Occurrence of "natural" benzodiazepines

There is accumulating evidence that benzodiazepines (BZD)--agents widely used as anxiolytics and hypnotics-could be regarded as "natural" drugs since they have been found in trace amounts also in plants, various tissues of different animal species and even humans. The biosynthesis of such BZD is still unknown and the hypothesis is favoured that they may be of plant origin. Besides diazepam (D) and its major metabolite desmethyldiazepam (DD) several other BZD (e.g. delorazepam, deschloro-diazepam, delormetazepam, isodiazepam, lormetazepam, oxazepam) could be detected. In some cases identification of these compounds was accomplished by specific mass spectrometry (GC-MS) and for quantification various methods have been applied resulting in different concentrations which range for D from about 0.005 to 1 ng/g and for DD from 0.01 to 0.5 ng/g. It is very unlikely that these trace amounts exert any direct pharmacological effects and at the moment only speculations upon their physiological/biological role are possible. Recently BZD-receptor binding activity equivalent to surprisingly high levels of more than 900 ng/ml was found in cerebrospinal fluid of patients with advanced hepatic encephalopathy. As long as the structure of this binding activity has not been elucidated no firm conclusions can be drawn from these findings. If pertinent analytical problems (e.g. drug-free biological material; exact quantification by internal standard techniques) are solved and if the source(s) of BZD are established it might be possible to answer also the critical question whether "endogenous" or "natural" BZD play any (in-) direct role in the regulation of CNS activity.     

Cellulases immobilization on chitosan-coated magnetic nanoparticles: application for <Emphasis Type="Italic">Agave Atrovirens</Emphasis> lignocellulosic biomass hydrolysis

In the present study, Trichoderma reesei cellulase was covalently immobilized on chitosan-coated magnetic nanoparticles using glutaraldehyde as a coupling agent. The average diameter of magnetic nanoparticles before and after enzyme immobilization was about 8 and 10 nm, respectively. The immobilized enzyme retained about 37 % of its initial activity, and also showed better thermal and storage stability than free enzyme. Immobilized cellulase retained about 80 % of its activity after 15 cycles of carboxymethylcellulose hydrolysis and was easily separated with the application of an external magnetic field. However, in this reaction, K _m was increased eight times. The immobilized enzyme was able to hydrolyze lignocellulosic material from Agave atrovirens leaves with yield close to the amount detected with free enzyme and it was re-used in vegetal material conversion up to four cycles with 50 % of activity decrease. This provides an opportunity to reduce the enzyme consumption during lignocellulosic material saccharification for bioethanol production.     

A modified method of isolation of "tight" 70S ribosomes from Escherichia coli highly active at different stages of the elongation cycle

The functional activity of the wide-spread "tight" 70S ribosomes is usually equal to 55-80%. We show here that the inactive fraction of this type of ribosomes is virtually blocked by residual endogenous RNA's. These RNA's are shown to be removable by introducing an additional stage in the isolation procedure including: 1. short heating (15 min, 37 degrees C) of "tight" 70S under dissociation conditions, i. e. in a buffer containing 3 mM MgCl2 and 200 mM NH4Cl; 2. washing off endogenous RNA's on a sucrose density gradient in the same buffer; 3. final selection of purified "tight" 70S on the sucrose gradient containing 5 mM MgCl2 and 50 mM NH4Cl. "Tight" 70S ribosomes isolated by such a procedure are 90-100% active with respect to tRNA binding (including the factor-dependent one), peptide bond synthesis and translocation.     

Pharmacological profile of the "triple" monoamine neurotransmitter uptake inhibitor, DOV 102,677

1. The molecular and behavioral pharmacology of DOV 102,677 is characterized. 2. This characterization was performed using radioligand binding and neurotransmitter uptake assays targeting the monoamine neurotransmitter receptors. In addition, the effects of DOV 102,677 on extracellular neurotransmitter levels were investigated using in vivo microdialysis. Finally, the effects of DOV 102,677 in the forced swim test, locomotor function, and response to prepulse inhibition was investigated.3. DOV 102,677 is a novel, "triple" uptake inhibitor that suppresses [(3)H]dopamine (DA), [(3)H]norepinephrine (NE) and [(3)H]serotonin (5-HT) uptake by recombinant human transporters with IC(50) values of 129, 103 and 133 nM, respectively. Radioligand binding to the dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporters is inhibited with k (i) values of 222, 1030, and 740 nM, respectively. DOV 102,677 (20 mg/kg IP) increased extracellular levels of DA and 5-HT in the prefrontal cortex to 320 and 280% above baseline 100 min after administration. DA levels were stably increased for the duration (240 min) of the study, but serotonin levels declined to baseline by 200 min after administration. NE levels increased linearly to a maximum of 348% at 240 min post-dosing. Consistent with these increases in NE levels, the density of beta-adrenoceptors was selectively decreased in the cortex of rats treated with DOV 102,677 (20 mg/kg per day, PO, 35 days). 4. DOV 102,677 dose-dependently reduced the amount of time spent immobile by rats in the forced swim test, a model predictive of antidepressant activity, with a minimum effective dose (MED) of 20 mg/kg and a maximal efficacy comparable to imipramine. This decrease in immobility time did not appear to result from increased motor activity. Further, DOV 102,677 was as effective as methylphenidate in reducing the amplitude of the startle response in juvenile mice, without notably altering motor activity. 5. In summary, DOV 102,677 is an orally active, "balanced" inhibitor of DAT, NET and SERT with therapeutic versatility in treating neuropsychiatric disorders beyond depression.     

Solubilization of Quisqualate-Sensitive [3H]Glutamate Binding Activity from Rat Retina

Binding activity of a putative central neurotransmitter, L-glutamic acid, was examined in the supernatant preparations solubilized from rat retinal membranes by Nonidet P-40. [3H]Glutamate binding activity increased linearly with increasing concentrations of the solubilized proteins up to 15 micrograms. The binding activity reached an equilibrium within 10 min at 2 degrees C, while increasing with incubation time up to 60 min at 30 degrees C. Addition of an excess of nonradioactive glutamate rapidly decreased the activity at 30 degrees C. Scatchard analysis revealed that the solubilized retinal binding activity consisted of a single component with a KD of 0.25 microM and a Bmax of 57.4 pmol/mg protein. The solubilized binding activity exhibited a stereospecificity and a structure selectivity to L-glutamate, and was abolished by quisqualate, L-glutamate diethyl ester, and DL-2-amino-3-phosphonopropionate. None of the other agonists and antagonists for the central excitatory amino acid receptors affected the binding activity. Reduction of incubation temperature from 30 degrees C to 2 degrees C resulted in a drastic attenuation of the binding activity due to decrement of the number of the apparent binding sites. Cation-exchange column chromatography revealed that unidentified radioactive material was in fact formed during the incubation of [3H]glutamate with the retinal preparations at 30 degrees C. These results suggest that retinal [3H]glutamate binding activity may be derived at least in part from the quisqualate-sensitive membranous enzyme with a stereospecific and structure-selective high affinity for the central neurotransmitter.     

On the bound state of alkali cations in subcellular preparations of rat liver.

Gel filtration and ion-specific electrodes were used together with atomic absorption spectrophotometry in a search for substances in rat liver which are capable of binding alkali cations. In the cytosol, a material which binds K specifically and reduces the ion activity of potassium can be detected. The binding material, which may be destroyed by alpha-chymotrypsin, has been purified about 100-fold. Its molecular weight is around 5 x 10(3). 1 muval K becomes bound per 20-40 amino acid residues; the total binding capacity may amount to 10-15% binding of the K in rat-liver cytoplasm. Washed nuclear residues, consisting mainly of chromatin, are capable of binding Na in a cation-specific mode. DNA and RNA are ruled out as binding material, so it is assumed to consist of protein, and would then contain about 20 amino acid residues per Na. The cation-binding processes are discussed with regard to nucleo-cytoplasmic sequestration of Na and K, with consequences for the cellular chemi-osmotic gradients, and for the regulation of gene activity in the nucleus.     

Partially purified "activated" receptor-glucocorticoid complex from rat liver: regulation of nuclear, chromatin, and DNA-cellulose binding of "activated" complex by pyrophosphate and ATP

The effects of PP1 and ATP on nuclear binding of the "activated" receptor-[3H]-triamcinolone acetonide (TA) complex purified about 3,000-fold from adrenalectomized rat liver were investigated. ATP at up to 5 mM did not affect nuclear binding of the "activated" complex, but PP1 at 2-7 mM greatly enhanced it. However, ATP in the presence of PP1 decreased nuclear binding dose-dependently. Similar results were obtained in the case of chromatin binding, but PP1 alone did not alter DNA-cellulose binding of the "activated" complex, suggesting that the binding sites for chromatin and DNA on the "activated" complex are different. Furthermore, PP1 enhanced ATP-agarose binding of the "activated" complex, indicating that the PP1 binding site(s) on the receptor is different from the ATP binding site(s). The physicochemical properties of the "activated" receptor-glucocorticoid complex bound with ATP and/or PP1 were examined by sucrose density gradient centrifugation and Sephadex G-150 gel filtration. There was no detectable change in the sedimentation coefficient or molecular weight (about 4.2S; Mr approximately equal to 98,000) on binding with ATP and/or PP1. These results suggest that the binding of PP1 and PP1 plus ATP to the "activated" complex caused some allosteric change of the acceptor binding sites of the receptor, resulting in increase or decrease in its binding to nuclei, chromatin, or DNA.     

The effect of wheat germ agglutinin on sialyl and galactosyltransferases of rat liver Golgi membranes.

The sialyltransferase and galactosyltransferase activities of the Golgi-rich fraction from rat liver were enhanced by the binding of wheat germ agglutinin (WGA). The sialytransferase was more sensitive than the galactosyltransferase to the WGA. Maximal stimulation of the galactosyltransferase activity resulted from the binding of 60--80 micrograms WGA to the Golgi membrane, while only 40 micrograms of WGA produced a maximal enhancement in the sialyltransferase activity. Within 5 min of WGA binding, the Golgi sialytransferase activity was doubled. After the initial binding of WGA to the Golgi fraction, the galactosyltransferase activity was decreased by 30%. However, in 15 min the activity was doubled by the binding of WGA. The activities of both enzymes were further enhanced by incubation for up to 90 min. The stimulation of both sialyltransferase and galactosyltransferase activities by WGA was reversed by N-acetyl-D-glucosamine (GlcNAc), the specific inhibitor of agglutination by WGA. Complete reversal of the enhanced activity was observed after 20--30 min in the presence of 1 micromol GlcNAc. The association constant for the binding of WGA to the Golgi membranes was calculated to be 4.16 X 10(-6) M from a Steck-Wallach plot. The 'n' value or mean binding sites was calculated as 5.26 X 10(-5) M/mg of Golgi membrane protein.     

UV-B对不同发育时期离体蓝莓主要果实品质及相关酶活性的影响

该研究以幼果期、白果期、转色期的离体‘北陆’蓝莓果实为试材,设置0(CK)、5、10、15min紫外光辐照处理,24h后取样分析蓝莓果实中可溶性糖、总酚、类黄酮和花青苷含量,以及苯丙氨酸裂解酶(PAL)和查尔酮异构酶(CHI)活性的变化,探究UV-B紫外照射处理对不同发育时期蓝莓主要果实品质及相关酶活的影响。结果显示:(1)对于幼果期蓝莓,5min UV-B处理可显著增加果实内可溶性糖含量;10min UV-B处理果实PAL活性增加效果最为显著;15min UV-B处理对果实总酚和花青苷积累的促进作用最大,但显著降低了类黄酮含量和CHI活性。(2)对于白果期蓝莓,5min UV-B处理显著增加了果实类黄酮含量和CHI活性,10min处理使果实可溶性糖和总酚含量较对照分别增加25%和18%;15min处理对果实花青苷含量和PAL活性影响作用最大。(3)对于转色期蓝莓,各处理除果实可溶性糖及类黄酮含量降低外,其余物质含量均显著增加。(4)UV-B处理并未改变果实发育过程中可溶性糖、总酚、类黄酮和花青苷含量及PAL、CHI酶活性的积累规律。(5)蓝莓果内PAL活性与其可溶性糖、总酚和类黄酮的积累呈极显著正相关关系,而CHI活性仅与其可溶性糖呈极显著正相关。研究表明,UV-B辐照处理促进了幼果期和白果期可溶性糖的积累,也能促进不同发育时期蓝莓果实总酚和花青苷及白果期类黄酮的积累,对蓝莓果实主要品质能够产生积极的影响。     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Glycerol-Releasing Activity of Histamine-Sensitizing Factor of Bordetella pertussis for Rat Adipocytes In Vitro

Histamine-sensitizing factor (HSF) purified from Bordetella pertussis induced specifically the release of glycerol from rat epididymal adipocytes in vitro. The most sensitive and reproducible results were obtained by using 1 to 2 × 10⁵ adipocytes/tube from rats weighing 150 to 200 g, and by incubation at 37 C for 180 min. After a lag period of about 60 min, HSF-treated adipocytes released glycerol in increasing amounts between 60 and 240 min, depending on the dose of HSF. A close correlation between the glycerol-releasing (GR) activity of HSF for adipocytes and histamine-sensitizing or leukocytosis-promoting activity in mice was observed. The GR activity was inactivated by heating at 56 C for 60 min, 63 C for 30 min or 96 C for 10 min. The adipocytes washed out with a Krebs-Ringer bicarbonate buffer immediately after being exposed to HSF for 1 to 3 min manifested about 75% of the total GR activity induced by HSF, and those washed out after being exposed for 30 min or longer had full activity. Anti-HSF serum neutralized the activity when it was added to adipocytes simultaneously with HSF, but did not when it was added 30 min after being exposed to HSF. By using both native and ¹²⁵I-labeled HSF, the ratio of binding of HSF to adipocytes was estimated to be 10 to 15% of the total HSF per 2 × 10⁵ cells/tube, and to be about 1,000 molecules of HSF per cell to induce the release of glycerol. The GR activity induced with 10 ng of HSF was inhibited by addition of insulin at a dose of over 1 μIU/tube, but not by concanavalin A.     

The aging of a brain soluble fraction modifies its effect on the activity of neuronal Na+, K+-ATPase

In previous work we presented evidence showing that a brain soluble fraction was necessary to observe the stimulation of membrane Na+,K+-ATPase activity by catecholamines. Preliminary experiments suggested to us that the soluble fraction by itself was able to modify this enzyme activity. In the present study we have assayed the activity of synaptosomal Na+,K+-ATPase in the presence of a soluble fraction (aqueous supernatant after 100,000 g 30 min) prepared from rat cerebral cortex. The soluble fraction was used at different times after its preparation and different conditions in the incubation period previous to the enzyme assay were tested. It was observed that the enzyme activity increased 70% in the presence of a "0 min" soluble fraction. This effect was not found: a) in the presence of a "30 min" soluble fraction or b) when the membranes plus a "0 min" soluble fraction were incubated for 30 min (15 min at 37 degrees C + 15 min at 0 degree C) before the ATPase assay. In the presence of a "60 min" or "24 h" soluble fraction Na+,K+-ATPase activity was inhibited 50%. Results obtained indicate that Na+,K+-ATPase activity of synaptosomal membranes can be stimulated, inhibited or unchanged, depending on the aging of the soluble fraction.     

Sequence requirements in the catalytic core of the "10-23" DNA enzyme

A systematic mutagenesis study of the "10-23" DNA enzyme was performed to analyze the sequence requirements of its catalytic domain. Therefore, each of the 15 core nucleotides was substituted separately by the remaining three naturally occurring nucleotides. Changes at the borders of the catalytic domain led to a dramatic loss of enzymatic activity, whereas several nucleotides in between could be exchanged without severe effects. Thymidine at position 8 had the lowest degree of conservation and its substitution by any of the other three nucleotides caused only a minor loss of activity. In addition to the standard nucleotides (adenosine, guanosine, thymidine, or cytidine) modified nucleotides were used to gain further information about the role of individual functional groups. Again, thymidine at position 8 as well as some other nucleotides could be substituted by inosine without severe effects on the catalytic activity. For two positions, additional experiments with 2-aminopurine and deoxypurine, respectively, were performed to obtain information about the specific role of functional groups. In addition to sequence-function relationships of the DNA enzyme, this study provides information about suitable sites to introduce modified nucleotides for further functional studies or for internal stabilization of the DNA enzyme against endonucleolytic attack.     

Binding of lectins to "young" and "old" human erythrocytes

"Old" human erythrocytes showed a 21.2% decrease in cell surface area and a 2% decrease in the number of WGA receptor sites, but a 27% increase in the distribution density of the WGA (lectin) receptor site, when compared with "young" human erythrocytes. For a list of lectin abbreviations, see Materials and methods). Both "young" and "old" erythrocytes exhibited very weak binding activity for 125I-labeled PNA, but there was no difference in binding activity for PNA between "young" erythrocytes and "old" ones. Compared with "young" erythrocytes, decreases in the number and distribution density of receptor sites for five lectins including LPA, Con A, RCA-II, SBA and BPA on the cell surface were observed in aged erythrocytes. "Old" erythrocytes also showed a decrease in the number of PHA-E receptor sites, while the distribution density of the same receptor site remained unchanged. In view of these and other observations, it is thought that human erythrocyte aging is accompanied by elimination of some glycoconjugates which have affinity for six lectins, LPA, Con A, RCA-II, PHA-E, SBA and BPA, whereas no WGA receptor-containing glycoconjugates are released from erythrocyte membranes. Elimination of the glycoconjugates results in shrinkage of erythrocytes to reduce their cell surface areas.     

Near-infrared optical imaging of ovarian cancer xenografts with novel alpha 3-integrin binding peptide "OA02"

Through screening of random one-bead one-compound (OBOC) libraries, we previously identified cyclic peptides with the cDGXGXXc motif that bind to alpha3 integrin subunit on ovarian adenocarcinoma cell lines ES-2, SKOV-3, and CaOV-3. We subsequently synthesized two secondary libraries based on this motif and identified new peptides that bound with a higher affinity to these cell lines. One of the peptides identified from the 20% "down-substituted" focused library was the c-dGHCitGPQ-c ("OA02") peptide. The goal of this study was to determine whether this peptide labeled with near-infrared probes could be detected after intravenous injection in ovarian tumor-bearing mice and if it would selectively localize in the tumor. Three different forms of this peptide were synthesized, "OA02"-biotin (noncovalently linked to streptavidin-Cy5.5); "OA02"-Cy5.5 and "OA02"-AlexaFluo 680. Using a KODAK IS2000MM image station, these peptide probes were used at the near-infrared (NIR) spectra to image nude mice bearing ES-2 (alpha3 integrin positive) and Raji (alpha3 integrin negative) xenografts. The peptide probe displayed highly specific tumor uptake within 15 min, which lasted for 70 min for "OA02"-Cy5.5 and "OA02"-AlexaFluo 680 and for 24 hours for "OA02"-biotin-streptavidin-Cy5.5. Some kidney and bladder signal were noted. Prior injection with anti-alpha3 monoclonal antibody blocked the binding of this peptide to the ES-2 tumors.     

Receptor dynamics of closely related ligands: "fast'' and "slow'' interferons.

Two related human alpha interferons with 83% homology in their primary sequences show a similar specific activity on nonhuman cells, but a striking difference on human cells, on which alpha-1 shows 1-5% of the specific molar activity displayed by alpha-2. Both interferons were labelled with 125I, and their binding kinetics followed on growing cultures of the human Burkitt line Daudi. Binding of alpha-1 showed slower rates of association and faster rates of dissociation implying that differences in apparent binding affinity were responsible for the differences in specific molar activity. However, binding was shown to reach steady-state rather than an equilibrium, so differences in the dynamics of the ligand-receptor complexes may represent amplification of differences in the initial binding constant. alpha-2, but not alpha-1, induces a marked loss of binding sites leading to a high affinity steady-state binding. Inhibition of cell multiplication by both interferons depends on a continued stimulation by free ligands at steady-state. It is proposed that the differences in specific molar activity are, in the main, kinetic and cause alpha-1 and alpha-2 to behave respectively as "slow' and "fast' interferons.     

Removal of "tightly bound" nucleotides from soluble mitochondrial adenosine triphosphatase (F1).

Soluble mitochondrial ATPase (F1) from beef heart prepared in this laboratory contained approximately 1.8 mol of ADP and 0 mol of ATP/mol of F1 which were not removed by repeated precipitation of the enzyme with ammonium sulfate solution or by gel filtration in low ionic strength buffer containing EDTA. This enzyme had full coupling activity. Treatment of the enzyme with trypsin (5 mug/mg of F1 for 3 min) reduced the "tightly bound" ADP to zero, abolished coupling activity, but had no effect on the ATPase activity, stability, or membrane-binding capability of the F1. When the trypsin concentration was varied between 0 and 5 mug/mg of F1, tightly bound ADP was removed to varying degrees, and a correlation was seen between amount of residual tightly bound ADP and residual coupling activity. Gel filtration of the native F1 in high ionic strength buffer containing EDTA also caused complete loss of tightly bound ADP and coupling ability, whereas ATPase activity, stability, and membrane-binding capability were retained. The ADP-depleted F1 preparations were unable to rebind normal amounts of ADP or any ATP in simple reloading experiments. The results strongly suggest that tightly bound ADP is required for ATP synthesis and for energy-coupled ATP hydrolysis on F1. The results also suggest that ATP synthesis and energy-linked ATP hydrolysis rather than involving one nucleotide binding site on F1, involve a series or "cluster" of sites. The ATP hydrolysis site may represent one component of this cluster. The results show that nonenergy-coupled ATP hydrolysis on F1 can occur in the absence of tightly bound ADP or ATP.     

Solubilization of Peripheral-Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.     

Chronic antidepressants and GABA "B" receptors: a GABA hypothesis of antidepressant drug action

Amitryptyline (10 mg/kg), desipramine (5 mg/kg), citalopram (10 mg/kg) and viloxazine (10 mg/kg) were administered to rats either acutely (decapitation 1 hr after i.p. injection) or subacutely (by subcutaneous minipump implantation for 18 days followed by decapitation 24 hr after removal). After acute administration there was not any consistent alteration in GABA levels, GAD activity, 3H GABA "A" or 3H-GABA "B" receptor binding or 3H-nipecotic acid binding to the recognition site for GABA uptake in the frontal cortex or hippocampus. Upon subacute antidepressant drug infusion, GABA levels, GAD activity and 3H-GABA-"A" binding showed only scattered differences in drug treated animals as compared to saline treated rats. However, 3H-GABA "B" binding in the frontal cortex was consistently elevated after all drug treatments (in % of control: amitryptyline = 155%; desipramine = 151%; citalopram = 173%; viloxazine = 189%). Scatchard analysis showed that this was due to a Bmax increase without an effect in Kd. These findings were reproduced by subacute administration of pargyline, a MAO inhibitor. These data suggest that GABA "B" receptors may be involved in the mechanism of action of antidepressant drugs and provide a link between GABAergic and monoaminergic hypotheses of depression.     

Protease-Induced Formation of the Sperm Acrosomal Filament

Filament extension during the sperm acrosome reaction in Sicyonia ingentis is triggered by an egg trypsin-like protease whose action can be mimicked using trypsin. Using biotinylated trypsin and either a fluorescently-labeled or colloidal gold-labeled antibody to biotin, trypsin binding was localized to the anterior granule of the sperm which is exposed upon acrosomal exocytosis. The binding was to proteinaceous material at the base of the granule juxtaposed to the inner acrosomal membrane. Other labeled proteins also bound in the same pattern but only in the presence of unlabeled trypsin; non-proteolytic proteins did not induce filament formation. Binding of all proteins tested occurred slowly over a period of about 30 min. A minimum of 30 min of trypsin exposure was required in order to trigger filament formation, and increasing trypsin concentration did not reduce this time requirement. These results indicate that the protease slowly uncovers a binding site for itself (or other proteins), and then its proteolytic activity is again required to induce filament formation. The protease kallikrein appeared to be a more potent inducer than trypsin, while thrombin and clostripain had no apparent inducing activity.