosterol and 7-dehydrocholesterol) were better raft stabilizers than cholesterol. For vesicles containing SM/cerebroside and SM/cer mixtures the effect of precursor upon raft stability was small, although the relative effects of different precursors were the same. Using both detergent resistance and a novel assay involving fluorescence quenching induced by certain sterols we found cholesterol precursors were displaced from cer-rich rafts, and could displace cer from rafts. Precursor displacement by cer was inversely correlated to precursor raft-stabilizing abilities, whereas precursor displacement of cer was greatest for the most highly raft-stabilizing precursors. These observations support the hypothesis that sterols and cer compete for raft-association (Megha, and London, E. (2004) J. Biol. Chem. 279, 9997-10004). The results of this study have important implications for how precursors might alter raft structure and function in cells, and for the Bloch hypothesis, which postulates that sterol properties are gradually optimized for function along the biosynthetic pathway.     

Role of corticosteroids in distal acidification of amiloride-treated rats.

The role of amiloride-dependent sodium channels in the action of adrenal cortical steroids on urine-blood PCO2 (U-B PCO2) differences was studied in bicarbonate-infused and amiloride-treated adrenalectomized rats. U-B PCO2 was significantly reduced by amiloride in bicarbonate-infused control rats. Adrenalectomy further reduced U-B PCO2 in amiloride-treated, bicarbonate-infused rats (from 27.9 +/- 1.82 mmHg in sham-operated rats to 21.3 +/- 1.58 mmHg in adrenalectomized (ADX) rats) (1 mmHg = 133.322 Pa). Acute administration of corticosterone and 18-hydroxycorticosterone (18-OH-B), but not of aldosterone, caused recovery of U-B PCO2 to the level of sham-operated animals treated with amiloride. Aldosterone did not affect U-B PCO2 in the presence of amiloride (21.9 mmHg ADX group vs. 20.98 mmHg aldosterone group). Results are compatible with aldosterone affecting distal H ion secretion mostly by a sodium and potential difference dependent mechanism, while corticosterone and 18-OH-B should act by other mechanisms (e.g., increased luminal buffer level).     

Thermodynamic stability of a kappaI immunoglobulin light chain: relevance to multiple myeloma

Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry. The protein was found to contain a cysteinyl post-translational modification at Cys(214). Protein stability and conformation of MM-kappaI as a function of temperature or denaturant conditions at pH 7.4 and 4.8 were investigated. At pH 4.8, calorimetry demonstrated that MM-kappaI undergoes an incomplete, cooperative, partially reversible thermal unfolding with increased unfolding temperature and calorimetric enthalpy as compared to pH 7.4. Secondary and tertiary structural analyses provided evidence to support the presence of unfolding intermediates. Chemical denaturation resulted in more extensive protein unfolding. The stability of MM-kappaI was reduced and protein unfolding was irreversible at pH 4.8, thus suggesting that different pathways are utilized in thermal and chemical unfolding.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

Effects of ACTH on the last step of aldosterone biosynthesis

The production of tritiated aldosterone and tritiated SM (a saponifiable 18-hydroxycorticosterone derivative) by rat adrenals were studied at various incubation times in absence or presence of two concentrations of ACTH. Tritiated 18-hydroxycorticosterone or 18-deoxyaldosterone served as precursors. The lower ACTH concentration (150 pM) increased the production of tritiated aldosterone. Whereas, the higher ACTH concentration (1.5 microM) stimulated tritiated aldosterone production at shorter incubation time (30 min), while after 60 min it inhibited. This time dependency would reflect variations in the levels of endogenous steroids. On the other hand, the effects of ACTH on tritiated SM production were opposite to those on tritiated aldosterone. In effect, while 150 pM ACTH inhibited SM production, 1.5 microM ACTH stimulated it. These results suggest that ACTH promotes opposite effects on the productions of aldosterone and SM and therefore both productions would be coordinated under the regulation of ACTH.     

Development and characterization of antisera to 18-hydroxy-corticosterone and 18-hydroxy-11-deoxycorticosterone and radioimmunoassay for serum 18-hydroxycorticosterone

A method for the production of the haptens 18-hydroxy-11-deoxycorticosterone-3-(O-carboxymethyl)-oxime (18-OH-DOC-3-CMO) and 18-hydroxycorticosterone-3-(O-carboxymethyl)-oxime (18-OH-B-3-CMO) is described. The formation of the oximes was studied in kinetic experiments. They were prepared at pH 1.6 in methanol/HC1 using a short reaction time. Antisera were raised in rabbits using serum albumin conjugates. The highly specific antisera were used at a final dilution of 1: 79 000 (18-OH-DOC) and 1: 43 000 (18-OH-B); the affinity constants were 1.2 × 10¹⁰l/mol and 8.1 × 10⁹l/mol, respectively. The radioimmunoassay procedure for 18-OH-B in serum involves purification by paper chromatography. The intra- and interassay precision was 7.3% and 12.3%, respectively. The mean serum 18-OH-B level (± S.D.) for normal male and female ambulatory subjects (n = 40) on a normal sodium diet was 0.802 ± 0.262 nmol/l. After 60 minutes of recumbency, the serum 18-OH-B level was 0.313 ± 0.061 nmol/l (n = 6) for men.     

pH-dependent fusion of vesicular stomatitis virus with Vero cells. Measurement by dequenching of octadecyl rhodamine fluorescence

We have studied fusion between membranes of vesicular stomatitis virus (VSV) and Vero cells using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We could identify the two pathways of fusion by the kinetics of R18 dequenching, effects of inhibitors, temperature dependence, and dependence on osmotic pressure. Fusion at the plasma membrane began immediately after lowering the pH below 6 and showed an approximately exponential time course, whereas fusion via the endocytic pathway (pH 7.4) became apparent after a time delay of about 2 min. Fusion via the endocytic pathway was attenuated by treating cells with metabolic inhibitors and agents that raise the pH of the endocytic vesicle. A 10-fold excess of unlabeled virus arrested R18VSV entry via the endocytic pathway, whereas R18 dequenching below pH 6 (fusion at the plasma membrane) was not affected by the presence of unlabeled virus. The temperature dependence for fusion at pH 7.4 (in the endosome) was much steeper than that for fusion at pH 5.9 (with the plasma membrane). Fusion via the endocytic pathway was attenuated at hypo-osmotic pressures, whereas fusion at the plasma membrane was not affected by this treatment. The pH profile of Vero-VSV fusion at the plasma membrane, as measured by the dequenching method, paralleled that observed for VSV-induced cell-cell fusion. Fusion was blocked by adding neutralizing antibody to the Vero-VSV complexes. Activation of the fusion process by lowering the pH was reversible, in that the rate of fusion was arrested by raising the pH back to 7.4. The observation that pH-dependent fusion occurred at similar rates with fragments and with intact cells indicates that pH, voltage, or osmotic gradients are not required for viral fusion.     

Selection of a chemically defined medium for culturing adult newt forelimb regenerates.

Three commercially available tissue-culture media were evaluated for their ability to support continued growth and differentiation of 14-day regenerates of adult newt forelimbs. Serums, embryo extracts, egg ultrafiltrates, and antimicrobial agents were avoided in this analysis. The hormones insulin and L-thyroxine were added to these chemically defined media to enhance continued cellular metabolism and growth. The optimum conditions appeared to be cultivated at 25 degrees C (pH 7.2 to 7.4) in media osmotically adjusted to conditions approximating amphibian blood values (i.e. 225 m0SM for 199, 244 m0SM for CMRL-1066, and 262 m0SM FOR L-15).     

A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins

The interactions of sodium dodecyl sulfate with a number of proteins were examined at a variety of pH values ranging from 4.8 to 11.6 The dodecyl sulfate-induced precipitation of some of these proteins was observed within a relatively limited range of total dodecyl sulfate concentration. Most of the basic proteins precipitated at low pH but as the isoelectric point of the protein was approached the amount of protein that precipitated decreased. Bovine myelin basic protein was unique in that it precipitated at all pH values examined both above and below its isoelectric point. Thus, the dodecyl sulfate-induced precipitation of myelin basic protein appears to be different from the dodecyl sulfate-induced precipitation of most proteins. A comparison of protein precipitation at equivalent dodecyl sulfate: protein molar or weight ratios revealed very little difference in the precipitation behavior of the proteins studied. When the bovine myelin basic protein was cleaved at its single tryptophan residue, the N-terminal fragment (1–115) formed insoluble dodecyl sulfate complexes at pH values ranging from 4.8 to 9.2. The C-terminal fragment (116–169) precipitated almost completely at pH 4.8 but to a lesser extent at pH 7.4 and 9.2 Equimolar mixtures of the N- and C-terminal fragments precipitated in the presence of dodecyl sulfate at pH 7.4 and 9.2 to an extent greater than the C-terminal fragment alone but comparable to the N-terminal fragment alone or the whole basic protein. These results suggest: (a) that the mechanism by which dodecyl sulfate induces the precipitation of myelin basic protein may be unique compared to other proteins and (b) that the intact myelin basic protein is not necessary for its precipitation by dodecyl sulfate.     

Adsorption and Degradation of Doxorubicin from Aqueous Solution in Polypropylene Containers

The purpose of this study was to examine doxorubicin adsorption in polypropylene containers as a function of pH and drug concentration based on anecdotal evidence of such adsorption. Doxorubicin loss was first examined in high-performance liquid chromatography (HPLC) glass inserts by UV absorbance to determine appropriate pH and time durations for subsequent analysis. Doxorubicin loss was then investigated in polypropylene microcentrifuge tubes at different pH values and starting drug concentration at 37°C over 48 h using HPLC with fluorescent detection. Doxorubicin concentrations was essentially constant in HPLC glass inserts at pH 4.8 up to 12 h but declined 5% at pH 7.4 by 3 h. The percent doxorubicin adsorption was calculated in polypropylene microcentrifuge tubes from extrapolations to zero time and was the least at pH 4.8, but increased with pH values 6.5 and 7.4, and decreased with drug concentration to reach a maximum adsorption of 45% in 2.0 μg/mL at pH 7.4 and 37°C. Degradation rate constants, ranging from 0.0021 to 0.019 h⁻¹, also increased with pH in these studies. Determinations of low amounts of doxorubicin in polypropylene containers at pH 7.4 may be underestimated if adsorption and degradation issues are not taken into account.KEY WORDS: adsorption, analysis, chemical stability, doxorubicin, glass, HPLC, polypropylene     

The influence of cholesterol precursor – desmosterol – on artificial lipid membranes

The disorders in cholesterol biosynthesis pathway and various diseases manifest in the accumulation of cholesterol precursors in the human tissues and cellular membranes. In this paper the effect of desmosterol – one of cholesterol precursors – on model lipid membranes was studied. The investigations were performed for binary SM/desmo and POPC/desmo and ternary SM/POPC/desmo monolayers. Moreover, the experiments based on the gradual substitution of cholesterol by desmosterol in SM/POPC/chol = 1:1:1 system were done. The obtained results allowed one to conclude that desmosterol is of lower domains promoting and stabilizing properties and packs less tightly with the lipids in monolayers. Moreover, desmosterol probably could replace cholesterol in model membranes, but only at its low proportion in the system (2%), however, at a higher degree of cholesterol substitution a significant decrease of the monolayer stability and packing and alterations in the film morphology were detected. The results collected in this work together with those from previous experiments allowed one to analyze the effect of a double bond in the sterol side chain as well as its position in the ring system on membrane activity of the molecule and to verify Bloch hypothesis.     

Synthesis and activity of new inhibitors of aldosterone biosynthesis

A new family of aldosterone biosynthesis inhibitors, designed as 18-mono-oxygenase, cytochrome-P450-dependent, potential Kcat inhibitors, is described. These compounds are progesterone derivatives substituted at the 18-methyl group. Preliminary results on the in vitro biological evaluation of these modified progesterones are presented. Aldosterone biosynthesis is completely inhibited by 18-vinyl progesterone 5 at a concentration of 0.8 microM and by 18-ethynyl progesterone 6 at 8 microM. It appears that products designed as alkylating agents for the prosthetic heme group are the most potent inhibitors in that series.     

Induced thermotolerance under nonisothermal treatments of a heat sensitive and a resistant strain of Staphylococcus aureus in media of different pH

AIMS: The aim was to assess the induced thermotolerance under nonisothermal treatments of two strains of Staphylococcus aureus in media of different pH. METHODS AND RESULTS: Staphylococcus aureus ATCC 25923 was more heat resistant than S. aureus ATCC 13565 at any pH investigated under isothermal conditions. At pH 7.4, the D58 value of the resistant strain was approx. 30 times greater. Both strains showed a higher heat resistance at pH 4.0 than at pH 7.4. In contrast, under nonisothermal treatments (0.5-2 degrees C min(-1)), both strains were more heat resistant when treated at pH 7.4 than at pH 4.0 due to heat adaptation at the higher pH. At the slowest heating up rate tested at pH 7.4, the initially heat-sensitive strain nearly reached the thermotolerance of the heat-resistant strain. CONCLUSIONS: The induced thermotolerance under nonisothermal treatments depended on the treatment medium pH and the microbial strain tested. The induced thermotolerance in a sensitive strain can be greater than in a heat-resistant strain, showing similar resistance under nonisothermal conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows data of interest about mechanisms of microbial resistance and adaptation to heat. Moreover, it contributes to the development of more adequate combined processes for food preservation.     

Origins of the differences in function of rat adrenal zona glomerulosa cells incubated as intact tissue and as collagenase-prepared cell suspensions

While in vitro incubation of dispersed cell preparations of adrenal cell types has been widely used as an experimental model, few studies have addressed the possibility that the enzymic and mechanical treatments involved may affect tissue functions. Using rat adrenal whole capsule tissue, consisting of glomerulosa cells still attached to the connective tissue capsule together with some fasciculata cells, and dispersed glomerulosa cell preparations formed by a variety of enzymic and incubation treatments, striking differences have been demonstrated between the functions of the various preparations in vitro. Under ACTH stimulation, whole capsules produced (ng per pair ± s.e.) 405 ± 35 ng aldosterone, 650 ± 60 ng 18-hydroxycorticosterone (18-OH-B) and 850 ± 90 ng corticosterone. In cells dispersed by collagenase incubation followed by repeated pipetting and filtration, aldosterone and 18-OH-B yields under ACTH stimulation fell to values less than 10% of those produced by whole tissue, whereas corticosterone values were unchanged. Omitting the filtration step gave a less well marked decline in aldosterone and 18-OH-B to 50% of intact tissue values. When the tissue was not dispersed after collagenase incubation, aldosterone and 18-OH-B outputs were similar in the two preparations. The decline in aldosterone and 18-OH-B is not attributable to loss in cell–cell contact alone, since short term culture of collagenase dispersed cells on contracting collagen discs did not restore the capacity to produce these steroids, and a decline in their output also occurred in similar culture of intact capsule tissue. In acute incubations, hyaluronidase had similar effects to collagenase, whereas trypsin, papain and a bacterial protease evoked aldosterone release during the preincubation period, but did not affect subsequent yields of aldosterone and 18-OH-B in incubations of dispersed (but not filtered tissue) in the presence of ACTH. Chymo-trypsin had no effect on preincubation but eliminated subsequent response to ACTH in all incubation conditions. Together with previously published data on the effects of trypsin, the results support the view that in intact rat adrenal glomerulosa tissue, aldosterone and 18-OH-B are sequestered into intracellular stores in the form of novel steroid-protein complexes. These are hydrolysed by trypsin and other preoteases with consequent release of steroid, but are virtually eliminated by conventional methods of cell suspension preparations, using collagenase preincubation with subsequent mechanical dispersal and filtration.     

Calcium binding constituents of the organic shell matrix from the freshwater snail Biomphalaria glabrata

The Ca2+ binding of an EDTA-free water-soluble (SM) and -insoluble (IM) organic matrix of the freshwater snail Biomphalaria glabrata was investigated, using a 45Ca2+ autoradiography after SDS-electrophoretical separation and a calcium binding assay. Electrophoresis of the SM showed a considerable amount of Alcian blue and Stains all positive material, regarded as glycosaminoglycans (GAGs) or proteoglycans (PGs). This part of the SM was slightly positive after 45Ca2+ autoradiography at pH 6.8. The Ca2+ binding increased, raising the pH to 7.4 and 8.0 and was especially strong when simulating the real conditions of the extrapallial space with a carbonate buffer of pH 7.4. The Ca2+ binding assay of the IM showed the same pH-dependency that was observed in the SM. The titration of the IM with Ca2+ at pH 8.0 lead to a dissociation constant of 7.5 x 10(-5) M. While Mg2+ displaced 45Ca2+ in the same way as nonradioactive Ca2+, an approximately 400-fold amount of Na+ was necessary to reduce the binding of 45Ca2+ to 50%. The Ca2+ binding of the organic matrix from the B. glabrata shell appears to be a process of low specificity, medium affinity and high pH-dependency. Apparently, acidic carbohydrate-rich PGs are the only calcium binding constituents of the organic shell matrix.