Chemical constituents from the bark of Hibiscus syriacus L

Seven constituents (I-VII) were isolated from the bark of Hibiscus syriacus and identified as nonanedioic acid (I), suberic acid (II), 1-octarcosanol (III), beta-sitosterol (IV), 1,22-docosanediol (V), betulin (VI) and erythrotriol (VII). VII was obtained from the plant for the first time, I, II, III and VI were isolated from Malvaceae plants for the first time.     

Metabolism of 1,3,7-trimethyldihydrouric acid in the rat: new metabolic pathway of caffeine

[1-CH3-14C] 1,3,7-trimethyldihydrouric acid which, in quantity, is the most important caffeine metabolite, was isolated and purified from the urine of rats fed with [1-CH3-14C] caffeine. The oral administration of this metabolite to rats showed that 1,3,7-trimethyldihydrouric acid was excreted unchanged in urine and was therefore an end product of caffeine metabolism. This result implies a new metabolic pathway of caffeine.     

Immunizing against depression and anxiety after spinal cord injury

We attempted to isolate lipolytic substances from the stem and rhizome of Sinomenium actum Rehder et Wilson by using high-performance liquid chromatography (HPLC). S-I and S-II were isolated from the fractions showing lipolytic activity. S-I and S-II were identified as caffeine and 1,7-dimethylxanthine, respectively, by direct comparison with authentic samples. Caffeine (S-I) dose-dependently stimulated lipolytic activity in isolated fat cells of rats, at concentrations of 500 to 1000 microM. 1,7-Dimethylxanthine (S-II) also stimulated lipolytic activity at concentrations of 500 to 1000 microM. Furthermore, we found that caffeine and 1,7-dimethylxanthine enhanced catecholamine-induced lipolysis at lower concentrations of 0.1 to 1 microM.     

Metabolism of N-ethyl-3-piperidyl benzilate in rats

The metabolic fate of N-ethyl-3-piperidyl benzilate (I) and its potential metabolites 3-piperidyl benzilate (II), N-ethyl-3-hydroxypiperidine (III), and 3-hydroxypiperidine (IV) was studied. Incubation of I with rat liver homogenates resulted in the formation of II and III. Only a trace of unchanged drug appeared in urine after intraperitoneal injection of I. Approximately 9% of the injected dose of I was excreted in urine as III and 2% in the form of metabolites that produced III after acid hydrolysis. After intraperitoneal injection of II in rats, 18% of the dose was excreted in urine as IV. Approximately 26% of the injected dose of III was present in urine as the unchanged drug, and 63% of the dose was excreted in the urine in the form of conjugates that produced III on acid hydrolysis. Urine of rats injected with IV contained approximately 50% of the injected dose as the unchanged drug and 50% of the dose in the form of a conjugate that produced IV on acid hydrolysis. The identity of the metabolites in extracts from urine was established by GLC-mass spectrometry. It is concluded that hydrolysis was one metabolic pathway for I and II. The major routes of elimination of these compounds are not yet known and may include excretion in feces or metabolic transformations resulting in the degradation of the piperidine ring.     

Transformation of Escherichia coli with foreign DNA by electroporation

A new alkaloid, named desmodimine, C12H15NO4, gum, and a new natural product, named desmodilactone, C8H13NO3, mp 84-85 degrees C have been isolated from the aerial parts of Desmodium styracifolium (Osbeck) Merr. belonging Leguminosae. On the basis of spectral analysis their structures were deduced as formula I and II. In addition, lupenone (III), lupeol (IV), tritriacontane (V), stearic acid (VI), eicosanoic acid eicosyl ester (VIII) and beta-sitosterol (VII) were isolated for the first time from this plant.     

Microbial transformation of beta-sitosterol and stigmasterol into 26-oxygenated derivatives

The genetically modified Mycobacterium sp. BCS 396 strain has been used to transform sterols with stigmastane side chain in order to obtain 26-oxidized metabolites. beta-Sitosterol (I) was transformed to 4-stigmasten-3-one (II), 26-hydroxy-4-stigmasten-3-one (III), and 3-oxo-4-stigmasten-26-oic acid (IV), while stigmasterol (V) was converted to 4,22-stigmastadien-3-one (VI), 6 beta-hydroxy-4,22-stigmastadien-3-one (VII), 26-hydroxy-4,22-stigmastadien-3-one (VIII), 3-oxo-4,22-stigmastadien-26-oic acid methyl ester (IX), and 3-oxo-1,4,22-stigmastatrien-26-oic acid methyl ester (X) with that strain. In both beta-sitosterol and stigmasterol, 26-oxidation generates the R-configuration on C-25.     

Ultrasonography of cystic thyroid nodules: sonographic-pathologic correlation

Retrospectively the ultrasonographic findings of 153 surgically resected cystic thyroid nodules were reviewed. The pathologic findings in this series revealed that 86% were degenerating benign adenomas or adenomatous goiters, and 14% were malignant tumors. The sonographic appearance of these lesions was classified into 7 groups as follows: type I: entirely cystic (less than 1cm), type II: cystic(more than 1cm) [II(a)], and with small polyp or dome-like elevation on the cyst wall [II(b)], type III: larger cyst with projection (more than 1cm) into the lumen, type IV: cyst with a peripherally localized solid component, type V: irregularly mixed cystic and solid components, type VI: a solid mass with multiple crescentic cysts [VI(a)], or round cysts [VI(b)], type VII: a solid mass with only one or two cysts. Pathologic correlation revealed that malignancy in this series ranged from 80% in type III and V to only 4% in type II, where most of the lesions in this group were composed of granulation tissue in degenerating adenomatous polyps and cyst walls. Lesions in type IV showed malignancy rate of 40%. Type III showed characteristic sonographic findings seen in cystic papillary carcinomas (CPCs), with multiple punctate echogenic foci in large pedunculated projections. The typical psammomatous calcifications specific in this group were confirmed in 6 of the 8 type III CPCs. The multiple crescentic cysts in type VI(a) lesions were characteristic sonographic signs seen in adenomatous goiters, representing the pathologic finding of cysts forming around each of multiple adenomatous nodules in this group. Type VII represented non specific appearing lesions, included adenomas, adenomatous goiters, CPCs and follicular carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of photosensitive fatty acids into phospholipids of Escherichia coli and irradiation-dependent cross-linking of phospholipids to membrane proteins

In an approach to the study of phospholipid-protein interactions in biological membranes, the photoactivable fatty acids, omega-(m-azidophenoxy)-undecanoic acid (I) and omega-(m-diazirinophenoxy)-hexadecanoic acid (II), were incorporated biosynthetically into the phospholipids of the Escherichia coli fatty acid auxotroph, strain K1060-B5. The extent of incorporation of the two fatty acids was 43% and 21%, respectively, of the total fatty acid content of the phospholipids. Membrane vesicles prepared from cells grown on the fatty acid supplements and [32P]H3PO4 were irradiated at suitable wavelengths to generate the reactive nitrene or carbene intermediates. Subsequent analysis of solubilized membrane proteins by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis indicated cross-linking between radioactive phospholipids and an number of proteins. A corresponding experiment with cells grown on oleic acid showed only trace amounts of covalently cross-linked phospholipid-protein adducts. While the extent of cross-linking in vesicles from cells grown on I was only 3 times the background level observed for oleic acid-grown cells, cells grown on II showed 30 times this amount. The present results, together with the previously observed nonreactivity of the nitrene generated from I to undergo C-H insertion, show that the use of carbene precursors such as II is promising for chemical analysis of specific phospholipid-protein interactions in bacterial membranes under biologically meaningful conditions.     

Purification and properties of two isozymes of gamma-glutamyltranspeptidase from Bacillus subtilis TAM-4

Two isozymes of gamma-glutamyltranspeptidase, GGT-A and GGT-B, were purified to electrophoretic homogeneity from a culture broth of Bacillus subtilis TAM-4, which produces poly(gamma-glutamic acid) (PGA) de novo. GGT-A was composed of three subunits with molecular weights of 23,000 (I), 39,000 (II), and 40,000 (III). GGT-B was composed of two subunits with molecular weight of 22,000 (I) and 39,000 (II). The N-terminal amino acid sequences of GGT-A subunit I and GGT-B subunit I were very similar. GGT-A subunit II and GGT-B subunit II had an identical N-terminal amino acid sequence. That of GGT-A subunit III showed no similarity to the other subunits. Both GGTs had similar enzymatic properties (optimum pH and temperature: pH 8.8 and 55 degrees C) but showed a significantly different thermal stability at 55 degrees C. Both GGT-A and -B used D-gamma-glutamyl-p-nitroanilide as well as the L-isomer as the gamma-glutamyl donor and used various amino acids and peptides as the acceptor. It was also found that the PGA produced by the strain was hydrolyzed to glutamic acid by its own GGTs.     

Renal handling and plasma elimination kinetics of carbonic anhydrase isoenzymes I, II and III in the rat

Using a radio-immunosorbent technique, the levels of the carbonic anhydrase (CA) isoenzymes CA I, II and III in plasma (1-3 micrograms ml-1), lymph (0.5-1.6 micrograms ml-1) and urine (0.03-0.06 micrograms ml-1), were determined in the rat. The renal clearances of CA I, II and III were 11 +/- 3, 42 +/- 11 and 35 +/- 4 nl min-1 (g kidney wet wt)-1 (n = 4-5), respectively. After a single i.v. injection of purified native or 125I-labelled isoenzymes, the elimination of CA I, and CA III from plasma followed a bi-exponential decline, with half-times of 7 and 9 min for the rapid phase and 112 min for the slow phase, respectively. Nephrectomy decreased the rapid phase and the build-up of catabolites. Therefore, the rapid phase of CA I and III elimination is probably explained by filtration of unbound isoenzyme at the glomeruli and subsequent degradation by the proximal tubules. The plasma elimination curve for CA II was different and followed a mono-exponential decline, with a half-time of 210 min both in normal and nephrectomized animals. This indicates that CA II is not filtered at the glomeruli. However, in acute renal failure, with leaking tubular cells, CA II was excreted into the urine. The slow elimination of the major part of the isoenzymes from plasma is explained by the binding of CA I, II and III to a plasma protein, immunochemically similar to transferrin, forming a macromolecular complex with a mol wt of 114 +/- 2 kDa.     

Interaction of Oct-1 and automodification domain of poly(ADP-ribose) synthetase

Ataxia telangiectasia (AT) cells display a profound sensitivity to ionizing radiation, exhibiting more frequent chromosomal breaks, increased micronuclei formation and abnormal DNA repair kinetics following exposure. Despite the recent cloning of the ATM gene there remains a need for a simple and rapid means of discriminating AT heterozygotes from normal individuals. Caffeine (1,3,7-trimethyl xanthine), known to inhibit the repair of double-strand DNA breaks following ionizing radiation, increases the frequency of radiation induced chromosomal breaks in normal cells. Here we report that caffeine potentiates the induction of chromosomal breaks in G2 arrested AT heterozygote and normal lymphoblastoid cells, but not in homozygous AT lymphoblastoid cells. This observation parallels the findings reported by others that caffeine fails to potentiate the effect of ionizing radiation in radiation-sensitive yeast strains and radiation sensitive CHO cells. It also suggests that caffeine may somehow mimic the effect of the ATM gene product in normal cells. We also report that caffeine is unlikely to be useful in helping to discriminate AT heterozygotes from normal individuals.     

ON and OFF channels of the frog optic tectum revealed by current source density analysis

The spatiotemporal patterns of excitatory synaptic activity in response to diffuse lightON and OFF stimuli were examined by means of current source density (CSD) analysis. The qualitative and quantitative analyses obtained from 24 depth profiles for each stimulus revealed obviously different distributions of synaptic activity in the laminar structure. Two or three dominant current sinks I, II, and III were evoked in response to diffuse light ON stimulation. Sink I was observed at the bottom of the retinorecipient layer. Both sinks II and III, showing an identical spatial pattern, were observed just above sink I. On the other hand, diffuse light OFF stimulation elicited up to six current sinks IV, V, VI, VII, VIII, and IX. Sink IV was observed at the bottom of the retinorecipient layer. Sink V was observed in the most superficial layer. Both sinks VI and VIII were located between the two preceding sinks. Finally, sinks VII and IX occurred below the retinorecipient layer. Five electrically evoked current sinks A, B, C, D, and E, characterized in our previous study, were also recognized in the present quantitative analysis. A statistical analysis revealed that, in visually evoked responses, statistical differences in the spatial distribution were not present between sinks I and IV, and sinks II and VIII (P < 0.05). The analysis also showed that, in electrically evoked responses, only a pair of sinks C and E exhibit virtually identical spatial distribution (P < 0.05). Based on well-known properties of the retinal ganglion cells, possible neuronal mechanisms underlying each of current sinks in the ON and OFF channels and their functional meanings were considered. Sink I reflects the excitatory monosynaptic activity derived from R3 retinal ganglion cells. Sink IV reflects the excitatory monosynaptic activity derived from both R3 and R4 cells. Sinks V, VI, VII, and IX may be composed of successive polysynaptic excitatory potentials derived from convergence of inputs from both R3 and R4 cells. We concluded that the early four sinks play in particular an important role in eliciting avoidance behavior. On the other hand, sinks II, III, and VIII reflect excitatory synaptic activities derived from - retinal fibers of another type having slow conduction velocity. These late current sinks were suggested to mediate prey catching and its facilitation.     

Molecular forms of gastrin in antral mucosa, plasma and gastric juice during vagal stimulation of anesthetized cats

Gastrin was released by electrical vagal stimulation in anesthetized cats. Antral mucosa, blood and gastric juice samples collected during vagal stimulation were subjected to gel filtration in order to characterize the different molecular forms of gastrin. In antral mucosa component III (gastrin-17) predominated. Besides, the antrum contained 5 per cent component II (gastrin-34, "big" gastrin), 1 per cent component I and trace amounts of component IV (gastrin-14 or "mini" gastrin). Immediately after vagal stimulation, component III (gastrin-17) appeared in the gastric venous effluent followed within a few minutes by component IV (gastrin-14). Component I and II (gastrin-34) were not detectable in any of the plasma samples. We suggest that component III (gastrin-17) is released from the antral mucosa and is then rapidly metabolized to component IV (gastrin-14) possibly to a significant extent in the fundic region of the stomach. Large amounts of component III (gastrin-17) were found in the vagally-induced gastric juice. Only very small amounts of degradation products were present, indicating that cat gastrin is relatively resistant to peptic degradation and acid hydrolysis.     

Interferon alpha with ribavirin for the treatment of chronic hepatitis C in non-responders or relapsers to interferon monotherapy

The ability of different serotypes of group B streptococci (GBS) to induce septic arthritis in mice was compared. Types II, III, IV, V, VI and VII GBS were investigated. A highly capsulate strain of type III GBS, COH1, and its mutants, COH1-11 (lacking capsular sialic acid) and COH1-13 (non-capsulate), obtained by transposon insertional mutagenesis, were used to assess the role of type-specific polysaccharide on the induction of arthritis. At an intravenous dose of 10(7) cfu/mouse, reference strains of types II, III, IV, VI and VII and type III strain COH1 induced arthritis with an incidence ranging from 70 to 90%. For type V and strain COH1-11, 10(8) cfu/mouse was required to obtain a 50% incidence of arthritis; lesions were not evident with strain COH1-13. The presence of the capsule played a major role in the induction of GBS septic arthritis. The presence and amount of sialic acid in capsular polysaccharide influenced the incidence of articular lesions. The bacterial dose affected the manifestations of arthritis; the less virulent strains of GBS also induced articular lesions when an adequate number of micro-organisms reached the joints.     

Membrane traffic and control of proximal tubular sodium phosphate (Na/Pi)-cotransport

Phosphate (P(i)) is freely filtered at the glomerular capillaries and largely reabsorbed in the proximal tubule by a Na-dependent, secondary active transport mechanism. Two different brush border membrane Na/P(i)-cotransporters have recently been "cloned" (type I and type II). Only the type II transporter undergoes physiological regulation (e.g., diet, acid/base, parathyroid hormone); it is also involved in pathophysiological alterations of renal Pi-handling (e.g., X-linked hypophosphatemia). In recent experiments on rats and on tissue culture cells (Opossum kidney cells, OK cells) id was documented that manoeuvres leading to increased uptake involve membrane insertion (fast changes) and new synthesis of type II transporters (slow changes), whereas decreased Na/Pi-cotransport activity is associated with their specific membrane retrieval (fast changes) and lysosomal degradation (slow changes).     

The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases

We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum. The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases. Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized. Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin. Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities. In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak. A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C. However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus. In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A. These findings suggest that both enzymes from P. occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.     

Substrate specificity of T4 RNA-ligase: the role of a purine nucleotide base in forming a covalent AMP-RNA-ligase complex

The NTP binding site of bacteriophage T4 RNA-ligase (EC 6.5.1.3) was studied using several ATP analogs modified in the purine moiety of the nucleotide at positions 1, 2, 6 and 8: adenosine-N'-oxide 5'-triphosphate (I), 6-chloropurine riboside 5'-triphosphate (II), 6-mercaptopurine riboside 5'-triphosphate (III), 1,N6-ethenoadenosine 5'-triphosphate (IV), 8-sulphoadenosine 5'-triphosphate (V), 8-bromoadenosine 5'-triphosphate (VI), inosine 5'-triphosphate (VII) and guanosine 5'-triphosphate (VIII). For all of the ATP analogs under study a reversible inhibition was demonstrated. These analogs appeared to be competitive inhibitors of the T4 RNA-ligase adenylation reaction and only V, VI and VII were efficient as substrates. The kinetic parameters for the competitive inhibition, (I50, Ki) were determined. A putative structure for the T4 RNA-ligase active site was proposed.     

Ontogeny of the endocrine cells of the intestine and rectum of sea bass (Dicentrarchus labrax L.): an ultrastructural study

Several endocrine cell types were ultrastructurally characterized during the differentiation of the intestine and rectum of sea bass (Dicentrarchus labrax L.) larvae. Only one cell type (type I) was found in the posterior region of the undifferentiated gut of 5-day-old larvae (phase I). Types V and VI were found in both the intestine and rectum, types II, III and IV in the intestine, and types VII and VIII in the rectum of 9- and 12-day-old larvae (phase II), the rectum alone showing signs of functional differentiation. In phase III larvae, in which both the intestine and rectum were differentiated, types IX, X, XI, XII, XIII, XIV and XV were found in the intestine, only types X, XI and XII being seen in the rectum. Besides these, a new cell type, XVI, was observed in the intestine of 55- and 60-day-old larvae (phase IV), in which the digestive tract was completely differentiated. The endocrine cells appearing in phases I and II showed very scarce secretory granules and the ultrastructural features of undifferentiated cells. Some endocrine cell types in the earliest developmental stages were related to some of those found later. A maturational process of the endocrine cell types paralleled the differentiation of the intestine and rectum, with an apparent increase in the number of secretory granules accompanying organelle development.     

Characterization of engF, a gene for a non-cellulosomal Clostridium cellulovorans endoglucanase

A new Clostridium cellulovorans (strain ATCC 35296) endoglucanase gene engF has been isolated and sequenced. The gene contains 1671 bp and codes for a protein containing 557 amino acids and a mass of 60.1 kDa. A putative signal peptide of 29 amino acids is present and the mature protein has a mass of 57.1 kDa. EngF does not have amino acid sequence homology to previously isolated EngB and EngD, but does show sequence homology to family 5 glycosyl hydrolases from Bacillus, Erwinia carotovora, and C. acetobutylicum species. EngF is not a component of the cellulosome and does not contain a duplicated sequence (DS) at its C-terminal region. EngF is capable of binding to cellulose and hydrolyzing carboxymethylcellulose but not xylan. The cellulose binding domain (CBD) differs from types I, II and III CBDs and no obvious homology has been found to other CBD types. The maximum activity of EngF occurs at pH 5.5 and at 47 degrees C. Its properties suggest that EngF plays an ancillary role in the degradation of cellulosic materials.     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

The role of metabotropic (mGluRs) and N-methyl-D-aspartate (NMDA) glutamate receptors on 5-hydroxytryptamine (5-HT) release has been studied in rat periaqueductal gray (PAG) matter by using in vivo microdialysis. (1S,3R)-aminocyclopentane- 1,3-dicarboxylic acid [(IS,3R)-ACPD; 0.5 or 1 mM], a group I/group II mGluRs agonist, increased the dialysate 5-HT concentration. (2S)-alpha-ethylglutamic acid (EGlu; 1 mM), an antagonist of group II mGluRs, but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), an antagonist of group I mGluRs, antagonized the 1S,3R-ACPD-induced effect. (S)-3,5-dihydroxyphenylglycine (DHPG; 0.5 and 1 mM), an agonist of group I mGluRs, did not modify dialysate 5-HT. (2S, 3S, 4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I; 0.5 and I mM), an agonist of group II mGluRs, increased extracellular 5-HT. This effect was antagonized by EGlu. Similarly, L-serine-O-phosphate (L-SOP; 1 and 10 mM), an agonist of group III mGluRs, increased extracellular 5-HT and this effect was antagonized by (RS)-(alpha-methylserine O-phosphate (M-SOP; 1 mM), an antagonist of group III mGluRs. Out of the several N-methylD-aspartate concentrations used (NMDA; 10, 50, 100, 500 and 1000 microM) only the 50 microM infusion significantly decreased dialysate 5-HT. The GABA(A) receptor agonist, bicuculline (30 microM), increased 5-HT release on its own and antagonized the decrease caused by the opiate antagonist, naloxone (2 mM), as well as the increases caused by CCG-I or L-SOP. These data show that stimulation of PAG's group II/group II mGluRs increases 5-HT release, while stimulation of NMDA glutamate receptors may decrease it. We speculate that glutamate does not modulate 5-HT release in the PAG directly, but via activation of tonically active GABAergic interneurons.