Partition Assay Research Articles

Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans

Cell surface hydrophobicity (CSH) of blastoconidia and blastoconidia bearing germ tubes of Candida albicans ATCC 26555 was monitored by assessing attachment of polystyrene microspheres to the cell surface, and we found that mature hyphae were significantly hydrophobic. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) or proteases abolished or significantly reduced attachment of latex beads to hyphae. This effect paralleled an obvious reduction in CSH of the entire cell population, as measured by an aqueous-hydrocarbon biphasic partitioning assay. Analysis of the cell wall material released by Zymolyase and adsorbed on polystyrene microspheres indicated that germ tube-specific cell wall proteins and mannoproteins with apparent molecular masses of 20 to 67 kDa may be responsible for the hydrophobicity of hyphae. Zymolyase released from blastoconidia cell walls a different set of proteins and mannoproteins that were able to adsorb to polystyrene microbeads. Such molecular species might in turn be responsible for the CSH exhibited by blastoconidium populations as determined by the biphasic partitioning assay, although these probably hydrophobic components can be masked on the surface of blastoconidia, as the latter had no or very few latex microspheres attached to their surfaces. Treatment of cells of both C. albicans morphologies with 2-mercaptoethanol released qualitatively distinct species of polystyrene-adsorbed proteins and mannoproteins from yeast and mycelial cells. These observations suggested that hydrophobic proteins and mannoproteins that could be associated with CSH are bound to the cell wall structure through diverse types of linkages.

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity in Bacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity inBacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.

Use of puromycin N-acetyltransferase (PAC) as a new reporter gene in transgenic animals

Puromycin N-acetyltransferase (PAC) is a bacterial enzyme produced by Streptomyces alboniger, which has been purified and well characterized (1). PAC inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. Its gene has been cloned (2), and its use as a reporter gene in transient expression of transfected cells has been described (3, 4). We have made different lines of transgenic mice with pac as a reporter gene. These lines have been made with different parts of the uteroglobin 5'-flanking region (5), the pac coding sequence and the SV40 polyadenylation signal. Transgenic mice were detected by slot-blot of genomic DNA and their tissues were extracted, minced and frozen at — 80°C until they were used. Tissue homogenization was made in 200 /il of TGE buffer. After a microfuge centriftigation (3', 4°C) supernatants were employed for PAC assay. Two kind of controls were applied to avoid variations among different experiments on different days: Non transgenic tissues were employed in all PAC assays and two samples of each homogenate, with and without the substrate puromycin, were always used. The reaction mixture for PAC assays contains: 10 tl of an acetyl CoA mix (for 100, /tl; 50 /il 1 M Tris-HCl pH 8.0, 35 /tl 2 mM acetyl CoA and 15 /tl [H]acetyl CoA); 10 /tl 10 mM ATP; 2.5 /tl 100 mM MgCl2; 0.3 units of acetyl CoA synthetase; 10 /il tissue homogenate; + / 5 /tl 2 mM puromycin and H2O to get a final volume of 50 /il. The reaction was performed at 33°C for 1 hour. Two main methods have been used for CAT assay and both of them can be used with PAC as well: A classic chromatographic method (6) and a recently called two phase partition assay (7). — For the chromatographic assay, the N-acetylpuromycin was extracted twice with 500 /tl ethyl acetate. After partial evaporation, volumes of 20 /tl were applied to a TLC aluminum sheet of silica gel 60F254 which were developed with methanol-ethyl acetate 1:3 (v/v). The spot of N-acetylpuromycin and the adjacent zones as controls were cut and counted. For non chromatographic assay, 200 /tl of 5 M NaCl-0.1 M Na2B4C<7 and 1 ml of scintillation liquid were added and counted for 5 min. Both assays work equally well in our hands, although the former allows more confidence when measuring low PAC activity. The system described here functions in a reproducible fashion and is quite sensitive, even working with the not very strong uteroglobin promoter (see table below). The use of pac as a reporter gene for transgenic animals, reported here for the first time, could be based on different aspects: (i) PAC exhibits an enzymatic activity not present in eukaryotic systems, at least in the different tissues analyzed in control animals (brain, lung, heart, liver, intestine, kidney, spleen, skeletal muscle, oviduct, uterus, testis and male tract), (ii) The product of the reaction, N-acetylpuromycin, shows a chromatographic behavior different from the non acetylated form. This characteristic allows the use of similar techniques than those applied to detect chloramphenicol acetyltransferase (CAT) activity in cultured cells and in tissues of transgenic animals, (iii) As puromycin is toxic for animal cells, its inactivation by PAC could be used to select cells expressing PAC, i.e. embryonic stem cells transfected with PAC constructs, and use them to create transgenic animals by microinjection to the blastocyst, as it has been described before for gene constructs bearing the neomycin resistance gene (8). The use of PAC in this task is simpler compared to neo constructs as it allows to use just one gene both as a reporter and to confer antibiotic resistance, (iv) The use of PAC might bypass some new problems found with the use of CAT since a CAT-like activity has been recently described in eukaryotic cells (7, 9).

Applicatlon of the two-phase partition assay for chloramphenicol acetyl transferase (CAT) to transfections with simian virus 40-CAT plasmids

The two-phase partition assay for chloramphenicol acetyl transferase (CAT) was tested for its applicability in transfection experiments involving CAT-expression plasmids containing simian virus 40 (SV40) transcriptional control elements. The assay was shown to be simple, sensitive, reproducible, and, with the modification described, cost-effective. However, CAT transfection experiments themsel ves gave highly variable results. Yields of CAT per microgram of protein in pSV2-CAT transfections of CV-1 cells varied by 60%. This variablity could not be explained by variation in the quality of DNAs used, differences in growth states of the cells at transfection, the culture medium employed, or the time at which transfected cells were extracted for CAT assay.

Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein.

Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.

New approaches to the diagnosis of tuberculosis in childhood with special reference to neurotuberculosis

Bacteriological diagnosis of tuberculosis in childhood is often unsuccessful owing to the difficulty in obtaining suitable specimens. Many attempts have been made to diagnose tuberculosis immunologically but with very limited success. Positive tuberculin reactions may be the result of nonspecific sensitization while negative reactions occur in undernourished children. Serodiagnostic tests suffer from problems of specificity, even when very specific antigens are used, and are often least helpful in diagnostically difficult cases. Detection of antigen has proved to be of more value, especially with clean specimens such as cerebrospinal and pleural fluids. Detection of specific components of Mycobacterium tuberculosis by linked gas chromatography and mass spectroscopy is very sensitive and specific but the equipment is very costly. Detection of specific DNA sequences of M. tuberculosis in specimens by use of labelled 'DNA probes' is rather insensitive although the sensitivity may be increased greatly by use of the polymerase chain reaction to amplify small amounts of the specific DNA. Non specific indicators of tuberculosis are generally unhelpful although the bromide partition test and assay of the enzyme adenosine deaminase in cerebrospinal fluid appear to be of value in the diagnosis of tuberculous meningitis. More research is required to develop a simple, specific and automated test for tuberculosis in childhood.

A comparison of BKV, JCV, and SV 40 transcriptional enhancers in primate cells

The activities of the transcriptional enhancers of the prototype human polyomaviruses, BKV (Dun) and JCV (MAD-1), were compared with that of the rhesus virus SV 40 in Old World African green monkey kidney cells (CV-1) and in New World owl monkey kidney cells (OMK). Enhancer activities were tested in CAT-expression plasmids. CAT activity in transfected cell extracts were measured by the novel two-phase partition assay. The activities of the BKV and JCV enhancers were 45% and 20% that of SV 40, respectively, in CV-1 cells. The effectiveness of each viral enhancer was less in OMK cells than in CV-1 cells, as would be expected if the viruses co-evolved with their natural hosts. In CV-1 cells, the BKV enhancer was more active than that of JCV, but in OMK cells the reverse was true. This result is significant in view of the fact that JCV, but not BKV, is oncogenic in owl monkeys.

A soluble fatty acyl – acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.

Surface structures, haemagglutination and cell surface hydrophobicity of Bacteroides fragilis strains

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2.1 +/- 0.25 nm and appeared to be 'curly'. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 Showed phase variation of fimbriae as expression of fimbriae was repressed at 20 degrees C and in early exponential phase at 37 degrees C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7.9 and 23.9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6.6 to 52.1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.

Surface-associated properties of Streptococcus milleri group strains and their potential relation to pathogenesis

Thirty strains from the Streptococcus milleri (anginosus) group (SMG) obtained from various sources were tested for a range of characters that could be associated with pathogenicity and the results were compared with those for type strains of S. sanguis, S. mutans and S. pyogenes. The SMG strains were heterogeneous in all tests. Most (18) belonged to one of the Lancefield groups with group F predominating. Adherence of strains isolated from abscesses to buccal epithelial cells was greater than that of other strains (p = 0.033). Compared with strains of S. sanguis, SMG strains were generally not aggregated by human saliva. They differed from the type strain of S. pyogenes in their relative ability to bind fibrinogen and fibronectin; they were less effective in binding fibrinogen (0.33-4.28% cf. 22% for S. pyogenes) and generally more effective in binding fibronectin (0.49-12.37% cf. 0.95%). Strains isolated from infections were statistically better at binding fibronectin than other strains (p less than 0.001). The ability of strains to adhere to saliva-coated hydroxyapatite (SHA) varied 10-fold, from 0.16-16.35%. The amount of fibronectin bound by SMG strains correlated with their ability to adhere to SHA (p less than 0.001). The hydrophobicity of the strains, as measured in the hexadecane partition assay, ranged from 0.0% to 99.0%. Some strains carried both positive and negative cell-surface charges and some strains with a highly hydrophobic cell surface also possessed a relatively high cell-surface charge. A minority of strains possessed a net positive cell-surface charge. Neither hydrophobicity nor cell-surface charge was related to the capacity of strains to adhere to SHA. Strains of SMG co-aggregated weakly with strains of Veillonella parvula, V. dispar, Actinomyces viscosus and A. naeslundii.

Production of methyl farnesoate by the mandibular organs of the mud crab, Scylla serrata: Validation of a radiochemical assay

Parameters for the validation of a radiochemical assay to monitor the biosynthesis and release of methyl farnesoate (MF) by the mandibular organs (MO) of the mud crab, Scylla serrata, have been defined. On the basis of HPLC analysis, MF appeared to be the exclusive radiolabeled product of release, using [ 3H- methyl]methionine as precursor. HPLC of isooctane extracts of medium in which MO had been maintained similarly revealed that MF was the exclusive 3H-product and indicates that the isooctane partition assay can be employed to monitor MF release. The time course of MF biosynthesis and release suggested that the endogenous l-methionine pool is large, because both cumulative release and rate of release increased over an 8-hr incubation but remained constant between 8 and 16 hr. Glandular content of MF did not appear to stabilize until 6–8 hr after the start of the incubation. Cumulative MF release, as a function of glandular content or biosynthesis, increased gradually with time, but even after 16 hr the majority of biosynthesized MF was retained within the MO rather than released. l-Methionine concentration in the incubation medium influenced both MF biosynthesis and release, although the dose dependency was more apparent with biosynthesis. Although it was difficult to associate strictly a level of biosynthesis with l-methionine concentration, low rates of MF biosynthesis were observed at concentrations of less than 25 μ M. Because high levels of biosynthesis were observed consistently between 60 and 70 μ M l-met, all subsequent incubations employed a concentration of 65 μ M. MO of S. serrata show a striking asymmetry in MF biosynthesis and release which is not time dependent. Hence the comparison of right and left glands in assessing the effects of experimental treatments is precluded.

A partition assay for the simultaneous determination of insect juvenile hormone esterase and epoxide hydrolase activity

A partition assay was developed to measure insect juvenile hormone (JH) I and III metabolism in biological samples containing both JH esterase and JH epoxide hydrolase activity. The assay utilizes commercially available radiochain 3H-labeled JH as substrate and the selective JH esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone. JH partitions into an isooctane phase and the metabolites JH acid, JH diol, and JH diol-acid into aqueous methanol after incubation of JH substrate with inhibited and uninhibited sample. The assay provides a time-and cost-efficient alternative to the currently available thin-layer chromatography method for the measurement of JH esterase and epoxide hydrolase activity.

A comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius.

Fibrillar and fimbriate strains of Streptococcus salivarius were compared for their ability to adhere to buccal epithelial cells and saliva-coated hydroxyapatite beads, and for their ability to coaggregate with Veillonella strains. The fibrillar Lancefield group K strains adhered statistically significantly better to both buccal epithelial cells and saliva-coated hydroxyapatite beads than the fimbriate strains, which lacked the Lancefield group K antigen. After 1 h the fibrillar strains coaggregated statistically significantly better than the fimbriate strains with V. parvula strain V1, but after 24 h, coaggregation both of fibrillar and of fimbriate strains reached approximately 90%. Freshly isolated Veillonella strains all coaggregated with the S. salivarius strains, but the percentage coaggregation varied considerably after 1 h depending on the Veillonella strain. Coaggregation was independent of the presence of Ca2+. S. salivarius strain HB-V5, a mutant of strain HB that had lost the Veillonella-binding protein, coaggregated weakly with V. parvula strain V1, but coaggregated very well with other wild-type veillonellae, suggesting the presence of an alternative mechanism for Veillonella-binding for strain HB. Fibrillar strains were, therefore, more adhesive to oral surfaces and coaggregated with veillonellae after 1 h better than the fimbriate S. salivarius strains. Both fibrillar and fimbriate strains were highly hydrophobic in the hexadecane-buffer partition assay.

A polystyrene microsphere assay for detecting surface hydrophobicity variations within Candida albicans populations

Adherence of microbial pathogens to host cell surfaces may involve hydrophobic interactions. Here, we describe the development of an assay for detecting cell surface hydrophobicity of populations and individual cells of the opportunistic fungal pathogen Candida albicans. The assay involves mixing polystyrene latex microspheres with cells and subsequent enumeration of cell-attached microspheres. Similar levels of hydrophobicity within a population of yeast cells were obtained with the microsphere assay and with a commonly used aqueous-hydrocarbon biphasic partitioning assay. Various buffers were found to support detection of surface hydrophobicity with the microsphere assay. Complex fungal growth media did not. Serum in test media prevented microsphere attachment. A unique advantage of the assay compared to others is that individual cells can be assessed for surface hydrophobicity. Within a population of C. albicans yeast cells, strongly, moderately and weakly hydrophobic cells were observed. Within some pairs of mother-daughter cells, only one cell was hydrophobic. Germ tbes and hyphae were hydrophobic regardless of the hydrophobic status of the parent cell. These results indicate that the microsphere assay is a useful test evaluating cell surface hydrophobicity of C. albicans.

A-CAM: a 135-kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies.

The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane-bound plaques of the fascia adherens. In cultured chick lens cells, A-CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A-binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451-1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction-specific membrane glycoprotein that is involved in intercellular adhesion in these sites.

Epoxide hydrolysis in the cytosol of rat liver, kidney, and testis: Measurement in the presence of glutathione and the effect of dietary clofibrate

The hydrolysis of trans- and cis-stilbene oxide and benzo[ a]pyrene-4,5-oxide was measured in cytosol and microsomes of liver, kidney, and testis of control and clofibrate-fed rats. Significant levels of nonprotein sulfhydryls were detected in cytosol from liver (4.6 mM) and testis (1.5 mM). Glutathione was moderately stable in these fractions and interfered with the partition assays as conjugates were retained in the aqueous phase along with diols. When the products were separated by thin-layer chromatography, significant amounts of glutathione-conjugates were found to have been formed in the cytosol of liver and testis. Overnight dialysis or preincubation of cytosol with 0.5 mM diethylmaleate eliminated conjugate formation without affecting diol production. In dialyzed cytosol from clofibrate-fed rats (0.5%, 14 days), the rates of hydrolysis of trans-stilbene oxide were 506, 171, and 96% of controls for liver, kidney, and testis, respectively, and 126% of controls in liver microsomes. Rates of hydrolysis of cis-stilbene oxide were 149, 172, and 96% of controls in microsomes and 154, 124, and 91% of controls in cytosols from livers, kidneys, and testis of clofibrate-fed rats respectively. Hydrolysis of benzo[ a]pyrene-4,5-oxide was similar to that of cis-stilbene oxide. Conjugation of the cis-stilbene oxide with glutathione was detected in cytosols from all three tissues with lesser amounts in the microsomes from liver and kidneys. After clofibrate treatment, the rates of this activity were 200, 173, and 95% of controls in cytosol from liver, kidneys and testis, and 203 and 202% of controls in microsomes from liver and kidneys respectively. These results indicate that epoxide hydrolysis and conjugation in rat liver and kidney are responsive to clofibrate treatment and support other evidence which suggests that hydrolysis of cis- and trans-stilbene oxides in cytosol is catalyzed, in part, by distinct enzymes.

Juvenile hormone and juvenile hormone esterase in adult females of the mosquito Aedes aegypti

Juvenile hormone III levels and juvenile hormone esterase activity were measured in whole body extracts and haemolymph, respectively, of female Aedes aegypti. The amount of juvenile hormone, determined by coupled gas chromatography-mass spectrometry, rose over the first 2 days after emergence from 0.7 to 7.5 ng/g, and then slowly fell over the next 5 days in females not given a blood meal. In females fed blood, juvenile hormone levels fell during the first 3 h to 2.3 ng/g. The rate of decline then slowed so that levels had reached their lowest point (0.4 ng/g) by 24 h after the blood meal. By 48 h, levels started to rise again until 96 h when they were equivalent to pre-blood meal levels. Juvenile hormone esterase activity in the haemolymph of females was measured with a partition assay. The esterase activity showed small fluctuations in unfed animals. In females fed blood on the 3rd day after emergence, the juvenile hormone esterase activity rose slowly to a peak at 36 h. At 42 h it began to decline, and by 66 h it had returned to pre-blood meal levels. Thus, juvenile hormone levels and juvenile hormone esterase activity were inversely correlated after a blood meal. Both the ovary and fat body produce juvenile hormone esterase in organ culture. Juvenile hormone III acid was the only metabolite produced after incubation of haemolymph with racemic-labelled juvenile hormone III. Juvenile hormone acid, diol, and acid diol were the main metabolic products seen in whole animal extracts after topical application of labelled hormone. About 25% of topically applied, labelled juvenile hormone appears in the haemolymph as the acid diol, and 50% of this is excreted in the urine immediately after the blood meal. Topical application of BEPAT ( S-benzyl- O-ethyl phosphoramidothiolate), a specific inhibitor of juvenile hormone esterase, resulted in the absence of juvenile hormone acid and a reduction in the acid diol. Both BEPAT and methoprene, a juvenile hormone analogue, caused a reduction in egg hatch when applied topically 30 h after a blood meal, demonstrating that the decline in juvenile hormone levels after a blood meal is necessary for normal egg development and suggesting that the decline is mediated, at least in part, by juvenile hormone esterase.

Reevaluation of the hydrophobic nature of the 110‐kD calmodulin‐, actin‐, and membrane‐binding protein of the intestinal microvillus

A complex of calmodulin (CM) and the 110-kD (110K) subunit composes the helical array of cross-bridges linking the microvillus actin filament bundle with the membrane. The hydrophobic properties of the 110K protein, assessed by the detergent phase partitioning assay [Bordier C: J Biol Chem 256:1604, 1981], are highly dependent on the solution conditions used in its isolation. The ATP-dissociable 110K-CM complex [Howe and Mooseker: J Cell Biol 97:974, 1983] exhibits hydrophilic characteristics in this assay. In contrast, the 110K subunit extracted from brush borders by Triton X-100, sodium dodecyl sulfate, and sodium pyrophosphate (detergent-treated 110K) [Glenney JR, Glenney P: Cell 37:743, 1984] behaves as a hydrophobic protein. However, because the soluble hydrophilic 110K-CM can be rendered hydrophobic by treating the complex with the same detergent and salt conditions used in the preparation of detergent-treated 110K, the properties of detergent-treated 110K seem likely to be an effect of the solution conditions on its native conformation, sedimentability, or exposure of binding domains. In addition, the detergent-treated 110K is devoid of calmodulin and no longer exhibits the actin-binding activity characteristic of the ATP-dissociable 110K-CM and of the intact complex in situ. With two partially purified preparations of the 110K subunit exhibiting such dramatically distinct properties, it seems premature to define the nature of the 110K subunit's association with the membrane at this time.

Fucosterol epoxide lyase of insects: Synthesis of labeled substrates and development of a partition assay

Fucosterol epoxide labeled with tritium in the C-29 methyl has been synthesized and employed in the development of a partition assay which allows the rapid determination of fucosterol epoxide lyase activity in vitro in homogenates of insect tissues. An independent synthesis of [24- 14C]fucosterol epoxide provided a control substrate to evaluate nondealkylative transfer of labeled steroid to the aqueous layer during the enzyme assay. The diastereomeric 24 R,28 R- and 24 S,28 S-[29- 3H]fucosterol epoxides were obtained via HPLC separation of their benzoate esters. Homogenates of the midgut tissue of larval tobacco hornworms ( Manduca sexta) were examined at pH 5 to 9 in several buffer systems, and at temperatures of 7 to 67°C in phosphate buffer. Optimal activity was found using pH 7.4, 76 m m phosphate buffer at 37°C. The 24 R,28 R diastereomer of fucosterol epoxide was metabolized at a rate at least 100 times that of the 24 S,28 S isomer by this enzyme system.