Monobody adapter for functional antibody display on nanoparticles for adaptable targeted delivery applications.

Vascular endothelial cells (ECs) play a central role in the pathophysiology of many diseases. The use of targeted nanoparticles (NPs) to deliver therapeutics to ECs could dramatically improve efficacy by providing elevated and sustained intracellular drug levels. However, achieving sufficient levels of NP targeting in human settings remains elusive. Here, we overcome this barrier by engineering a monobody adapter that presents antibodies on the NP surface in a manner that fully preserves their antigen-binding function. This system improves targeting efficacy in cultured ECs under flow by >1000-fold over conventional antibody immobilization using amine coupling and enables robust delivery of NPs to the ECs of human kidneys undergoing ex vivo perfusion, a clinical setting used for organ transplant. Our monobody adapter also enables a simple plug-and-play capacity that facilitates the evaluation of a diverse array of targeted NPs. This technology has the potential to simplify and possibly accelerate both the development and clinical translation of EC-targeted nanomedicines.

Primary intracranial solitary leptomeningeal glioma: a report of 3 cases.

Primary intracranial solitary leptomeningeal gliomas are exceedingly rare. We, therefore, performed a detailed clinical, radiological and pathological analysis to better characterize these tumors in 3 patients (33- and 72-year-old men and a 72-year-old woman). Two of the tumors were located in the frontal region and 1 in the temporal region. Magnetic resonance imaging revealed a well circumscribed large lesion (maximal diameter 4 - 6 cm) with peritumoral edema, mixed low- and isosignal intensity on T1-weighted images, hypersignal intensity on T2-weighted images and non-homogeneous contrast enhancement. External carotid angiography demonstrated a vascular supply to these tumors via branches of the middle meningeal artery. Gross total resection was achieved in all patients. The pathological diagnosis was glioblastoma in 2 patients and oligodendroglioma in 1. The MIB-1 nuclear labeling index ranged from 11.8% - 23.6% (mean 18.2%). Local tumor recurrence was documented in 2 patients after 8 and 11 months, respectively. The other patient with glioblastoma developed a metastasis to the femur 39 months after craniotomy. A definitive diagnosis can be made by careful radiological assessment and histopathological examination.

N-benzylimidazole for preparation of S9 fraction with multi-induction of metabolizing enzymes in short-term genotoxicity assays.

To evaluate the usefulness of N-benzylimidazole (BI) as an inducer with wide spectrum detection of precarcinogens in short-term bioassays, hepatic levels of cytochrome P-450 (CYP) and mutagenic activation of various carcinogens in Wistar and Sprague-Dawley rats orally treated with BI and BI plus ethanol or acetone were compared with those in the same strains of rats treated with 3-methylcholanthrene (MC), phenobarbital (PB) and polychlorobiphenyls (PCB). Immunoblot analyses for microsomal CYP proteins revealed a marked induction by BI in the levels of CYP1A1, CYP2B1 and constitutive CYP1A2 (approximately 11-fold), 2B2 (approximately 21-fold), 2E1 (1.5-fold) and 3A2 (4-fold) in rats of both strains. These levels were comparable with those induced by MC and PB, but were less than the CYP1A1/2 and 2B1 levels induced by PCB, while CYP2B2 was at the same level. In contrast, the level of CYP2E1 was clearly higher in BI-treated rats. The combinations of BI and acetone or ethanol specifically induced CYP2E1 (4-fold) and 2B1 (1.7-fold) levels when compared with BI alone in Wistar rats. The combined treatments also elevated mutagenic activities of eight heterocyclic amines (HCAs), aflatoxin B(1) (AFB(1)), benzo[a]pyrene and 2-aminofluorene in strain TA98 up to 14.3-, 5.1-, 2.8- and 2.1-fold above the untreated group, respectively, and those of five N-nitrosamines in strain TA100 up to 19.1-fold. Induction of specific CYP species responsible for activation of HCAs, AFB(1) and N-nitrosamines was confirmed by application of several CYP inhibitors. In addition, BI induced activities of both MC- and PB-inducible UDP-glucuronyltransferases towards 4-nitrophenol and testosterone. These results demonstrate that BI has a bifunctional action, with wide spectrum induction of phase I and II enzymes, and combined treatment with ethanol or acetone would be a pertinent inducer for metabolic enzymes in in vitro bioassays, the potential being comparable with or superior to other typical ones.

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet.

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.

Lack of change in the levels of liver and kidney cytochrome P-450 isozymes in p53+/- knockout mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine.

We have previously shown that p53(+/-) knockout mice are highly sensitive to urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in spite of a lack of effects of p53 heterozygosity on N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) excretion in urine. To determine the influence of p53 deficiency on in vitro formation of BCPN, mutagenicity of BBN and BCPN and levels of several cytochrome P450 (CYP) isozymes, groups of five p53(+/-) knockout and wild-type mice (littermates), as well as animals of the C57BL/6 parental strain, were administered 0.025% BBN in their drinking water for 4 weeks. The livers and kidneys were then used for analyses of BBN metabolism, western immunoblotting and Ames liquid incubation. BBN treatment caused a slight decrease in BCPN formation in the livers of C57BL/6 mice, but there was no significant difference between p53 knockout, wild-type and C57BL/6 mice. In kidney BCPN formation in p53 knockout mice was 33-46% less than that in their wild-type counterparts. Using anti-rat CYP antibodies, CYP1A2, 2B9/10, 2E1 and 3A11/13 were constitutively detected in liver microsomes and CYP2E1 and 3A11/13 in the kidney. Densitometric determination of these CYP proteins revealed no significant variation in levels detected in both tissues among the four groups of mice. BBN and BCPN were not mutagenic for Salmonella typhimurium TA100 in either the absence or presence of liver S9 from untreated mice and rats and from p53 knockout mice treated with BBN. In conclusion, p53 deficiency and BBN had no enhancing effects on metabolism of BBN to BCPN and expression of the CYP isozymes typically responsible for activation of environmental carcinogens, including both of the N-nitrosamines tested, and their mutagenicity, indicating that the high susceptibility of p53(+/-) knockout mice is not attributable to metabolic activation in liver and kidney by CYP isozymes or urinary excretion of BCPN.

Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface.

It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions.

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain.

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.

Quantitative analysis of bowel gas using plain abdominal radiograph in patients with irritable bowel syndrome.

OBJECTIVE: Ideally, the diagnosis of irritable bowel syndrome (IBS) would be achieved using a minimal number of procedures. It is presumed that bowel gas is related to IBS, and it is easily visualized by plain abdominal radiograph. In the present study, to clarify the relationship between IBS and the quantity of bowel gas, the measured bowel gas volume using plain abdominal radiographs was compared with the pathology of IBS. METHODS: Plain abdominal radiographs were digitized and transmitted to a computer (computed radiography) in 30 IBS patients and 30 normal controls. The quantity of bowel gas, determined as the pixel value on images and standardized by physique, was defined as the gas volume score (GVS). Using the mean +/- 2SD of GVS in the control group as the normal score, IBS patients were divided into three groups: high, normal, and low. To examine the sequential reproducibility of a similar quantity of bowel gas, a second plain abdominal radiography was performed about 2 months later, and the GVS were compared. The colonic transit time was determined using radiopaque markers. RESULTS: There was a strong correlation between the quantities of bowel gas measured by two independent gastroenterologists. The mean GVS of IBS patients was significantly higher than that in the control group (p < 0.001). The sequential reproducibility was recognized in all 10 IBS patients. There was no significance between colonic transit time and GVS, nor between symptoms and GVS. CONCLUSIONS: Abdominal gas was analyzed objectively by using GVS, and GVS was considered to represent a useful tool for the diagnosis of IBS.

Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet.

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.

Design of single-layer beta-sheets without a hydrophobic core.

The hydrophobic effect is the main thermodynamic driving force in the folding of water-soluble proteins. Exclusion of nonpolar moieties from aqueous solvent results in the formation of a hydrophobic core in a protein, which has been generally considered essential for specifying and stabilizing the folded structures of proteins. Outer surface protein A (OspA) from Borrelia burgdorferi contains a three-stranded beta-sheet segment which connects two globular domains. Although this single-layer beta-sheet segment is exposed to solvent on both faces and thus does not contain a hydrophobic core, the segment has a high conformational stability. Here we report the engineering of OspA variants that contain larger single-layer beta-sheets (comprising five and seven beta-strands) by duplicating a beta-hairpin unit within the beta-sheet. Nuclear magnetic resonance and small-angle X-ray scattering analyses reveal that these extended single-layer beta-sheets are formed as designed, and amide hydrogen-deuterium exchange and chemical denaturation show that they are stable. Thus, interactions within the beta-hairpin unit and those between adjacent units, which do not involve the formation of a hydrophobic core, are sufficient to specify and stabilize the single-layer beta-sheet structure. Our results provide an expanded view of protein folding, misfolding and design.

Effect of cigarette smoke on the mutagenic activation of environmental carcinogens by rodent liver.

In order to assess the effect of cigarette smoke (CS) on metabolic enzymes, male hamsters and rats were exposed for two weeks to smoke produced in a Hamburg type II smoking machine. The livers were then used for Ames liquid incubation and western immunoblot assays. Mutagenic activities of seven heterocyclic amines (HCAs) in Salmonella typhimurium TA98 in the presence of rat or hamster liver S9 were elevated up to 3.7 times above controls (including sham smoke control). Enhancement of mutagenic activities of PhIP and aflatoxin B(1) was observed only in CS-exposed hamster, whereas no significant alteration of mutagenicity was observed with 2-aminofluorene, benzo[a]pyrene, and 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene in strain TA98 or with six N-nitrosodialkylamines in strain TA100. 7,8-Benzoflavone and/or furafylline considerably inhibited the mutagenic activation of IQ and Trp-P-1 in the presence of liver S9 from untreated hamsters and sham smoke- or CS-exposed hamsters and rats, indicating the predominant involvement of hamster cytochrome P450 (CYP) 1A enzymes in the metabolic activation of HCAs. In addition, the data suggest that CS-exposure may selectively induce hepatic CYP1A1/1A2 isoforms. Western immunoblot analyses of liver microsomes using anti-rat CYP antibodies revealed that CS-exposure increased the levels of hamster CYP1A2 (3.9-fold) and rat CYP1A2 (3.0-fold) and CYP1A1, without significant change in the levels of CYP2E1 and CYP2B and 3A isoforms in each species. The presently observed selective induction of HCA activation and CYP isozymes due to CS supports the idea that CS may contribute to enhancing effects on initiation by carcinogens which are metabolically activated by hepatic CYP1A1/1A2. In conjunction with results observed for smokers, the present findings indicate that the hamster is a good animal for studies with CS, and that cigarette smoking in combination with intake of heating protein-rich foods as a life style may markedly contribute to the human carcinogenesis by HCAs.

ScoC regulates peptide transport and sporulation initiation in Bacillus subtilis.

Oligopeptides are transported into Bacillus subtilis by two ABC transport systems, App and Opp. Transcription of the operon encoding the Opp system was found to occur during exponential growth, whereas the app operon was induced at the onset of stationary phase. Transcription of both operons was completely curtailed by overproduction of the ScoC regulator from a multicopy plasmid and was enhanced in strains with the scoC locus deleted. ScoC, a member of the MarR family of transcription regulators, is known from previous studies to be a negative regulator of sporulation and of protease production that acts by binding directly to the promoters of the genes it regulates. Since peptide transport is essential for inactivation of the negative regulation of sporulation by Rap phosphatases, the control of ScoC transcription repression activity plays a crucial role in the initiation of sporulation.

The fibronectin type III domain as a scaffold for novel binding proteins.

The fibronectin type III domain (FN3) is a small autonomous folding unit which occurs in many animal proteins involving in ligand binding. The beta-sandwich structure of FN3 closely resembles that of immunoglobulin domains. We have prepared a phage display library of FN3 in which residues in two surface loops were randomized. We have selected mutant FN3s which bind to a test ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble protein and its properties were investigated in detail. Heteronuclear NMR characterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutations at 12 out of 94 residues. These results clearly show the potential of FN3 as a scaffold for engineering novel binding proteins.

High susceptibility of p53(+/-) knockout mice in N-butyl-N-(4-hydroxybutyl)nitrosamine urinary bladder carcinogenesis and lack of frequent mutation in residual allele.

The loss of p53 functions is considered to compromise the growth-suppression machinery of the cell and facilitate neoplastic change. In humans, genetic alteration in the p53 gene is one of the most frequently observed molecular changes in tumors, including urinary bladder carcinomas. We have investigated the susceptibility of heterozygote p53 knockout mice to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in terms of urinary bladder tumor induction. Both p53(+/-) knockout mice and C57BL/6 original parent strain were administered 0, 0.002, 0.004, 0.0075 and 0.025% BBN in the drinking water for 20 weeks. As compared with the C57BL/6 strain, greater lesion yields were observed in knockout mice after 20 weeks of treatment. Transitional cell carcinomas were found in 9 (75%) and 12 (100%) of each 12 mice of the 0.0075 and 0.025% BBN treatment groups, respectively, whereas only 1 (11%) and 6 (67%) of each 9 of the C57BL/6 mice demonstrated tumors. Preneoplastic lesions (dysplasia) were also observed more frequently in the lower dose groups in the knockout mice than C57BL/6 mice. PCR single-strand conformation polymorphism analysis followed by DNA direct sequencing of the p53 gene (exons 5-8) extracted from bladder tumors demonstrated mutations in 3 of 11 (27.3%; exon 7) and 8 of 29 (27.6%; exons 5-8) tumors in C57BL/6 and knockout mice, respectively. There was no significant difference in the mutation rates at the residual p53 gene between the two cases. All mutations observed in knockout mice were restricted to the normal allele, and none were present in the gene-targeted null allele. In a separate experiment, 5-bromo-2'-deoxyuridine labeling indices after treatment with BBN for 2 or 4 weeks were significantly higher in knockout mice than wild-type mice. Measurement of the urinary concentration of N-butyl-N-(3-carboxypropyl)nitrosamine, a proximate carcinogenic metabolite, revealed no significant differences between knockout and original parent strain after administration of 0.0075% BBN in the drinking water for 4 weeks. In conclusion, knockout mice are distinctly more sensitive to urinary bladder carcinogenesis induced by BBN than their original parent strain, as evidenced by elevated DNA synthesis during carcinogen administration and an increased tumor yield. The high susceptibility of p53 knockout mice appeared to be related to the high level of cell proliferation rather than that of N-butyl-N-(3-carboxypropyl)nitrosamine in the urine or that of mutations at the p53 gene.

[Relationship between mitral valve echo score and hemodynamic variables in patients with mitral stenosis].

Mitral valve echo score has been proposed as a predictor or of the outcome of balloon mitral valvotomy in patients with mitral stenosis. The relationship between mitral echo score and the hemodynamic variables was evaluated. In 41 patients with pure mitral stenosis (nine men and 32 women, aged 57.9 +/- 9.4 years), mitral echo score was estimated from two-dimensional echocardiographic findings, and mitral valve area was measured by planimetry on the two-dimensional short-axis view. Apex phonocardiography and continuous-wave Doppler echocardiographic recording of transmittral flow were simultaneously performed to measure left atrial/left ventricular mean transmittral pressure gradient, pressure half-time and (Q-1)-(2-OS) interval. Linear regression analysis revealed that both mitral echo score and mitral valve area were significantly correlated with mean transmittral gradient (r = 0.522, p = 0.0005 and r = -0.651, p < 0.0001, respectively), pressure half-time (r = 0.491, p < 0.005 and r = -0.757, p < 0.0001) and (Q-1)-(2-OS) interval (r = 0.551, p < 0.0005 and r = -0.487, p < 0.005, respectively). Mitral echo score has a significant correlation with hemodynamic variables, which were comparable to but slightly different from mitral valve area, in patients with mitral stenosis.

A stable single-layer beta-sheet without a hydrophobic core.

Outer surface protein A from the Lyme disease spirochete Borrelia burgdorferi contains a single-layer beta-sheet connecting the N- and C-terminal globular domains. The central beta-sheet consists largely of polar amino acids and is solvent-exposed on both faces, which so far appears to be unique among known protein structures. We show that the single-layer beta-sheet segment is surprisingly stable (deltaG for hydrogen exchange is approximately 8 kcal mol(-1) at 45 degrees C). Possible factors contributing to the stability of the single-layer beta-sheet are discussed based on an analysis of the crystal structure.

A case of small cell carcinoma of the stomach.

A case of small cell carcinoma of the stomach is reported. A 53-year-old male was referred to our hospital for elective surgery for gastric cancer. Pre-operative examinations revealed no metastases. Gastrectomy was performed curatively, and there were no gross findings of metastases. Histologically, the tumor was composed of intermediate-sized cells with hyper-chromatic nuclei and scanty cytoplasm. These cells were argyrophilic and positive for chromogranin A. A small portion of the tumor consisted of conventional adenocarcinoma (signet ring cell carcinoma and tubular adenocarcinoma). No lymph node metastasis was observed microscopically. However, 7 months after the operation, splenic and hepatic metastases were detected, and the patient died very soon thereafter. Small cell carcinoma of the stomach is a very rare disease. In literature, only 15 cases have been cured surgically. Among them, only one case had been diagnosed as small cell carcinoma before the operation, which suggests the difficulty of pre-operative diagnosis. The prognosis of this disease is very poor compared with the common type of gastric carcinoma. Considering the poor prognosis of this particular disease, adjuvant chemotherapy might be mandatory in all cases even if surgically curative resection is performed.

The importance of the real-time fluoroscopic intraoperative direct cholangiogram in the laparoscopic cholecystectomy using a new instrument.

BACKGROUND/AIMS: Laparoscopic cholecystectomy (LC) has become an accepted standard operative technique for gallstone treatment worldwide. On the other hand, complications, such as bile duct injuries, have been reported recently with the expansion of indication for LC. Intraoperative cholangiogram (IOC), to minimize the risk of bile duct injury, is now considered to be essential for safe LC. There are disadvantages to IOC such as increased operating time, the possibility of bile duct injury and the difficulties of manipulation. MATERIAL AND METHODS: We have developed a method for real-time fluoroscopic cholangiograms using a new instrument designed by our group for safe LC. First, a round-tip stylet is inserted through a sheath to coax it gently through the spiral valves of the cystic duct. Secondly, the stylet is removed and the cholangiogram catheter is inserted smoothly. Digital C-arm fluoroscopy provides "real-time" imaging of biliary tree. As a result, we became able to obtain a clear cholangiogram easily in a very short time. RESULTS: In the first 136 patients, direct cholangiograms were attempted in 106 cases and successfully completed in 102 cases (96.2%). CONCLUSION: With the development of real-time fluoroscopic intraoperative direct cholangiogram, we are able to cope with bile duct injuries and anomalies, and unsuspected bile duct stones.