Desensitization of prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Desensitization of prostaglandin (PG) F₂ receptor-mediated phosphoinositide (PI) hydrolysis was investigated in cultured rat astrocytes. Prolonged exposure of astrocytes differentiated by dibutyryl cyclic AMP-treatment to PGF₂ caused the desensitization of subsequent PGF₂-induced PI hydrolysis. The desensitization was time- and PGF₂ dose-dependent; maximal decrease in the PI hydrolysis was observed after exposure to 10 M PGF₂ for 4 h and the degree of the desensitization was 31.7±2.7% of control. Pretreatment with either PGD₂ or PGE₂ also induced the desensitization of subsequent PGF₂-stimulated PI hydrolysis and conversely pretreatment of PGF₂ decreased the PI responses to PGD₂ and PGE₂. The desensitization prevented by phloretin and was reversible upon removal of the agonist. Protein synthesis inhibitors blocked the recovery of the desensitization. Treatment of the cells with phorbol 12-myristate 13-acetate had no effect on the desensitization. These results suggest that prolonged exposure of the astrocytes to PGF₂ caused the desensitization of the receptors.     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy

Hypoxia has been shown to activate the pleiotropic cytokine TNF in the lung. TNF in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNF in chronic hypoxia we exposed TNF null (TNF–/–) and wild-type mice to three weeks of hypobaric hypoxia (10% O₂). Equivalent erythocytosis (Hematocrit increased by 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice ( 75%), yet was unaltered in the TNF–/– mice. Concomitantly right ventricular hypertrophy was attenuated in the TNF–/– mice (35 ± 5% increase) when compared to wild-type mice (124 ± 6% increase p < 0.001, n 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNF is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNF signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNF.     

Immunohistochemical demonstration of prostaglandins in various tissues of the rat

To demonstrate the tissue localization of prostaglandin (PG) E₂, PGF₂ and 6-keto-PGF₁ (a stable metabolite of PGI₂) various tissues, including decalcified periodontal tissue of 7-week-old male Wistar strain rats, were immunohistochemically examined using a streptavidin-biotin complex method. Besides tissue macrophages and endothelial cells in various tissues, hepatocytes, renal tubular cells, and parietal and chief cells in the gastric mucosa showed a positive reaction for the various PGs examined. PGs were demonstrated in the cytoplasm or in association with the cell membrane. We generally observed no difference between the localization patterns of PGE₂-, PGF₂-, and 6-keto-PGF₁-positive cells in these tissues. However, in the periodontal ligament and alveolar bone, 6-keto-PGF₁ was localized in the cytoplasm of osteocytes, osteoblasts, cementocytes, and cementoblasts, while no reaction for PGE₂ or PGF₂ was revealed in these cells. We demonstrated the immunohistochemical localization of PGs in various rat tissues including decalcified periodontal tissue and discuss the important roles of PGs in the modulation of their normal functions in these tissues.     

Production of thermostable amylolytic enzymes by Thermococcus hydrothermalis

A cell extract of Thermococcus hydrothermalis, grown for 6 h, gave -glucosidase activity at 14.9 U/l, degrading oligosaccharides and maltose. -Amylase, -glucosidase and pullulanase activities were detected at 289 U/l, 13.5 U/l and 30 U/l respectively in the culture medium after 24 h growth of the archaeum. All of three enzymes, characterised by a half-life time of 1 to 5 h at 95°C, degraded both the (14) and (16) linkages of polysaccharides and the (14) linkages of oligosaccharides. © Rapid Science Ltd. 1998     

Prostaglandins differently regulate FGF-2 and FGF receptor expression and induce nuclear translocation in osteoblasts via MAPK kinase

We have previously reported that prostaglandin F₂ (PGF₂) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF₂ or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF₂ stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF₂. Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF₂. Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF₂ exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF₂ stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process.This research was supported by grants from University of Camerino and Fondazione Carima Italy and by National Institutes of Health Grant AR-46025 (to M.M.H)     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

High affinity insulin binding in the human placenta insulin receptor requires αβ heterodimeric subunit interactions

Summary Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0mm DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot.¹²⁵I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an heterodimeric insulin receptor complex from the ₂₂ heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional heterodimeric insulin receptor complex. (ii) the heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the ₂₂ association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the heterodimers.Abbreviations used are ATP adenosine 5-triphosphate - DTT dithiothreitol - SDS sodium dodecyl sulfate - DSS disuccinimidyl suberate - NEM N-ethylmaleimide - IGF-I insulin-like growth factor-I - EDTA ethylenediaminetetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

Translational arrest in hypoxic potato tubers is correlated with the aberrant association of elongation factor EF-1α with polysomes

Translation elongation factor EF-1 became stably associated with potato tuber polysomes at the onset of hypoxia, coincident with a sharp rise in lactate and decrease in tissue pH. This aberrant association of EF-1 with polysomes also occurred when aerobic tuber extracts were acidified in vitro. Upon resumption of protein synthesis, an increase in the steady-state levels of EF-1, and expression of an EF-1/GUS transgene was observed. These results indicate that translational arrest results from to the failure of EF-1 to dissociate from ribosomes during the elongation cycle, and that restoration of protein synthesis is coordinated with expression of EF-1.     

High frequency of triplicated α-globin loci and absence or low frequency of α thalassemia in Polynesian Samoans

Summary Most of the population in certain areas of Melanesia have one -globin gene deletion ( thal₂). It is thought that the high frequencies of thal₂ in this population is due to a selective advantage given by malaria infection to carriers of thal₂. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to ³²P-labeled -globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated -globin loci type 1, while none had thal₂. On digestion with Bgl II the third -globin gene was found in an additional 3.7kb fragment in all seven Samoans with triplicated -globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying -globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had thal₂ or thal₁. Only two of the Malaysians had triplicated -globin loci.     

Variant MoFe proteins of Azotobacter vinelandii: effects of carbon monoxide on electron paramagnetic resonance spectra generated during enzyme turnover

The resting state of wild-type nitrogenase MoFe protein exhibits an S=3/2 electron paramagnetic resonance (EPR) signal originating from the FeMo cofactor, the enzymes active site. When nitrogenase turns over under CO, this signal disappears and one (sometimes two) of three new EPR signals, which also arise from the FeMo cofactor, appears, depending on the CO concentration. The appearance and properties of these CO-inducible EPR signals, which were also generated with variant MoFe proteins (R96Q, R96K, Q191K, R359K, R96K/R359K, R277C, R277H, and nifV) that are impacted around the FeMo cofactor, have been investigated. No new CO-induced EPR signals arise from any variant, suggesting that no new CO-binding sites are produced by the substitutions. All variant proteins, except R277H, produce the lo-CO signal; all, except Q191K, produce the hi(5)-CO signal; but only two (R96Q and nifV) exhibit the hi-CO signal. FeMo cofactors environment clearly dictates which CO-induced EPR signals are generated; however, none of these EPR signals correlate with CO inhibition of H₂ evolution observed with some of these variants. CO inhibition of H₂ evolution is, therefore, due to CO binding to a different site(s) from those responsible for the CO-induced EPR signals. Some resting-state variants have overlapping S=3/2 EPR signals, whose intensities simultaneously decrease under turnover conditions, indicating that all FeMo cofactor conformations are catalytically active. Moreover, these variants produce a similar number of hi(5)-CO signals after turnover under CO to the number of resting-state S=3/2 signals. The FeMo cofactor associated with the hi(5)-CO signal likely contains two bridging CO molecules.     

Growth arrest and induction of apoptosis in breast cancer cells by antisense depletion of protein kinase A-RIα subunit: p53-independent mechanism of action

The enhanced expression of the RI subunit of cyclic AMP-dependent protein kinase type 1 (PKA-I) has been correlated with cancer cell growth. We have investigated the effects of sequence-specific inhibition of RI gene expression on the growth of MCF-7 human breast cancer cells. We report that RI antisense treatment results in a reduction in RI expression at both mRNA and protein levels and inhibition of cell growth. The growth inhibition was accompanied by changes in cell morphology, cleavage of poly(ADP-ribose) polymerase (PARP) and appearance of apoptotic nuclei. In addition, bcl-2 protein level was reduced and p53 expression increased in growth arrested cells. Interestingly, RI antisense inhibited cell viability and induced apoptosis in the absence of p53, suggesting that these actions of RI antisense are exerted independent of p53. In contrast, two- and four-base mismatched control oligonucleotides had no effect on either cell growth or morphology. These results demonstrate that the RI antisense, which efficiently depletes the growth stimulatory molecule RI, induces cell differentiation and apoptosis, providing a new approach to combat breast cancer cell growth.     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Integrins: cell adhesives and modulators of cell function

Summary Integrins encompass a family of cell-surface molecules which play a crucial role in cell-cell and cell-extracellular matrix interaction. Of these heterodimeric transmembrane glycoproteins (consisting of an and chain) as yet at least 20 different types have been described, all with a different pattern of reactivity with extracellular matrix components. In this review the cell and tissue distribution of the integrins is discussed, with special emphasis on immunohistochemical localization of the ₁ integrins and the ₆₄ integrin. The ₁ integrins comprise a subfamily in which eight chains combine with one (the ₁) chain. The ₂₁, ₃₁ and ₆₁ and the ₆₄ integrins are expressed on a wide variety of epithelia on the basolateral surface or exclusively on the basal surface facing the basement membrane (e.g. ₆₁ and ₆₄). Leucocyte integrins, which share a common ₂ chain, occur almost exclusively on white blood cells and their precursors. The vitronectin receptors, which share a common _v chain, occur in a wide variety of cell types. Integrins play a major role in the interaction of the cell with the extracellular matrix in order to create and maintain tissue architecture. It has become clear, however, that through integrin-ligand interaction cell function is also modulated. Furthermore, in pathological conditions integrins play a role of some significance. Integrins mediate leucocyte traffic in developing inflammatory processes and function in neoplastic growth when it comes to invasion and metastasis.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Eicosanoid production in experimental alcoholic liver disease is related to vitamin E levels and lipid peroxidation

We investigated the association between vitamin E, lipid peroxidation and eicosanoid production in experimental alcoholic liver injury. We used the intragastric feeding rat model in which animals were fed corn oil and ethanol (CO+E) and corn oil and dextrose (CO+D) for 2 and 4 week periods. At sacrifice, we measured plasma levels of alpha-tocopherol, 8-isoprostane, thromboxane B₂(TXB₂) and 6-ketoprostaglandin F₁(6-KetoPGF₁). Animals fed CO+E had significantly lower concentrations of -tocopherol and higher concentrations of 8 isoprostane at both 2 and 4 weeks. a significant inverse correlation was seen between -tocopherol concentrations and the TXB₂: PGF₁ ratio (r=0.72, p<0.01). A positive correlation was seen between the TXB₂: PGF₁ ratio and 8 isoprostane levels (r=0.84, p<0.001). These results suggest that vitamin E depletion and enhanced lipid peroxidation may affect eicosanoid metabolism in experimental alcoholic liver disease in such a way so as to increase the thromboxane to prostacyclin ration.Abbreviations PGH₂ Prostaglandin H₂ - PGL₂ Prostacyclin - CO+E Corn Oil and Ethanol - CO+D Corn Oil and Dextrose - TXB₂ Thromboxane B₂ - TXA₂ Thromboxane A₂     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.