Maximizing Kolmogorov Complexity for accurate and robust bright field cell segmentation.

Background: Analysis of cellular processes with microscopic bright field defocused imaging has the advantage of low phototoxicity and minimal sample preparation. However bright field images lack the contrast and nuclei reporting available with florescent approaches and therefore present a challenge to methods that segment and track the live cells. Moreover, such methods must be robust to systemic and random noise, variability in experimental configuration, and the multiple unknowns in the biological system under study.

Stomatal responses to humidity in isolated epidermes.

The ability of guard cells to hydrate and dehydrate from the surrounding air was investigated using isolated epidermes of Tradescantia pallida and Vicia faba. Stomata were found to respond to the water vapour pressure on the outside and inside of the epidermis, but the response was more sensitive to the inside vapour pressure, and occurred in the presence or absence of living, turgid epidermal cells. Experiments using helium-oxygen air showed that guard cells hydrated and dehydrated entirely from water vapour, suggesting that there was no significant transfer of water from the epidermal tissue to the guard cells.

The role of the mesophyll in stomatal responses to light and CO2.

Stomatal responses to light and CO(2) were investigated using isolated epidermes of Tradescantia pallida, Vicia faba and Pisum sativum. Stomata in leaves of T. pallida and P.

Membrane trafficking and osmotically induced volume changes in guard cells.

Guard cells rapidly adjust their plasma membrane surface area while responding to osmotically induced volume changes. Previous studies have shown that this process is associated with membrane internalization and remobilization. To investigate how guard cells maintain membrane integrity during rapid volume changes, the effects of two membrane trafficking inhibitors on the response of intact guard cells of Vicia faba to osmotic treatments were studied.

Cellular calcium mobilization in response to phosphoinositide delivery.

Intracellular calcium [Ca(2+)](i) is mobilized in many cell types in response to activation of phosphoinositide (PIP(n)) signaling pathways involving PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3). To further explore the relationship between increases in intracellular PIP(n) concentrations and mobilization of [Ca(2+)](i), each of the seven phosphorylated phosphoinositides (PIP(n)s) were delivered into cells and the metabolism and physiological effects of the exogenously administered PIP(n)s were determined. The efficient cellular delivery of fluorophore-tagged and native PIP(n)s was accomplished using histone protein, neomycin, and dendrimeric polyamines.

Metastasis suppression by breast cancer metastasis suppressor 1 involves reduction of phosphoinositide signaling in MDA-MB-435 breast carcinoma cells.

Several molecules that suppress metastasis without suppressing tumorigenicity have been identified, but their mechanisms of action have not yet been determined. Many block growth at the secondary site, suggesting involvement in how cells respond to signals from the extracellular milieu. Breast cancer metastasis suppressor 1 (BRMS1)-transfected MDA-MB-435 cells were examined for modifications of phosphoinositide signaling as a potential mechanism for metastasis suppression.

Changes in surface area of intact guard cells are correlated with membrane internalization.

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis-that surface area remains approximately constant because of changes in shape-has not been investigated.

A real-time fluorogenic phospholipase A(2) assay for biochemical and cellular activity measurements.

A fluorogenic analog of the PLA(2) substrate PC, named Dabcyl-BODIPY-PC, or simply DBPC, was synthesized with a fluorescence quencher (Dabcyl, 4-[(4-[N,N-dimethylamino]phenyl)azo]benzoic acid) in the sn-1 acyl chain and a BODIPY fluor in the sn-2 acyl chain. DBPC was recognized by sPLA(2) from each of the four sources examined (bee venom, human synovial fluid, cobra venom, and bovine pancreas). A dramatic and quantifiable fluorescence enhancement of DBPC occurred upon phospholipase digestion both in the presence and absence of excess PC.

A monoclonal antibody to visualize PtdIns(3,4,5)P(3) in cells.

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsP(n)) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsP(n) quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsP(n)s. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P(3) immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P(3) IgM, and immunodetection of PtdIns(3,4,5)P(3) in stimulated mammalian cells.