Identification and Quantification of AKT Isoforms and Phosphoforms in Breast Cancer Using a Novel Nanofluidic Immunoassay

Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoPro^TM 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.Activation of the PI3K-AKT signaling pathway is one of the most common events in cancer (1, 2). Pathway activation can confer a number of advantages to the cancer cells, including enhanced proliferation and survival (1, 2). Multiple mechanisms exist by which the pathway may become activated, including amplification or activation of receptor tyrosine kinases (e.g. ERBB2 in breast and EGFR in lung tumors), mutation of the catalytic or regulatory subunits of PI3K (e.g. PIK3CA in colorectal and breast tumors), loss of the negative regulator PTEN (e.g. mutation in prostate and melanoma), and gain of function of AKT (e.g. amplification or mutation in breast and pancreatic tumors) (reviewed in Refs. 1 and 2).AKT represents a central node in the PI3K signaling cascade (3). AKT is recruited to the cell membrane via its pleckstrin homology domain when PI3K phosphorylates PIP₂ to form PIP₃ (4, 5). Following recruitment, AKT is phosphorylated by PDK1 and the rictor-mTOR complex, resulting in conformational changes and activation of the protein (5–8). Multiple studies have shown that the phosphorylation of AKT leads to the phosphorylation and activation of downstream effectors of the signaling pathway, such as mTOR complex 1 and S6K (reviewed in Ref. 1). The central role of this pathway in cancer is further underscored by the efforts of multiple pharmaceutical companies that have developed inhibitors against AKT as potential anti-oncogenic therapeutics (9).Despite the importance of AKT in growth and survival signaling in cancer, there are surprisingly few data that address the specific roles played in growth and survival by the multiple AKT family members (AKT-1, -2, and -3) and different phosphorylation and putative phosphorylation sites that can potentially activate the protein. Western blot analysis has been the foundation of most AKT studies, but in many cases pan-AKT antibodies have been employed that fail to distinguish between the different AKT isoforms. Recent siRNA silencing studies have indicated distinct functions for different AKT family members within a cell (10, 11). Moreover, there is evidence in breast cancer that the three isoforms exhibit different localizations and therefore must have at least partially distinct functions (12). Similarly, evidence is mounting for multiple phosphorylation sites in AKT beyond the two most studied phosphorylation events (Thr-308 and Ser-473) (5–8). Phosphorylation at serine and threonine residues at Thr-72 and Ser-246 may be required for the activation or regulation of kinase activity (13). The functional significance of constitutive phosphorylation of Ser-124 and Thr-450 is still unknown (14). Finally, there is evidence that phosphorylation of tyrosine residues at Tyr-315 and Tyr-326 is required for full kinase activity (15).Analysis of such phospho- and isoform-specific activation often requires complicated in-depth analyses using large quantities of proteins, purified recombinant protein, immunoprecipitation, incorporation of ³²P isotopes, and/or mass spectroscopy, which makes such studies more difficult to perform and not easily adaptable to clinical specimens. Thus, better methods are required for the accurate assessment of both phosphoform and isoform usage in cells with an activated PI3K-AKT pathway and the effects of pathway inhibitors using relatively small amounts of starting material. We describe here the development of such an assay using nanocapillary-based isoelectric focusing (16). This approach allows the separation of AKT into distinct peaks that correspond to different iso- and phosphoforms using a small amount of starting material and a single pan-specific antibody. This approach should allow for more accurate determinations of isoform usage in different cell types, as well as of changes in phosphorylation states in response to pathway inhibition, including in clinical specimens.     

Liposomes and Liposome-like Vesicles for Drug and DNA Delivery to Mitochondria

Mitochondrial research is presently one of the fastest growing disciplines in biomedicine. Since the early 1990s, it has become increasingly evident that mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Most remarkably, mitochondria, the “power house” of the cell, have also become accepted as the “motor of cell death” reflecting their recognized key role during apoptosis. Based on these recent exciting developments in mitochondrial research, increasing pharmacological efforts have been made leading to the emergence of “Mitochondrial Medicine” as a whole new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, colloidal vectors, i.e., delivery systems, need to be developed able to selectively transport biologically active molecules to and into mitochondria within living human cells. Here we review ongoing efforts in our laboratory directed toward the development of different phospholipid- and non-phospholipid-based mitochondriotropic drug carrier systems.     

Fine genetic mapping of RXopJ4, a bacterial spot disease resistance locus from Solanum pennellii LA716

The RXopJ4 resistance locus from the wild accession Solanum pennellii (Sp) LA716 confers resistance to bacterial spot disease of tomato (S. lycopersicum, Sl) caused by Xanthomonas perforans (Xp). RXopJ4 resistance depends on recognition of the pathogen type III effector protein XopJ4. We used a collection of Sp introgression lines (ILs) to narrow the RXopJ4 locus to a 4.2-Mb segment on the long arm of chromosome 6, encompassed by the ILs 6-2 and 6-2-2. We then adapted or developed a collection of 14 molecular markers to map on a segregating F₂ population from a cross between the susceptible parent Sl FL8000 and the resistant parent RXopJ4 8000 OC₇. In the F₂ population, a 190-kb segment between the markers J350 and J352 cosegregated with resistance. This fine mapping will enable both the identification of candidate genes and the detection of resistant plants using cosegregating markers. The RXopJ4 resistance gene(s), in combination with other recently characterized genes and a quantitative trait locus (QTL) for bacterial spot disease resistance, will likely be an effective tool for the development of durable resistance in cultivated tomato.     

Optically active antifungal azoles: synthesis and antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol

A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (11a-n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (12a-n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine demonstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The compound 12d showed MIC value of 0.12 microg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25 microg/mL for C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5 microg/mL for C. glabrata, C. krusei ATCC 6258, which is comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25-0.5 microg/mL) against Candida spp. and strong anticryptococcal activity (MIC; 0.25 microg/mL) against C. neoformans.     

A hierarchy of lipid constructs for the sperm plasma membrane

We have presented a series of lipid constructs as models of the sperm plasma membrane. We also isolated the plasma membrane from rabbit sperm cells and characterized the lipid composition. The behavior of these various membrane systems was evaluated using a vesicle leakage assay, in which surfactant (nonoxynol-9, N-9; or benzalkonium chloride, BZK) exposure induced membrane permeabilization. These studies shed light on the relative importance and significance of particular components present in the lipid constructs. In particular, a highly unsaturated phospholipid component characterized by an ether-linkage to position 1 of the glycerol backbone (as opposed to the more conventional ester linkage) as well as the presence of sulfogalactosyl ceramide were found to have an effect on the surfactant-induced leakage response. The presence of cholesterol had the greatest influence on membrane behavior. The construct series also demonstrated the ability of the surfactants studied to discriminate between different membrane systems. We found that N-9 displayed little sensitivity to membrane composition while BZK showed specific behavior with the various membrane systems.     

Trafficking of intracerebroventricularly injected antisense oligonucleotides in the mouse brain

Intracerebroventricular (icv) delivery of therapeutic molecules directly into the brain parenchyma has attracted considerable attention because of the advantage of bypassing the blood-brain barrier. Exogenous icv administration of antisense oligodeoxynucleotides (AS-ODNs) has been implicated in modifying gene expression within the targeted brain area. The biodistribution, tissue penetration, and stability of exogenously administered AS-ODNs are the major determinants with regard to their potential utility as agents for modifying gene expression. This report examined the distribution and clearance of labeled AS-ODNs with the aim of exploring the feasibility of icv administration of AS-ODNs as a targeted treatment approach to Alzheimer's disease. A single icv injection of fluorescein-labeled 2'-O-(methoxy) ethyl (2'MOE) ribosyl-modified AS-ODNs directed at the beta-secretase cleavage site of beta-amyloid precursor protein (APP) mRNA into the mouse brain showed rapid uptake by 15 minutes, overall gradual spread and retention by 30 minutes to 3 hours, and complete clearance by 8 hours postinjection. Labeled AS-ODNs were observed to penetrate across the cell membrane and accumulate in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cell populations. Current study provides a basic pattern of uptake, distribution, and stability of AS-ODNs in the mouse brain.     

Basidiobolomycosis of the Nose and Face: A Case Report and a Mini-Review of Unusual Cases of Basidiobolomycosis

Background  

Subcutaneous zygomycosis is a chronic infection caused by fungus of the order Entomophthorales. It can have varying presentations and presents in the nose and face area with gradually progressing subcutaneous swelling that may be difficult to diagnose unless a strong suspicion of fungal involvement is maintained. We present a case of subcutaneous zygomycosis in a 35-year-old male patient, resident of a North Indian state. The patient was diagnosed to be suffering from subcutaneous zygomycosis, the causative agent being Basidiobolus ranarum identified on culture and lactophenol cotton blue mount preparation. He responded well to treatment with Itraconazole and Terbinafine and is asymptomatic on follow-up.