In vitro secretion of ACTH, beta-endorphin and beta-lipotropin in Cushing's disease and Nelson's syndrome

Tissue from histologically confirmed ACTH cell adenomas in Cushing's disease (CD) and Nelson's syndrome (NS) was gained by transsphenoidal surgery. Combined enzymatic and mechanic agitation of tumor tissue yielded a cell suspension. Aliquots of the cell suspension were transferred to superfusion chambers immediately after isolation and investigated for ACTH and beta-endorphin production. Feedback action of cortisol (CO) and dexamethasone on basal hormone production and on lysine vasopressin (LVP) induced ACTH secretion were studied. Adenomatous tissue and anterior lobe tissue from the same patient in CD could be investigated simultaneously in 4 cases. The paraadenomatous tissue showed depression of basal and LVP-induced ACTH secretion. In all adenomatous tissues investigated there was missing or reduced suppression of basal ACTH secretion by physiological levels of CO. CO not only failed to suppress LVP-induced ACTH secretion but also seemed to enhance LVP stimulation in some experiments. This study confirms former results, that a missing or inversed feedback action or glucocorticoids in adenoma cells is a mechanism involved in the pathological ACTH secretion in CD and NS. Bioassayable and immunoreactive ACTH from media of superfusion and short-term static incubation were compared with beta-endorphin and beta-LPH in an assay detecting these two peptides with equimolar sensitivity. Secretory patterns were basically parallel but great differences showed in quantities of hormones secreted. In addition, Sephadex G-50 gel chromatography was performed to separate beta-endorphin from beta-LPH and to calculate the ratios. These profiles show great variations between different adenomas.     

In vitro killing of Babesia bovis by the ornithine analog alpha-monofluoromethyldehydroornithine methyl ester

The effects of the 2 ornithine decarboxylase inhibitors alpha-difluoromethylornithine (DFMO) and alpha-monofluoromethyldehydroornithine methyl ester (delta MFMO-ME) on growth of Babesia bovis blood stages in vitro were tested. The DFMO had no apparent effects on in vitro growth of B. bovis nor on the morphology of the parasite at concentrations up to 20 mM. In contrast, delta MFMO-ME had cytotoxic effects on B. bovis at 0.5 mM which were more pronounced at 5 mM. delta MFMO-ME caused both a decrease in percentage parasitized erythrocytes and a degeneration of parasites after 12 h exposure, and the magnitude of both effects was dose-dependent. The effects of delta MFMO-ME were not reversible for B. bovis precultured for 12 h (5 mM) or 24 h (0.5 mM) in drug before culturing the parasite in drug-free medium. Unexpectedly, 300 microM putrescine did not reverse the effects of delta MFMO-ME on B. bovis blood stage, raising the possibility that inhibition of ornithine decarboxylase is not responsible for these effects.     

In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor.

Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme involved in the biosynthesis of ether phospholipids, is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2. The human alkyl-dihydroxyacetonephosphate synthase precursor produced in vitro or expressed in Escherichia coli could be processed to a lower molecular weight protein by incubation at 37 degrees C with a guinea pig liver fraction, enriched in mitochondria, lysosomes, and peroxisomes. This lower molecular weight protein was identified as the mature human alkyl-dihydroxyacetonephosphate synthase by radiosequencing, indicating that the processing protease is present in this organellar fraction. Characterization of the processing protease indicated that it is a cysteine protease with a pH optimum of 6.5. Furthermore, it was demonstrated that exogenously added pre-alkyl-dihydroxyacetonephosphate synthase was imported and processed in purified peroxisomes in vitro. Processing of alkyl-dihydroxyacetonephosphate synthase did not increase the activity of the enzyme. This indicates that the presence of the presequence does not affect the activity of the enzyme.     

In vitro maturation and artificial activation of donkey oocytes

Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.     

In vitro uptake and stability study of pVEC and its all-D analog

A key step in the development of new hydrophilic pharmaceuticals is to get them through biological barriers. Cell-penetrating peptides, CPPs, have been shown previously to enter cells both in vitro and in vivo by a non-endocytotic mechanism and to be able to carry large cargo molecules with them. Recently, we showed that a small peptide, pVEC, from murine vascular endothelial cadherin, has the characteristics to be classified as a protein derived CPP. Here we have further investigated pVEC together with its all-D analog for cellular uptake, intra- and extracellular stability, and their enzymatic degradation. The two peptides, pVEC and all-D pVEC, translocate into aortic endothelial cells and murine fibroblasts by a non-endocytotic mechanism. In phosphate buffer, pVEC remains intact while the C-terminal lysine is quickly removed in human serum and serum-containing media. Both pVEC and pVEC without the C-terminal Lys were detected by mass spectrometry inside the two cell types tested. The pVEC half-life is 10.5 min in phosphate buffer containing 10 units of trypsin and 44.6 min in phosphate buffer containing 4.2 units of carboxypeptidase A and 18 units of carboxypeptidase B. In contrast topVEC, the all-D analog remains intact in serum and resists enzymatic degradation.     

In vitro synthesis and processing of tomato fruit polygalacturonase

The in vitro processing of tomato fruit polygalacturonase (PG) (poly[1,4-α-d-galacturonide]glucanohydrolase, EC 3.2.1.15) was studied. Complete chemical deglycosylation of a mixture of mature, purified PG 2A and PG 2B isozymes (45 and 46 kilodaltons; respectively) with trifluoromethane sulfonic acid yielded a single polypeptide of 42 kilodaltons. Similarly, N-terminal amino acid sequencing of the PG 2A/2B isozyme mixture yielded a single 21 amino acid N-terminal sequence, suggesting that the two isozymes result from differential post-translational processing of a single polypeptide. Translation of PG mRNA in vitro results in the synthesis of a single polypeptide with an apparent molecular weight of 54 kilodaltons. Nucleotide sequence analysis of a full-length PG cDNA clone indicates that the large size difference between the PG in vitro translation product and the mature isozymes is due to the presence of a 71 amino acid (8.2 kilodaltons) domain at the N-terminus of in vitro translated PG, consisting of a hydrophobic signal sequence followed by a highly charged prosequence. To determine the precise cleavage site of the signal sequence, PG mRNA was translated in vitro in the presence of canine pancreas microsomal membranes. This resulted in the production of two glycosylated PG processing intermediates with apparent molecular weights of 58 and 61 kilodaltons. The PG processing intermediates were shown to be sequestered within the lumen of the microsomal membranes by protease protection and centrifugational analysis. Deglycosylation of the PG processing intermediates with endoglycosidase H yielded a single polypeptide with an apparent molecular weight of 54 kilodaltons. The production of two distinct, glycosylated processing intermediates from the single in vitro translated PG polypeptide suggests a mechanism by which the differential glycosylation observed for the mature PG 2A and PG 2B isozymes may occur. Edman degradation of ³H-labeled 58 and 61 kilodalton PG processing intermediates indicates that the site of signal sequence cleavage is after amino acid 24 (serine). These results suggest that the proteolytic processing of PG occurs in at least two steps, the first being the co-translational removal of the 24 amino acid signal sequence and the second being the presumed post-translational removal of the remaining highly charged 47 amino acid prosequence.     

In vitro processing of E. coli tRNA precursors

Using E. coli tRNA precursors isolated from an RNAase P mutant strain, we have studied the steps required for the formation of tRNAs having a mature primary sequence in vitro. Our results suggest that at least three different enzymatic activities can participate in the processing of tRNA precursors.     

In vitro processing of pro-subtilisin produced in Escherichia coli

In a previous paper (Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864), we demonstrated that the pro-sequence consisting of 77 amino acid residues at the amino terminus of subtilisin is essential for the production of active subtilisin. When the aggregates of pro-subtilisin produced in Escherichia coli were solubilized in 6 M guanidine hydrochloride and dialyzed against 200 mM sodium phosphate buffer (pH 7.1 or 6.2), pro-subtilisin was efficiently processed to active subtilisin. When more than 14 residues were removed from the amino terminus of the pro-sequence, active subtilisin was no longer produced as in the in vivo experiments. Similarly, active subtilisin would not renature under the same conditions once solubilized in guanidine hydrochloride. When the aspartic acid residue at the active site (Asp32) was altered to asparagine, processing of mutant pro-subtilisin was not observed even in the presence of wild-type pro-subtilisin. Inhibitors such as phenylmethanesulfonyl fluoride or Streptomyces subtilisin inhibitor did not block the processing of wild-type pro-subtilisin. These facts indicate that processing or pro-subtilisin is carried out by an intramolecular, self-processing mechanism. When the sample was dialyzed against 20 mM sodium phosphate (pH 6.2), no active subtilisin was found, suggesting that the highly charged nature of the pro-sequence plays an important role in the process of refolding of denatured pro-subtilisin.     

In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase

A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose. Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B. subtilis PNPase gene (Luttinger et al ., 1996 — accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase). Processive 3'-to-5' exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site. Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure. A similar stall site was observed for an RNA that comprised the intergenic region between the B. subtilis rpsO and pnpA genes. The ability to initiate degradation of a substrate that had a stem structure at its 3' end differed for the B. subtilis and Escherichia coli PNPase enzymes.     

In vitro synthesis of artificial polysaccharides by glycosidases and glycosynthases

Artificial polysaccharides produced by in vitro enzymatic synthesis are new biomaterials with defined structures that either mimic natural polysaccharides or have unnatural structures and functionalities. This review summarizes recent developments in the in vitro polysaccharide synthesis by endo-glycosidases, grouped in two major strategies: (a) native retaining endo-glycosidases under kinetically controlled conditions (transglycosylation with activated glycosyl donors), and (b) glycosynthases, engineered glycosidases devoid of hydrolase activity but with high transglycosylation activity. Polysaccharides are obtained by enzymatic polymerization of simple glycosyl donors by repetitive condensation. This approach not only provides a powerful methodology to produce polysaccharides with defined structures and morphologies as novel biomaterials, but is also a valuable tool to analyze the mechanisms of polymerization and packing to acquire high-order molecular assemblies.     

In vitro and in vivo activity of an antibacterial peptide analog against uropathogens

The alarming rate of bacterial resistance induction highlights the clinical need for antimicrobial agents that act by novel modes of action. Based on the activity profile, the general tissue distribution and renal clearance of peptide-based drugs, we hypothesized that our newly developed pyrrhocoricin derivative would be able to fight resistant uropathogens in vitro and in vivo. Indeed, the Pip-pyrr-MeArg dimer killed all 11 urinary tract infection-related Escherichia coli and Klebsiella pneumoniae strains we studied in the sub-low micromolar concentration range. Almost all control antibiotics, including the currently leading trimethoprim-sulfametoxazole combination for urinary tract infection, remained without considerable activity against two or more of these bacterial strains. In a mouse ascending urinary tract infection model with E. coli CFT073 as pathogen, two doses of intravenous, subcutaneous or oral treatment with the Pip-pyrr-MeArg derivative reduced the bacterial counts in the kidneys, bladder and urine to varying levels. Statistically significant elimination or reduction of bacteria compared to untreated animals was observed at dual intravenous or subcutaneous doses of 0.4 or 10mg/kg, respectively. Serial passage of the same E. coli strain in the presence of sublethal doses of the designed peptide failed to generate resistant mutants. The Pip-pyrr-MeArg dimer showed no toxicity to COS-7 cells to the highest 500microM concentration studied.     

In vitro regeneration of Aristolochia tagala and production of artificial seeds

Protocols for in vitro plant multiplication from somatic tissues and production of artificial seeds through encapsulation of nodes were developed for Aristolochia tagala Cham., a rare and valuable medicinal plant, as a measure of conservation and as a prerequisite for genetic transformation procedure. A maximum number of adventitious shoots were regenerated from leaf-derived callus on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP; 2 μM), α-naphthaleneacetic acid (NAA; 0.5 μM), and phloroglucinol (PG; 10μM). Nodes collected from in vitro established shoot cultures were encapsulated in 3 % (m/v) sodium alginate and 1 % (m/v) calcium chloride. Multiple shoots were successfully regenerated from the encapsulated nodes cultured on MS medium supplemented with 3 μM BAP and 0.5 μM kinetin (KIN). Regenerated shoots from callus and artificial seeds were successfully rooted and acclimated to greenhouse conditions. Since roots of A. tagala are primarily used in traditional medicine, a protocol for regenerating roots directly from the leaf derived callus was also developed. Maximum root length was obtained when the callus was cultured in MS medium supplemented with KIN (1 μM), indole acetic acid (IAA; 0.5 μM), NAA (0.1 μM), and PG (10 μM). Biochemical parameters were studied in calli grown with and without PG in the medium to establish a correlation between these parameters and shoot morphogenesis. An increment of antioxidant enzymes (peroxidase and catalase) and metabolites (sugars and proteins), and a decrease in the amount of polyphenol oxidase was observed in the calli which were grown in the presence of PG.     

In vitro translation and processing of rat kidney gamma-glutamyl transpeptidase

Rat kidney gamma-glutamyl transpeptidase is composed of two nonidentical glycosylated subunits. The enzyme is localized on the lumenal surface of the brush-border membranes of proximal tubule epithelial cells; it is attached to the membranes via an NH2-terminal segment of the larger of the two subunits. Tissue-labeling experiments followed by immunoprecipitation with antibodies directed against the enzyme and its two subunits demonstrate that a glycosylated single chain precursor (Mr = 78,000), containing the elements of both the subunits, is initially synthesized. Pulse-chase studies in the presence of pactamycin, and inhibitor of protein synthesis initiation, indicate that the larger of the two subunits is located at the NH2 terminus of the Mr = 78,000 precursor. The initial events in the biosynthesis and processing of gamma-glutamyl transpeptidase were investigated by in vitro translation of rat kidney mRNA. Such translation results in the synthesis of a Mr = 63,000 unglycosylated polypeptide which has been shown immunologically to contain the domains for both subunits. The Mr = 63,000 species is processed to a Mr = 78,000 core-glycosylated polypeptide when translation of mRNA is carried out in the presence of dog pancreas microsomes. This processing does not appear to be associated with cleavage of an NH2-terminal leader sequence. The Mr = 78,000 polypeptide is integrated into the microsomal membranes with an orientation that is analogous to that found on the brush-border membranes. Glycosylation and membrane integration of transpeptidase are cotranslational events. Upon longer incubation, the Mr = 78,000 species sequestered within the microsomal vesicles is cleaved to species corresponding in size to the two subunits of the kidney enzyme.     

In vitro processing of amyloid precursor protein by cathepsin D

The formation of beta A4 amyloid in the brains of individuals with Alzheimer's disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimer's disease.