Frequency of Post Endoscopic Retrograde Cholangiopancreatography Acute Pancreatitis in an Academic Hospital of Bangladesh.

Post-ERCP acute pancreatitis (PEP) is a common and serious complication with high morbidity and mortality rates. There is a paucity of data on the frequency of PEP in a resource constraint setting like Bangladesh. Hence we have conducted a prospective study to determine the frequency of PEP and the factors associated with its occurrence. This prospective, observational study was carried out in Gastroenterology Department of Dhaka Medical College & Hospital, Dhaka, Bangladesh from April 2018 to December 2018 on the consecutive patients who underwent ERCP. PEP and its severity were diagnosed according to consensus definition. Serum lipase was done in all patients before procedure and 24 hours after procedure or if patient develops abdominal pain after the procedure which became earlier. Total 168 patients were included (mean age 46.97±14.35 years; male 72(43.0%). The most common indication of ERCP was choledocholithiasis 97(58.0%) followed by malignant biliary obstruction 45(27.0%), recurrent pyogenic cholangitis 8(5.0%), chronic pancreatitis 4(2.3%), biliary ascariasis 4(2.3%) and others 10(6.0%). Overall post ERCP complication rate was 46(27.3%) including cholangitis 29(17.3%), pancreatitis 16(9.5%), bleeding 12(7.1%), aspiration pneumonia 4(2.4%) and death 3(1.8%). Regarding the severity of PEP, 50.0%, 43.7% and 6.3% patients developed mild, moderate and severe pancreatitis respectively. Number of cannulation attempts >5 times [22(48%) vs. 17(14%); p=0.001], cannulation attempts duration more than 10 minutes [25(55%) vs. 27(22%); p=0.001], unintentional passage of guide wire into the pancreatic duct [8(17%) vs. 18(15%); p=0.001], pancreatic duct contrast injection [12(26%) vs. 2(1.6%); p=0.001] and pre-cut sphincterotomy [16(35%) vs. 6(4.9%); p=0.001] were significantly different between the patients who developed PEP compared to those who did not. In multiple logistic regressions analysis, pancreatic duct contrast injection was significantly associated with PEP [OR 25.523 with 95% CI (4.049- 100.0%)]. Around ten percent patients had developed PEP. Regarding the severity half of them were mild, 44.0% patients had moderate and 6.0% patient had severe type of pancreatitis. Difficult cannulation, unintentional passage of guide wire into the pancreas, pancreatic duct contrast injection and pre-cut sphincterotomy were associated with PEP. Among them pancreatic duct contrast injection had independent significance in the causation of PEP.

Effect of intravenous ondansetron on sensory and motor block after spinal anaesthesia with hyperbaric bupivacaine.

Ondansetron is a potent antiemetic and a competitive antagonist at serotonin receptors, which are also involved in modulation of pain. This study was designed to assess the effect of systemic ondansetron on sensory and motor block after subarachnoid anaesthesia with hyperbaric bupivacaine. Sixty patients undergoing transurethral resection of bladder tumours were randomly assigned to one of two groups. Group 1 received 2 ml (4 mg) of ondansetron whereas patients in group 2 received 2 ml of normal saline 15 minutes prior to administration of subarachnoid block with 2.5 ml of hyperbaric bupivacaine 0.5%. Time to attain peak sensory block (P = 0.27), time to two segment regression (P = 0.19) and time to regression to the S, dermatome (P = 0.84) did not significantly differ between the two groups. No differences in regression of sensory block were noted at any time. The mean duration of motor block also did not differ (P = 0.44), with similar regression of block at all time intervals except at 90 minutes. We concluded that intravenous ondansetron does not affect the intensity or duration of sensory and motor block after spinal anaesthesia with hyperbaric bupivacaine.

Elucidation of the mechanism of interaction of sheep spleen galectin-1 with splenocytes and its role in cell-matrix adhesion.

The binding of a 14 kDa beta-galactoside animal lectin to splenocytes has been studied in detail. The binding data show that there are two classes of binding sites on the cells for the lectin: a high-affinity site with a K(a) ranging from 1.1 x 10(6) to 5.1 x 10(5) M (-1) and a low affinity binding site with a K(a) ranging from 7.7 x 10(4) to 3.4 x 10(4) M (-1). The number of receptors per cell for the high- and low-affinity sites is 9 +/- 3 x 10(6) and 2.5 +/- 0.5 x 10(6), respectively. The temperature dependence of the K value yielded the thermodynamic parameters. The energetics of this interaction shows that, although this interaction is essentially enthalpically driven (DeltaH - 21 kJ lambdamol(-1)) for the high-affinity sites, there is a very favorable entropy contribution to the free energy of this interaction (-TDeltaS - 17.5 Jmol(-1)), suggesting that hydrophobic interaction may also be playing a role in this interaction. Lactose brought about a 20% inhibition of this interaction, whereas the glycoprotein asialofetuin brought about a 75% inhibition, suggesting that complex carbohydrate structures are involved in the binding of galectin-1 to splenocytes. Galectin-1 also mediated the binding and adhesion of splenocytes to the extracellular matrix glycoprotein laminin, suggesting a role for it in cell-matrix interactions.

Effect of pre-emptive analgesia on self-reported and biological measures of pain after tonsillectomy.

This prospective, double-blind, randomized study assessed effect of pre-emptive peritonsillar block in 30 ASA-I children, aged 6-12 years, of both sexes, scheduled for tonsillectomy. Patients were divided into three groups: those in group I received a sham block, whereas peritonsillar blocks with bupivacaine 0.25% were given to the children before tonsillectomy (group II) or immediately after surgery had been completed (group III). Constant pain, pain on swallowing, blood glucose, serum epinephrine and norepinephrine concentrations were measured immediately after surgery and 4h after operation. Patients in group I experienced more pain (P< 0.05) than those in groups II and III, both in the immediate postoperative period and over the next 4 h. Patients in groups II and III experienced similar pain (P> 0.05). The pain experienced when water was swallowed was similar to that of the constant pain. The rise of serum norepinephrine concentration in group II was significantly less (P< 0.05) compared to groups I and III. We found both pre-emptive and postoperative block to be equally effective in treating postoperative pain, with pre-emptive block being more effective in preventing the rise in norepinephrine concentration.

Comparative studies on kinetics of inhibition of protein synthesis in intact cells by ricin and a conjugate of ricin B-chain with momordin.

Ribosome inactivating proteins from plants have been widely used for the preparation of immunotoxins and hormonotoxins, which have potential application in the therapy of diseases such as cancer. However, these hybrid toxins have been found to be less cytotoxic than native ribosome inactivating proteins. Therefore, it is important to understand the factors that control the intrinsic toxicity of RIPs and the hybrid toxins prepared using them. Here, a hybrid toxin has been prepared by coupling ricin B-chain to momordin and the cytotoxicity of this hybrid toxin has been compared to that observed in case of native ricin. In the two cell types used here, thymocytes and macrophages, the conjugate was found to be about 40 fold less toxic than native ricin. Kinetics of inhibition of protein synthesis showed that prior to onset of inhibition the conjugate exhibits a longer lag phase than native ricin. The rates of inhibition of protein synthesis by the conjugate were also found to be slower than ricin. Analysis of the results suggests that in addition to cell surface binding, the B-chain of ricin facilitates another step in the transmembrane translocation of ricin A-chain to the cytosol.

Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding.

The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2K] and the A-subunit (Tm 324.6K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting DeltaHm (the molar transition enthalpy at Tm) against Tm yielded values for DeltaCp (change in excess heat capacity) of 27+/-2 kJ.mol-1.K-1 for the B-subunit and 20+/-1 kJ.mol-1.K-1 for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.

The effect of pH on the unfolding pathway and stability of ribosome-inactivating protein abrin-II.

The effect of pH on the unfolding pathway and the stability of the toxic protein abrin-II have been studied by increasing denaturant concentrations of guanidine hydrochloride and by monitoring the change in 8,1-anilino naphthalene sulfonic acid (ANS) fluorescence upon binding to the hydrophobic sites of the protein. Intrinsic protein fluorescence, far and near UV-circular dichroism (CD) spectroscopy and ANS binding studies reveal that the unfolding of abrin-II occurs through two intermediates at pH 7.2 and one intermediate at pH 4.5. At pH 7.2, the two subunits A and B of abrin-II unfold sequentially. The native protein is more stable at pH 4.5 than at pH 7.2. However, the stability of the abrin-II A-subunit is not affected by a change in pH. These observations may assist in an understanding of the physiologically relevant transmembrane translocation of the toxin.

Evolution of tetrameric lectin Ricinus communis agglutinin from two variant groups of ricin toxin dimers.

Seeds of Ricinus communis contain two types of lectins; the toxin ricin (approximately 60 kDa) and R. communis agglutinin (approximately 120 kDa). The toxin is a heterodimer composed of a toxic A subunit and a lectin B subunit, while R. communis agglutinin is a tetramer, constituted of two ricin-like dimers held together by non-covalent forces. The lactamyl Sepharose affinity-purified ricin consists of two major groups of variants designated ricin II and III [Hegde, R. & Podder, S. K. (1992) Eur. J. Biochem. 204, 155-164]. The purified A subunits of all the variants of ricins and R. communis agglutinin show heterogeneity in the molecular mass as shown for ricin before [Fulton, J. R., Blakey, C. D., Knowles, P. P., Uhr, J. W., Thorpe, P. E. & Vitetta, E. S. (1986) J. Biol. Chem. 261, 5314-5319]. Since the isoelectric points of the R. communis agglutinin variants fall between the isoelectric points of ricin II and III, we investigated the possibility that this lectin is made up of ricin II and III. The isoelectric points of the purified B subunits of R. communis agglutinin matched well with those of ricin II and III on urea-polyacrylamide isoelectric focussing gel. Further, two-dimensional electrophoretic analysis of the ricin constituants of R. communis agglutinin in the presence of 9 M urea, showed two protein bands, differing by nearly pH 2 in their isoelectric points, the more alkaline one corresponding to that of ricin III analyzed under the same conditions, while the other, although a higher molecular mass variant, corresponding well with ricin II in its isoelectric point. Based on these results and those obtained from adenine binding to A chains of both ricin and R. communis agglutinin, we provide a plausible evolutionary relationship between R. communis agglutinin and two groups of ricin variants; ricin II and III. The model predicts that one half of R. communis agglutinin is derived from ricin I and II, and the other half from ricin III. The results presented, contrary to the existing notion, unequivocally show that the two halves of R. communis agglutinin are not identical protein units, but differ both in surface charge and molecular mass.

Evaluation of the stoichiometry and energetics of carbohydrate binding to Ricinus communis agglutinin: a calorimetric study.

High-sensitivity isothermal titration calorimetry has been used to investigate the thermodynamics of binding of Ricinus communis agglutinin to galactose, lactose and their derivatives in the temperature range 280.5-298 K. The present study unequivocally establishes the carbohydrate-binding stoichiometry of the tetrameric agglutinin from castor bean as two, i.e. the (As-sB)2-type tetramer of the agglutinin has two equivalent sites that are non-interacting and independent. The site binding constants range from 2.2x10(3) M-1 at 282 K for galactose to 4.84x10(4) M-1 at 281 K for N-acetyl-lactosamine. The binding enthalpies range from -21.9 kJ. mol-1 at 293 K for 4-methylumbelliferyl-beta-galactoside to -50.2 kJ. mol-1 at 292.9 K for thiodigalactoside. The observation of limited entropy-enthalpy compensation for binding of the sugars to the lectin indicates that reorganization of water molecules plays an important role in binding. As the slope of the compensation plot is greater than unity, the reactions are largely enthalpically driven. These studies show that the stronger binding of N-acetyl-lactosamine than lactose is due to a favourable interaction between the acetamido group of the reducing-end N-acetylglucosamine of the former and the corresponding loci in the agglutinin molecule. Preferential binding of methyl-beta-galactoside over methyl-alpha-galactoside also indicates the apolar nature of the interaction with the methyl group of the former sugar.

A- and B-subunit variant distribution in the holoprotein variants of protein toxin abrin: variants of abrins I and III have constant toxic A subunits and variant lectin B subunits.

The cytotoxic lectin abrin shows more than 30 variant forms (R. Hegde, T. K. Maiti, and S. K. Podder, 1991, Anal. Biochem. 194, 101-109). The lectin B subunit as cause for variance in abrins I and III was detected by a combination of one- and two-dimensional electrophoresis and Western blotting. Intriguingly, in abrin I but not in abrin III, association of a single A subunit with the variant B subunits shifts the holoprotein pI toward the alkaline side indicating that the subunit association involves neutralization of few negative charges. The B-subunit variants of abrins I and III overlap in their pI, and the A-subunit association gives the holoproteins a distinctness on isoelectric focusing gel. The results were also confirmed by analyzing the pH titration curves. These differences in the subunit association pattern between abrins I and III are in corroboration with the previously observed differences in the kinetics of protein synthesis inactivation and accessibility of the disulfide bridge to reducing agents in the presence or absence of putative receptor (R. Hegde, A. Karande, and S. K. Podder, 1993 Eur. J. Biochem. 215, 411-419). Further, the genetic origin of variance was confirmed by peptide mapping of the individual subunit variants. Considering a theoretical value of 0.1 to 0.2 pI/charge, a 15-17 charge difference could be predicted between the variants of two extreme pIs. The fact that the A subunits are not shared between the groups was taken to interpret that the protein synthesized as prepro form is processed posttranslationally and the processing takes place only after the disulfide bond formation between A and B subunits. The N-terminal 16 amino acids of A subunits of abrins I and III showed 26% dissimilarity. The A subunits of abrins I and III did not react with concanavalin A, indicating that the heterogeneity in the molecular weight is because of differential processing but not because of glycosylation.

Unusual structural stability and ligand induced alterations in oligomerization of a galectin.

L-14, a 14-kDa S-type lectin shows the jelly roll tertiary structural fold akin to legume lectins yet, unlike them, it does not dissociate on thermal unfolding. In the absence of ligand L-14 displays denaturation transitions corresponding to tetrameric and octameric entities. The presence of complementary ligand reduces the association of L-14, which is in stark contrast with legume lectins where no alterations in quaternary structures are brought about by saccharides. From the magnitude of the increase in denaturation temperature induced by disaccharides the binding constants calculated from differential scanning calorimetry are comparable with those extrapolated from titration calorimetry indicating that L-14 interacts with ligands essentially in the folded state.

Basis for dosing time-dependent changes in the ocular and systemic absorption of topically applied timolol.

The objective of the present study was to determine the basis for dosing time-dependent changes in the ocular and systemic absorption of topically applied timolol in pigmented rabbits. The gamma scintigraphic technique was used to monitor the changes in precorneal solution retention following instillation. Changes in timolol concentration in the plasma over 120 min and in various anterior segment eye tissues at 30 min following the topical instillation of 25 microliters of 0.65% timolol maleate solutions at various dosing times were monitored using reversed phase HPLC. Corneal and conjunctival permeability at various dosing times was evaluated in the modified Ussing chamber. The results indicated that precorneal solution drainage was slowest at noon. Suppressing tear production by anesthesia led to an increase in ocular timolol absorption at 6 a.m. but not at other dosing times, in spite of the lowest corneal permeability then. There was no statistically significant dosing time influence on systemic timolol absorption following nasal or conjunctival dosing. In conclusion, possible diurnal changes in precorneal solution clearance may be the main factor underlying the diurnal changes in ocular as well as systemic timolol absorption in rabbits. In addition, diurnal changes in corneal permeability may also contribute to diurnal changes in ocular timolol absorption.

Experiences with fine needle aspiration cytology as a diagnostic procedure for breast lesions in a Calcutta Hospital.

Nine hundred and forty cases of breast lesions were subjected to fine needle aspiration. Nine hundred twenty-four of them were females and sixteen were males. Smears from the 60 female cases were rejected as unsatisfactory. Of the remaining 864 female cases (aged 15 to 72 years) 704 (81.4%) were benign. Histopathological correlations were done in 448 cases with diagnostic accuracy of 97.3%. One hundred and sixty (18.5%) out of 864 females were malignant and cytohistological correlation was done in 142 cases with 98.6% corroboration. Ten of the 16 male cases were histologically examined with full corroboration. Overall accuracy considering both males and females was 98.6%. Though there was underdiagnosis involving 12 cases of infiltrating ductal carcinoma when the lesions were small and deeply situated there was no overdiagnosis in this study.

Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin.

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.

Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer.

The entry of the plant toxin ricin and its A- and B-subunits in model membranes in the presence as well as absence of monosialoganglioside (GM1) has been studied. Dioleoylphosphatidylcholine and 5-, 10-, and 12-doxyl- or 9,10-dibromophosphatidylcholines serve as quenchers of intrinsic tryptophan fluorescence of the proteins. The parallax method of Chattopadhyay and London [(1987) Biochemistry 26, 39-45] has been employed to measure the average membrane penetration depth of tryptophans of ricin and its B-chain and the actual depth of the sole Trp 211 in the A-chain. The results indicate that both of the chains as well as intact ricin penetrate the membrane deeply and the C-terminal end of the A-chain is well inside the bilayer, especially at pH 4.5. An extrinsic probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS) has been attached to Cys 259 of the A-chain, and the kinetics of penetration has been followed by monitoring the increase in AEDANS fluorescence at 480 nm. The insertion follows first-order kinetics, and the rate constant is higher at a lower pH. The energy transfer distance analysis between Trp 211 and AEDANS points out that the conformation of the A-chain changes as it inserts into the membrane. CD studies indicate that the helicity of the proteins increases after penetration, which implies that some of the unordered structure in the native protein is converted to the ordered form during this process. Hydrophobic forces seem to be responsible for stabilizing a particular protein conformation inside the membrane.

Cogan's syndrome: a rare systemic vasculitis.

A 4 year old girl presented with keratitis and ataxia. Over the next two months she developed profound hearing loss, arthritis, and polychondritis. A diagnosis of Cogan's syndrome was made. The literature on the condition is reviewed and the importance of early diagnosis to prevent hearing loss is highlighted.