Electro-optic periodically poled lithium niobate Bragg modulator as a laser Q-switch.

We report an electro-optic Bragg modulator using a periodically poled lithium niobate (PPLN) crystal. We measured a half-wave voltage of 160 V when transmitting a 1064 nm laser through a 14.2 mm long, 780 microm thick, 20.13 microm period PPLN crystal at the Bragg angle. We also demonstrated a Q-switched Nd:YVO(4) laser using such a PPLN Bragg modulator as its Q-switch, producing 7.8 ns, 201 microJ pulses at a 10 kHz repetition rate when pumped by a 19.35 W diode laser at 808 nm.

Monolithically integrated laser Bragg Q-switch and wavelength converter in a PPLN crystal.

We report a periodically poled lithium niobate (PPLN) crystal for both temperature-insensitive laser Q-switching and temperature-tuned wavelength conversion. The PPLN crystal consists of two sections, a 20.3-mum period section functioning as an electro-optic Bragg grating for Qswitching a diode-pumped Nd:YVO4 laser at 1064 nm and a 31-mum-period section functioning as an optical parametric generator for down converting the generated 1064-nm laser. When driving the PPLN Bragg grating with 170-V voltage pulses, we measured 181 muJ pulse energy at 1064 nm from the Nd:YVO4 laser pumped by 20.4 W diode power. The 181-muJ pulsed laser was further converted into mid-infrared radiation in the monolithic PPLN crystal with 35% parametric efficiency. The wavelengths were broadly tunable in the range of 1.75-1.88 mum (signal) and 2.7-2.44 mum (idler) via temperature without affecting the performance of the PPLN Bragg Qswitch.

Molecular characterization and chromosomal mapping of porcine adipose differentiation-related protein (ADRP).

ADRP plays an important role in regulating lipid storage in various cells. We investigated the ADRP gene as a candidate gene for intramuscular fat deposition and marbling traits in pigs. A full-length transcript of porcine ADRP was cloned by RT-PCR and RACE. The porcine ADRP cDNA (1848 bp) contains a 1377-bp open reading frame, encoding a deduced protein of 459 amino acids, which has amino acid sequence identities of 89, 89, 82 and 81% with cattle, human, mouse and rat ADRP genes respectively. The genomic structure and sequence of the porcine ADRP were also analysed using a BAC clone of a Korean native pig. Pig ADRP comprises eight exons spanning approximately 13 kb and is located on chromosome 1 q2.3-q2.7 between microsatellite markers SW2185 and SW974. Several sequence variations were detected from nine different pig breeds. The biological role of this gene and the mapping localization indicated that the porcine ADRP is a possible candidate gene for fat deposition and marbling traits.

Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.

The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.

The physiological and pharmacological roles of cytochrome P450 isoenzymes.

The cytochrome P450 isoenzymes are a superfamily of haemoprotein enzymes that catalyse the metabolism of a large number of endogenous and exogenous compounds. Recently, the cytochrome isoenzymes have been shown to be important in the synthesis of steroid hormones and bile acids, the arachidonic acid cascade and in central nervous function. These enzymes are a major determinant of the pharmacokinetic behaviour of numerous drugs. Furthermore, alterations in cytochrome P450 activity have been implicated in some diseases.

Multiple changes in gene expression are associated with normal cell-induced modulation of the neoplastic phenotype.

Specific regulatory pathways in neoplastic cells seem to be responsive to control signals provided by the normal cell/tissue environment. The present experiments were designed to define, at the molecular level, the growth-regulatory signals in neoplastic cells that are associated with the modulation of expression of the neoplastic phenotype by normal cell populations. When cultured in the presence of normal cell-conditioned medium, a highly malignant rat tracheal carcinoma-derived cell population (IC-12) undergoes dramatic changes in morphology, and the anchorage-independent growth of these cells is inhibited. This phenomenon is termed normalization. The strategy adopted for elucidating the cellular/molecular changes associated with the induction of these phenotypic alterations was to define the differences in mRNA expression patterns between IC-12 populations exhibiting the neoplastic phenotype (wild-type cells) and those exhibiting the normalized phenotype. For this purpose, the differential display technique and subsequent Northern blot analyses were used. Once specific, differentially expressed genes were identified, the temporal sequence of altered gene expression was determined by monitoring the levels of mRNA expression after the addition of normal cell-conditioned medium. Some of the identified known genes are grouped into three general categories: (a) group I genes are those involved in cellular adhesion processes; (b) group II genes are those involved in signal transduction pathways; and (c) group III genes are those involved in transcriptional and translational processes. Genes that are differentially expressed during the normalization process seemed to exhibit characteristic temporal expression patterns after the addition of normal cell-conditioned medium. Identification of these differentially expressed genes and their associated cellular functions provide insight into some of those regulatory pathways in neoplastic cells that are amenable to regulation by normal cells. An analysis of the temporal sequence of altered gene expression provides further information that allows the identification of those genes that are likely to be critical upstream effectors regulating transcriptional regulatory events that result in the moderation of neoplastic behavior.

Emergence of undifferentiated rat tracheal cell carcinomas, but not squamous cell carcinomas, is associated with a loss of expression of E-cadherin and of gap junction communication.

A series of cells representing normal, non-tumorigenic cell lines, as well as differentiating neoplastic and undifferentiated neoplastic rat tracheal epithelial cell populations were evaluated for their ability to establish homologous and/or heterologous cell-cell gap junction communication in culture. Gap junction communication was evaluated by flow cytometric quantitation of the transfer of the fluorescent dye calcein from a donor to a recipient cell population via gap junctions. The data indicate that normal primary cultures of rat tracheal epithelial cells, as well as non-tumorigenic cell lines and squamous cell carcinomas cell populations, retain the ability to establish both homologous and heterologous gap junction communication. In all cases an average of >48% of recipient cells had acquired calcein label during a 5-h interval of co-culture of donor and recipient cells at confluent densities. Cells harvested directly from squamous cell carcinoma tumors exhibited similar levels of cell-cell communication. In contrast, cells giving rise to undifferentiated carcinomas, as well as cells harvested from undifferentiated carcinomas, exhibited very low levels or no homologous or heterologous cell-cell communication. Cell populations exhibiting distinctly different communication phenotypes were evaluated by Northern blot analysis for expression of connexins (Cx 26, 32 and 43) and E-cadherin. Neither communicating nor non-communicating cells expressed connexin 32. Those cell populations, which established functional gap junctions, expressed E-cadherin as well as connexin 26 and/or 43. In contrast, those cell populations that lacked the ability to communicate universally lacked expression of E-cadherin, and a quarter also lacked expression of detectable levels of connexin.

Selective serotonin reuptake inhibitors. Pharmacology and clinical implications in anaesthesia and critical care medicine.

The newer, selective serotonin reuptake inhibitors are increasingly used in psychiatry in both children and adults. Although they have fewer side-effects, they can cause significant physiological changes and drug interactions which have implications for the anaesthetist and the critical care physician, especially the potential to induce the serotonin syndrome.

A simple and rapid method for purification of oligodeoxyribonucleoside methylphosphonates.

An alternative dimethoxytrityl-on (dmt-on) method is described to purify hydrophobic oligodeoxyribonucleoside methyl-phosphonates (OM) with a phosphodiester linkage at the 5' end, instead of the conventional dmt-off method using a DEAE ion-exchange column. This method is modified from the reverse-phase method for purification of normal oligonucleotides.

Identification of uidA gene sequences in beta-D-glucuronidase-negative Escherichia coli.

A probe specific for the uidA gene of Escherichia coli hybridized with 112 of 116 E. coli isolates examined, including 31 beta-D-glucuronidase-negative and 12 enterohemorrhagic E. coli serotype O157:H7 isolates. Southern hybridizations confirmed the presence of a 900-bp HinfI fragment from the uidA gene in all isolates examined, suggesting that uidA gene sequences are present in most E. coli.

Effect of antibiotic pretreatment on glycoside/glycosidase-based colonic drug delivery.

The effect of antibiotic pretreatment on the intestinal distribution and hydrolysis of the prodrug prednisolone-beta-D-glucoside was studied in rats. A combination of neomycin, lincomycin, and metronidazole was administered twice daily by gastric intubation for three days to young adult male rats. On the fourth day, prednisolone-beta-D-glucoside was administered intragastrically. The distribution of prodrug and drug in the intestinal contents was significantly altered by the antibiotic treatment. In comparison with untreated rats, stomach to cecum transit time appeared to be reduced, and more prodrug was hydrolyzed in the small intestine. In addition, an appreciable amount of the dose was retained longer in the small intestine of treated animals. The total recovery of prodrug and drug was unaltered by the pretreatment. Possible explanations for the observed results are presented.

Proportion of beta-D-glucuronidase-negative Escherichia coli in human fecal samples.

Convenient assays and reports that almost all clinical isolates of Escherichia coli produce beta-D-glucuronidase (GUR) have led to great interest in the use of the enzyme for the rapid detection of the bacterium in water, food, and environmental samples. In these materials, E. coli serves as an indicator of possible fecal contamination. Therefore, it was crucial to examine the proportion of GUR-negative E. coli in human fecal samples. The bacterium was isolated from 35 samples, and a mean of 34% and a median of 15% were found to be GUR negative in lauryl sulfate tryptose broth with 4-methylumbelliferyl-beta-D-glucuronide. E. coli from three samples were temperature dependent for GUR production: very weakly positive at 37 degrees C but strongly positive at 44.5 degrees C. These results remind us of differences between fecal and clinical E. coli populations, of diversity in GUR regulation and expression in natural populations of E. coli, and of the need for caution in using GUR for the detection of fecal E. coli.

Pancreatobiliary and biliary control of fecal beta-glucuronidase activity in the rat.

Bile duct ligation experiments suggest that bile is an important regulator of intestinal beta-glucuronidase, an enzyme thought to be involved in colon carcinogenesis. Exclusion of pancreatobiliary secretions from the rat intestine significantly decreases glucuronidase activity (Roberton et al., Cancer Res., 42 (1982) 5165-5166). However, the separate roles of pancreatic and biliary secretions have not been examined. We ligated the bile ducts of rats at the hepatic duct, allowing pancreatic juice but not bile to enter the intestine, or at the Sphincter of Oddi, excluding pancreatic juice as well as bile. Fecal beta-glucuronidase activity was lowered in both cases, indicating that bile itself, and not pancreatic juice, is a major factor modulating beta-glucuronidase activity.

Effects of certain dietary fibers on apparent permeability of the rat intestine.

Apparent intestinal permeability was determined indirectly by orally administering a poorly absorbed dye, phenol red, to rats and measuring its recovery in feces and in urine. Increased apparent permeability was recognized by increased dye recovery in urine and by an increased ratio of urinary to fecal dye recovery. Guar gum, pectin, carrageenan type I (80% kappa, 20% lambda), carrageenan type II (iota) and cellulose were each fed at levels of 5 and 15% (wt/wt) of the diet for 31 d to male Fischer 344 rats. The average initial weight of rats was 230 g. Rats fed 15% guar gum gained significantly less weight than most of the other rats (P less than 0.05). Phenol red recovery was measured at 2 and 4 wk after the beginning of the experiment. At 2 wk urinary recoveries of phenol red were high in rats fed fiber-free and carrageenan type II diets, indicating increased apparent permeability. By 4 wk, adaptation had apparently taken place. Urinary dye recoveries were lower in every diet group, and most fiber-containing diet groups gave significantly lower recoveries than did the fiber-free group. Fecal recovery of phenol red was high in the cellulose, carrageenan I, and 5% carrageenan II groups, intermediate in the 5% pectin and 15% carrageenan II groups, and low in the fiber-free, guar gum and 15% pectin groups at both 2 and 4 wk. The ratio of phenol red recovery from urine to that from feces, another index of apparent intestinal permeability, was higher in the fiber-free diet group than in all the other groups. Rats fed 15% dietary fiber had higher average ratios than those fed the same fiber at 5%. These data are consistent with the hypothesis that intestinal permeability to foreign substances may be altered considerably by diet.

Drug glycosides: potential prodrugs for colon-specific drug delivery.

The influence of prodrug structure on specificity of glycoside/glycosidase based colon-specific drug delivery was studied by preparing nine steroid glycosides, measuring their relative lipophilicities, and hydrolyzing them with bacterial glycosidases from rat intestines. The 21-yl beta-D-glucosides and galactosides of dexamethasone, prednisolone, hydrocortisone, and fludrocortisone and the 21-yl beta-D-cellobioside of prednisolone were prepared by a modified Koenigs-Knorr reaction. The deacetylated glycoside prodrugs, along with the P-nitrophenyl derivatives of beta-D-glucoside, galactoside, and cellobioside, were subjected to hydrolysis by the contents of the rat stomach, proximal small intestine (PSI), distal small intestine (DSI), and cecum. All the prodrugs were hydrolyzed slowly by PSI and stomach contents, more rapidly by contents of the DSI, and most rapidly by cecal contents. This is the basis of the site-specific drug delivery reported earlier (Friend, D. R.; Chang, G. W. J. Med. Chem. 1984, 27, 261). Furthermore, the prodrugs themselves had very different susceptibilities to hydrolysis. Hydrolysis rates catalyzed by DSI contents decreased in the following order: prednisolon-21-yl beta-D-galactoside (10) greater than prednisolon-21-yl beta-D-glucoside (2) greater than prednisolon-21-yl beta-D-cellobioside (13) greater than dexamethason-21-yl beta-D-galactoside (9) greater than dexamethason-21-yl beta-D-glucoside (1). Hydrolysis of cellobioside 13 was only half that of glucoside 2 and one-fourth that of galactoside 10. Hydrolysis of all the prodrugs in cecal contents was rapid, with the exceptions of hydrocortison-21-yl beta-D-glucoside (5) and fludrocortison-21-yl beta-D-glucoside (7), which were hydrolyzed more slowly than the other glucoside prodrugs. Eadie-Hofstee plots for hydrolysis of the glucoside compounds suggested that bacterial beta-D-glucosidase activity in the colon may be more heterogeneous in nature than beta-D-galactosidase activity. Relative lipophilicities of the prodrugs and free steroids were compared by measuring their octanol-buffer partition coefficients (P). The logarithm of the P of cellobioside 13 (-0.56) was considerably lower than that of the other prodrugs, which ranged from 0.11 to 0.84. Log P of the free steroids ranged from 1.54 to 1.73. These relative rates of hydrolysis and relative lipophilicities, along with previously reported animal experiments, enable one to estimate the site specificity of glycoside prodrugs prior to extensive animal studies.

A colon-specific drug-delivery system based on drug glycosides and the glycosidases of colonic bacteria.

Steroid glycosides and the unique glycosidase activity of the colonic microflora form the basis of a new colon-specific drug-delivery system. Drug glycosides are hydrophilic and, thus, poorly absorbed from the small intestine. Once such a glycoside reaches the colon it can be cleaved by bacterial glycosidases, releasing the free drug to be absorbed by the colonic mucosa. This concept was illustrated with dexamethasone 21-beta-D-glucoside (1) and prednisolone 21-beta-D-glucoside (2), two prodrugs that may be useful in treating inflammatory bowel disease. Hydrolysis of the prodrugs by beta-glucosidase and fecal homogenates in vitro released the free steroids. Glucosides 1 and 2 were administered to rats intragastrically to determine when and where the free steroids were released. Unmodified dexamethasone (3) and prednisolone (4) were also given to rats intragastrically to compare absorption of the glucosides with the free steroids. Both glucosides were found to reach the rat lower intestine in 4-5 h, where they were rapidly hydrolyzed, releasing the free steroids. Delivery of steroid 3 (via glucoside 1) was more specific than that of steroid 4 (via glucoside 2): nearly 60% of an oral dose of glucoside 1 reached the cecum, whereas less than 15% of glucoside 2 reached the cecum. When free steroids 3 and 4 were administered orally, they were almost exclusively absorbed in the small intestine: less than 1% of an oral dose of each reached the cecum.

Effects of bran, lignin and deoxycholic acid on the permeability of the rat cecum and colon.

The ability of dietary fiber to modify the effects of a bile acid on permeability of the cecum and colon was studied. A cecal catheter, which permits administration of test materials to conscious, unrestrained rats over a period of several weeks, was designed. Rats were fed fiber-free diet or diets containing 20% bran or lignin. Permeability of the lower intestine was assessed indirectly by infusing polyethylene glycol (PEG) and measuring excretion of PEG in urine. Under these conditions there was little effect of diet on permeability of PEG. However, when sodium deoxycholate was infused with the PEG, permeability was increased in rats fed fiber-free and lignin diets. In contrast, rats fed the bran diet showed no such response to the bile acid. The interaction among type of dietary fiber, presence of bile acid and intestinal permeability may have important implications for the etiology of intestinal disease.

Effects of dietary fiber on fecal mucinase and beta-glucuronidase activity in rats.

Mucinase and beta-glucuronidase enable colon bacteria to degrade protective mucins and recycle glucuronide conjugates of toxins and carcinogens. The response of these bacterial enzymes to dietary fiber was studied in the laboratory rat. Fiber-free basal diet was mixed with guar gum, pectin, carrageenan, or cellulose at levels of 5 and 15%. These diets were fed for 21 days to groups of six male Fischer-344 rats having an average weight of 150 g. Mucinase and beta-glucuronidase activities were assayed in fresh rat feces. Rats fed 15% guar gum or pectin gained significantly (P less than 0.05) less weight than the other rats. Mucinase specific activity was highest in the fiber-free diet group and lowest in the 15% guar gum group. Total daily output of mucinase was highest in rats fed fiber-free diet or cellulose and lower in rats fed more readily fermentable fiber. Specific activity and total output of beta-glucuronidase were highest in rats fed fiber-free diet and significantly lower in those fed 15% fiber diets. These data are consistent with the hypothesis that some kinds of dietary fiber may play a role in the etiology of intestinal disease.