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NONPATENTED INVENTIONS:
PATENTED INVENTIONS:
NONPATENTED INVENTIONS: A method for preparing pancreatic tissue for structural (electron microscopy) and in-vitro functional studies in a manner that minimizes tissue damage and maximizes functional performance. This procedure is used beneficially with pancreatic tissue taken from guinea pig, rat or mouse. The procedure involves using a syringe and a needle to inject Krebs Ringer Bicarbonate (KRB) solution into the interstitial space thereby distending the tissue and revealing the smallest lobular structures perceived by the human eye. Then, using a pair of ophthalmic scissors, these lobular structures may be removed from the duct system and incubated in KRB under in vitro physiological conditions. Normally 5-10 lobules are incubated in a single Erlenmeyer flask at 37 degrees Celsius and in the presence of 95% oxygen and 5% carbon dioxide. In experiments that conduct pulse-chase studies that rely on rapid transfer of lobules from one flask to another or rapid washing conditions, a piece of nylon mesh cut into a 2 cm circle, may be used to attach the lobules to a single mesh disk in each incubation flask. The connective tissue surrounding the lobules attach spontaneously to the nylon mesh. Following attachment of the lobules to the nylon mesh, which occurs immediately during shaking in a temperature-controlled water-filled incubator, a pair of forceps may be used to pick up the group of lobules for washing or transferring manipulations without introducing injury to the tissue itself. Slide Presentation - Pancreatic Lobules Ultilizing enzymatic digestion with collagenase and vigorous shearing forces, pancreatic lobules can be further reduced to pancreatic acini. However, there is an important functional difference between lobules and acini. In pancreatic lobules the acinar lumen is sequestered from the incubation medium allowing use of ductal inhibitors to manipulate the fluid, electrolyte and pH conditions of the acinar lumen. In pancreatic acini the conditions of the acinar lumen can be directly controlled by the conditions of the incubation medium. These functional differences allowed Dr. Freedman and myself to discover the role of bicarbonate secretion from duct cells in regulating the dissolution of secretory enzymes during exocytic release and the cellular uptake of exocytic membranes (secretory granule membranes inserted into the APM) by activated endocytosis at the APM. Due to the fragile nature of tissue in the mouse pancreas, pancreatic acini obtained after treating with collagenase and shear forces, show relatively poor performance with high background levels of secretion. Thus, it is optimal to use pancreatic lobules when studying the mouse pancreas in vitro. Further, pancreatic lobules taken from mouse pancreas act more like acini in that the conditions of the acinar lumen appear to be directly controlled by the incubation medium. This observation suggests that small pancreatic ductile segments are severed during the tissue distension procedure. TWO DIMENSIONAL GEL ELECTROPHORESIS: Two Dimensional Gel Electrophoresis separating proteins by charge and size - 2D separation of proteins in a slab gel utilizing isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension allowed separation of proteins by charge and size, respectively. This procedure, developed independently by Dr. George Scheele and Dr. Patrick O'Farrell, allowed complex mixtures of proteins (up to 2000) to be separated by gel electrophoresis for the first time. Accordingly, it ushered in the era of "proteomics" twenty years before that term was coined. Invention of this technique allowed my laboratory to study the following biological processes for the first time:
Slide Presentation - 2D gel Electrophoresis MICROSOMAL MEMBRANE TRANSLOCATION MODELS: Microsomal membrane models that allowed elucidation of N-terminal signal sequences responsible for the sequestration of secretory enzymes in the RER. The membrane model that proved to show the greatest success was the dog pancreas microsomes for the following reasons: • Compared to other organs, pancreatic microsomal membranes show the highest polypeptide chain translocation efficiencies. • Dog pancreas was superior to pancreas from other species because of low RNase levels (0.5 ug RNase per gm of tissue) and extraordinarily high RNase inhibitor levels (200:1 molar excess over RNase) in the cytosol. Slide Presentation - Membrane Model for Protein Targeting & RER Sequestration in the Signal Peptide Hypothesis for Translocation of Presecretory Proteins into the RER A method by which purified RNase inhibitor is added to in-vitro translation systems to protect against contaminating RNase found in the translation components and the membrane fractions. Slide Presentation - Purified RNase Inhibitor PATENTED INVENTIONS: FLEX TECHNOLOGY FOR SYNTHESIS OF FULL-LENGTH GENE LIBRARIES: FLEX Technology for synthesizing full-length cDNA libraries - This patented technology utilizes poly-T priming followed by a reversible tailing procedure at the 5' end of single-strand cDNA to increase the representation of full-length double-stranded cDNAs from control rates of 10-20% to FLEX rates of 50-70%. This patent formed the basis for formation of Alphagene, Inc., a genomics company formed in 1993 and based in Boston , MA . • Scheele, G. and Fukuoka , S.-I. Synthesis of full-length, double-stranded DNA from a single-stranded linear DNA template. Patent no. 5,162,209, issued on 11-10-92. • Scheele, G. Synthesis of full-length double-stranded DNA from a single-stranded linear DNA template. Patent no. 5,643,766, issued on 7-1-97. PDF File - Flex Description BICARBONATE TREATMENT FOR CYSTIC FIBROSIS: While conducting scientific research at Harvard Medical School , my laboratory determined the ultimate biochemical deficiency in Cystic Fibrosis and published the mechanism of this deficiency in the exocrine pancreas in a number of scientific publications. Detailed information on these discoveries appear in this website under "Scientist" and "Pancreatic Dysfunction in Cystic Fibrosis is Due to Impairments in Bicarbonate Secretion". • Scheele, G. Treatment of pulmonary conditions associated with insufficient secretion of surfactant. Patent no. 5,863,563, issued on 1-26-99. This patent is held by Dr. George A. Scheele, the inventor. Slide Presentation - Pancreatic Dysfunction in Cystic Fibrosis A METHOD FOR DIAGNOSING PANCREATITIS A method for diagnosing pancreatitis, which measures Glycoprotein-2 (GP2) in the serum. • Scheele, G. and Fukuoka , S.I. A method for diagnosing pancreatitis. Patent no. 5,663,315, issued on 9-2-97. TOPICAL PREVENTION AND EARLY TREATMENT OF VIRAL INFECTIONS : Under the leadership of Dr. George A. Scheele, La Jolla Biosciences, LLC, has filed for three patents in the United States , with corresponding international filings, which claim the use of topical beta cyclodextrins for viricidal treatment of envelope viruses on topical surfaces of the body thereby preventing viral entry into the body. Drs. James E.K. Hildreth (Johns Hopkins Medical Institutions) and George A. Scheele (President, La Jolla Biosciences, LLC) are listed as co-inventors in the following three patent applications: 1. Prophylaxis, Systemic & Topical Treatments, Vaccines:
SUMMARY OF CLAIMS: • Broad protection covering use of cholesterol sequestering agents to prevent or treat infection by envelop viruses, including HIV, HTLV, Herpes, Hepatitis, Influenza & Pox viruses • Oral & IV therapeutics • Oral, IV, ID, IM & SubQ vaccines • Topical treatment of viral diseases (Herpes and Influenza) on all epithelial surfaces. • Protection for (myco-) bacterial, fungus, protozoan and non-envelope viruses (prevention of intracellular uptake) • Extracorporeal treatment of envelope viruses. 2. Protection/Decontamination/Sanitation Formulations for the Health Care Industry:
SUMMARY OF CLAIMS: • Broad protection covering use of cholesterol sequestering agents to reduce or prevent dermal transmission of envelope viruses, including HIV, HTLV, Herpes, Hepatitis, Influenza, SARS and Pox viruses. • Protect Medical workers, blood handlers and soldiers. • Gloves with an interior surface coated by BCD • Treating environmental surfaces • Uses of lotions, sprays and powders containing BCD. 3. Protection Technologies & Formulations for Blood Products:
SUMMARY OF CLAIMS: • Addition of cholesterol-sequestering agents to whole blood, plasma, packed red blood cells, platelet concentrates, gamma globulin, purified plasma factors, albumin and factors VIII, IX and VIIA. • Render viral-infected cells incapable of production of viable or functional viral particles • Inhibit cells from uptake of envelop and non-envelope viruses • Destroy free viral particles • Filtration of 2-HP-BCD treated white blood cells • Application of 2-HP-BCD to the blood circulation, cervical and vaginal canals to prevent envelope and non-envelope virus infection during childbirth to prevent maternal-to-fetal transmission of viruses. |
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