Interdomain interactions in the chimeric protein toxin sCD4(178)-PE40: a differential scanning calorimetry (DSC) study.

The thermal denaturation of the chimeric protein toxin known as sCD4(178)-PE40 (sCD4-PE40) was studied using differential scanning calorimetry (DSC). sCD4-PE40 consists of HIV-binding domains of the T-cell membrane protein known as CD4 and the cytotoxic domains of Pseudomonas exotoxin A (PE40). sCD4-PE40 undergoes two DSC transitions. An endothermic transition associated with unfolding of the CD4 and PE40 components occurs at approximately 46 degrees C in buffered saline at pH 6.5. An exothermic transition associated with precipitation of unfolded protein occurs at higher temperatures. Both transitions are irreversible. DSC studies of solutions of pH 5.0 to 9.5 indicate that sCD4-PE40 shows maximal thermal stability at around pH 6.5. Variable pH experiments are also presented on solutions of sCD4(183) and PE40 revealing how these components denature as independent structural entities. sCD4(183) denaturation occurs at significantly higher temperatures than does the CD4 component of sCD4-PE40. PE40 denaturation occurs at the same temperatures as sCD4-PE40. These results suggest that the native CD4 and PE40 components are independent and non-interacting entities in the chimeric sCD4-PE40 molecule and that unfolding of the less-stable PE40 component induces unfolding of the CD4 component. These destabilizing interdomain interactions of sCD4-PE40 are in contrast to the stabilizing interactions which apparently exist in wild-type Pseudomonas exotoxin A between its PE40 domains and the cell binding domain of the native toxin (analogous to the CD4 component in sCD4-PE40). Reasons are discussed why the type of interdomain interactions observed for sCD4-PE40 might be the norm for chimeric proteins.

Evidence for transbilayer, tail-to-tail cholesterol dimers in dipalmitoylglycerophosphocholine liposomes.

The behavior of multilamellar liposomes of 2,3-dipalmitoyl-sn-glycero-1-phosphocholine (DPPC) was studied by differential scanning calorimetry (DSC) in the presence of < or = 5 mol % of the amphiphilic solutes methyl oleate, cholesterol, pregnenolone, and dehydroandrosterone. The DSC thermograms indicate that the solutes are miscible only with the liquid-disordered (Id) phase, and not with the solid-ordered (so) phase. The slopes of the Tm vs solute concentration curves confirm this conclusion: It appears that the so-1d phase transition of DPPC, which corresponds to the melting of the phospholipid chains, can be treated as a simple melting process and, thus, could be used as a cryoscopic system. In that case, its melting point depression constant, Kf, can be calculated a priori from the experimentally measured heat of fusion per gram of DPPC, lf, and the temperature of the phase transition of pure DPPC, T(o), by the equation Kf = RTo2/(1000lf) = 12.3 +/- 0.9 K g M-1 cm3. With methyl oleate as the solute, the Tm vs methyl oleate concentration plot is linear, and from the slope we calculate Kf = 12.9 +/- 0.8 K g M-1 cm3. Thus, methyl oleate appears to form an ideal cryoscopic system with dipalmitoyllecithin liposomes: It is fully miscible with the 1d phase but is apparently insoluble in the s(o) phase. Pregnenolone and dehydroandrosterone also form ideal cryoscopic systems with dipalmitoyllecithin liposomes: The Tm vs solute concentration plots are linear and yield the correct MWs for these solutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of U-75875, a peptidomimetic inhibitor of retroviral proteases, on simian immunodeficiency virus infection in rhesus monkeys.

U-75875 inhibits human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus (SIV) proteases and blocks Gag-Pol protein processing and viral maturation and replication in vitro. Rhesus monkeys were treated with vehicle alone or with formulated U-75875 at doses of 7 or 20 mg/kg of body weight per day for 26 days by continuous intravenous infusion beginning 6 h prior to intravenous inoculation with 10 monkey 50% infectious doses of SIV Delta B670, and the monkeys were monitored until death. The effects of treatment on the level of SIV p26 antigenemia, the infectious virus titer in serum, and the level of proviral DNA in blood mononuclear cells evaluated by PCR were assessed. SIV infection of the controls resulted in an initial viral antigenemia that began 5 to 10 days postinoculation (p.i.), reached peak values on days 10 to 14 p.i., and lasted for more than 15 days. Proviral DNA was detectable in peripheral blood mononuclear cells by 7 to 11 days p.i., reached the mean peak level by 11 days p.i., and remained at high levels through day 24 p.i. Infectious virus was detected in serum from all of the infected controls by 24 days p.i. Treatment with U-75875 for 26 days resulted in a dose-related delay in the day of the peak level of antigenemia (P = 0.034). The level of proviral DNA in peripheral blood mononuclear cells at 11 days p.i. was significantly decreased in a dose-related fashion in the treated monkeys ( P

In vitro evaluation of the plasma and blood compatibility of a parenteral formulation for ditekiren, a novel renin inhibitor pseudopeptide.

Ditekiren (U-71038; Boc-Pro-Phe-N-MeHis-Leu-psi[CHOHCH2]-Val-Ile-(aminomethyl)pyridine ) is a potent renin inhibitor peptide and was formulated for clinical intravenous administration in acidified dextrose. This formulation of ditekiren was evaluated in vitro with human and monkey plasma as to its potential for forming a precipitate either of drug or of plasma proteins. Analysis by centrifugation showed that no drug precipitation occurred in plasma from either species at concentrations 25 times higher than anticipated in clinical studies. Results obtained by turbidimetry indicated that formulated ditekiren did not cause aggregation of human platelets or flocculation of proteins at concentrations approaching the solubility limit of the drug in plasma. Ditekiren or vehicle also caused no detectable lysis of red cells at concentrations representing 10 times the maximum clinical level. Therefore, ditekiren solutions as formulated are judged completely compatible with blood and plasma upon clinical intravenous administration.

Precipitation of the renin inhibitor ditekiren upon i.v. infusion; in vitro studies and their relationship to in vivo precipitation in the cynomolgus monkey.

Ditekiren is a pseudo-octapeptide being developed as an inhibitor of human renin. Preclinical drug safety studies with this drug involved continuous i.v. infusions through indwelling catheters in the right internal jugular vein of the cynomolgus monkey for up to 30 days. The following physiocochemical properties of ditekiren make it susceptible to intravascular precipitation immediately following iv infusion: (1) the water solubility of ditekiren is high at acidic pH where the drug is formulated (pH 4) but low at physiologic pH, and (2) the water solubility of ditekiren decreases by roughly 50% from room temperature (25 degrees C) to physiologic temperature (37 degrees C). Studies of 28- and 30-day infusion durations revealed intravascular precipitation in monkeys using drug solutions and rates of infusion that were expected to be precipitation-free, based on the solubility of ditekiren and assumptions about blood flow in the monkey right internal jugular vein. Therefore, an in vitro apparatus was used to study the relationship among the drug concentration in the infusate, the rate of infusion, and the occurrence of precipitation in a fluid stream of phosphate-buffered bovine serum albumin solution (a facsimile of plasma). Maximum rates of infusion without precipitation were determined for a range of concentrations of drug in two separate formulations. Infusion conditions identified by the in vitro method as precipitation-free were then tried in a definitive 14-day monkey study. Of 24 monkeys infused with solutions of ditekiren, none showed evidence of intravascular precipitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutralization of saxitoxin by anti-saxitoxin rabbit serum.

This study examined the ability of anti-saxitoxin rabbit serum to neutralize saxitoxin, both in vitro and in vivo. In vitro, two rabbit antisera decreased [3H]-saxitoxin binding to specific sites in rat brain membranes. The more potent of these sera, antiserum A, when combined with saxitoxin in vitro, decreased saxitoxin's lethal potency based on mouse bioassay. Antiserum A also neutralized saxitoxin in vivo, as illustrated by the fact that mice injected i.p. with antiserum A (1:4) survived a s.c. injection 1 hr later of 16.7 micrograms saxitoxin/kg (1 LD99). Finally, antiserum A prevented death when injected i.v. immediately after s.c. injection of 16.7 micrograms saxitoxin/kg, however, antiserum injected by the i.p. and i.m. routes caused no significant increase in survival. This study indicates that antiserum can neutralize saxitoxin both in vitro and in vivo.

A competitive displacement assay to detect saxitoxin and tetrodotoxin.

An assay is described which detects saxitoxin (STX) and tetrodotoxin (TTX) by their competitive displacement of [3H]saxitoxin from its receptor in rat brain membranes. The assay has a sensitivity of 0.15 ng STX/ml and 0.8 ng TTX/ml for buffer samples. The assay was also applied to detection of these toxins in unextracted human plasma and found to have a sensitivity of 0.5 ng STX/ml and 0.6 ng TTX/ml. The competitive displacement assay appears to be the most sensitive procedure yet for detection of STX and TTX.

Characterization of the calorimetric C transition of the human erythrocyte membrane.

The largest calorimetric endotherm of the human erythrocyte membrane, termed the C transition (68 degrees C), was shown to derive from the denaturation of the membrane-spanning domain of the anion transport protein, band 3. This identification was based on the following evidence: (i) the fluorescence properties of the highly specific covalent ligand of band 3, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, abruptly changed during the C transition; (ii) the potent, noncovalent inhibitor of anion transport, dipyridamole, was ejected from erythrocyte membranes during the C transition; (iii) the intrinsic fluorescence of the membrane-spanning domain of band 3 decreased suddenly at the temperature of the C transition; and (iv) the purified 53000-dalton, membrane-spanning domain of band 3 yielded the C transition upon reconstitution into egg phosphatidylcholine/bovine brain phosphatidylserine vesicles. Although lipid melting was shown not to contribute to the C endotherm, the thermal stability of band 3 was nevertheless observed to be sensitive to its lipid/detergent environment. The stability of the membrane-spanning domain of band 3 was also found to be unaffected by the presence or absence of glycophorin, suggesting that the putative complex between this region of band 3 and glycophorin is either weak or nonexistent.

The effect of anesthetic charge on anesthetic-phospholipid interactions.

Cationic and uncharged forms of a tertiary amine local anesthetic are reported to have different properties and potencies as nerve blocking agents. However, the relative capacities of each form of the local anesthetic to perturb the properties of different model membrane systems is unknown. For this reason we have studied the effects of uncharged lidocaine (high pH) and its quaternary amine analogue (W49091) on the phase transition properties of DMPS, DPPE and DPPC liposomes using high-sensitivity differential scanning calorimetry. We report that neutral lidocaine interacts similarly with all three phospholipids. This interaction results in a decrease in the temperature of the gel leads to liquid crystalline phase transition (Tm), an increase in the enthalpy of the transition (delta H), and a slight decrease in the cooperativity of melting. Quaternary lidocaine (W49091), on the other hand, interacts significantly with only DMPS; the result being again a decrease in the temperature of DMPS melting, an increase in delta H, and a slight decrease in the cooperativity of the phase transition. These results are interpreted to indicate that uncharged lidocaine enters the membrane during the DPPE and DPPC phase transitions. In the case of DMPS, an influx of both charged forms of lidocaine must occur at Tm. These anesthetic fluxes at the lipid's phase transition are suggested to be responsible for the observed elevated enthalpies of the respective transitions. The observation that the cationic form of lidocaine does not significantly modify the behavior of DPPC and DPPE liposomes suggests that these lipids are not important components of the anesthetic's site in nerve membranes. However, the dramatic perturbation of the properties of DMPS by W49091 suggests that phosphatidylserine may comprise part of this inhibitory site.