Binding of bilirubin with albumin-coupled liposomes: implications in the treatment of jaundice.

In the present study, we demonstrated the suitability of liposomes as a method of removing plasma bilirubin in hyperbilirubinemic rats. The liposomes have innate tendency to bind with bilirubin through hydrophobic interaction. Among different types of liposomes, the positively charged liposomes were found to have maximum affinity to free bilirubin. However, the entrapment or coupling of serum albumin on the surface of egg phosphatidylcholine liposomes can render a several-fold increase in their bilirubin binding capacity. The proteoliposomes were able to preferentially bind with bilirubin even in the presence of erythrocytes. Interestingly, these liposomes were found to displace bilirubin bound on the surface of erythrocytes as well. The results of the present study further demonstrate that albumin-bearing liposomes were equally effective in removing plasma bilirubin in experimental jaundiced animals. These observations indicate that liposome-mediated selective homing of excess plasma bilirubin to the liver cells (cf. hepatocytes) may help in the development of safer strategy for the treatment of hyperbilirubinemic conditions in the model animals.

Enhanced bilirubin binding to different mammalian erythrocytes in the presence of magnesium ions.

Effect of magnesium ions on the binding of bilirubin to erythrocytes of different mammalian species, namely, human, buffalo, goat and sheep was studied. Increase in the concentration of magnesium ions led to a gradual increase in the erythrocyte-bound bilirubin in both human and buffalo erythrocytes whereas in sheep and goat erythrocytes, the pronounced increase was found beyond 2.0 and 2.7 mM MgCl(2) concentrations respectively. Percentage increase in erythrocyte-bound bilirubin was found highest in human erythrocytes followed by buffalo and sheep erythrocytes and minimum in goat erythrocytes. These differences in the binding of bilirubin to different mammalian erythrocytes can be attributed to the differential shielding effect of metal ions which involves the masking of negatively charged phosphate of phospholipids found on the erythrocyte surface.

Gap junctional communication and regulation of the glycogenic response to insulin by cell density and glucocorticoids in cultured fetal rat hepatocytes.

Cell culture studies have revealed that metabolic functions of the adult hepatocyte are related to cell density. Development of the glycogenic response to insulin under glucocorticoid control was investigated in 15- and 18-day-old fetal rat hepatocytes plated at different cell densities. After culturing for 48 hours with glucocorticoids, the stimulatory effect of insulin on [14C]glucose incorporation into glycogen after 3 hours progressed from weak response (less than 1.4-fold) in sparse cultures to a maximal response in dense ones (3.0- to 4.5-fold), depending on the fetal stage. The response was always no more than 2.0-fold in the absence of glucocorticoids, even with dense cultures. Such a dual regulation pattern was not found for the glycogenolytic effect of glucagon similarly expressed regardless of culture conditions. When cells were clustered in limited circular regions of the dish, the insulin response was higher than for sparse cultures for a similar number of cells per culture. Using the scrape-loading technique with Lucifer Yellow CH, a positive dye transfer was obtained in clustered cultures providing that they were grown in the presence of glucocorticoids; insulin as well as glucagon stimulated twofold intercellular communication. Connexin32 (Cx32) and connexin26 (Cx26) protein levels were assayed by Western immunoblotting and developed according to age and exposure to glucocorticoids. Thus, glucocorticoids through development of gap junctions enabled establishment of intercellular communication that could be stimulated by insulin and glucagon in cultured fetal hepatocytes. Gap junction functioning and the biologic effect of insulin correlated closely.

Erythrocytes from healthy smokers bind more bilirubin than the erythrocytes from healthy non-smokers.

Cigarette smoking is an adverse prognostic factor for health. Its damaging effects on many enzymatic and cellular activities are well known. The present study was carried out to evaluate whether there is a difference in the binding of bilirubin to the erythrocytes from healthy smokers and non-smokers. The results suggest that the binding of bilirubin to the erythrocytes from healthy smokers as well as in vitro smoked erythrocytes is significantly higher than that of healthy non-smokers.

Interaction of bromocresol green with different serum albumins studied by fluorescence quenching.

The binding of bromocresol green to bovine serum albumin at micromolar concentrations leads to quenching of protein fluorescence. This property has been used here to study interaction of bromocresol green with bovine serum albumin as a function of pH and ionic strength. The transformation of fluorescence quench data obtained with bromocresol green into Scatchard plots yielded an association constant of 3.06 x 10(7) 1 M-1 and a binding capacity of about 1.0. The affinity of bromocresol green for bovine serum albumin remains virtually unchanged between pH 4.0 and 8.0 but decreases by about 7 fold with increase in ionic strength from 0.01 to 1.0. Six other serum albumins obtained from cat, dog, human, pig and sheep have also been studied for bromocresol green binding. Although all the albumins studied bind bromocresol green, they show considerable differences in their affinities towards the dye. It appears that despite a great degree of overall similarity in their structure and conformation, serum albumins from different species differ in their ligand binding properties.

Differential expression of migration inhibitory and migration stimulatory factors in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin. 1. Migration stimulatory factor(s) from T and B cells of immune spleen.

Expression of the lymphokine migration inhibition factor in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) response of their lymph node cells to phytohemagglutinin was found to be modulated by concomitant expression of migration stimulation factor(s) [MStF(s)]. The expression of both lymphokines was dependent on genetic character and the immunizing dose of antigen. In mice immunized 5 days earlier with 50 micrograms ovalbumin in Freund's complete adjuvant (Ova in FCA immune), migration inhibition factor, assessed with a sensitive photoelectric method, was well expressed by male spleen or lymph node 24-hour culture supernatants of Lo/PHA but Hi/PHA, especially female, expressed marked MStF(s) instead. Immunization with 500 micrograms Ova in FCA markedly enhanced expression of MStF(s) in Lo/PHA but inhibited it in Hi/PHA. MStF(s) of Ova in FCA immune spleens of the two lines were found to derive from both T and B cells, B cell activity being greater. Lo/PHA were by far better expressors of both T- and B-cell-derived MStF(s) as compared to Hi/PHA (p less than 0.01). Spleen cells of mice immunized with FCA alone also expressed MStF(s) but to lesser extent than Ova in FCA immune spleens, expression by Lo/PHA B cells being significantly higher than in Hi/PHA (p less than 0.05). The MStF(s) of Ova in FCA immune spleens was found to be non-immunoglobulin in nature.

Multiple ovulation and embryo transfer in Indian buffalo (Bubalus buoalis ).

This study was undertaken to identify the most effective dosage level of folltrooin for superovulation in buffalo. In addition, the effect of the day of estrus on superovulation and the use of exogenous GnRH were also studied. Eighty-three buffalo were treated with prostaglandin. A functional Corous luteum (CL) was palpated in only 73 buffalo 1 d before the superovulation treatment was initiated. One of eight treatments was used on the buffalo: Protocol I(n 8) 9 mg folltropin (PPFE) on Days 9 to 12 of the cycle; Protocol II(n 10) 18 mg PPFE on Days 9 to 12 of the cycle; Protocol III(n 9) 18 mg PPFE on Days 13 to 15 of the cycle; Protocol IV(n 9) 21.6 mg PPFE on Days 9 to 12 of the cycle; Protocol V(n 9) 21.6 mg PPFE with GnRH on Days 9 to 12 of the cycle; Protocol-VI(n 10) 25.2 mg PPFE on Days 9 to 12 of the cycle; Protocol VII(n 9) 28.8 mg PPFE on Days 9 to 12 of the cycle; Protocol VIII (n 9) 36 mg PPFE on Days 9 to 12 of the cycle. The highest ovulation rate was observed in Protocol VI (x 5.3+/-0.79) which is significantly higher (P < 0.01) than other Protocols. Maxium embryos (x 3.7) were recovered using Protocol III. Whereas, highest number of transferable embryos (x 2.2) were recovered from Protocol V. Use of GnRH and superovulation treatment on Days 13 to 15 has no advantageous effect on ovulation rate. In all, 41 embryos were transferred to 35 recioients: nine buffalo became pregnant; 59 embryos were frozen; 12 were thawed; nine good frozen-thawed embryos were transferred to eight recidients, three of which were diagnosed pregnant.

Immunoprotection by beta-1,3 glucan antigen combination in Plasmodium berghei infection in mice.

In an attempt to protect mice against experimental infection with P. berghei, mice were immunized against soluble extract of P. berghei in combination with beta-1,3 glucan or FCA and also independently. Mice immunized against P. berghei antigen-glucan developed well defined cell mediated and humoral immune responses, while mice injected with antigen FCA or antigen alone developed only an antibody response. Antigen-glucan immunization afforded a high degree of immune protection to the host against the challenge with live parasites.

Immunization of guinea pigs against Entamoeba histolytica using glucan as an adjuvant.

Beta 1-3 polyglucose or glucan, an extract of cell wall of Saccharomyces cerevisiae, has been successfully employed in this laboratory as an effective immunopotentiator in experimental studies on amoebiasis. An antigen extract from Entamoeba histolytica was combined with beta, 1-3 glucan for immunizing guinea pigs. In order to study the effectiveness of such vaccine preparations, several batches of guinea pigs were immunized with amoeba antigen alone, and in combination with various immunoadjuvants. Antigen inoculations were carried out via intraperitoneal route. Protective immune responses were obtained against amoeba antigen by using glucan as an adjuvant partner. The study showed that glucan can be safely used as an effective immune enhancer.