Ratna Bose

Infrared spectroscopic study of diabetic platelets

In the present study, we use infrared difference spectroscopy to correlate spectroscopic changes with biochemical and physical profiles in diabetic platelets. The IR spectroscopic analysis confirms the increased glycosylation of diabetic platelets compared to that of control platelets. The protein composition and/or structure in diabetic platelets also changed. Furthermore, the overall content of membrane lipids in diabetic platelets increased compared to that in control platelets as did the lipid peroxidation while their membrane fluidity decreased. To investigate the chronic hyperglycemia effect on platelet membranes, we also incubated the platelets with a high concentration of glucose. Key changes observed in the glucose-incubated platelets were increased glycosylation of platelet membranes, increased lipid content and peroxidation, however, the membrane fluidity was not altered. Our results provide direct information on the overall changes in diabetic platelets, and suggest that IR spectroscopy may have a place in the characterization of platelets in diabetes.

Purified human early pregnancy factor from preimplantation embryo possesses immunosuppressive properties

This study was undertaken to determine whether early pregnancy factor secreted by preimplantation embryos has immunosuppressive properties. Human early pregnancy factor was purified from embryo growth media of in vitro fertilized ova with ion-exchange and gel filtration chromatography. During each step of purification the fractions were tested for (1) early pregnancy factor activity with the rosette inhibition assay, (2) immunosuppressive properties with a concanavalin A-stimulated lymphocyte proliferation assay, and (3) purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that (1) human early pregnancy factor has a basic molecular weight of 14 kd, (2) early pregnancy factor has immunosuppressive activity, (3) polymers of early pregnancy factor also appear to be present in the embryo growth media, and (4) immunosuppressive factors other than early pregnancy factor are also secreted by preimplantation human embryos. Early pregnancy factor and other factor(s) produced by the preimplantation embryo may play a role in suppressing maternal cellular immune responses, thereby preventing maternal rejection of the embryo.

Calcium channels in smooth muscle

The molecular and biophysical characterization of Ca2+ channels in smooth muscle has lagged behind that in cardiac and skeletal muscles. This is caused by the difficulties in isolating viable cells and applying electrophysiological techniques to smooth muscle cells. The heterogeneity of smooth muscle cells from different organs also adds to the complexity. In spite of these problems there has been an increasing number of reports on Ca2+ channels in smooth muscle. This review provides an overview of this area. As in skeletal muscles, L (long-acting) and T (transient) voltage-dependent Ca2+ channels are found in smooth muscle; the former is better characterized. L channel is regulated by voltage, Ca2+, cyclic nucleotides, and protein kinase C. Ca2+ also influxes through receptor-operated channels that are voltage insensitive. The receptor-operated and T channels are insensitive to dihydropyridine. Clinically, Ca2+ blockers are extensively used for diseases of the cardiovascular system, with more limited use in the gastrointestinal tract. Further understanding of the properties of Ca2+ channels could lead to development of selective Ca2+ channel blockers for the gastrointestinal tract.

The antiproliferative properties of tamoxifen and phenothiazines may be mediated by a unique histamine receptor (?H3) distinct from the calmodulin-binding site

SummaryN,N-diethyl-2-[(4-phenylmethyl)-phenoxyl]-ethanamine HCl (DPPE), a novel histamine antagonist (?H3), which selectively binds with high affinity to the antiestrogen-binding site (AEBS/?H3), inhibits the activity of calmodulin-dependent myosin light chain kinase (MLCK) only at concentrations >1 mM, as opposed to tamoxifen (TAM), which has an IC50=4 μM in the same assay. This suggests that the antiestrogen-binding site is distinct from the site on calmodulin which binds TAM and phenothiazines. However, at an in vitro concentration of 1×10-6M, the antiproliferative effects of DPPE and several phenothiazines, which also compete for binding to AEBS/?H3, are about equal; this suggests that affinity for AEBS/?H3 rather than that for the calmodulin-binding site may correlate with clinically relevant antigrowth effects of these compounds.

Molecular and functional characterization of the human platelet Na+/Ca2+ exchangers

BACKGROUND AND PURPOSE The Na+/Ca2+ exchanger is a bi‐directional transporter that plays an important role in maintaining the concentration of cytosolic Ca2+ ([Ca2+]i) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na+/Ca2+ exchangers: K+‐independent Na+/Ca2+ exchanger (NCX) and K+‐dependent Na+/Ca2+ exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs.

Taurine indirectly increases [Ca]i by inducing Ca2+ influx through the Na+-Ca2+ exchanger

Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na+-Ca2+ exchange. Therefore, we investigated the effect of β-alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2′,4′-dimethylbenzamil), a Na+-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3 Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 μM of the blocker of the taurine-Na+ cotransporter, β-alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of β-alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na+-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of β-alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na+-Ca2+ exchanger.

Human embryo associated immunosuppressor factor(s) from pre-and post-implantation stages share some similarities

The present study demonstrates that embryo associated immunosuppressive factor(s) (EASF) secreted by the human embryo at pre- and post-implantation stages share some similarities. Human EASF was partially purified from embryo growth media of in vitro fertilized ova and from first trimester pregnancy sera. Non-pregnancy sera were fractionated in parallel. During each step of purification the fractions were tested for immunosuppressive properties using concanavalin A-stimulated lymphocyte proliferation assay. Analysis of EASF positive fractions on SDS-PAGE identified 14 kDa and 24 kDa molecules in embryo growth media and pregnancy sera. No such molecules were found in control sera, suggesting that these factors are embryo associated. The relationship of pre- and post-implantation EASF was also analyzed by EASF binding assay using murine anti-EASF antibody, which was raised against EASF isolated from embryo growth media. Results show that murine antibody bound to EASF purified from pregnancy sera, but not to identical fractions from control sera, indicating that these post-implantation EASF possess some similarity with pre-implantation EASF. Results also indicate that a species of suppressor factors present in embryo growth media and pregnancy sera were unique for their origin. Presence of these three EASFs at various stages of gestation may play a role in suppressing maternal cellular immune responses thereby preventing maternal rejection of the embryo.

The partially purified pre-implantation suppressor factor may be one of several factors to play a role in successful pregnancy

Embryo-associated immunosuppressor factor (EASF) is detected by its suppressive properties in the concanavalin A (ConA)-induced lymphocyte proliferation assay. EASF was partially purified from human embryo growth media of in vitro fertilized ova (pre-implantation EASF) as three fractions. The aim of the present study was to determine whether the EASF isolated from human embryo growth media is similar to the EASF secreted by the pre-embryo, which has been shown to be associated with successful pregnancy. EASF activity was measured in the purified pre-implantation EASF fractions and in a total of 24 individual embryo growth media obtained from 10 patients undergoing in vitro fertilization and embryo transfer, where 6 patients achieved successful pregnancy and 4 did not. The results show that: (i) all three EASF fractions and the individual embryo growth media from patients who became pregnant were suppressive when added to the early phase of ConA-supplemented cultures; this was not seen with the embryo growth media from patients who failed to become pregnant, suggesting that the purified pre-implantation EASF may be one of several factors responsible for successful pregnancy; and (ii) some embryo growth media, irrespective of the pregnancy outcome of the patient, showed an irreversible immunosuppressive effect on ConA-induced lymphocyte proliferation, whereas none of the purified EASF fractions did; this could be due to the loss of activity during purification.

Immunosuppressive activity in human embryo growth media is associated with successful pregnancy: Effect of gonadotropin releasing hormone agonist (GnRHa) treatment of patients undergoingin vitro fertilization and embryo transfer (IVF-ET)

The aim of the present study was to determine whether the secretion of embryo-associated immunosuppressor factor (EASF) by preimplantation embryo correlates with pregnancy outcome and whether this relationship is influenced by pretreatment of gonadotropin releasing hormone agonist (GnRHa) in patients undergoingin vitro fertilization and embryo transfer (IVF-ET). EASF activity was measured using concanavalin A-induced human lymphocyte proliferation assay in 256 embryo growth media obtained from 61 patients undergoing IVF-ET. EASF activity was then correlated with GnRHa treatment and pregnancy outcome in these IVF patients. Results indicate that (i) the presence of immunosuppressive activity in human embryo growth media is associated with success of pregnancy in GnRHa nontreated patients and (ii) serum factors present in GnRHa-treated patients may affect the EASF secretion by preembryosin vitro.

Mechanisms of frequency-induced potentiation of contractions in isolated rat atria

SummaryMechanisms underlying the potentiation of contractions after periods of high frequency stimulation (post-stimulation potentiation; PSP) and periods of rest (rest potentiation; RP) were investigated in isolated rat atria. Transmembrane action potentials were not changed during PSP and RP and were superimposable upon the pre-test action potentials. However, the45Ca content of atrial strips was significantly increased during PSP, which indicates a net gain in intracellular Ca.45Ca content was not changed during RP. PSP and RP were increased in magnitude in atria pre-treated with gallopamil (2.5 μmol/l). This effect was due to a greater depression by gallopamil of the pre-test contractions than the potentiated post-test contractions. In contrast, PSP was abolished in atria exposed to 7.5 mmol/l [Ca]o and a transient depression of the post-test contractions was seen. RP was also abolished by high Ca medium, but contractions were not depressed after periods of rest. RP, but not PSP, was unmasked when gallopamil was added to high Ca medium to decrease the size of the basal contractions. Conversely, ryanodine (100 nmol/l) abolished RP but did not affect PSP. With ryanodine present, PSP was greatly increased when the extracellular Ca concentration was increased to 5 mmol/l, whereas RP remained abolished. These results suggest that PSP may reflect an increased transsarcolemmal influx of extracellular Ca, possibly mediated through Na-Ca exchange. In contrast, the mechanism suggested for RP is a transient increase in contractile Ca resulting from an intracellular redistribution of Ca to release sites in the sarcoplasmic reticulum.