Applications of the ChromaPhor Protein Recovery System.

ChromaPhor protein stain binds to proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the mini-gel format. Because proteins are not fixed in the gel by ChromaPhor staining, they may be quickly and easily recovered using the ChromaPhor Protein Recovery System for use in further protein studies. We studied the recovery yields obtained using this system to recover proteins of various sizes from gel bands containing 2 micrograms of protein.

Assessment of gene expression and peptide secretion from individual cells.

We have developed an assay that allows one to monitor gene expression in and peptide secretion from individual cells. By combining the reverse hemolytic plaque with in situ hybridization, investigators can quantitate simultaneously the level of gene expression and the level of secretion of a peptide. The method can be used in any system in which an appropriate antibody for the reverse hemolytic plaque assay and probes complementary to the mRNA of interest are available.

Vasoactive intestinal polypeptide reduces hydrochloric acid-induced duodenal mucosal permeability.

The duodenum in anesthetized rats was perfused with HCl, and mucosal integrity was assessed by measuring the clearance of 51Cr-labeled EDTA from blood to lumen and/or by morphological examination (lesion score). Duodenal blood flow was determined by laser Doppler flowmetry and luminal alkalinization as well as H+ disappearance by backtitration. Intravenous infusion of vasoactive intestinal polypeptide (VIP; 13.

The use of ERCP in the management of common bile duct stones in patients undergoing laparoscopic cholecystectomy.

The purpose of this study was to evaluate the indications and results of endoscopic retrograde cholangiopancreatography (ERCP) for gallstone disease since the advent of laparoscopic cholecystectomy. In our personal series of 410 consecutive cases of laparoscopic cholecystectomy, we found 17 common bile duct (CBD) stones; seven were identified by preop ERCP, nine at laparoscopy by intraoperative cholangiography, and one postop by ERCP. We have performed preop ERCP in 21 patients (5.

Voice therapy practices and techniques: a survey of voice clinicians.

Members of the American Speech-Language-Hearing Association Special Interest Division on Voice were asked to rate statements which emanated from a previously conducted national survey regarding the practice of voice therapy (Larson and Mueller, 1991). Respondents rated eight data-based statements on a five-point scale as to agreement/disagreement. Results indicated strong agreement with previous findings with the exception of the item addressing treatment time.

Epidermal growth factor accelerates connective tissue wound healing in the perforated rat mesentery.

Epidermal growth factor (EGF) has been reported to stimulate healing of wounds in skin, cornea, and gastric mucosa. In the present study, we further investigate the effect of endogenous and exogenous EGF in healing of connective tissue wounds using the rat perforated mesentery model. Healing of mesenteric perforations is accomplished by the connective tissue fibroblasts since there are no interfering variables such as interactions of epithelial cells, desiccation, or foreign materials such as sutures or subcutaneous implants.

Western blots using stained protein gels.

A general method is described for the electrophoretic transfer of proteins from stained gels to membranes and subsequent Western detection of specific proteins on the stained membranes. Proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gels are stained using either of two different methods followed by electrophoretic transfer to nitrocellulose or Immobilon-P membranes. The transferred proteins remain stained during immunodetection, providing a set of background markers for protein location and size determination.

Cocaine-induced behavioral sensitization and D1 dopamine receptor function in rat nucleus accumbens and striatum.

Based on electrophysiological data showing that repeated cocaine administration produces persistent enhancement of D1 dopamine (DA) receptor-mediated responses in nucleus accumbens (NAc), we investigated whether changes in neurochemical properties of these receptors resulted when rats were injected with cocaine (15 mg/kg) for 6 days followed by a 7-day abstinence period. D1 DA receptor density and affinities for either [3H]SCH 23390 or DA were similar between NAc and striatum and between saline and cocaine treatment groups. DA-stimulated adenylyl cyclase activity was 1.

Simultaneous monitoring of pituitary hormone secretion and gene expression within individual cells.

We have recently developed a method to simultaneously quantitate the level of gene expression and the level of secretion of a peptide from individual cells. Our approach has been to combine the reverse hemolytic plaque assay sequentially with in situ hybridization. We present data to show how we have used the pituitary lactotroph as a model to demonstrate the power of this technique.

Multipractice analysis of laparoscopic cholecystectomy in 1,983 patients.

We analyzed the results of laparoscopic cholecystectomy in 1,983 patients from a variety of practice settings in order to evaluate a large, cross-sectional experience for this new procedure. Twenty general surgeons from 9 clinics in 4 states examined the records and outcome of their laparoscopic cholecystectomy patients through March 1991. In 88 patients (4.

Deantigenation of human erythrocytes by bacterial glycosidases--evidence for the noninvolvement of medium-sized glycosphingolipids in the Dolichos biflorus lectin hemagglutination.

Fresh human A1 erythrocytes, washed and pretreated in phosphate buffer with or without papain, were incubated at 37 degrees C with blood group-degrading enzymes from the human fecal Ruminococcus torques strain IX-70. The effects were assayed as changes in hemagglutination patterns, and blood group activities of alkali stable glycolipid extracts from the enzyme-treated cells using Dolichos biflorus anti-A1 lectin, Ulex europaeus type 1 anti-H lectin, and various monoclonal anti-A antibodies. Hemolysis was negligible (less than or equal to 1% after 6 h), and the osmotic fragility increased slightly only after papain treatment.

Gonadotropin concentrations, follicular development, and luteal function in pituitary stalk-transfected ewes treated with bovine follicular fluid.

Two experiments, each arranged as a 2 x 2 factorial, were conducted in ewes to examine direct effects of bovine follicular fluid (bFF) on follicular development and luteal function and to further characterize follicular development and luteal function after pituitary stalk transection (SS). In Exp. 1, ewes were sham-operated or SS on d 6 of an estrous cycle and received 5 ml of saline or bFF three times daily on d 5 through 11 of the same cycle.

Altered response to growth rate changes in Kluyveromyces lactis versus Saccharomyces cerevisiae as demonstrated by heterologous expression of ribosomal protein 59 (CRY1).

We report the cloning, characterization and preliminary analysis of the regulation of the gene coding for ribosomal protein 59 (RP59) from the budding yeast Kluyveromyces lactis. The RP59 gene is present as a single copy, contains an intron within the amino terminal coding portion of the gene, and harbors conserved S. cerevisiae splicing signals.

Enhancing effects of bile salts on the degradation of glycosphingolipids by glycosidases from bacteria of the human fecal flora.

Different concentrations of ionic and non-ionic detergents were examined for optimization of the in vitro degradations of intestinal glycosphingolipids by alpha- and beta-glycosidases from human fecal bacteria. In 5 mM Triton X-100 the enzymes hydrolyzed glycosphingolipids with lactoseries type 1 and 2 chains essentially to lactosylceramide (LacCer). In 5 mM sodium di- and trihydroxy bile salts lactosylceramide was degraded to glycosylceramide (GlcCer) in varying extent by enzymes from all five strains.

Inhibitors of arachidonic acid metabolism: effects on rat striatal dopamine release and uptake.

The purpose of this study was to investigate the possibility that arachidonic acid metabolites mediate D-2 dopamine (DA) receptor inhibition of striatal DA release. The phospholipase A2 inhibitor p-bromophenacyl bromide (BPB; 10 microM) increased electrically evoked overflow of endogenous DA from rat striatal slices and appeared to partially block the modulatory effects of the D-2 DA receptor agonist N-0437 on this release. However, BPB also increased spontaneous DA outflow in a dose-dependent manner.

The effect of procedural variations on the S/Z ratio of adults.

The effect of elicitation order, number of required trials, and selection of /s/ and /z/ values utilized to calculate the s/z ratio in adult subjects was examined. Results support the conclusion that a single phoneme production trial is a valid and efficient method for obtaining the s/z ratio. Procedural and clinical implications are discussed.

Endoscopically placed nasoenteral feeding tubes. Indications and techniques.

We reviewed indications, techniques, and results for endoscopic placement of nasoenteral feeding tubes by analyzing records of 29 hospitalized patients who had undergone 41 tube placements. Indications included gastric stasis after gastric surgery (11 patients), gastroparesis and reflux or both in critically ill trauma patients (10), stricture secondary to vertical banded gastroplasty (3), and partial gastric outlet obstruction (5). Two endoscopic techniques were used.

Age-related alterations in prolactin secretion by individual cells as assessed by the reverse hemolytic plaque assay.

The objectives of this study were to determine: 1) if lactotropes from old rats, compared to those from young rats, secrete a greater amount of prolactin (PRL) per cell, 2) if the percentage of pituitary cells secreting PRL changes with age; and 3) how estradiol (E2), dopamine (DA), or thyrotropin-releasing hormone (TRH), or the combination of these factors influences both of these parameters in old rats. To meet these objectives we used the reverse hemolytic plaque assay (RHPA), because this method allows us to determine both the percentage of pituitary cells secreting prolactin during the experimental period and the amount of hormone released by each secreting pituitary cell. These parameters were measured in young (2-3 mo old) or old (17-19 mo old) female Sprague-Dawley rats.

Interruption of professional and home activity after laparoscopic cholecystectomy among French and American patients.

With a laparoscopic approach, patients can undergo cholecystectomy with a shorter hospitalization, minimal pain, and quicker recovery. It has not been demonstrated, however, that patients actually return to work after laparoscopic cholecystectomy faster than the traditional 4- to 6-week absence from work after a standard open procedure. A survey of 104 French and 84 American patients undergoing laparoscopic cholecystectomy revealed that postoperative discomfort was completely resolved in 2 weeks in 73% of French and 93% of American patients.

The receptor repertoire defines the host range for attaching Escherichia coli strains that recognize globo-A.

Escherichia coli strains which colonize the human urinary tract express lectins specific for different members of the globoseries of glycolipids, e.g., globotetraosylceramide and globo-A.

Measurement of peptide secretion and gene expression in the same cell.

A combined reverse hemolytic plaque-in situ hybridization assay was developed to allow analysis of the relationship between peptide secretion and gene expression within individual cells. We used the pituitary lactotroph as a model system, but this strategy should be widely applicable. It can be used to test hypotheses regarding if and when peptide secretion and gene expression are coupled in any system in which antibodies to the secreted peptide and probes complementary to the mRNA are available.

Regional differences in the appearance of adult-type glycosphingolipids in the small intestine of inbred rats at weaning time.

The small intestine of 15- to 33-day-old rats was cut into four segments: duodenum, proximal jejunum, distal jejunum, and ileum. Neutral glycosphingolipids and gangliosides were purified from each segment and analyzed by thin-layer chromatography in order to study the developmental appearance of adult-type glycolipids at each level of the small intestine. Type 1 A-6 glycolipid was first detected in the ileum at 15 days and subsequently in the jejunum and duodenum at 19 days of age.

Transient expression of type 2 chain in A-active hexaglycosylceramide of rat small intestine at weaning time. Demonstration by affinity chromatography and ceramide glycanase hydrolysis of A-active glycosphingolipids followed by gas chromatography and mass spectrometry of permethylated hexasaccharides.

The small intestine of 15- to 23-day-old rats was cut into four segments from the duodenum to the ileum. Neutral glycosphingolipids were purified from each segment and submitted to thin-layer chromatography and immunostaining with the A005 monoclonal anti-A antibody. This antibody detected an hexaglycosylceramide located mainly in the duodenum during the postnatal development.

Bacteria of the human intestinal microbiota produce glycosidases specific for lacto-series glycosphingolipids.

Five strains of human fecal bacteria, of the Ruminococcus and Bifidobacterium genera, produce extracellular alpha- and beta-glycosidases that degrade intestinal mucin oligosaccharides and glycosphingolipids of the lacto-series type 1 chain. We have tested the activities and substrate specificities of these enzymes using para-nitrophenyl glycosides and glycosphingolipids of different core chains (lacto, neolacto, globo, isoglobo, galabio, and ganglio), carrying different blood group determinants (A, H, X, Y, Forssman, and para-Forssman), and with different degrees of sialylation (mono- to tetra-sialo). Lactotetraosylceramide and neolactotetraosylceramide were the only core glycosphingolipids degraded by enzymes from these strains, resulting in lactosylceramide and glucosylceramide as the major end products.