Hepatic organic anion transport kinetics and bile flow during short-term total parenteral nutrition in the rabbit

Plasma disappearance of sulfobromophthalein (BSP) after an intravenous bolus (5 mg/kg) was determined in six lab chow-fed (LCF) rabbits and in six rabbits maintained on total parenteral nutrition (TPN) for 5 days. A common bile duct cannula enabled measurements of bile flow and biliary BSP excretion. Compartmental analysis of the biexponential plasma disappearance curve yielded three fractional transfer rates, plasma to liver (hepatic uptake), liver to plasma (reflux), and liver to bile (canalicular excretion). The transfer rates for hepatic uptake were 0.253 +/- 0.061/min for LCF and 0.147 +/- 0.040/min for TPN (P less than 0.01) and for the canalicular excretion of BSP were 0.038 +/- 0.019/min for LCF and 0.019 +/- 0.002/min for TPN (P less than 0.05). Model-computed rates for BSP excretion in bile over 60 min were lower with TPN (61%) than with LCF (80%); the measured excretory rates were 53% for TPN rabbits and 75% of injected dose for LCF animals. Basal biliary flow was reduced by 50% in the TPN group. With a two-compartmental model, assuming two pools and three transfer rates, we have demonstrated for the first time significant decreases in hepatic uptake and canalicular excretion of the organic anion BSP during TPN. A decrease in hepatic blood flow due to the enteral fast of TPN could have contributed in part to the decreased hepatic uptake. But, because the second exponent of the biexponential curve is independent of hepatic blood flow, the decrease in liver to bile transfer rate is a true approximation of a diminished canalicular excretory capacity during TPN. It is concluded that the movement of organic anions along the hepatic BSP/bilirubin transport system is impaired early during TPN.     

Intradermal administration of lipopolysaccharide in treatment of human cancer

 Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m² dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans. Received: 3 August 1995 / Accepted: 2 April 1996     

Polyamines, both common and uncommon, under heat stress in rice (Oryza sativa) callus

Enhanced production and accumulation of free and conjugated polyamines as well as increased activities of their biosynthetic enzymes in plants have been associated with heat stress. Perchloric acid-soluble free, as well as conjugated polyamines, and their metabolic enzymes were studied under 45°C heat stress in callus raised from heat-tolerant and -sensitive rice cultivars. The levels of free and conjugated polyamines, as well as arginine decarboxylase (EC 4.1.1.19) and polyamine oxidase (EC 1.4.34) activities were higher in tolerant than in sensitive callus under non-stressed conditions. Heat stress caused greater accumulation of free and conjugated polyamines in callus of the heat-tolerant cultivar N22 than in that of the heat-sensitive cultivar IR8. In particular, the uncommon polyamines norspermidine and norspermine were detected in cv. N22, which increased appreciably during stress, but they were not detected in callus of cv. IR8. Arginine decarboxylase and polyamine oxidase activities increased to a larger extent in N22 than in IR8 callus during stress, activities that were well correlated with the increased levels of common and uncommon polyamines. Increased levels of transglutaminase activity indicated the high titre of conjugated polyamines.     

Longitudinal study of a heteroplasmic 3460 Leber hereditary optic neuropathy family by multiplexed primer-extension analysis and nucleotide sequencing.

Nucleotide-sequencing and multiplexed primer-extension assays have been used to quantitate the mutant-allele frequency in 14 maternal relatives, spanning three generations, from a family that is heteroplasmic for the primary Leber hereditary optic neuropathy (LHON) mutation at nucleotide 3460 of the mitochondrial genome. There was excellent agreement between the values that were obtained with the two different methods. The longitudinal study shows that the mutant-allele frequency was constant within individual family members over a sampling period of 3.5 years. Second, although there was an overall increase in the mutant-allele frequency in successive generations, segregation in the direction of the mutant allele was not invariant, and there was one instance in which there was a significant decrease in the frequency from parent to offspring. From these two sets of results, and from previous studies of heteroplasmic LHON families, we conclude that there is no evidence for a marked selective pressure that determines the replication, segregation, or transmission of primary LHON mutations to white blood cells and platelets. Instead, the mtDNA molecules are most likely to replicate and segregate under conditions of random drift at the cellular level. Finally, the pattern of transmission in this maternal lineage is compatible with a developmental bottleneck model in which the number of mitochondrial units of segregation in the female germ line is relatively small in relation to the number of mtDNA molecules within a cell. However, this is not an invariant pattern for humans, and simple models of mitochondrial gene transmission are inappropriate at the present time.     

Optical Fiber-Based In Situ Spectroscopy of Pigmented Single Colonies

We have adapted a commercially available fiber-optic spectroradiometer with diode array detection to record reflection and absorption spectra from single, 1-mm-diameter bacterial colonies. A careful assessment of the performance of the spectroradiometer for this application is reported. In a model study employing colonies from various phototrophic bacteria, we show that the reflectance spectra are reliable within the range of 450 to 820 nm, whereas the transmission spectra yield accurate peak intensities and absorption maxima from 400 to 900 nm. For screening of populations of about 10(sup4) colonies, fiber-optic transmission spectroscopy provides an attractive and inexpensive alternative to present techniques based on charge-coupled device imaging technology.     

Immunoblotting identifies and antigen recognized by anti gp63 in the immune complexes of Indian kala-azar patient sera

In SDS-PAGE the immune complexes (IC) of kala-azar patient sera showed intense bands at 55 kDa and 20 kDa corresponding to heavy and light chains of immunoglobulins. In immunoblot experiment, kala-azar and normal IC after treatment with patient sera showed multiple bands of which the band at 55 kDa was most prominent in kala-azar IC. It is known that in kala-azar sera antihuman IgG is present, so the heavy band at 55 kDa region may be due to higher amount of IgG and/or other antigen(s) present at that region. Immunoblot experiments of kala-azar IC with anti gp63 also developed a major band at 55 kDa. It suggests that the antigen (55 kDa) and gp63 have common antigenic epitope (s). Normal IC did not react with anti gp63 indicating absence of this antigen in normal IC. Antigenic similarity between the IC antigen (55 kDa) and gp63 indicated that the former antigen may have been processed from gp63. In summary, identification of a parasite antigen (55 kDa) in IC of kala-azar patients sera may be useful in developing a serodiagnostic assay for visceral leishmaniasis. (Mol Cell Biochem130: 11–17, 1994)Abbreviations IC Immune Complexes - PEG Polyethylene Glycol (Mol wt 8000) - PBS Phosphate Buffer Saline - VL Visceral Leishmaniasis - AVL American Visceral Leishmaniasis - IgG Immunoglobulin G - TBS Tris Buffer Saline - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - gp63 A leishmanial surface glycoprotein of molecular mass 63,000 - TEMED N,N,N,N-Tetramethylethylenediamine     

Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism.

A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis. The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S. Chattopadhyay, A. Mukherjee, and S. Ghosh, J. Bacteriol. 175:3240-3243, 1993) has now been completely sequenced. The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively. The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus. A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene. Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source. The reason for this is not clear. A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A. brasilense.     

Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules.

We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small clusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, and without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces.     

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.