The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.

Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three protoxins, CryIA(b), CryIC, and CryID, and the CryIC protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) protoxin in the former. The interactions of structurally related protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.     

Response to and expression of amphiregulin by ovarian carcinoma and normal ovarian surface epithelial cells: nuclear localization of endogenous amphiregulin

Amphiregulin (AR) is a polypeptide growth regulator which has sequence homology to the epidermal growth factor-related family of ligands and contains putative nuclear targeting sequences. Human ovarian carcinoma cell lines and their normal counterparts, ovarian surface epithelial cells (OSEs), were assessed for their ability to respond to and express AR. Addition of exogenous AR (8-200 pM) inhibited the growth of 2 of 3 OSE specimens and 3 of the 6 carcinoma cell lines indicating that AR has the potential to inhibit the growth of normal cells, in addition to carcinoma cells. In contrast, concentrations of AR ranging from 1-5 nM stimulated the growth of all 3 of the OSEs and 4 of the 6 carcinoma cell lines. Immunocytochemical staining of the cells using antipeptide antibodies directed against residues 8-26 of AR indicated that all cells expressed AR and that the staining was localized to the nucleus. The nuclear staining of AR was concentrated in the nucleolus of the carcinoma cells, whereas the staining was diffuse in the nucleus of the OSEs. These results suggest that AR may play a growth regulatory role in the nucleus of cells and this role may be different in normal and malignant epithelial cells.     

Chemical modifications and amino acid substitutions in recombinant hirudin that increase hirudin-thrombin affinity

Recombinant hirudin (r-hirudin), unlike the naturally occurring leech protein, lacks a sulfate ester on Tyr-63 which reduces its binding affinity to thrombin by 3-10-fold. We demonstrate that nitration or iodination of Tyr-63 restores hirudin-thrombin affinity to levels similar to or exceeding that of the natural inhibitor. In contrast, nitration of Tyr-3 reduces the affinity of hirudin for thrombin. These chemical modifications results in multiple reaction products that are readily separated by reverse-phase HPLC. The mechanism of the observed changes in thrombin affinity may involve a reduction in the pK of the hydroxyl group of tyrosine due to substitution of the electrophilic iodo or nitro group on the phenyl ring, resulting in an increased negative charge at neutral pH. For Tyr-63, this effect mimics the sulfatotyrosine of natural hirudin, leading to an increased thrombin affinity at the anion-binding exosite. For Tyr-3, the increased polarity may destabilize its interaction within the apolar-binding site of thrombin. Substitution of the highly conserved Tyr-3 residue with Phe or Trp not only enables specific and quantitative chemical modification at Tyr-63 but also independently increases hirudin-thrombin affinity. Kinetic analysis of thrombin inhibition showed that enhanced binding by r-hirudin(nitro-Tyr-63) is due to an increase in the association rate between hirudin and thrombin whereas the reduced binding of r-hirudin(nitro-Tyr-3) results from a large increase in the dissociation rate. These observations indicate that specific segments within both the amino- and carboxy-terminal regions of hirudin interact with thrombin.     

Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N2-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9).(G10-T11-A12-C-13-F14-C15 -A16-T17-G-18) X.F 9-mer duplex. Two-dimensional NMR experiments establish that the X.F 9-mer helix is right-handed with Watson-Crick A.T and G.C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4.C15 and G6.C13 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4.C15 and G6.C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N2-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.     

Identification of signaling states of a sensory receptor by modulation of lifetimes of stimulus-induced conformations: the case of sensory rhodopsin II

Lifetimes of stimulus-induced conformations of the phototaxis receptor sensory rhodopsin II (SR-II) from Halobacterium halobium are modulated with seven receptor analogues. By monitoring the receptor dynamics in vitro and physiological responses of the cell in vivo, we observe receptor signaling efficiency increases with decreasing cycling frequency (turnover number) of the receptor. The results demonstrate that modulating lifetimes of protein conformations at the SR-II photoactivation site with chromophore analogues alters the lifetime of the active conformation at the signaling site. We further explore the relationship between photocycle intermediates and the signaling efficiency by analyzing the time-averaged concentrations of the two long-lived spectral intermediates of the SR-II photocycle: S-II350 and S-II530. The results are consistent with the signaling site being activated during formation of S-II350, but not reset by the transition of S-II350 into S-II530; rather deactivation appears to require subsequent decay of S-II530. The results indicate the structural changes at the photoactivation site in the S-II350----S-II530 transition do not reset the signaling site. The procedure used here, applicable in principle to any photoactivated or ligand-activated receptor, provides an initial approach to identify structural alterations key to the receptor activation process.     

Hypercholesterolaemia: simvastatin and pravastatin alter cholesterol metabolism by different mechanisms

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin, reduced low-density-lipoprotein (LDL) cholesterol in hypercholesterolaemic patients by 40% (P less than 0.001). The reduction in LDL cholesterol was accompanied by a significant decrease in the esterified/free cholesterol ratio of the patients' LDL from 2.51 +/- 0.13 to 2.06 +/- 0.14 (P less than 0.01). This change led to a significant increase (P less than 0.05) in the capacity of the LDL to suppress [14C]acetate incorporation into cholesterol in mononuclear leucocytes. Furthermore, [14C]acetate incorporation into the patients mononuclear leucocytes was significantly lower (P less than 0.02) following drug treatment (117 +/- 22 vs. 162 +/- 29 nmol/mg cell protein). Comparison of simvastatin with another HMG-CoA reductase inhibitor pravastatin, showed similar reduction in LDL cholesterol. Pravastatin treatment however, did not result in a reduction in the LDL esterified/free cholesterol ratio or in the changes in cellular cholesterol synthesis and its regulation by LDL which accompanied simvastatin treatment. The activity of the enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT) in patients' mononuclear cells remained unchanged after treatment with either drug. Results of the study show that while the drugs are equally effective in lowering LDL cholesterol, simvastatin has additional compositional effects on LDL which increase its capacity to regulate mononuclear leucocyte cholesterologenesis.     

Cholesterol transport between cells and high-density lipoproteins

Various types of studies in humans and animals suggest strongly that HDL is anti-atherogenic. The anti-atherogenic potential of HDL is thought to be due to its participation in reverse cholesterol transport, the process by which cholesterol is removed from non-hepatic cells and transported to the liver for elimination from the body. Extensive studies in cell culture systems have demonstrated that HDL is an important mediator of sterol transport between cells and the plasma compartment. The topic of this review is the mechanisms that account for sterol movement between HDL and cells. The most prominent and easily measured aspect of sterol movement between HDL and cells is the rapid bidirectional transfer of cholesterol between the lipoprotein and the plasma membrane. This movement occurs by unmediated diffusion, and in most situations its rate in each direction is limited by the rate of desorption of sterol molecules from the donor surface into the adjacent water phase. The net transfer of sterol mass out of cells occurs when there is either a relative enrichment of sterol within the plasma membrane or a depletion of sterol in HDL. Recent studies suggest that certain minor subfractions of HDL (with pre-beta mobility on agarose gel electrophoresis and containing apoprotein A-I but no apo A-II) are unusually efficient at promoting efflux of cell sterol. To what extent efflux to these HDL fractions is balanced by influx from the lipoprotein has not yet been established clearly. The prevention and reversal of atherosclerosis require the mobilization of cholesterol from internal (non-plasma membrane) cellular locations. To some extent, this may involve the retroendocytosis of HDL. However, most mobilization probably involves the transport of internal sterol to the plasma membrane, followed by desorption to extracellular HDL. Several laboratories are investigating the transport of sterol from intracellular locations to the plasma membrane. Studies on biosynthetic sterol (probably originating mostly in the smooth endoplasmic reticulum) suggest that there is rapid transport to the plasma membrane in lipid-rich vesicles. Important features of this transport are that it bypasses the Golgi apparatus and may be positively regulated by the specific binding of HDL to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Season but not age affects Sertoli cell number in adult stallions.

To evaluate the effect of age and season on Sertoli cell number per paired testes, ratio of germ cells per Sertoli cell, and daily sperm production, testes were obtained from 184 adult (4-20 yr) stallions at slaughter throughout one year. Numbers of Sertoli cells or germ cells were derived from nuclear volume density, volume of individual nuclei, and parenchymal volume. Germ cell to Sertoli cell ratios were calculated from cell numbers. Regression analysis was used to detect age-related differences in the breeding season (May-Jul) or throughout the year. A two-way analysis of variance was used to evaluate time periods (Nov-Jan, Feb-Apr, May-Jul, and Aug-Oct) and age groups (4-5.5, 6-12.5, or 13-20 yr). Paired parenchymal weight and daily sperm production per horse increased significantly with age. Neither regression nor analysis of variance revealed an effect of age on Sertoli cell number. While season contributed (p less than 0.01) to variation in Sertoli cell number per horse, there was no (p greater than 0.05) age x season interaction or age effect on Sertoli cell number. In testes obtained from adult stallions, age had no effect on the number of Sertoli cells per horse, the ratio of maturation-phase spermatids to Sertoli cells, or the ratio of all stage VIII germ cells to Sertoli cells. Given no age effect within a given season on Sertoli cell number per horse, the number of Sertoli cells in the recrudesced testis of the breeding season probably is not significantly different for a given stallion between 4 and 20 yr of age.     

Relaxation dynamics of the gel to liquid-crystalline transition of phosphatidylcholine bilayers. Effects of chainlength and vesicle size.

The relaxation kinetics of the gel to liquid-crystalline transition of five phosphatidylcholine (DC14PC to DC18PC) bilayer dispersions have been investigated using volume perturbation calorimetry, a steady-state technique which subjects a sample to sinusoidal changes in volume. Temperature and pressure responses to the volume perturbation are measured to monitor the relaxation to a new equilibrium position. The amplitude demodulation and phase shift of these observables are analyzed with respect to the perturbation frequency to yield relaxation times and amplitudes. In the limit of low perturbation frequency, the temperature and pressure responses are proportional to the equilibrium excess heat capacity and bulk modulus, respectively. At all temperatures, the thermal response data are consistent with a single primary relaxation process of the lipid. The less accurate bulk modulus data exhibit two relaxation times, but it is not clear whether they reflect lipid processes or are characteristic of the instrument. The observed thermal relaxation behavior of all multilamellar vesicles are quantitatively similar. The relaxation times vary from approximately 50 ms to 4 s, with a pronounced maximum at a temperature just greater than Tm, the temperature of the excess heat capacity maximum. Large unilamellar vesicles also exhibit a single relaxation process, but without a pronounced maximum in the relaxation time. Their relaxation time is approximately 80 ms over most of the transition range.