Catabolism of [4-14C]testosterone by subcellular fractions of human prostate

1. Incubation conditions were established in experiments with human-prostate homogenates for almost complete conversion of [4-(14)C]testosterone into at least ten transformation products. 2. Whole homogenates of tissue with benign hypertrophy were shown to contain 3alpha-, 3beta- and 17beta-hydroxy steroid dehydrogenases, Delta(4)-3-oxo steroid 5alpha- and 5beta- reductases and unidentified hydroxylases. 3. Most of the 17beta-hydroxy steroid-dehydrogenase activity was located in the mitochondria, which showed little other activity. 4. The 3alpha- and 3beta-hydroxy steroid dehydrogenases and the 5beta-reductase were located in the high-speed supernatant and required supplementation with NADPH for activity. 5. The 5alpha-reductase was located in both microsomal and high-speed supernatant fractions and also required supplementation with NADPH.     

Transient Stimulation of Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase and Histone Acetylation in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Apparent Induction of Host Ribonucleic Acid Synthesis

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn²⁺-(NH₄)₂SO₄ and Mg²⁺-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.     

Kinetic study of Lactococcus lactis strains (SLT6 and SLT10) growth on papain-hydrolysed whey

The effect of controlled whey hydrolysis by papain on growth of two lactic acid bacteria isolated from artisanal Leben: Lactococcus lactis var. diacetylactis (SLT6 and SLT10) was investigated. The higher biomass and maximum specific growth rate (μ _max) were obtained after 30 min of hydrolysis. HPLC analysis of peptides showed that whey hydrolysis reduced the amount of peptides of MW > 400 Da and increased those peptides of MW < 400 Da. The two studied strains exhibited different peptide requirements. The pH-controlled batch cultures in 30 min hydrolysed whey followed the Monod kinetic for growth and for lactate production. The values of the key kinetic constants were: maximum specific growth rate (μ _max), 1.08 and 0.56 h^?1; yield biomass on lactose (Y _x/s), 0.20 and 0.18 g g^?1 and saturation constant K _s, 4.2 and 2.8 g L^?1 for SLT6 and SLT10, respectively. When compared with batch experimental data, the model provided good predictions for growth, lactose utilisation and lactate production profiles.     

Deletion of Running-Induced Hippocampal Neurogenesis by Irradiation Prevents Development of an Anxious Phenotype in Mice

Recent evidence postulates a role of hippocampal neurogenesis in anxiety behavior. Here we report that elevated levels of neurogenesis elicit increased anxiety in rodents. Mice performing voluntary wheel running displayed both highly elevated levels of neurogenesis and increased anxiety in three different anxiety-like paradigms: the open field, elevated O-maze, and dark-light box. Reducing neurogenesis by focalized irradiation of the hippocampus abolished this exercise-induced increase of anxiety, suggesting a direct implication of hippocampal neurogenesis in this phenotype. On the other hand, irradiated mice explored less frequently the lit compartment of the dark-light box test irrespective of wheel running, suggesting that irradiation per se induced anxiety as well. Thus, our data suggest that intermediate levels of neurogenesis are related to the lowest levels of anxiety. Moreover, using c-Fos immunocytochemistry as cellular activity marker, we observed significantly different induction patterns between runners and sedentary controls when exposed to a strong anxiogenic stimulus. Again, this effect was altered by irradiation. In contrast, the well-known induction of brain-derived neurotrophic factor (BDNF) by voluntary exercise was not disrupted by focal irradiation, indicating that hippocampal BDNF levels were not correlated with anxiety under our experimental conditions. In summary, our data demonstrate to our knowledge for the first time that increased neurogenesis has a causative implication in the induction of anxiety.     

Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli

Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)His] as an antigen. From them, those reacting equally with His-tagged and wild-type TetA(B) were selected and named TCL-1. Cysteine-scanning mutants were used to determine the TCL-1 binding site on the TetA(B) protein. First, 12 Cys mutants of TetA(B) in which one residue in a protruding loop region was replaced by cysteine were constructed. Western blot analysis revealed the binding of TCL-1 to all of these Cys-mutants except for R186C. Then, we constructed 13 cysteine-scanning mutants, F179C to T191C. Among them, eight mutants, F179C to T182C, N184C, and T189C to T191C, exhibited TCL-1 binding, whereas the other five, K183C, T185C, R186C, D187C, and N188C, exhibited no or lower TCL-1 binding. These results clearly indicate that the sequence recognized by TCL-1 is 183Lys-X-Thr-Arg-Asp-Asn188 in the central loop region of TetA(B). TCL-1 is the first reported antibody that binds to a region other than the C-terminus of TetA(B), and the recognized amino acid sequence was identified.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Cloning of rat ABCA7 and its preferential expression in platelets

We cloned the full-length cDNA of a rat orthologue of ABCA7 (rABCA7) from rat platelets. The cDNA of rABCA7 is 6510bp in length and encodes a protein of 2170 amino acids. The amino acid sequence of rABCA7 exhibits homology to those of mouse ABCA7 (92.5% identical in amino acid sequence) and human ABCA7 (76.6%). We obtained two clones of monoclonal antibodies against rABCA7 recognizing different epitopes. Analysis of CHO cells stably expressing rABCA7 by confocal laser-scanning microscopy indicated that rABCA7 is mainly located in the plasma membrane. Western blot analysis of rat tissues revealed that rABCA7 was preferentially expressed in platelets and that its apparent molecular mass was 250kDa. This is the first report of the tissue distribution of rABCA7 at the protein level and is the first reported case of ABC transporters being expressed in platelets, suggesting their important role in platelet function.     

A novel method for study of protein folding kinetics by monitoring diffusion coefficient in time domain

Molecular diffusion process after the photo-induced electron injection to ferric cytochrome c (Fe(III) cyt c) in guanidine hydrochloride (GdnHCl) 3.5 M buffer solution is studied by the time-resolved transient grating technique. Circular dichroism studies have revealed that Fe(III) cyt c is unfolded under this condition but the reduced form, Fe(II) cyt c, is folded. Hence, this pulsed laser-induced reduction should initiate the folding process of cyt c. The observed transient grating signal shows prominent features, which have never been observed before. Based on several characteristic points, we concluded that the apparent diffusion coefficient (D) of Fe(II) cyt c after the reduction is time dependent, which must be associated with the protein folding dynamics. This time-dependent apparent D should reflect either the continuous time development of the hydrodynamic radius or population change of the unfolded and folded states during the folding dynamics. This is the first observation of the time-dependent apparent D during any chemical reaction, and this time-dependent measurement of D should be a unique and powerful way to study the protein folding kinetics from a viewpoint of the protein's shape or the protein-water intermolecular interaction.