Prefabricated buccal mucosa-lined flap in an animal model that could be used for vaginal reconstruction

Congenital vaginal aplasia, gynecological tumor excision, and male-to-female sex surgery are three clinical conditions in which the plastic surgeon is involved in vaginal reconstruction. Skin-lined or skin-grafted local flaps are currently used, but for many reasons, keratinized skin is not the ideal lining for such a moist cavity because it leads to dryness, desiccation, maceration of the skin, and even hair growth in the cavity. The purpose of this study was to create a subcutaneous cavity lined with mucosa in an area with a predictable blood supply. The abdominal area supplied by the deep circumflex iliac vessels was chosen. Six minipigs were used. Strips of tongue buccal mucosa formed the lining; if additional tissue was required, it was taken from the mucosal aspect of the cheek. The mucosa was expanded by using multiple stab incisions. The mucosa was sutured onto the fascia supplied by the deep circumflex iliac vessels, and the skin incision was closed over a silicone sheet to prevent adhesion to the underlying mucosa. This was left for 1 week to allow the mucosa to take. The prefabricated fascial flap was rolled over a silicone stent and was closed longitudinally to form a cylindrical shape. The flap was placed in a subcutaneous pocket in the right inguinal area. The caudal end was left open and was sutured to the surrounding skin. The silicone stent was used to keep the cavity patent and to prevent adhesions in the early stage of the healing process. Regular digital examination was performed to assess patency and contour; endoscopy allowed assessment of mucosa viability. This method of producing a mucosa-lined flap may provide a solution to the difficult problem of vaginal reconstruction.     

Role of methylation in aerotaxis in Bacillus subtilis.

Taxis to oxygen (aerotaxis) in Bacillus subtilis was characterized in a capillary assay and in a temporal assay in which the concentration of oxygen in a flow chamber was changed abruptly. A strong aerophilic response was present, but there was no aerophobic response to high concentrations of oxygen. Adaptation to a step increase in oxygen concentration was impaired when B. subtilis cells were depleted of methionine to prevent methylation of the methyl-accepting chemotaxis proteins. There was a transient increase in methanol release when wild-type B. subtilis, but not a cheR mutant that was deficient in methyltransferase activity, was stimulated by a step increase or a step decrease in oxygen concentration. The methanol released was quantitatively correlated with demethylation of methyl-accepting chemotaxis proteins. This indicated that methylation is involved in aerotaxis in B. subtilis in contrast to aerotaxis in Escherichia coli and Salmonella typhimurium, which is methylation independent.     

Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus.

The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.     

Female-induced sexual arousal in male mice and rats: behavioral and testosterone response

Exposure of a male mouse to a female mouse separated from it by a holed partition induced specific behavior and an increase in blood testosterone in the male. The male made more approaches to the partition and spent more time at it. The time spent by the male mouse over the first 10 min at the partition, behind which an estrus female was placed, was increased sixfold compared to the time spent by a male mouse exposed to the vacant neighboring compartment; and 1.5-fold compared to that spent by a male mouse exposed to a nonreceptive female or a male. Increased blood testosterone level was detected at 20 min of exposure to a receptive female in winter and at 40 min in summer. No variation in blood testosterone levels in the male mouse exposed to a nonreceptive female or a male was observed. Similar response to a receptive female placed in the neighboring compartment was shown in a male rat. The time spent by the male rat at the partition was 12 times higher when there was an estrus female behind it than in control. Blood testosterone in the male rat increased in response to a female rat and did not change in response to a male rat indicating female-induced motivation. It was concluded that the partition time might serve as a quantitative measure of sexual motivation in the males and that the model of female-induced sexual arousal used was suitable for studying both motivational and hormonal components of sexual arousal in male mice and rats.     

Psittacosis in Manitoba

Paired blood specimens submitted to the Virus Laboratory, between January and September 1970, from 200 patients with a lower respiratory tract infection were examined for antibodies to the psittacosis-human pneumonitis group antigen. A high static titre of antibodies was found in four patients and a rising titre in three. The illness varied in the seven patients, tending to be severe, chronic and recurrent. All patients recovered following a course of antibiotic therapy which was repeated in four; treatment tended to be inadequate since the diagnosis of psittacosis was made retrospectively. The likely source of the infection in four of the patients was budgerigars, in one it was pigeons, in one it was a canary as well as a pigeon, and in one the source was not identified. Two additional patients had a fairly high static titre of antibodies to the psittacosis-human pneumonitis antigen but were excluded from this report since they showed a diagnostic increase in antibodies to a respiratory virus during the course of their illness; both patients had an avian contact and are being followed up.     

Ribonuclease deficiency in lymphocytes of patients with chronic lymphocytic leukemia (CLL)

Ribonuclease (RNase) activity in the lymphocytes of 20 chronic lymphocytic leukemia (CLL) patients and 10 normal subjects was studied. It was found that in the lymphocytes of the control subjects the RNase activity could be detected in the pH range 4.5 to 8.6, inclusive. The RNase activity versus pH profile of normal lymphocytes consists of an acid RNase peak at pH 6.5 and alkaline RNase peak at pH 7.8. When treated with pCMB an inhibitor-bound RNase activity was revealed. The peak of this activity lay between pH 6.7 to 7.0. Liberating the inhibitor-bound RNase activity changed the RNase activity-pH profile, yielding one peak curve with a maximum at pH 7.0. RNase activity in CLL lymphocytes was remarkably lower than that in normal lymphocytes. The acid RNase in 80% of the CLL patients was lower by a factor of ten. Likewise, a many fold decrease in alkaline RNase activity (in some cases down to the zero level) was observed in CLL lymphocytes. However, in 70% of CLL patients, a level of the inhibitor-bound RNase activity was similar to that found in normal lymphocytes. In 20% of the studied CLL patients, a remarkable decrease in both free alkaline and inhibitor-bound RNase activity was observed. When poly-C was used as a substrate for determining RNase activity, a decrease to approximately 15% in CLL lymphocytes was observed, when poly-U was used instead of poly-C, a decrease to 65% was found only as compared with normal lymphocytes. This may suggest that CLL lymphocytes are deficient in a poly-C specific RNase which displays its activity within a neutral and alkaline pH range.     

A Nuclear Stain for Escherichia Coli And Related Organisms

The dye base of new fuchsin was precipitated by adding potassium hydroxide to the dye solution. The precipitate was filtered out and washed with water. It was then suspended in water, brought into solution and adjusted to a pH of about 5.0 with nitric acid. The staining solution was prepared by adding 0.3 ml. of a 14% aqueous solution of pyrogallol and 0.1 ml. of a 1% aqueous solution of boric acid to 3.0 ml. of the dye solution. Smears of cells were made in water on a slide and allowed to dry before covering with the staining solution which was also permitted to air dry. The smear was then washed in water and mordanted for 5-20 seconds in a 0.1% aqueous solution of mercuric nitrate. After rinsing in water, the smear was air dried. When dry, the slide was placed on a 50° C. warm plate for a few seconds before covering with a very thin film of a 5% aqueous solution of nigrosin which had a pH of about 5.0.     

Manometric biosensor for on-line measurement of milk urea

Performance of a prototype sensor for on-line measurement of urea in milk during milking was evaluated. The sensor was based on a manometric assay of the carbon dioxide generated by the enzymatic hydrolysis of urea. Temperature compensation of the sensor was described briefly, and was shown to be effective. The calibration of the sensor was described and resulted in a standard calibration error of about 0.15 mM of urea. The standard error of the sensor in milk was shown to be about 0.25 mM (given a physiological range of about 2-7 mM in cow milk). The sensor was simple, inexpensive, suffered from no interferences in raw milk, and completed a measurement cycle in about 5 min (less than the time to milk a typical cow). A custom made sampling device, whereby milk was passively collected from the milk line under vacuum, was shown to collect an ample volume within 10 s to run a test with the sensor. No measurable bubbles or foam were introduced from the sampling mechanism so that the milk sampled was not diminished in density compared to samples taken by other methods.     

Molecular Characterization of Recombinant Mus a 5 Allergen from Banana Fruit

Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55 % of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.     

Inhalation anesthesia in adult cattle

An inhalation technique was used for anesthesia during ileal cannulation in five adult cows. Following sedation with intravenous acepromazine, anesthesia was induced intravenously with thiopental sodium in 5% glyceryl guaiacolate solution. Endotracheal intubation was performed and anesthesia maintained with halothane in oxygen via a circle system with a precision vaporizer. In all cases, induction was smooth and no difficulties were experienced during the maintenance of anesthesia. Total anesthesia time was 1.5 to 2.5 hours. Following completion of the surgical procedure, which was performed with the animal in left lateral recumbency, each cow was rolled to a sternal position and supported, if necessary. The endotracheal tube was left in place, with oxygen administration continued, until the animal was able to swallow. Recoveries were rapid and all animals were ambulatory within 30 minutes after completion of the surgery. The only post-operative complication due to anesthesia was transient mouth soreness in two cases, attributed to the use of a mouth speculum during intubation.     

Prototype symbolization in hooded crows

The ability in prototype symbolization was studied in four crows trained in matching concept. The experimental approach was characterized by the absence of direct training providing an association between the pictured arrays and Arabic numerals. Experimental sessions were divided into demonstrative and test phases. In the demonstrative phases, the sample and the reinforced stimulus belonged to the same category: if the sample was a pictured array, the reinforced stimulus was also the pictured array, if the sample was a numeral, the reinforced stimulus was likewise a numeral. Matching-to-sample competence was sufficient to perform this task successfully. In case of a correct choice a crow received some mealworms, their number was equal to the number of items in the pictured array or the Arabic numeral. In the test phases, the sample and the comparison stimuli were of different categories. If the sample was a pictured array, the comparison stimuli were numerals; if the sample was a numeral, the comparisons were the pictured arrays. Such tests required the birds to choose a numeric symbol corresponding to the pictured array or to choose the array of pictured items numerically equal to the Arabic symbol. All four crows performed this task successfully. Birds were able to set up a correspondence between the Arabic numeral from 1 to 4 and the number of elements in pictured arrays by way of mental juxtaposition of the earlier received information. Similar experiments showed that crows are likely to be able to perform mental operations with numerals analogous to simple arithmetic addition.     

Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes

Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.     

Immunolocalization of four antioxidant enzymes in digestive glands of mollusks and crustaceans and fish liver

The aim of this work was to determine the immunolocalization of the antioxidant enzymes catalase, Cu,Zn-superoxide dismutase (SOD), Mn-SOD, and glutathione peroxidase (GPX) in the bivalve mollusks Mytilus galloprovincialis and Crassostrea sp., the crab Carcinus maenas, and the teleostean fish Mugil cephalus. By immunoblotting, crossreactivity between antibodies and the corresponding proteins in the digestive gland/hepatopancreas of invertebrates and the fish liver was demonstrated. Immunohistochemical studies showed that the stomach epithelium was strongly immunostained for catalase in mollusks. In crabs, ducts showed stronger immunostaining than tubules and in mullet hepatocytes the reaction appeared in discrete granules corresponding to peroxisomes. With regard to Cu,Zn-SOD, the apex of the tubule cells in mussels and crabs was distinctly immunostained, whereas in oysters the reaction was more marked in ducts and in mullet liver a uniform diffuse cytoplasmic staining was found. Mn-SOD was strongly positive in mollusk and crab ducts and in mullet periportal hepatocytes. Finally, GPX was not detected in mussels while in oysters a slight reaction was noted in all cell types. In crabs, connective tissue cells and the apex of duct cells were immunostained, but in mullet liver only erythrocytes appeared reactive. Immunoelectron microscopy revealed that catalase was localized in peroxisomes with a dense labeling in fish and less intense labeling in invertebrates. Cu,Zn-SOD was mainly a cytosolic protein although additional positive subcellular sites (peroxisomes, nuclei) were also observed, while Mn-SOD was restricted to mitochondria. GPX was localized in the cytosol, nucleus, and lysosomes, occurring also in peroxisomes of the fish liver. The results presented here provide a basis for future application of the immunodetection techniques to study the possible differential induction of antioxidant enzymes in aquatic organisms subjected to oxidative stress as a result of exposure to environmental pollutants.     

Probing the yeast 5 S RNA-protein complex by fluorescence and controlled proteolytic digestion

The nature of the interaction between the RNA and the protein component in the yeast 5 S rRNA-L1a complex was assessed using fluorescence and controlled proteolytic and RNase digestion. (a) Influence of L1a on the RNA conformation was monitored by ethidium fluorescence and controlled RNase T1 digestion. The complex was digested with alpha-chymotrypsin, Staphylococcus aureus protease V8, subtilisin, or trypsin. Both termini of L1a in the complex were readily accessible to proteases. Proteolytic digestion of the complex resulted in a reduction in fluorescence intensity if ethidium was added after proteolysis. No change was observed when ethidium was allowed to react with the complex prior to proteolysis. Neither the rate of proteolysis nor the resultant peptide pattern was affected by the presence of ethidium. T1 digestion of intact RNP and trypsin-treated RNP produced different oligonucleotide patterns. Both the fluorescence and the T1 digestion data suggest that the conformation of the RNA moiety was influenced by the protein. (b) Influence of the RNA molecule on L1a conformation in the complex was monitored by limited proteolysis. Whereas the protein in the complex was relatively sensitive to proteases, free protein was completely resistant to digestion under identical conditions. The trypsin sensitivity of L1a in complexes containing different truncated 5 S RNA molecules was studied also. Upon removal of residues 31-49 of the 5 S RNA molecule, L1a in the complex became resistant to proteolysis. These results are interpreted in a model in which specific regions of both the RNA and the protein are involved in the interaction.     

Cloning and expression in Escherichia coli of the gene for extracellular phospholipase A1 from Serratia liquefaciens.

From a genomic library of Serratia liquefaciens, a cloned DNA fragment comprising a two-gene operon was isolated and expressed in Escherichia coli. One of the gene products was identified as a phospholipase A1, and the enzyme was found to be excreted to the outer environment from S. liquefaciens as well as from E. coli. Both genes were sequenced, and the relationship between open reading frames in the DNA sequence and in vitro-expressed polypeptides was established. The length of the phospholipase polypeptide was found to be 319 amino acids. In the amino-terminal end of the coding sequence was a stretch of about 20 hydrophobic amino acids, but, in contrast to consensus signal peptides, no basic residues were present. The length of the second polypeptide was 227 amino acids. It was found that expression of the phospholipase gene in both E. coli and S. liquefaciens was growth phase regulated (late expression).     

Long-term transpiration change with rainfall decline in a Mediterranean Quercus ilex forest

In the Mediterranean basin, precipitation is expected to decline as a consequence of climate change. The response of a Quercus ilex forest in southern France to such a decline in water availability was studied using a 4-year throughfall exclusion experiment. Seasonal courses of sap flow and leaf water potential were obtained from 2004 to 2007 and used to characterize tree water relations in a control and a dry treatment. The experiment reduced the average precipitation input to the soil by 29%, and resulted in a 23% reduction in annual transpiration. Soil water potential was significantly lower in the dry treatment only during summer drought, but transpiration was reduced all year round even during well-watered periods. Despite a tight stomatal control over transpiration, whole-tree hydraulic conductance was found to be lower in the trees growing in the driest conditions. This reduction in water transport capacity was observed jointly with a reduction in leaf transpiring area. Canopy leaf area decreased by 18% in the dry treatment as a consequence of the throughfall exclusion, which was found to validate the ecohydrological equilibrium theory.     

Studies on the mechanism of castanospermine inhibition of alpha- and beta-glucosidases

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is an indolizidine alkaloid that was isolated from the Australian plant, Castanospermum australe. This alkaloid was found to be a potent inhibitor of lysosomal alpha- and beta-glucosidases. In this report, the mechanism of inhibition of amyloglucosidase (an exo-1,4-alpha-glucosidase) and almond emulsin beta-glucosidase was examined. Castanospermine proved to be a competitive inhibitor of amyloglucosidase at both pH 4.5 and 6.0 when assayed with the p-nitrophenyl-alpha-D-glucoside. It was also a competitive inhibitor of almond emulsin beta-glucosidase at pH 6.5, but in this case previous studies had shown that inhibition was of the mixed type at pH 4.5 to 5.0. Th pH of the incubation mixture had a marked effect on the inhibition. Thus, in all cases, castanospermine was a much better inhibitor at pH 6.0 to 6.5 than it was at lower pH values. The pK for castanospermine was found to be 6.09, indicating that the alkaloid was probably more active in the unprotonated form. This was also suggested by the fact that the N-oxide of castanospermine, while still a competitive inhibitor, was 50 to 100 times less active than was castanospermine, and its activity was not markedly altered by pH. These results probably explain why castanospermine is a good inhibitor of the glycoprotein processing enzyme, glucosidase I, since this is a neutral enzyme.     

A novel three-compartmented electrochemical bioreactor for enrichment of strict anaerobes based on metabolite production

In this study, a novel three-compartmented electrochemical bioreactor (3-CEB) was designed in an effort to overcome the disadvantages of the two-compartmented electrochemical bioreactor (2-CEB) separated with a cation-selective membrane for enrichment of strict anaerobes. The 3-CEB was comprised of an anode, outlet, and a cathode compartment. The outlet compartment was positioned between the anode and cathode compartment, and it was separated with the anode side by a rubber plate and with the cathode side by a porous glass membrane. A platinum wire bridging the anode and outlet compartment operated as a redox passage, however, through which no material could permeate. Butyrate fermentation bacteria were enriched on the basis of the metabolite production. Butyrate generated by strict anaerobes was significantly more abundant in the 3-CEB than in the 2-CEB. Acetic acid and lactic acid generated by facultative anaerobes was relatively higher in the 2-CEB than in the 3-CEB. Meanwhile, butyrate was not generated in the bioreactor utilized for the control test, to which the electrochemical potential was not charged. In a continuous culture using the 3-CEB, the majority of the glucose was fermented to butyrate, and the acetate additionally supplied to the bacterial culture was metabolically reduced to butyrate. More lactate than butyrate was generated from glucose in the 2-CEB.