Anticancer, Inc.

The United States of America, As Represented by the Secretary, Department of Health and Human Services

Mingxu Xu

Yuying Tan

Lingna Li

Qinghong Han

Li Tang

Ping Jiang

Meng Yang

Shigeo Yagi

Kenji Miki

Huiyan Guo

Valeryi Lishko

Shukuan Li

Zhijian Yang

Xinghua Sun

Levy Kopelovich

Eligio Reynoso

Quinghong Han

Xiao-Ming MD Li

Ming MD Zhao

Meng MD Yang

Eligio Reynose

A method is described to identify secreted proteins identified with stages of malignancy of cancer. The proteins are initially identified by trapping them with a fluorescent protein containing vector that can insert in any gene. The secreted proteins are initially identified by their fluorescence. Secreted proteins identifying tumors with specific degrees of malignancy are isolated to determine if they can serve as markers of cancer progression.

Methods for identifying markers for early-stage human cancer, cancer progression and recurrence

Immunocompromised rodents that have been modified to express a fluorescent protein in substantially all tissues are described. These rodents are useful as models for gene expression, tumor progression and angiogenesis. Also provided are model systems where heterologous tissues fluorescing in a first color are transplanted into hosts that have been modified to fluoresce in substantially all tissues with a second color.

Immunocompromised rodents as dual color tumor models

Infection bycan be treated by administering methioninase optionally in combination with additional therapeutic agents, such as antibiotics.

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Methods of obtaining faithful expression libraries from tissue samples comprise extraction of RNA from intact tissue cultured in three-dimensional sponge-gel based histocultures.

Expression profiling based on histocultures

A method to determine a total cysteine in biological fluids utilizes similarly treated portions of the fluid with a homocysteinase and a non-specific desulfurase.

Total cysteine assay

Methods for inhibiting the growth of tumor cells by combination treatment with a selenium-containing prodrug and an enzyme for which it is a substrate are described. The treatment also enhances the effect of anti-tumor agents.

Selenium-containing pro-drugs for cancer therapy

Living cells can be stably modified to emit different colors from the cytoplasm and nucleus, thus permitting analysis of the status of said cells and the effect of agents on said cells either by visual or instrumentally-aided observation. These observations may be made, if desired, in real time. In addition, rates of proliferation and drug sensitivities can be determined in vitro in real time by the use of cells modified to express a single fluorescent protein and observing fluorescence intensity as a function of time.

Real time measurement of cellular responses

The invention provides novel methods relating to SAM detection and production as well as a novel SAHH enzymatic activity for use in such methods. Additional methods, compositions, and kits relating to the novel SAHH are also provided.

High expression and production of high specific activity recombinant S-adenosyl homocysteinase (SAHH) and improved assays for S-adenosylmethionine (SAM)

A method to detect and measure the concentration of hydrogen peroxide comprises adding reagents consisting essentially of peroxidase and a dialkyl phenylenediamine and detecting any color generated by the interaction of hydrogen peroxide with these reagents.

Method to determine hydrogen peroxide

Compositions and methods which ameliorate the virulence of bacterial infection are described wherein the active ingredient modulates transmethylation reactions in bacterial cells. Particularly useful compounds are inhibitors of S-adenosyl methionine synthetase (SAMS), of S-adenosyl homocysteine hydrolase (SAHH) and of transmethylases.

Modulators of methylation for control of bacterial virulence

A depletion method to inhibit tumor growth includes introducing a viral expression system capable of expressing methioninase or a fusion protein containing methioninase into a tumor contained in a vertebrate subject or cells thereof. The fusion protein may contain a fluorescent protein to permit monitoring of the completeness of the depletion method. The fusion protein can be used in vivo as well as in vitro screening protocols which employ the viral expression system. The expression system includes control sequences and means to integrate the nucleotide sequence into the genome of a host cell for expression. The method may also include treating the cells with isolated methioninase and/or with a therapeutic cell. The depletion method can be with other known therapies, such as maintaining the animal having the tumor on a methionine depleted diet.

Methioninase gene therapy for tumor treatment

Enzymatic methods to determine the concentration of pyridoxal 5&prime;-phosphate (PLP) in biological fluids are described. The methods of the invention are useful to assess risk for cardiovascular disease. The assay can be a homogeneous assay using the ability of PLP to function as a co-enzyme for homocysteinase and related enzymes and measuring the products of the reaction preferably spectrophotometrically. The invention also includes improvements in sensitivity of assays for measuring hydrogen sulfide production by measuring fluorescence as opposed to absorbance of the oxidized product of H S with N,N-dialkyl p-phenylene diamine.

Homogeneous enzymatic assay for vitamin B6 and improvements in H2S detection

Biological fluid assay methods

Novel enzymatic methods to determine the concentration of homocysteine in biological fluids are described. In a typical embodiment of the invention, the biological fluid sample is from a patient, and the methods of the invention are useful to assess risk for cardiovascular disease. The novel methods of the invention involve use of particular homocysteinase enzymes that permit the determination of homocysteine concentrations in biological samples without interference from the concentrations of cysteine and/or of methionine that are routinely present in such samples. There is also provided a diagnostic kit for use in determining the amount of homocysteine in a biological sample comprising (a) a homocysteinase having the aforementioned characteristics, and (b) at least one reagent capable of being used to determine the amount of product formed in the homocysteinase reaction. In a further aspect, the homocysteinase is provided as a chimeric molecule that comprises amino acid subsequences derived from, or patterned on, more than one homocysteinase, and which is typically produced from a chimeric polynucleotide that encodes therefor. Additional enhancements in homocysteine assay methodology include use of the enzyme .gamma.-glutamylcysteine synthetase to further limit any interference from cysteine present in the biological samples.

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La Jolla, CA, United States of America

Mingxu Xu