Bone marrow mononuclear cells versus mesenchymal stem cells from adipose tissue on bone healing in an Old World primate: can this be extrapolated to humans? / Células mononucleares de medula óssea versus células-tronco mesenquimais de tecido adiposo em cicatrização de lesão óssea em primata: pode ser extrapolado para humanos?

In veterinary medicine, the cell therapy is still unexplored and there are many unanswered questions that researchers tend to extrapolate to humans in an attempt to treat certain injuries. Investigating this subject in nonhuman primates turns out to be an unparalleled opportunity to better understand the dynamics of stem cells against some diseases. Thus, we aimed to compare the efficiency of bone marrow mononuclear cells (BMMCs) and mesenchymal stem cells (MSCs) from adipose tissue of Chlorocebus aethiops in induced bone injury. Ten animals were used, male adults subjected, to bone injury the iliac crests. The MSCs were isolated by and cultured. In an autologous manner, the BMMCs were infused in the right iliac crest, and MSCs from adipose tissue in the left iliac crest. After 4.8 months, the right iliac crests fully reconstructed, while left iliac crest continued to have obvious bone defects for up to 5.8 months after cell infusion. The best option for treatment of injuries with bone tissue loss in old world primates is to use autologous MSCs from adipose tissue, suggesting we can extrapolate the results to humans, since there is phylogenetic proximity between species.(AU)

Na medicina veterinária, a terapia celular ainda é inexplorada e há muitas perguntas não respondidas, o que leva os pesquisadores a uma tendência a estender a terapia para os seres humanos, na tentativa de tratar certas lesões. Investigar esse assunto em primatas não humanos revela-se uma oportunidade sem precedentes para compreender melhor a dinâmica das células-tronco contra algumas doenças. Assim, objetivou-se comparar a eficiência das células mononucleares de medula óssea (BMMCs) e das células-tronco mesenquimais (MSCs) do tecido adiposo de Chlorocebus aetiops na lesão óssea induzida. Foram utilizados 10 animais, adultos do sexo masculino, submetidos à lesão óssea nas cristas ilíacas. As MSCs foram isoladas e cultivadas; de forma autóloga, as BMMCs foram infundidas na crista ilíaca direita e as MSCs de tecido adiposo na crista ilíaca esquerda. Após 4,8 meses, a crista ilíaca direita foi totalmente reconstruída, enquanto a crista ilíaca esquerda continuou apresentando defeito ósseo evidente por até 5,8 meses após a infusão. A melhor opção para o tratamento de lesões com perda de tecido ósseo em primatas do Velho Mundo é a utilização de MSCs autólogas de tecido adiposo, sugerindo que se podem estender os resultados para seres humanos, uma vez que há proximidade filogenética entre as espécies.(AU)

Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization, and multipotentiality.

Adult stem cells are known for their plasticity and their potential to differentiate into several different cell types; these characteristics have implications for cell therapy and reproductive biotechnologies. In this study, we report on the isolation and characterization of mesenchymal stem cells (MSC) derived from bovine and buffalo adipose tissue. Cells isolated using enzymatic digestion of bovine and buffalo adipose-tissue biopsy samples were grown in vitro for at least 15 passages, verifying their capacity to proliferate. These cells were also subjected to immunophenotypic characterization for the presence of CD90, CD105, and CD79, and the absence of CD45, CD34, and CD73, which are positive and negative markers of MSC, respectively. To prove their multipotency, the cells were induced to differentiate into three different cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (Chondrogenic-Alcian Blue, Osteogenic-Alizarin Red, and Adipogenic-Oil-Red O, respectively) to confirm differentiation. Gene expression analysis of pluripotency-related genes was also conducted. Our results suggest that adipose tissue from bovines and buffalos can be used as a source of MSC, making adipose tissue-derived cells an interesting option for cell therapy and regenerative medicine. Additionally, these findings have implications for reproductive biotechnology because the use of MSC as nuclear donors has been linked to an increase in the efficiency of nuclear transfer.

Description of bovine major histocompatibility complex class IIa haplotypes using parthenogenetic embryo-derived cells.

In this study, we report an approach to characterize individual BoLA haplotypes using cells from parthenogenetic bovine embryos derived from slaughterhouse ovaries. Eight of the 15 parthenogenetic embryos so obtained had not undergone meiotic recombination on the BoLA region and were suitable to describe BoLA haplotypes. Detailed analysis of the BoLA class IIa region identified seven different class IIa haplotypes, including six not previously described and two new alleles of BoLA-DQA and one BoLA-DQB. Our method provided reliable sources of homozygous DNA to describe BoLA haplotypes.

Maturação in vitro de oócitos bovinos em meios suplementados com quercetina e seu efeito sobre o desenvolvimento embrionário / In vitro maturation of bovine oocytes in medium supplemented with quercetin, and its effect on embryonic development

A quercetina é um flavonoide, amplamente encontrada em frutas, vegetais, grãos, flores, com elevada concentração no vinho tinto, e tem sido caracterizada funcionalmente pela atividade antioxidante. Para avaliação da maturação nuclear e do desenvolvimento embrionário bovino, os oócitos foram maturados por 22h na presença de quercetina (0,4, 2, 10 e 50µM), cisteamina (100µM) e na ausência dos antioxidantes. Os oócitos maturados foram corados com Hoechst para avaliação da maturação in vitro. Para avaliação do desenvolvimento embrionário, os oócitos foram fertilizados e cultivados in vitro, as taxas de desenvolvimento embrionário foram determinadas no sétimo dia de cultivo e o percentual de eclosão e o número de células dos embriões no oitavo dia. Os níveis de glutationa (GSH) dos oócitos foram mensurados por emissão de fluorescência com CMF2HC. A porcentagem de maturação nuclear (±89%) não diferiu entre os grupos. O desenvolvimento embrionário variou entre os tratamentos, o percentual de blastocisto foi superior (P<0,05) nos grupos tratados com 0,4, 2, 10 e 50∝M de quercetina (56,9, 59,5, 53,6 e 49,6%, respectivamente) e com 100∝M de cisteamina (50,4%) em relação ao grupo controle (42,3%). Na comparação entre os dois antioxidantes, a quercetina (0,4 e 2µM) foi superior na produção de embriões (56,9 e 59,5%, respectivamente) em comparação com cisteamina (50,4%). As taxas de embriões eclodidos foram similares (P>0,05) entre os grupos (±63,0%). O número médio de células dos embriões também foi similar entre os grupos (±233). Os níveis intracelulares de GSH foram superiores nos oócitos maturados com cisteamina, mas similares entre os oócitos tratados com quercetina e o controle. A suplementação da maturação in vitro com antioxidantes melhora as taxas de blastocistos. A quercetina foi superior à cisteamina, que, por sua vez, foi superior ao controle. Mas os níveis de GSH foram superiores somente nos oócitos tratados com cisteamina.

Quercetin is a flavonoid widely found in fruit, vegetables, grains and flowers, with a high concentration in red wine, and has been functionally characterized by its antioxidant activity. For assessment of nuclear maturation and bovine embryo, oocytes were matured for 22h in the presence of quercetin (0.4, 2, 10 and 50µM), cysteamine (100µM) and in the absence of antioxidants. The matured oocytes were stained with Hoechst to evaluate the in vitro maturation. To assess embryonic development, oocytes were fertilized and cultured in vitro and rates of embryo development were obtained in the seventh day of culture and the percentage of hatching and the number of cells on eighth day embryos. The levels of glutathione (GSH) of the oocytes were measured by fluorescence emission with CMF2HC. The percentage of nuclear maturation (±89%) did not differ between groups. Embryonic development varied between treatments, the percentage of blastocyst was higher (P<0.05) in the groups treated with 0.4, 2, 10 and 50∝M of quercetin (56.9, 59.5, 53.6 and 49.6%, respectively) and 100 ∝M cysteamine (50.4%) compared to the control group (42.3%). Comparing the two antioxidants, quercetin (0.4 to 2µM) was superior in embryo production (56.9 and 59.5% respectively) compared with cysteamine (50.4%). The rates of hatched embryos were similar (P>0.05) between groups (±63.0%). The average number of embryo cells was also similar in both groups (±233). The intracellular GSH levels were higher in oocytes matured with cysteamine, but similar between the oocytes treated with quercetin and control. The supplementation of matured in vitro with antioxidants improves blastocyst rates. Quercetin was greater than cysteamine, which in turn was superior to the control. However, GSH levels were higher in oocytes treated only with cysteamine.

Effect of triiodothyronine on developmental competence of bovine oocytes.

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRß, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development.

Short-term culture of ovarian cortical strips from capuchin monkeys (Sapajus apella): a morphological, viability, and molecular study of preantral follicular development in vitro.

The aim of this study was to evaluate whether an in vitro culture (IVC) medium containing either or not ß-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) could be able to promote the development of capuchin monkeys' preantral follicles enclosed in ovarian cortical strips. Follicular viability after IVC was similar to control (89.32%). Primordial follicle recruitment to primary stage was not reached with IVC, but the rate of secondary follicle formation was increased in the medium supplemented with BME, BMP4, and PMSG (44.86%) when compared to IVC control (9.20%). In the medium supplemented with BME, BMP4, and PMSG, contrary to other media, anti-müllerian hormone-messenger RNA (mRNA) expression in ovarian tissue was upregulated (3.4-fold), while that of growth differentiation factor-9 was maintained. The BMP4-mRNA expression, however, appeared downregulated in all cultured tissues. Our findings show a favorable effect of BME, BMP4, and PMSG on the in vitro development of secondary follicles from capuchin monkeys.

Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella).

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

Characterization of folliculogenesis and the occurrence of apoptosis in the development of the bovine fetal ovary.

The aim of this research was to perform in situ quantification, morphometry evaluation, and apoptosis analysis of ovarian follicular wall cells in mechanically isolated follicles obtained from ovaries of bovine fetuses (Bos taurus indicus) between 3 and 9 months of age. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The number of isolated follicles increased from 3 months onward (102.5 ± 141.1, mean ± SEM), peaked at 6 months (12855.0 ± 9030.1), and then decreased by 7 months (3208.7 ± 3249.5), consistent with atresia occurring at these stages. Follicular density was greatest at 4 months, consistent with a sudden boost in follicular activity independent of a corresponding increase in ovarian size. Antral follicles were first observed at 5 months. As fetal age increased, there was a tendency for the percentage of primordial and primary follicles to decrease, and the percentage of secondary follicles to increase. However, the high variability (P < 0.05) for all follicle populations up to 5 months of age precluded further interpretation of these results. Oocyte diameter increased from the primordial (23.6 ± 4.4 µm) to the secondary follicular stages (38.0 ± 14.9 µm). Apoptosis was observed in ovaries from all fetal ages analyzed. We concluded that preantral follicles could be isolated from bovine fetuses by 3 months of age, with apoptosis affecting ovarian follicular dynamics throughout fetal life.

Evaluation of apoptosis as a mechanism of follicular cell atresia in the ovaries of cattle (bos indicus) and buffalo (bubalus bubalis) fetuses

The objective of this study was to investigate the occurrence of apoptosis in the ovaries of cattle and buffalo fetuses between 4 and 8 months old by the terminal deoxynucleotidyltransferase - mediated dUTP nick end labeling (TUNEL) assay . Histological analysis of the ovarian t issue showed that apoptosis occurred at all ages evaluated , presenting a similar pattern among different fetal stages in both species . Within species, secondary follicles displayed a higher (P 0.05) of apoptotic follicular cells among the three follicular classes compared . C omparing resul ts between species, secondary follicles had a higher (P < 0.05) mean number of TUN EL positive cells in bovine fetuses; however , this difference was proportional to the larger number of follicular cells present in secondary follicles in this species . In summary, the TUNEL method was effective for the detection of apoptosis in the support ing cells of ovarian follicles from bovine and buffalo fetuses with apoptosis occurring at similar rates in both species between 4 and 8 months of gestational age. Further studies are needed to better understand the dynamics of apoptosis as a regulator of follicular atresia in fetal ovaries from these species, as well as the potential involvement of the oocyte in this process.

Quantification, morphology and ultrastructure of preantral follicles of buffalo (Bubalus bubalis) foetuses.

The objective of this study was to determine the number, morphology and ultrastructure of preantral ovarian follicles of buffalo (Bubalus bubalis) foetuses at different ages. Quantification revealed number of primordial, primary and secondary follicles of 48,857 ± 17,506, 26,000 ± 20,452, 18,428 ± 10,875 and 18,375 ± 19,690, 225 ± 349, 326 ± 288 at 12-34 cm and 35-60 cm crown rump length (CRL), respectively. Follicular diameter values were 28.9 (± 3.4), 34.7 (± 5.9) and 59.4 (± 12.6) µm; oocyte diameters were 21.7 (± 2.8), 24.3 (± 3.4) and 33.0 (± 7.7) µm, and the numbers of follicular cells in the follicle equatorial section were 7.1 (± 1.4), 12.0 (± 2.4) and 13.8 (± 2.4) for primordial, primary and secondary follicles, respectively. The primordial follicle consisted of an oocyte surrounded by a layer of flattened follicular cells with a normally eccentric oocyte nucleus. Dispersed Golgi complex, smooth endoplasmic reticulum, rounded mitochondria and several lipid vesicles were observed in the cytoplasm and cell junctions between the follicle cell membranes and the oocyte. This work describes the number, morphometry and ultrastructure of preantral follicles of buffalo foetuses, concluding that folliculogenesis is established between 8 and 34 cm CRL and that follicle number varies individually and according to age and that further studies are needed in this species.

Isolation, follicular density, and culture of preantral follicles of buffalo fetuses of different ages.

The aim of the present study was to determine the most desirable ovarian tissue section thickness to isolate preantral follicles (Experiment I), determine follicular density (follicles/mm(2) of cortex) of ovaries of fetal buffalo of different ages (Experiment II), and cultivate preantral follicles of buffalo fetuses (Experiment III). In Experiment I, ovary sections with different thicknesses (25, 50, 75, and 100 microm) had 415.0+/-285.2, 457.5+/-341.9, 585.0+/-309.3, and 685.0+/-278.8 isolated preantral follicles, respectively. In Experiment II, the follicular density of 46 buffalo fetuses with ages between 3 and 8 months was estimated to be between 0 and 7220, with means of 0.0, 2070.7+/-2190.3, 2570.8+/-1796.6, 2298.1+/-2286.5, 1277.5+/-1074.9, and 643.6+/-543.9 throughout the age range studied. The follicular density of 5-month-old fetuses was greatest, coinciding with the largest number of follicles isolated at this age. In Experiment III, preantral follicles isolated from the ovaries of buffalo fetuses aged from 5 to 9 months old were cultivated individually for 7 days in four different media: basic medium (Minimal Essential Medium (MEM), 10% SFB, kanamycin, pyruvate, glutamine, hypoxanthine) with additional ITS and FSH 0.5mg/ml (treatment 1); basic medium with FSH and EGF 100 ng/ml (treatment 2); basic medium with additional ITS, FSH, and EGF (treatment 3); basic medium supplemented with ITS and EGF (treatment 4). Integrity and morphological features, viability, and increase in diameter of follicles cultured in vitro were evaluated individually with an inverted microscope and an ocular micrometer. The results showed that follicle structure and form were maintained during culture. Growth and survival rates of treatments 1, 2, and 3 over 7 day culture were 23.25+/-17.06, 33.75+/-26.19, and 43.75+/-31.73 microm, and 31.3+/-22.7, 22.06+/-8.13, and 28.92+/-21.32%, respectively. However, neither growth nor survival was observed in treatment 4. In conclusion, this study showed that preantral follicles of buffalo fetuses can be cultured in vitro, and that FSH is essential for follicle survival.

Cryopreservation of caprine ovarian tissue using dimethylsulphoxide and propanediol.

The caprine ovary is a rich source of potentially viable immature oocytes enclosed in preantral follicles (PF). Previous experiments showed that these oocytes can be successfully cryopreserved in ovarian tissue of several species. However, until now, no information about the caprine PF cryopreservation is available in the literature. The aim of the present research was to evaluate the structural and ultrastructural characteristics of caprine PF after treatment and cryopreservation of ovarian tissue with 1.5 and 3 M dimethylsulphoxide (DMSO) and propanediol (PROH). One fragment of ovarian tissue was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four fragments were equilibrated at 20 degrees C/20 min in 1.8 ml of minimum essential medium (MEM) containing 1.5 or 3 M DMSO or PROH for the toxicity test, and the other four fragments were slowly frozen in each cryoprotectant at the concentrations previously described. After toxicity test and freezing/thawing procedures, the ovarian fragments were fixed for histological examination. The results showed that after toxicity test and cryopreservation of ovarian tissue using both cryoprotectants, the percentage of normal PF was less (P < 0.05) as compared with the control group. The present study revealed that the percentage of normal PF after toxicity test and cryopreservation in 1.5 M DSMO was significantly greater (P < 0.05) as compared with results obtained with 3 M DMSO or 1.5 and 3 M PROH. This result was confirmed by transmission electron microscopy, which showed that the PF were preserved in a higher quality state with 1.5 M DMSO. In conclusion, the present study demonstrated that caprine PF can be cryopreserved in ovarian tissue using 1.5 M DMSO.

Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol.

Cryopreservation of ovarian tissue may be a potential alternative for the conservation of genetically superior animals, including high milk- and meat-producing goat breeds. However, until now, no information was available concerning the cryopreservation of preantral follicles (PF) enclosed in caprine ovarian tissue. The objective of the present study was to evaluate the structural and ultrastructural characteristics of caprine PF after exposure to and cryopreservation of ovarian tissue in 1.5 and 3M glycerol (GLY) and ethylene glycol (EG). At the slaughterhouse, each ovarian pair from five adult mixed breed goats was divided into nine fragments and randomly distributed into treatment groups. One fragment was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four of the ovarian fragments were equilibrated at 20 degrees C for 20 min in 1.8 ml of MEM containing 1.5 or 3M GLY or EG for a toxicity test and the final four fragments were slowly frozen using these cryoprotectants at the concentrations above. After toxicity testing and freezing/thawing, the ovarian fragments were fixed for histological examination. Histological analysis showed that after toxicity testing and cryopreservation of the ovarian tissue in GLY or EG at both concentrations, the percentage of normal PF was significantly lower than controls. Ultrastructural analysis of PF frozen in 1.5 and 3M GLY, as well as 3M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue.

Mechanical isolation of capuchin monkey (Cebus apella) preantral ovarian follicles

The aim of this study was to adapt a mechanical procedure for the isolation of intact preantral follicles from Cebus apella ovaries. The interval effect of serial sections of the tissue chopper was tested on a number of preantral follicles isolated from ovaries (n=6) of three C. apella females, two prepubertal and one adult. Ovaries were divided into four equal parts and fragmented with a tissue chopper, adjusted for serial sections at intervals of 250, 500, 750 and 1,000æm, respectively. Isolated follicles were counted in a Neubauer's chamber and classified as primordial, primary or secondary. The number (mean±SE) of preantral follicles isolated from 1/4 ovary varied from 68,330+17,590 (at the 1,000æm cut interval) to 300,830+111,460 (at the 500æm cut interval. The mean diameter of the isolated preantral follicles varied from 11.6æm to 27.8æm.

Generation, Microwave Spectrum, Barrier to Internal Rotation of Methyl Group, and ab Initio MO Calculation of syn-2-Nitrosopropene, syn-CH(2)&dbond;C(CH(3))&bond;N&dbond;O.

syn-2-Nitrosopropene was generated, in the gas phase, by chemical reaction of 1-chloro-2-(hydroxyimino)propane with K(2)CO(3) and identified by microwave spectroscopy. The microwave spectrum of the reaction product was observed in the frequency range from 8.0 to 40.0 GHz. The rotational constants (MHz) were determined as A = 8744.09(6), B = 4846.07(2), and C = 3177.84(3) for CH(2)&dbond;C(CH(3))&bond;(14)NO (normal species) and A = 8664.36(5), B = 4822.15(3), and C = 3157.04(3) for CH(2)&dbond;C(CH(3))&bond;(15)NO ((15)N species) in the ground vibrational state. The values of the planar moment (P(cc) = (I(a) + I(b) - I(c))/2) obtained for the normal and (15)N species were 1.525(1) and 1.526(1) u Å(2), respectively. This suggests that the nitrogen atom lies in or is close to the ab inertial plane of the molecule and shows also that only two hydrogen atoms are located symmetrically out of the symmetry plane. The reaction product was determined to be syn-2-nitrosopropene by comparing the observed and calculated rotational constants, kappa (Ray's asymmetry parameter) and r(s) coordinates of the nitrogen atom. The dipole moments (D) were determined to be µ(a) = 2.43(5), µ(b) = 1.12(7), and µ(total) = 2.67(7). The barrier heights of the internal rotation owing to the methyl group of the normal species in the ground and first excited torsional states were determined to be 1750(50) and 1740(50) cal/mol (1 cal/mol = 4.184 J/mol), respectively. The (14)N nuclear quadrupole coupling constants (MHz) were determined to be chi(aa) = 0.25(21), chi(bb) = -7.11(40), and chi(cc) = 6.85(61). Two vibrational excited states were observed and the vibrational frequencies (cm(-1)) of the C-N and C-C torsional modes were determined to be 160(40) and 175(40), respectively. The lifetime of syn-2-nitrosopropene was found to be ca. 2 min in the waveguide cell. Copyright 2000 Academic Press.

Microwave Spectrum and Molecular Conformation of (E)-Benzaldehyde Oxime.

The microwave spectrum of (E)-benzaldehyde oxime, C(6)H(5)-CH&dbond;NOH and C(6)H(5)-CH&dbond;NOD, has been observed in the frequency range from 26.5 to 40.0 GHz. The spectrum of the ground vibrational state was assigned and fitted to the Watson's A-reduced Hamiltonian to obtain these rotational and centrifugal distortion constants: A = 5183.13(29) MHz, B = 895.367(3) MHz, C = 763.819(3) MHz, Delta(J) = 0.019(3) kHz, and Delta(JK) = 0.204(7) kHz for the normal species, and A = 5158.4(23) MHz, B = 869.44(2) MHz, C = 744.34(2) MHz, Delta(J) = 0.023(2) kHz, and Delta(JK) = 0.193(7) kHz for the deuterated species. The values of the DeltaI (=I(c) - I(a) - I(b)) obtained for the normal and deuterated species were -0.295(6) and -0.28(5) a.m.u. Å(2), respectively. The molecular conformation of this molecule was a planar one in which the values of the dihedral angles, CCNO and CNOH, were almost 180 degrees and 180 degrees, respectively. Copyright 1999 Academic Press.

Microwave Spectrum and Molecular Conformation of delta-Valerolactam.

Microwave rotational spectra for normal and N-deuterated species of delta-valerolactam (2-piperidinone) were observed in the frequency range from 8 to 40 GHz. The spectra of the ground vibrational states for two isotopic species and the excited vibrational states for normal species were assigned, and the rotational constants (MHz) in ground vibrational state were determined using the Watson A-reduced Hamiltonian: A = 4590.96(11), B = 2495.03(2), C = 1731.06(2) for normal species and A = 4436.79(13), B = 2484.49(1), C = 1703.83(1) for deuterated species, respectively. The comparison of the rotational constants and r(s) coordinates of the imino hydrogen atom with ones from the ab initio MO calculation at the MP2/6-31G(d, p) level of theory led to the conclusion that the spectra assigned were due to a half-chair conformer. One vibrationally excited state was observed and its vibrational frequency was 95(64) cm(-1). Copyright 1999 Academic Press.

Generation, Microwave Spectrum, and Ab Initio MO Calculation of trans-1-Nitrosopropene, CH3CH&dbond;CH-NO (syn form).

trans-1-Nitrosopropene (syn form) was generated in the gas phase by pyrolysis of 1-chloro-1-methyl-2-(hydroxyimino)ethane and identified by microwave spectroscopy. The microwave spectrum of the pyrolysate was observed in the frequency range from 8.0 to 40.0 GHz. The rotational constants (MHz) were determined as A = 34 025(390), B = 2315.62(2), and C = 2198.54(2) for CH3CH&dbond;CH-NO (normal species) and A = 34 012(530), B = 2300.04(3), and C = 2182.70(2) for CH3CH&dbond;CH-15NO (15N isotopic species) in the ground vibrational state. The values of planar moment (Pcc = (Ia + Ib - Ic)/2) obtained for the normal and 15N isotopic species were found to be 1.62(10) and 1.52(13) uÅ2, respectively. These values are almost the same within the limit of errors. This suggests that the nitrogen atom lies in or is close to the ab inertial plane of the molecule and shows also that only two hydrogen atoms are located symmetrically out of the symmetry plane. The pyrolysate was determined to be trans-1-nitrosopropene by comparing the observed and calculated rotational constants, kappa (Ray's asymmetric parameter), and rs coordinates of the nitrogen atom. One vibrationally excited state was observed and assigned to the C-N torsional mode (158(50) cm-1). Interesting pyrolysates such as hydrogen cyanide and acetaldehyde were also detected during the pyrolysis of the precursor. The lifetime of trans-1-nitrosopropene is found to be ca. 5 s in the waveguide cell. Copyright 1999 Academic Press.

The Microwave Spectrum of n-Butyraldehyde Oxime.

The microwave spectrum of n-butyraldehyde oxime was observed in the frequency region 26.5-40 GHz. Four rotational conformers were found to exist in the gas phase; among these, two conformers belonged to the (E)-geometrical isomer and the other two to the (Z)-geometrical isomer. The microwave spectrum attributed to one of these two rotational conformers of (E)-butyraldehyde oxime was analyzed, and its rotational constants for the ground vibrational state were determined: A = 15883(379), B = 1269.97(1), C = 1251.60(1) MHz. The conformational structure of the molecule is discussed, referring to the rotational constants obtained and the ab initio molecular orbital calculation. Copyright 1998 Academic Press.

Spontaneous rupture of an intrahepatic bile duct with biloma treated by percutaneous drainage and endoscopic sphincterotomy.

A case of spontaneous rupture of an intrahepatic bile duct with biloma formation treated by percutaneous drainage and endoscopic sphincterotomy is reported. A 73-yr-old woman was admitted with fever and abdominal pain. There was no past history of abdominal surgery, instrumentation, or trauma. Ultrasound and computed tomography revealed a massive fluid collection in the abdominal cavity. Endoscopic retrograde cholangiography demonstrated extravasation of contrast medium from a distal biliary radicle in the left lobe of the liver. After successful treatment by percutaneous drainage and endoscopic sphincterotomy, the patient did well. Ultrasound and computed tomography showed resolution of the biloma. Nontraumatic bilomas are very rare: in fact, only 24 cases of spontaneous biloma have been reported. Endoscopic treatment for patients with spontaneous bilomas can be safe and effective, and should be considered.