PUBLICATIONS in SCIENTIFIC JOURNALS:
and Human RetroViruses:
Robert Neurath, Nathan Strict, Robert Fields and Shibo Jiang:
Peptides Mimicking Selected Disulfide Loops in HIV-1 gp120, Other Than V3,
Do not Elicit Virus-Neutralizing Antibodies. AIDS Research and Human RetroViruses, 7: 657-662, 1991.
The positions of all 9 intrachain disulfide bonds within the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1) have been established recently. Peptides expected to mimic some of the disulfide-bonded domains [(120-133)-(203-221); (133-138)-(164-203); (224-254); (391-425) and (385-392)-(425-452)] were synthesized. All peptides, except (120-133)-(203-221), elicited in immunized rabbits relatively high levels of antibodies reacting with gp120 in enzyme-linked immunosorbent assay (ELISA) and/or Western immunoblot assays. However, these antibodies failed to neutralize the infectivity of HIV-1. Combined with earlier reports concerning other gp120 loop peptides, these results confirm the uniqueness of the V3 (303-338) loop in encompassing a principal determinant(s) involved in virus neutralization.
Journal of Interferon Research:
Robert Fields, Tom M. Mariano, Stanley Stein and Sidney Pestka: Recombinant rat and murine immune interferons are phosphorylated at a single site, Ser132. J. Interferon Research, 8: 549-557, 1988.
Recombinant rat (Ra) and murine (Mu) immune interferons (IFN-gamma) were found to be phosphorylated by bovine heart muscle cAMP-dependent protein kinase at a single site, in contrast to human (Hu) IFN-gamma, which was reported to be phosphorylated at two different serine residues. Chromatography of a Staphylococcal aureus V8 protease digest of Ra or MuIFN-gamma indicated that the site of phosphorylation was in the carboxy-terminal undecamer fragment of the protein. Due to inherent problems in measuring both phenylthiohydantoin-serine (PTH-serine) and PTH-phosphoserine with an automated sequenator, a novel approach, involving partial Edman degradation of aliquots of the peptide followed by high performance liquid chromatography (HPLC) analysis, was developed. It was determined that Ser132 is the exclusive site of phosphorylation for both IFNs.
D.P. Abriola, Robert Fields, Stanley Stein, A.D. Mackerall and R. Pietruszko: Active site of human liver aldehyde dehydrogenase. Biochemistry, 26: 5679, 1987.
Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 188.8.131.52) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.
Robert Fields: Measurement of amino groups in proteins and peptides. Biochem J., 124: 581, 1971.
A technique is examined for determining amino groups with 2,4,6-trinitrobenzenesulphonic acid, in which the extinction at 420nm of sulphite complexes of the trinitrophenylated amino groups is measured. The sensitivity of the method is 5-200nmol of amino group. The method is especially suitable for checking the extent of blocking or unblocking of amino groups in proteins and peptides, owing to the short time required for reaction (5min at room temperature). The reaction of the reagent with thiol groups has been studied and was found to proceed 30-50 times faster than with in-amino groups of model compounds. The in(420) of a trinitrophenylated thiol group was found to be 2250m(-1).cm(-1). The reaction with several amino acids, peptides and proteins is presented. The in(420) of a typical alpha-amino group was found to be 22000m(-1).cm(-1) and that of an in-amino group, 19200m(-1).cm(-1). Difficulties inherent in the analysis of constituent amino group reactions in proteins are discussed.
Robert Fields, H.B.F. Dixon, Gillian Law and Chie Yui: Purification of RNase T-1 by DEAE cellulose chromatography. Biochem J., 121: 591, 1971.
A procedure is described for isolating the enzyme ribonuclease T1 from Takadiastase, an extract of the mould Aspergillus oryzae. It involves an initial concentration of the enzyme by adsorption on DEAE-cellulose followed by gradient elution. Later the enzyme is chromatographed on the same adsorbent with an eluent of constant composition. Yields of 350–380mg of ribonuclease T1 from 500g of Takadiastase were obtained.
Robert Fields and H.B.F. Dixon: Micromethod for the determination of reactive carbonyl groups in proteins and peptides using 2,4-DNP-hydrazine. Biochem J., 121: 587, 1971.
A method is described for determining carbonyl groups that is especially suitable for use with proteins and peptides. It involves the determination of the extinction at 370nm of a sample solution after adding 2,4-dinitrophenylhydrazine. The reaction of 2,4-dinitrophenylhydrazine with pyruvoylglycine and with transaminated ribonuclease T(1) is presented; the isolation of protein hydrazones is discussed.
Robert Fields and H.B.F. Dixon: A spectrophometric method for the micro-determination of periodate. Biochem J., 108: 883, 1968.
A method is described for measuring the concentration of periodate over the range 0·2–20μm by adding 1,2-di-(p-dimethylaminophenyl)ethane-1,2-diol to a sample solution. Periodate cleaves this compound to from two molecules of p-dimethylaminobenzaldehyde, the extinction of which is then read at 352mμ. The method has been used to follow the course of periodate oxidations of serine methyl ester, ribonuclease A and ribonuclease S-protein. Addition of the reagent stops further periodate reaction by reducing the remaining periodate to iodate. The presence of protein does not interfere with the assay.
PUBLICATIONS in SCIENTIFIC REFERENCE BOOKS:
Methods in Enzymology:
Robert Fields: The rapid determination of amino groups with TNBS. In “Methods in Enzymology”, Vol. XXV, Part B: 464, 1971.
Methods in Enzymology:
H.B.F. Dixon and Robert Fields: Specific modification of NH2-terminal residues by transamination. In “Methods in Enzymology”, Vol. XXV, B: 409, 1971.
Fibrinogen 3. Biochemistry, Biological Functions, Gene Regulation and Expression
2005 - Takako Fukumoto, Kasthuri Venkateswaran and Robert Fields: “Removal of Enzyme Inhibitors and Interferents from DNA Samples Using a Portable Robotic Instrument”; American Society of Microbiology, Atlanta, GA, USA.
2005 – J.C. Bruckner, R.C. Yalamanchili, R. Fields, R. Sumner and K. Venkateswaran: “Systematic Sampling and Processing of Nucleic Acids for Low Biomass Samples”; American Soc. of Microbiology, Atlanta, GA, USA.
2001 - Robert Fields, Taku Sugawara, Larry Mole and Mark Holodniy: “Fully Automated RNA and DNA Sample Prep Using a Multi-Channel Cartridge-Based Purification System”; CHI Conference, "Genomic Sample Preparation”, Boston, MA, USA.
2000 - J.P. Spencer, R. Fields, E.A. Henchal, D.R. Shoemaker and F.K. Knauert: “A Fully Automated Instrument for Preparing Bacillus anthracis DNA for PCR”; US Army Diagnostics Conference, Ft. Detrick, MD, USA.
1998 - Robert Fields, Fred K. Knauert, Bernard Courtney, & Erik A. Henchal: “Fully Automated Purification of Bacterial DNA In a Sealed Device”; RNA/DNA Diagnostics Conference - Washington, D.C., USA.
1994 - Robert Fields: “Rapid Detection of HIV-1 DNA in a Sealed Containment Cassette”; Tenth International Congress on AIDS, Yokohama, Japan
1991 - Robert Fields: “Automated Peptide Sequencing:
Two-fold increase in sensitivity by a instrument modification, using fine-bore
ETFE tubing”; Protein Society Meeting, San Diego,
Director of Microchemistry 1987–1993
Laboratory Head, DNA/Protein CORE Facility
Procedures and Lab Instrumentation:
DNA, RNA, Protein, Peptide, Carbohydrate Isolation and Purification
HPLC (Hewlett Packard Model 1090), Diode Array Detector 190nm - 720nm (Hewlett Packard ) with UV-Visible spectrum plotter, HPLC Fluorescence Detector (McPherson), HPLC (Hewlett Packard Model 1084) with UV Detector (LKB 2138) Speed Vac, Centrifuges, Freeze Dry Machines
DNA/RNA Oligonucleotide Synthesis and Purification
4-Column DNA/RNA Synthesizer (ABI Model 394-08) with multi-
fraction collector for trityl analysis; Single column DNA
ABI 391 PCR Mate EP) with fraction collector
Peptide Synthesis; instruments for this service included:
FMOC Chemistry (Advanced ChemTech Model 350 MPS) Solid-phase Multiple Peptide synthesis system FMOC chemistry (Milligen Biosearch Model 9600) Solid-phase Peptide synthesis system, one gram capacity.
Automated DNA Sequencing and PCR
Applied Biosystems Automated DNA Sequencer (ABI Model
Perkin Elmer (Model 9600) Thermal Cycler, Heating blocks, Gel casting equipment and Microfuge
Automated Protein Sequencing and Compositional Analysis
Applied Biosystems Protein Sequencer (ABI 477A) Applied Biosystems PTH amino acid analyzer (ABI 120A), Integrator (Spectra Physics), Amino Acid Analyzer (Beckman 6300)
Synthesis of synthetic peptide immunogens: peptide dendrimers, branched chain artificial proteins, iodination of proteins with 32P; radioimmuno assays; scintillation counting; computation of binding affinity
Informatics and Software analysis
reading frame selection, codon usage, hydrophobicity, amphiphilicity
analysis, DNA primer selection, amino acid pattern recognition,
identification of restriction and phosphorylation sites, protein
translation, secondary structure and antigenicity analysis,
determination of membrane spanning regions peptide binding sites.
DNASIS, PROSIS, DNA-STAR, MACAW.
Center for Advanced Biotechnology and Medicine 1986-1987 Piscataway, NJ
Research Associate Interferon and enzyme mechanism
University of Cambridge 1969-1971
Fellow, Science Research Council Ribonuclease enzyme mechanism of action
• Created a novel spectrophometric method for the micro-determination
of periodate, aldehydes and ketones
• Developed new method for quantitatively determining amino groups
in proteins using TNBS
1997 – 2008 IGene Diagnostics, Inc. (Founder and CEO)
1993 – 1997 ITI Molecular Diagnostics, Inc. (Founder and CEO)
1971 – 1985 Omnifit, Limited (Founder & Managing Director)
IGENE DIAGNOSTICS, INC. 1997 – 2008
Chief Executive Officer Analytical Instrument Development
ITI MOLECULAR DIAGNOSTICS, INC 1993 – 1997
New York, NY
Chief Executive Officer HIV Viral Load, R&D
Secured Angel funding; created a working prototype for a high speed PCR thermal cycler; filed PCT patent applications. Viral RNA was purified from patients’ whole blood, then high-speed RT-PCR was then carried out to provide a viral load measurement.
Omnifit, Limited 1971-1983
Founder and Managing Director OMNIFIT® Chromatography Equipment
Created the Omnifit® brand of chemically inert analytical chromatography
products; secured venture funding; supervised R&D, patenting,
manufacturing and marketing of products; set up subsidiary sales offices
in USA and Japan, established worldwide dealerships in France, Germany,
Switzerland, Holland and 10 other countries. Company was sold in 1983.
US Patent No. 3,880,452, entitled “CONNECTORS”.
US Patent No. 3,889,712, entitled “VALVES”.
US Patent No. 4,165,893, entitled “CONNECTOR FOR TUBING”.
US Patent No. 4,291,903, entitled “HIGH PRESSURE
US Patent No. 4,335,739, entitled “SLIDER VALVES”.
U.S. Patent No 5,840,573, entitled “MOLECULAR
AND METHOD OF USE”.
PCT International Patent, entitled “BIOMOLECULAR
granted in UK, France and Germany
PCT International Patent, entitled “MOLECULAR ANALYSER
AND METHOD OF USE”, granted in Canada, Australia, UK, France,
Twenty-eight additional patents granted in the USA, Australia, England,
Germany, France and Japan.
Technological Innovator of the Year, annual UK entrepreneurial award,
Technical Development Capital Ltd.; 1975
CRADA, for developing DNA isolation and purification instruments,
with U.S. Army, 1997-1998
Research Contract for portable DNA fluorescent sequence detection
instrumentation, U.S. Army, 1999
Research Grant A9C 904630 for portable DNA purification instrumentation,
U.S. Federal Bureau of Investigation, 1999
Research Contract for DNA purification instrumentation, NASA Jet
Propulsion Laboratory, 2002
Contract PO-0000164 for portable DNA purification instrumentation,
U.S. Air Force, 2003
Contract 609344 for portable DNA purification instrumentation,
Sandia National Laboratory, 2006