Efficacy and safety of using premedication with simethicone/Pronase during upper gastrointestinal endoscopy examination with sedation: A single center, prospective, single blinded, randomized controlled trial.

BACKGROUND AND AIM: To investigate the efficacy and safety of premedication with simethicone/Pronase during esophagogastroduodenoscopy (EGD) with sedation. METHODS: Six hundred and ten patients were randomly allocated to two groups based on type of premedication given. Premedication used in the control group was 10 mL lidocaine hydrochloride mucilage (LHM, N = 314) and premedication used in the intervention group was 80 mL simethicone/Pronase solution plus 10 mL lidocaine hydrochloride mucilage (SP/LHM, N = 296). EGD was done under sedation. Visibility scores, number of mucosal areas that needed cleansing, water consumption for cleansing, time taken for examination, diminutive lesions, pathological diagnosis, patients' gag reflex and oxygenation (pulse oximetry) were recorded. RESULTS: SP/LHM has significantly lower total visibility score than LHM (7.978 ± 1.526 vs 6.348 ± 1.097, P < 0.01). During the procedure, number of intragastric areas that needed cleansing and amount of water consumed were significantly less in the SP/LHM than in the LHM group (P < 0.01). In SP/LHM (P = 0.01), endoscopy procedure duration was significantly longer. Although there was no significant difference in rate of detection of diminutive lesions between LHM and SP/LHM, the endoscopist carried out more biopsies in SP/LHM. This led to a higher rate of diagnosis of atrophic gastritis (P = 0.014) and intestinal metaplasia (P = 0.024). There was no significant difference in gag reflex (P = 0.604) and oxygenation during the endoscopy procedure for either group of patients. CONCLUSION: Routine use of premedication with simethicone/Pronase should be recommended during EGD with sedation.

A method for the assessment of DNA damage in individual, one day old, zebrafish embryo (Danio rerio), without prior cell isolation.

Zebrafish (Danio rerio) embryos are increasingly used as an experimental model in toxicology for the detection of lethal and sub-lethal effects of diverse chemicals. DNA damage, an early biomarker of long-term effects such as mutagenesis and carcinogenesis, is commonly assessed in vitro and in vivo using the comet assay - single cell gel electrophoresis. Here we describe a new rapid method for the detection of DNA strand breaks in individual, one day old, zebrafish embryos, without the need for prior cell isolation. After the completed spawning, the embryos were exposed to non-toxic concentrations of model genotoxic compounds for 24h. The embryos were then treated with Pronase E, embedded on microscope slides and squashed to release the cells. After alkaline electrophoresis, the nuclei were stained with ethydium bromide and analyzed by fluorescence microscopy. Preparation of slides by the described method resulted in well separated cell nuclei with low background DNA damage. A significant increase in DNA damage was detected after exposure to the model genotoxic compounds, methylmethan sulphonate (MMS) and benzo(a)pyrene (BaP), while no DNA damage was induced by NaCl. Our method proved to be sensitive and suitable for the detection of DNA damage in one day old zebrafish embryos, suggesting it could serve as a useful tool for monitoring the genotoxic potential of chemicals and environmental pollutants.

Spontaneous and pronase-induced HER2 truncation increases the trastuzumab binding capacity of breast cancer tissues and cell lines.

A subgroup of HER2-overexpressing breast tumours co-expresses p95(HER2), a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95(HER2) expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2-positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95(HER2) expression. Immunohistochemistry was performed on parallel formalin-fixed, paraffin-embedded sections of the same case series using antibodies directed against either the intra- or extra-cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185(HER2)-positive/p95(HER2)-positive samples than in the p185(HER2)-positive/p95(HER2)-negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95(HER2)-positive cases. Conversely, the percentage of 2+ scored cells was higher inp95(HER2)-negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2-HER3 dimers was studied using a proximity-ligation assay. In vitro experiments showed that short-term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2-HER3 dimers, and did not interfere with pertuzumab-binding capacity. In conclusion, the presence of p95(HER2 as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab.

[A new method for isolation and culture of tracheal epithelial cell of mice].

Since most of the respiratory epithelial cell lines are descended from neoplastic tissues or have been fused with neoplastic cells when being selected to a cell line, their biological behaviors are far different from normal respiratory epithelial cells. Aiming at better reflecting the biological properties of epithelial cells under respiratory pathological conditions, our study probed into a new isolation technique and culture method of mice respiratory epithelial cell. Pronase was applied for cell isolation from mouse trachea, and a modified medium with collagen-coated plate for primary culture. The ciliary movement can be observed under microscope, which demonstrates that the original biological functions of the tracheal epithelial cell have been reserved with our method. The research presents the efficacy, convenience and reliability of the separation technique and culture method established by this study, and laying preferable condition for cell models of respiratory diseases. Meanwhile, this method may be used for other animal models.

In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br.

This work evaluated the in vitro cytotoxic activity of laticifer proteins (LP) recovered from the latex of the medicinal plant Calotropis procera. The LP displayed considerable cytotoxicity with IC(50) values ranging from 0.42 to 1.36 microg/ml to SF295 and MDA-MB-435 cell lines, respectively. In healthy peripheral blood mononuclear cells exposed to LP (10 microg/ml) for 72 h, no noticeable effects on viability or cell morphology were seen. The fractionating of LP on an ion exchange chromatography gave rise to a new fraction (PI) that retained almost all cytotoxicity. The cytotoxic effects of both LP and PI were diminished when previously treated with pronase, or 2-mercaptoethanol, suggesting a protein nature of active molecules, however, pre-incubation with dithiothreitol (DTT) only reduced PI activity. PI did not exhibit cysteine proteinase activity, indicating that cysteine proteinases, abundantly found in LP, are not implicated in LP cytotoxicity. In this study, using HL-60 cell as a model, LP was shown to inhibit DNA synthesis. This is probably due to alterations in the topology of DNA, since it was observed that LP is able to interfere in topoisomerase I activity by somehow acting upon DNA. LP provoked reduction in cell number but it did not cause any significant increase in the number of non-viable cells. These findings corroborated with the morphologic analysis, where cells treated with LP showed morphology of apoptotic process with abundant vacuoles, chromatin condensation and fragmentation of the nuclei. The results of this study suggests that LP is a target for DNA topoisomerase I triggering apoptosis in cancer cell lines.

The effect of engineered disulfide bonds on the stability of Drosophila melanogaster acetylcholinesterase.

BACKGROUND: Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use in biosensors for detection of these insecticides. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, its stability has to be improved for extensive utilization. RESULTS: To create a disulfide bond that could increase the stability of the Drosophila melanogaster acetylcholinesterase, we selected seven positions taking into account first the distance between Cbeta of two residues, in which newly introduced cysteines will form the new disulfide bond and second the conservation of the residues in the cholinesterase family. Most disulfide bonds tested did not increase and even decreased the stability of the protein. However, one engineered disulfide bridge, I327C/D375C showed significant stability increase toward denaturation by temperature (170 fold at 50 degrees C), urea, organic solvent and provided resistance to protease degradation. The new disulfide bridge links the N-terminal domain (first 356 aa) to the C-terminal domain. The quantities produced by this mutant were the same as in wild-type flies. CONCLUSION: Addition of a disulfide bridge may either stabilize or unstabilize proteins. One bond out of the 7 tested provided significant stabilisation.

Modifications to improve the efficiency of zona-free mouse nuclear transfer.

In the present study, some modifications were made to the zona-free nuclear transfer technique in the mouse in order to achieve greater efficiency. Firstly, a 1-h interval was allowed between cumulus removal and zona pellucida digestion. Secondly, acid Tyrode's was selected for zona pellucida removal, because contrary to pronase, it allows embryo survival during parthenogenic activation in the absence of calcium. Even when the exposure time to pronase was reduced to as little as 1 min or washed with fetal calf serum to inhibit the enzyme, the percentage of lysis during activation in the absence of calcium was still very high. Thirdly, electrofusion was performed at room temperature (21 degrees C), instead of 30 degrees C as in our previous experiments. Finally, embryos were cultured in groups of 12-15, instead of individually, using a "well of the wells" system during activation and culture. When compared, parthenogenic activated control embryos showed an increase in the development to blastocyst when cultured in pairs instead of individually. By the end of the experiments and using embryonic stem (ES) cells, there was a significant increase in fusion rate (1.5-fold increase) and in development to morula/blastocyst from cleaved reconstructed embryos (1.5-fold increase) when compared with the results before the modifications. A 2.4-fold increase in overall efficiency was achieved from the oocyte to morula/blastocyst stages.

Characterization of transferrin receptor-dependent GaC-Tf-FeN transport in human leukemic HL60 cells.

BACKGROUND: Understanding the uptake of GaC-Tf-FeN by cells will provide key insights into studies on transferrin-mediated drug delivery. METHODS: The mechanism of GaC-Tf-FeN transporting into and out of HL60 cells has been investigated by comparing transports between GaC-Tf-FeN and apoTf by means of 125I-labeled transferrin. RESULTS: An association constant for GaC-Tf-FeN was 2 times that for apoTf. GaC-Tf-FeN and apoTf of cell surface-bound displayed similar kinetics during the uptake, but the release rates of internalized GaC-Tf-FeN and apoTf from cells were different which showed characteristic disparate. The release continued to occur during the incubation of GaC-Tf-FeN in the presence of nonradioactive apoTf. Neither NaN3 nor NH4Cl could completely block internalization of GaC-Tf-FeN, but they prevented the release of GaC-Tf-FeN from the cells. Excess cold unlabeled apoTf could overcome the block in the release due to NH4Cl but not NaN3. The binding and internalization of GaC-Tf-FeN could be competitively inhibited by nonradioactive apoTf. It implies that both bind to the same receptor on the membrane and the localization of GaC-Tf-FeN resembles that of apoTf inside cells. Pretreated cells with pronase abolished the binding of GaC-Tf-FeN significantly. CONCLUSION: On the basis of these findings, we proposed the "transferrin receptor" for the mechanism of GaC-Tf-FeN transport by HL60 cells.

Biochemical characterisation of the trehalase of thermophilic fungi: an enzyme with mixed properties of neutral and acid trehalase.

The trehalases from some thermophilic fungi, such as Humicola grisea, Scytalidium thermophilum, or Chaetomium thermophilum, possess mixed properties in comparison with those of the two main groups of trehalases: acid and neutral trehalases. Such as acid trehalases these enzymes are highly thermostable extracellular glycoproteins, which act at acidic pH. However, these enzymes are activated by calcium or manganese, and as a result inhibited by chelators and by ATP, properties typical of neutral trehalases. Here we extended the biochemical characterisation of these enzymes, by assaying their activity at acid and neutral pH. The acid activity (25-30% of total) was assayed in McIlvaine buffer at pH 4.5. Under these conditions the enzyme was neither activated by calcium nor inhibited by EDTA or ATP. The neutral activity was estimated in MES buffer at pH 6.5, after subtracting the activity resistant to EDTA inhibition. The neutral activity was activated by calcium and inhibited by ATP. On the other hand, the acid activity was more thermostable than the neutral activity, had a higher temperature optimum, exhibited a lower K(m), and different sensitivity to several ions and other substances. Apparently, these trehalases represent a new class of trehalases. More knowledge is needed about the molecular structure of this protein and its corresponding gene, to clarify the structural and evolutionary relationship of this trehalase to the conventional trehalases.

Apotransferrin is internalized and distributed in the same way as holotransferrin in K562 cells.

Transferrin (Tf), a naturally existing protein, has received considerable attention in the area of drug targeting since it is biodegradable, non-toxic, and non-immunogenic. The efficient cellular uptake of Tf shows it has potential in the delivery of anti-cancer drugs, proteins, and therapeutic genes into proliferating malignant cells that overexpress transferrin receptor (TfR). In human serum, about 30% of Tf exists in the iron-saturated form (Fe(2)-Tf) and the remainder exists as apotransferrin (apo-Tf). Understanding the uptake of apo-Tf by cells will provide key insights into studies on Tf-mediated drug delivery. In the present study, we investigated visually the transport of apo-Tf into K562 cells and its intracellular localization by laser-scanning confocal microscopy (LSCM) and flow cytometry analysis (FCA). It was found that, like Fe(2)-Tf, apo-Tf can be taken up into the cells. The process is time- and temperature-dependent, competitively inhibited by Fe(2)-Tf, and significantly abolished by pronase pretreatment. Visual evidence showed that the transport of apo-Tf into K562 cells is a TfR-mediated process. Furthermore, the investigations using optical-slicing technique demonstrated that the distribution of apo-Tf is similar to that of Fe(2)-Tf, both appearing in the perinuclear region in ball-in-bowl shape.

Distribution and characterization of hemolytic activity by an oral anaerobe from the Streptococcus milleri group.

Some oral anaerobes from the Streptococcus milleri strain group were found to secrete human specific hemolytic toxin, which was detected when bacteria were cultured in Todd-Hewitt broth and Brain Heart Infusion broth. The toxin elicited by the Streptococcus intermedius strain was partially fractionated by ammonium sulfate precipitation. Preincubation with glutathione or cysteine showed significant inhibiting effects; however, no effects were seen with dithiothreitol or beta-mercaptoethanol, and cholesterol was a weak inhibitor. Five kinds of protease inhibitor had no effect on the hemolytic activity, and rabbit preimmune and immune sera against the bacterial cells showed weak inhibition at a similar level. Digestion with trypsin, chymotrypsin, proteinase-K, subtilisin and pronase-P brought about a rise in activity, followed by a decrease during long-term incubation. Other enzymes tested showed no effects. Further, the presence of the intermedilysin gene in the portion with hemolytic activity was not identified by polymerase chain reaction.

Human platelets produce granulocyte-macrophage colony-stimulating factor and delay eosinophil apoptosis.

An association between eosinophils and platelets has been described in several diseases, most notably asthma. Although the mechanisms through which platelets influence eosinophil behavior are not well defined, platelets seem to contribute to the selective accumulation of eosinophils at sites of allergic inflammation by virtue of their ability to produce eosinophil chemotactic factors. We report here for the first time that platelets delay apoptosis, thus enhancing eosinophil survival. A marked inhibition of spontaneous apoptosis was observed using eosinophil:platelet ratios of 1:50, 1:25, 1:10, and 1:5. Moreover, promotion of eosinophil apoptosis by either pronase or dexamethasone was also inhibited greatly in the presence of platelets. The antiapoptotic effect mediated by platelets was dependent on the release of soluble products and was significantly inhibited by neutralizing antibodies directed to GM-CSF. Studies performed by flow cytometry, directed to analyze the cellular source of this cytokine, demonstrated that intracytoplasmic GM-CSF is present in resting platelets. Moreover, GM-CSF was found in platelet supernatants, at concentrations able to prevent eosinophil apoptosis. Our findings support a novel mechanism through which platelets may contribute to eosinophil accumulation at allergic inflammatory sites.

Alterations of CMP-NeuAc:asialofetuin sialyltransferase activities in human colorectal adenocarcinoma.

It has been reported that surface glycoconjugates in tumour cells have more complex and more heavily sialylated sugar chains in glycoproteins. Here, we analysed CMP-NeuAc:asialofetuin sialyltransferase activities in total cell membranes from colorectal cancer tissue with respect to normal adjacent tissue, finding enhanced sialyltransferase activities. Determination of the kinetic parameters for the donor substrate, CMP-NeuAc, revealed that the apparent K(m) in tumour tissue was decreased as regards to the normal value. Furthermore, we failed to find any correlation between this activity and the characteristics of the samples such as the age or sex of the patient, or Dukes' stage of the tumour. In order to know which sialyltransferase activity was altered, we studied the incorporation of NeuAc into N- and O-linked chains from asialofetuin. The results allow us to conclude that it is on N-linked chains where the activity is enhanced. With respect to alpha(2,3)- and alpha(2,6)-NeuAc linkages, we observed that enhanced activity in tumour tissues affects both linkages equally.

Type III intestinal metaplasia and carcinoma express an identical carbohydrate-epitope revealed by a novel monoclonal antibody (2D11).

A monoclonal antibody (2D11, IgG2b) obtained by immunizing mice with a mucin fraction of the human gastric mucosa reacted specifically to intestinal metaplasia of human gastric mucosa and fetal intestinal mucosa but not to normal adult gastric, small intestinal or colonic mucosa in immunohistochemical staining. The results of Western blotting indicated that 2D11 recognized the high molecular weight glycoprotein(s) (mucin) of the stomach. Treatment of the antigens with sodium periodate abolished their reactivity to 2D11, and digestion of the antigens with beta-galactosidase reduced their reactivity to 2D11. Digestion of the antigens with pronase had no effect, however, suggesting that 2D11 recognizes the oligosugar moiety but not the peptide moiety of the antigens. Further immunohistochemical investigation showed that the reactivity of 2D11 was restricted to the Type IotaIotaIota intestinal metaplasia that is identified by a characteristic staining pattern with the high iron diamine-Alcian blue stain. 2D11 also reacted in high frequency to adenocarcinomas of the stomach (66.7%), pancreas (66.7%) and gallbladder (50.0%), but in low frequency to those in lung (8.3%) and colon (11.1%). It is of interest that 2D11 reacted to very restricted regions of the gastric adenocarcinomas. All monoclonal antibodies to mucin polypeptides (MUC1, 2, 3, 5AC and 6) examined stained intestinal metaplasia and carcinomas in a different pattern from 2D11 in immunohistochemistry. These facts indicate that Type IotaIotaIota intestinal metaplasia and carcinomas express carbohydrate chains identical to those expressed in the fetal intestinal mucosa, suggesting that both of them are closely related to fetal intestinal mucosa.

Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells.

Actinobacillus pleuropneumoniae biovar 1 serotypes 2, 5a, 9 and 10 strains were tested for their ability to adhere to alveolar epithelial cells in culture. For the serotypes 5a, 9 and 10 strains, optimal adherence was observed after growth of bacterial cells in a NAD-restricted medium (0.001% NAD). This condition was also associated with the expression of a 55 kDa outer membrane protein (OMP) and of fimbriae. For the serotype 2 strain, adherence and expression of fimbriae and a 55 kDa OMP was less influenced by the growth conditions. The N-terminal amino acid sequence of the 55 kDa OMP had no homology with any known sequence, suggesting that it is an as yet unknown protein. Adherence capabilities were significantly reduced following treatment of the bacteria with proteolytic enzymes or heat. These findings suggest that proteins are involved in adhesion. The hydrophobic bond-breaking agent tetramethylurea was unable to inhibit the adherence of A. pleuropneumoniae to alveolar epithelial cells. Treatment of the bacteria with sodium metaperiodate resulted in lower adhesion scores for the serotypes 2 and 9 strains but the inhibition of adhesion was clearly lower than after treatment with proteolytic enzymes. This indicates that, besides proteins, carbohydrates might also be involved in adhesion of A. pleuropneumoniae to alveolar epithelial cells. The finding that inhibition of adhesion was very high when bacteria were treated with a combination of sodium metaperiodate and pronase also suggests that more than one adhesin is involved.

Design of high essential amino acid proteins: two design strategies for improving protease resistance of the nutritious MB-1 protein.

Protein design is currently used for the creation of new proteins with desirable traits. In our lab, we focus on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations we face in this endeavour is achieving stable proteins despite a highly biased amino acid content. We report here the synthesis and characterisation of two mutants derived from our MB-1 designer protein. The first mutant contains a disulphide bridge designed to cross-link remote segments of the polypeptide chain. The second one is a Tyr62-Trp mutant, where position 62 is buried in the core of the protein. Both mutants were found to be largely helical as per design, and based on thermal denaturation experiments, were substantially more stable than the MB-1 parent molecule. Enhancement of conformational stability in MB-1Trp translated into an impressive improvement of its ability to resist proteolytic degradation. Furthermore, digestion experiments intended to model degradation of proteins in a cow's rumen revealed that MB-1Trp's resistance to degradation compared to that of cytochrome c. Design strategies used for these mutants are discussed with regards to their applicability in creating efficient nutritional proteins.

The cytoplasmic amino-terminus of the Latent Membrane Protein-1 of Epstein-Barr Virus: relationship between transmembrane orientation and effector functions of the carboxy-terminus and transmembrane domain.

The Latent Membrane Protein 1 (LMP-1) protein of Epstein-Barr virus (EBV) is localized in the plasma membrane of the infected cell. LMP-1 possesses a hydrophobic membrane spanning domain, and charged, intracellular amino- and carboxy-termini. Two models have been proposed for the contribution of the amino-terminus to LMP-1's function: (i) as an effector domain, interacting with cellular proteins, or (ii) as a structural domain dictating the correct orientation of transmembrane domains and thereby positioning LMP-1's critical effector domains (i.e. the carboxy-terminus). However, no studies to date have addressed directly the structural contributions of LMP-1's cytoplasmic amino-terminus to function. This study was designed to determine if LMP-1's cytoplasmic amino-terminus (N-terminus) encodes information required solely for maintenance of proper topological orientation. We have constructed LMP-1 chimeras in which the cytoplasmic N-terminus of LMP-1 is replaced with an unrelated domain of similar size and charge, but of different primary sequence. Retention of the charged amino-terminal (N-terminal) cytoplasmic domain and first predicted transmembrane domain was required for correct transmembrane topology. The absolute primary sequence of the cytoplasmic N-terminus was not critical for LMP-1's cytoskeletal association, turnover, plasma membrane patching, oligomerization, Tumor Necrosis Factor Receptor-associated factor (TRAF) binding, NF-kappaB activation, rodent cell transformation and cytostatic activity. Furthermore, our results point to the hydrophobic transmembrane domain, independent of the cytoplasmic domains, as the primary LMP-1 domain mediating oligomerization, patching and cytoskeletal association. The cytoplasmic amino-terminus provides the structural information whereby proper transmembrane orientation is achieved.

Serine proteases increase oxidative stress in lung cells.

Several serine proteases are directly cytotoxic. We investigated whether the cytotoxic effects of proteases are associated with increased levels of reactive oxygen species (ROS) in cells. We found that treatment of lung fibroblasts or bronchial epithelial cells with relatively high concentrations (0.1--100 U/ml) of neutrophil elastase, trypsin, and Pronase increased ROS levels in the mitochondria and cytoplasm. The protease-induced increase in ROS was associated with oxidative cellular injury as determined by generation of 8-hydroxy-2'-deoxyguanosine and malonaldehyde plus 4-hydroxyalkenal. The protease-induced increase in ROS was not merely due to cell detachment because the proteases also caused an increase in ROS in suspended cells, which precluded attachment to the extracellular matrix. The protease-induced increase in ROS appears to contribute to cytotoxicity because cell death induced by proteases was attenuated by treatment with catalase, a decomposer of H(2)O(2), and accelerated by treatment with aminotriazole, a catalase inhibitor. These results suggest that several proteases increase oxidative stress, indicating a direct interaction between proteases and ROS in mediating cytotoxicity.

Energy status of nonmatured and in vitro-matured domestic cat oocytes and of different stages of in vitro-produced embryos: enzymatic removal of the zona pellucida increases adenosine triphosphate content and total cell number of blastocysts.

In this study, we evaluated the adenosine triphosphate (ATP) content of individual domestic cat oocytes before and after in vitro maturation and of different stages of in vitro-produced embryos. To investigate the effects of assisted-hatching technique on the ATP content and total cell number, the zona pellucida of in vitro-produced blastocysts and expanded blastocysts (recovered 144 h postinsemination [hpi]) was completely removed by pronase treatment. The average (mean +/- SEM) ATP content of nonmatured oocytes (3.47 +/- 0.18 pmol) was significantly (P < 0.01) higher than that of in vitro-matured oocytes (2.17 +/- 0.10 pmol). After in vitro fertilization and culture, the ATP content of two-cell stages (24 hpi) was 1.17 +/- 0.08 pmol, which increased to 1.47 +/- 0.19 and 1.88 +/- 0.32 pmol at the four- (40 hpi) and eight-cell (48 hpi) stages, respectively. The ATP content then decreased to 1.48 +/- 0.10 pmol in 16-cell embryos (64 hpi), reaching a minimum of 0.49 +/- 0.04 pmol at the morula stage (120 hpi). Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 +/- 0.09, 1.79 +/- 0.01, and 4.17 +/- 0.21 pmol, respectively. After enzymatic removal of the zona pellucida (ERZP) at 144 hpi, ATP content and total cell numbers of blastocysts (4.15 +/- 0.37 pmol of ATP, 328.3 +/- 48.5 cells) and expanded blastocysts (5.81 +/- 0.54 pmol of ATP, 430.1 +/- 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 +/- 0.09 pmol of ATP, 65.3 +/- 4.6 cells; expanded blastocysts: 1.79 +/- 0.11 pmol of ATP, 121.4 +/- 6.5 cells). Our study describes, to our knowledge for the first time, changes in the energy status of domestic cat oocytes before and after maturation and during in vitro development after fertilization. The ERZP markedly increased the ATP content and total cell number of blastocyst stages, suggesting that this technique may improve the quality and viability of in vitro-produced domestic cat embryos.

Mass, charge, and subcellular localization of a unique secretory product identified by the basophil-specific antibody BB1.

BACKGROUND: BB1 is a basophil-specific mAb (Lab Invest 1999;79:27-38). The identity of the corresponding antigen has not been determined, but it gives a granular appearance on staining and is secreted on activation of basophils. OBJECTIVE: We sought to further characterize the basophilspecific antigen identified by BB1. METHODS: Intracellular localization was determined by flow cytometry and by immunogold labeling and electron microscopy. Physical chemical properties were investigated by gel filtration chromatography and preparative isoelectric focusing. RESULTS: In flow cytometry, permeabilization of cells increased immunofluorescence 100-fold, confirming the predominantly intracellular localization of the antigen. It was further localized to the secretory granules by immunoelectron microscopy. Double labeling with a CD63-specific antibody demonstrated selective binding of BB1 to the granule matrix. Gel filtration chromatography indicated that the antigen is secreted as a complex of approximately 5 x 10(6) d, which was well resolved from the 210-kd supramolecular complex containing tryptase. The antigen was degraded by pronase. Isoelectric focusing indicated a highly basic protein with an isoelectric point of 9.6. CONCLUSION: With its granule localization, release on cell activation, and unique properties, the antigen identified by BB1 could be a novel mediator of allergic disease. We propose the name basogranulin for this novel basophil-specific protein.