Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

Historical and Life History Factors in the Biogeography of Mayflies

SYNOPSIS. Cladistic analysis of mayflies suggests that dispersalwas very asymmetrical after the new land connection betweenNorth and South America. Twenty-one genera apparently movedfrom South to North and Central America, but there is good evidencefor only one North American genus moving into the south. Testablepredictions are possible once the boreal (Laurasian) or austral(Gondwanian) designations are made. For example, Paracloeodes,a genus of austral origin, was predicted to be found in SouthAmerica, and in North America in warm rivers north and eastof its known distribution. These predictions have been confirmed.Other characteristics of tropical mayflies, such as length oflarval period and emergence and mating patterns, may be usedto predict habitats and characteristics of present North Americangenera. Merger events and consequent dispersal of organismshave profound influences on distributional patterns, and fromsuch information, biologically useful generalities can be made.     

An Ethnographic Filmflam: Giving Gifts, Doing Research, and Videotaping the Native Subject/Object

Using the discussion of self-reflexivity as an organizing principle, this article examines how mobilizing digital video technology during fieldwork opens up empirical and theoretical space for reconceptualizing the relationship between anthropologists and informants. Placing the field of visual anthropology into critical conversation with long-standing theoretical arguments about the objectivist limitations of native anthropologists, I argue that the slipperiness of nativity as an anthropological designation helps to provide analytical tools for examining filmmaking as a kind of gift-giving process between native ethnographic filmmakers and the subjects of their films. This article highlights some of the ways in which my own filmic and videographic exploits in Harlem, New York, mark integral connections between seeing and being the proverbial other, probing social exchanges predicated on the usefulness of low-budget digital technology as a means of fostering politically and epistemologically valuable ethnographic collaborations.     

The Spontaneous Formation of Antheridia in Onoclea is not Blocked by Light

Onoclea sensibilis gametophytes were grown from spores on ashedsoil and agar to determine if the spontaneous formation of antheridiacan be blocked by light. Under most conditions, dark-grown gametophytesformed antheridia later than or at the same time as gametophytesgrown in the light. Under no circumstances was there a rapidonset of maleness in the dark. These results contradict thehypothesis that, in Onoclea, antheridiogen is required to inducemaleness because light inhibits the formation of antheridia.In the light, antheridia formed on heart-shaped thalli. In darkness,antheridia formed on filamentous gametophytes. The timing ofonset of maleness was affected by temperature and the presenceof sucrose. The effect of sucrose on the comparison betweenlight and dark treatments depended on both substrate and temperature Onoclea sensibilis, L., sensitive fern, fern gametophytes, sexuality, light-induced block     

Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.     

Eosinophils in a tri-cell multicellular tumor spheroid (MTS)/endothelium complex.

Eosinophils have been found in infiltrates of many different cancers. It is still unclear as to whether they are passive bystanders in the cellular milieu or active cellular agents in host responses. Thus their harmful or helpful nature remains equivocal. We have developed an in vitro tri-cell model of eosinophils, MCF-7 breast tumor cell spheroids and HUVEC endothelial cells to examine the binding and association of eosinophils with both the tumor and the endothelia and the ensuing action of the tumor. Eosinophils bound very rapidly to the tumor spheroid and remained tightly bound throughout the 24 hr culture period. Histological staining of the tri-cell complex revealed highly granulated eosinophils as well as large amounts of degranulated protein diffused throughout the spheroid. IL-5 treatment of eosinophil: MTS complexes resulted in destruction of the tumor cells, particularly those which had grown out from the spheroid onto the endothelial cells. Eosinophils, pretreated with IL-5 before interaction with the tumor or endothelial cells, bound aggressively to the endothelial cells, thereby preventing tumor attachment. This eosinophil tri-cell tumor model system mimics clinical observations with regards to binding to epithelial and endothelial cells, dispersal of granular proteins throughout the tumor and also tumor destruction. Because it closely mirrors in vivo cellular interactions, it allows one to study more closely the mechanism(s) of eosinophil killing, the modulation of eosinophil activity and the testing of therapeutic interventions. The accommodation of the model to tumor invasion, using metastatic tumor cells and extracellular matrices such as matrigel, will help to elucidate a role for eosinophils (and their mediators) in cancer invasion and metastasis.     

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus

Folate receptor (FR)-β has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid–derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a “macrophage-rich” model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.     

Immunocytochemical and Cytochemical Localization of Photosystems I and II

Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P₇₀₀ chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase-antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P₇₀₀ chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae.