Robert M. Wollman

Detection of epithelial ovarian cancer using 1H-NMR-based metabonomics

Currently available serum biomarkers are insufficiently reliable to distinguish patients with epithelial ovarian cancer (EOC) from healthy individuals. Metabonomics, the study of metabolic processes in biologic systems, is based on the use of 1H‐NMR spectroscopy and multivariate statistics for biochemical data generation and interpretation and may provide a characteristic fingerprint in disease. In an effort to examine the utility of the metabonomic approach for discriminating sera from women with EOC from healthy controls, we performed 1H‐NMR spectroscopic analysis on preoperative serum specimens obtained from 38 patients with EOC, 12 patients with benign ovarian cysts and 53 healthy women. After data reduction, we applied both unsupervised Principal Component Analysis (PCA) and supervised Soft Independent Modeling of Class Analogy (SIMCA) for pattern recognition. The sensitivity and specificity tradeoffs were summarized for each variable using the area under the receiver‐operating characteristic (ROC) curve. In addition, we analyzed the regions of NMR spectra that most strongly influence separation of sera of EOC patients from healthy controls. PCA analysis allowed correct separation of all serum specimens from 38 patients with EOC (100%) from all of the 21 premenopausal normal samples (100%) and from all the sera from patients with benign ovarian disease (100%). In addition, it was possible to correctly separate 37 of 38 (97.4%) cancer specimens from 31 of 32 (97%) postmenopausal control sera. SIMCA analysis using the Coomans plot demonstrated that sera classes from patients with EOC, benign ovarian cysts and the postmenopausal healthy controls did not share multivariate space, providing validation for the class separation. ROC analysis indicated that the sera from patients with and without disease could be identified with 100% sensitivity and specificity at the 1H‐NMR regions 2.77 parts per million (ppm) and 2.04 ppm from the origin (AUC of ROC curve = 1.0). In addition, the regression coefficients most influential for the EOC samples compared to postmenopausal controls lie around δ3.7 ppm (due mainly to sugar hydrogens). Other loadings most influential for the EOC samples lie around δ2.25 ppm and δ1.18 ppm. These findings indicate that 1H‐NMR metabonomic analysis of serum achieves complete separation of EOC patients from healthy controls. The metabonomic approach deserves further evaluation as a potential novel strategy for the early detection of epithelial ovarian cancer.

Using Conformational Analysis to Identify Structurally Conserved Regions of MAP Peptides that Exhibit Cellular Attachment Ability

A natural bioadhesive obtained from Mytilus edulis , mussel adhesive protein, MAP or mefp-1, is frequently used for cellular attachment. MAP is approximately 114 kD, and generally composed of repeating decapeptide units, A-K-P-S-Y-Hyp-Hyp-T-DOPA-K, MAP-RD. Prior nuclear magnetic resonance (NMR) spectroscopy and molecular modeling of MAP-RD revealed an overall bent-helix. NMR spectroscopy and molecular modeling of a MAP fourteen residue peptide, P-S-Y-Hyp-Hyp-T-Y-K-A-K-P-S-Y-Hyp, MAP-14, are presented. Additionally, a molecular model built and minimized from MAP-RD and MAP-14 produced a twenty-six-residue MAP peptide, (MAP-26), that maintained regional structural consistency with both MAP-RD and MAP-14. Multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy and ellipsometry of the MAP-14 as well as that of L-DOPA-containing MAP-14, (MAP-14(D)), showed uniform film formation near a monolayer in thickness was L-DOPA dependent. Significantly more undifferentiated leukocyte cells (MOLT-4) attached to and spread on MAP-14 (D) films (applied at 2 w g cm m 2 ) compared with intact MAP, MAP-RD or tissue culture treated polystyrene, indicating a cellular binding domain presence in the MAP-14 sequence. The culmination of biophysical data indicate the lysine-alanine-lysine (K-A-K) sequence as structurally conserved and responsible for the cellular attachment ability noted for MAP14(D) and ultimately MAP.

A Monte Carlo simulation of hydration of xanthine-derivatives and their stacked forms

Results on a Monte Carlo simulation of the hydration of monomer and possible stacked dimer forms of a purine alkaloid series in 200- and 400-water molecule clusters are presented. Investigation of different purine stacked dimers in a 200-water molecule cluster reveals that for caffeine there exists one, for theophylline two and for theobromine four dimers are energetically favorable. For caffeine, the same energetically favored stacked dimer form is observed in both the 200- and 400-water molecule cluster. The main factor stabilizing the preferred dimer stacks is the change in the interaction between water molecules of the monomer cluster and those water molecules in the dimer cluster.

A novel tumor necrosis factor-α inhibitory protein, TIP-B1

TIP-B1, a novel TNF inhibitory protein, has been identified, purified and characterized from cytosolic extracts of TNF-treated human fibroblasts, and a partial TIP-B1 cDNA clone has been obtained. The (27 kDa pI 4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids), and the nucleotide sequence of the corresponding cDNA clone. TNF-sensitive cells, when exposed to TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Thus, this TIP-B1 treatment effectively makes these cells TNF-resistant. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. TIP-B1 does not interfere with the interactions between TNF and the TNF receptors based on flow cytometric analysis of the cellular binding of biotinylated TNF. These and other data indicate that TIP-B1 is not a soluble TNF receptor, nor an anti-TNF antibody, nor a protease that degrades TNF, yet TIP-BI functions when added exogenously to cells. Thus, TIP-B1 is not one of the proteins previously reported to be involved in resistance to TNF. The fact that incubation of the newly discovered novel TIP-B1 with TNF-sensitive cells protects them from TNF-induced cell death, including TNF-mediated apoptosis, makes TIP-B1 a candidate for therapeutic modulation of TNF-induced effects.

Synthesis, structure, and conformation of anti-tumor agents in the solid and solution states: hydroxyl derivatives of Ftorafur.

ABSTRACT The pyrimidine antimetabolite Ftorafur [FT; 5-fluoro-1-(tetrahydro-2-furyl)uracil] has shown significant antitumor activity in several adenocarcinomas with a spectrum of activity similar to, but less toxic than, 5-fluorouracil (5-FU). It is considered as a prodrug that acts as a depot form of 5-FU, and hence the two drugs exhibit a similar spectrum ofchemotherapeutic activity. Ftorafur is metabolized in animals and humans when hydroxyl groups are introduced into the tetrahydrofuran moiety. These metabolites are also thought to be as active as ftorafur but less toxic than 5-FU. Hydroxyl derivatives: 2′-hydroxyftorafur (III), 3′-hydroxyftorafur (IV) and 2′,3′-dihydroxyftorafur (II) were synthesized and X-ray and NMR studies of these hydroxyl derivatives were undertaken in our laboratories to study the structural and conformational features of Ftorafur and its metabolites in the solid and solution states. X-ray crystallographic investigations were carried out with data collected on a CAD-4 diffractometer. The structures were solved and refined using the SDP crystallographic package of Enraf-Nonius on PDP 11/34 and Microvax computers. All of the compounds studied had the base in the anti conformation. The glycosidic torsion angles varied from −20 to 60 degrees. There is an inverse correlation between the glycosyl bond distances and the χ angle. Molecules with a lower χ angle have a larger bond distance and vice versa. The sugar rings show a wide variation of conformations ranging from C2′-endo through C3′-endo to C4′-exo. The crystal structures are stabilized by hydrogen bonds involving the base nitrogen atom N3 and the hydroxyl oxygen atoms of the sugar rings as donors and the keto oxygens O2 and O4 of the base and the hydroxyl oxygen atoms O2′ and O3′ as acceptors. The NMR studies were carried out on Brüker 400 and 600 MHz instruments. Simulated proton spectra were obtained through Laocoon, and pseudorotational parameters were solved by Pseurot. Presence of syn or anti forms was demonstrated with the use of NOE experiments. The glycosyl conformations in solution vary more widely than in the solid state. The conformations of the sugar molecules are in agreement with the values obtained in the solid state. The studies of the structure and conformation in the solid and solution states give a model for the Ftorafur molecule that could be used in structure, function and biological activity correlation studies.

Structural and Biophysical Characterization of a Cyclic Bioadhesive with Cell Attachment Ability

Structural and cellular attachment analysis identified overall bent helical regions of adhesive peptides identified within mussel adhesive protein (MAP) capable of also attaching cells. DOPA (L-DOPA, 3,4-dihydroxyphenylalanine) is frequently identified and credited for the attachment ability of several marine proteins. Newly designed cyclic peptides (DOPA-G-G-C-G-K-A-K-G-C [cyc-DOPA] & Y-G-G-C-G-K-A-K-G-C [cyc-Y]) derived from structurally conserved regions of several MAP peptides were examined to assist in the understanding of both surface and cellular attachment. Solution-state proton nuclear magnetic resonance (NMR) spectroscopy coupled with molecular modeling and dynamics revealed minimal differences in the structures of the proposed cellular attachment domain within these two peptides. Multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy, ellipsometry, and advancing contact angle analyses showed that formation of thin films by these peptides was L-DOPA- and pH-dependent. When compared with control surfaces, undifferentiated leukocyte cells (MOLT-4) significantly attached and spread onto films created from the cyc-DOPA. The culmination of these structural, biophysical, and cellular attachment techniques reveal a conformation of cyc-DOPA that is capable of both adsorbing to surfaces and then attaching cells that spread. This work supports the sequence K-A-K as the cellular attachment domain, especially when held in a reliable structural conformation.