Lyndon L. Larcom

Three-dimensional bioluminescence tomography with model-based reconstruction

A model-based image reconstruction method, bioluminescence tomography (BLT), is described. BLT has the potential to spatially resolve bioluminescence associated with gene expression in vivo, thus, offering a tomographic molecular imaging method. The three-dimensional spatial map of reporter genes is recovered using a diffusion equation model-based, finite element reconstruction algorithm. The imaging method is demonstrated using both numerical simulations and phantom experiments.

Anticarcinogenic Activity of Strawberry, Blueberry, and Raspberry Extracts to Breast and Cervical Cancer Cells

Freeze-dried fruits of two strawberry cultivars, Sweet Charlie and Carlsbad, and two blueberry cultivars, Tifblue and Premier were sequentially extracted with hexane, 50% hexane/ethyl acetate, ethyl acetate, ethanol, and 70% acetone/water at ambient temperature. Each extract was tested separately for in vitro anticancer activity on cervical and breast cancer cell lines. Ethanol extracts from all four fruits strongly inhibited CaSki and SiHa cervical cancer cell lines and MCF-7 and T47-D breast cancer cell lines. An unfractionated aqueous extract of raspberry and the ethanol extract of Premier blueberry significantly inhibited mutagenesis by both direct-acting and metabolically activated carcinogens.

Measurement of particle size distribution in mammalian cells in vitro by use of polarized light spectroscopy

We demonstrate the feasibility of measuring the particle size distribution (PSD) of internal cell structures in vitro. We use polarized light spectroscopy to probe the internal morphology of mammalian breast cancer (MCF7) and cervical cancer (Siha) cells. We find that graphing the least-squared error versus the scatterer size provides insight into cell scattering. A nonlinear optimization scheme is used to determine the PSD iteratively. The results suggest that 2-microm particles (possibly the mitochondria) contribute most to the scattering. Other subcellular structures, such as the nucleoli and the nucleus, may also contribute significantly. We reconstruct the PSD of the mitochondria, as verified by optical microscopy. We also demonstrate the angle dependence of the PSD.

Quantitative fluorescence of DNA-intercalated ethidium bromide on agarose gels

Techniques for analyzing DNA distributions on agarose gels are examined by both two-dimensional and one-dimensional methods. It is demonstrated that very large errors in DNA concentration occur in such analyses unless (i) the electrophoresis is performed in a careful, reproducible manner, (ii) the films are calibrated with an internal standard, (iii) high resolution densitometry is used for analyzing the films, and (iv) appropriate background controls are used to determine the baselines for integration. Two-dimensional scanning produces more accurate results than one-dimensional scanning, but in cases where the bands are relatively uniform, the one-dimensional analysis gives good results. A technique for determining accurate distributions is described.

Comparative effects of eight varieties of blackberry on mutagenesis

Abstract Diets containing large amounts of fruits and vegetables are known to decrease the probability of developing cancer. The chemical composition of fruits can vary with their genetic characteristics and the environmental conditions under which they are cultivated. Because of this variability, different varieties of the same fruit could be expected to have different effects on processes leading to carcinogenesis. Blackberries have been shown to have anti-carcinogenic potential. Since somatic mutations play a major role in the initiation and progression of cancer, we have compared eight varieties of blackberry grown under the same conditions for their abilities to inhibit carcinogen-induced mutagenesis. Using the Ames assay, we have measured the effects of each of the eight varieties on: 1) mutation induction by 2-amino anthracene (2AA), 2) mutation induction by methyl methanesulfonate (MMS) and 3) cell survival. All varieties were found to strongly suppress 2AA mutagenesis, but have minimal effect on MMS mutagenesis. Experiments were performed with berry juice and with homogenized berries. In addition, berries extracts were acidified to simulate changes which might be caused by the digestive process.

Chronic lymphocytic leukemia lymphocytes lack the capacity to repair UVC-induced lesions.

Cells from chronic lymphocytic leukemia (CLL) patients and from healthy individuals were irradiated with UVC and incubated for varying periods of time. The number of single strand breaks and alkali-labile sites was determined by comet analysis. Unirradiated CLL and healthy cells exhibited no significant numbers of single strand breaks. The extent of DNA damage was found to increase with dose for both healthy and CLL cells. However, the CLL cells had much more extensive DNA fragmentation than healthy cells at each dose. Deoxyribonucleoside supplemented medium inhibited comet formation in both cell types. Thymidine alone produced the same effect. In healthy cells, repair of lesions was complete after 4 h of incubation as indicated by the absence of comet formation. The CLL cells exhibited no significant repair even after 48 h. CLL lymphocytes are killed by very low doses of UVC radiation. The results reported here suggest that this hypersensitivity results from the inability of CLL cells to repair UVC-induced DNA damage and a contributing factor is the low amounts of intracellular deoxyribonucleosides.

Milk stimulates growth of prostate cancer cells in culture.

Concern has been expressed about the fact that cows’ milk contains estrogens and could stimulate the growth of hormone-sensitive tumors. In this study, organic cows’ milk and two commercial substitutes were digested in vitro and tested for their effects on the growth of cultures of prostate and breast cancer cells. Cows’ milk stimulated the growth of LNCaP prostate cancer cells in each of 14 separate experiments, producing an average increase in growth rate of over 30%. In contrast, almond milk suppressed the growth of these cells by over 30%. Neither cows’ milk nor almond milk affected the growth of MCF-7 breast cancer cells or AsPC-1 pancreatic cancer cells significantly. Soy milk increased the growth rate of the breast cancer cells. These data indicate that prostate and breast cancer patients should be cautioned about the possible promotional effects of commercial dairy products and their substitutes.

Diffusion of carbon nanotubes with single-molecule fluorescence microscopy

Single walled carbon nanotubes (SWNTs) are a promising gene and drug delivery system since their physical dimensions mimic nucleic acids. Towards this aim, the hydrophobicity of SWNTs was averted by coating with ribonucleic acid (RNA) polymer [poly(rU)] or bovine serum albumin (BSA) and the consequent diffusion of these synthetic-biomolecular hybrids was studied by single-molecule fluorescence microscopy. The diffusion coefficient for SWNT-poly(rU) was measured at 0.374±0.045μm2∕s and for SWNT-BSA it was 0.442±0.046μm2∕s. Our diffusion study provides a fundamental guidance to gene delivery using SWNT as transporters.

Red raspberries have antioxidant effects that play a minor role in the killing of stomach and colon cancer cells.

Berries and berry extracts possess properties that make them important in the prevention of cancer. The high antioxidant levels of these extracts play a role, but components of the berries can have other effects on cell replication and survival. We chose to test the hypothesis that (i) although the antioxidant capacity of raspberry extracts is important for inhibiting the proliferation of tumor cells, other characteristics of the berry extracts are responsible for a major part of their antiproliferative activity, and that (ii) the relative importance of the antioxidant effect can depend on the cell type being studied. The aim of this study was to assess the relative roles of low pH and high antioxidant levels in the killing of 3 cell types by an aqueous extract from Meeker red raspberries. Stomach, colon, and breast cancer cells were treated with berry extract and with HCl and ascorbic acid solutions of the same pH. A dilution of 7.5% ascorbic acid solution, of the same pH and slightly higher antioxidant concentration than the berry extract, killed less than 10% of the stomach and colon cancer cells. In contrast, the berry extract at this same dilution killed more than 90% of these cells. Antioxidants played a more significant role in the killing of breast cancer cells, however. For these cells, approximately 50% of the killing could be attributed to antioxidant effects. We conclude that the antioxidant effect plays a minor role in the killing of 2 gastrointestinal cell types, but its role in inactivating a breast cancer cell line is much more significant. No evidence of apoptosis was observed, and caspase activation did not contribute to cell killing by the extract.

Molecular typing and fluconazole susceptibility of urinary Candida glabrata isolates from hospitalized patients

At our community teaching hospital between August 1994 and August 1995, Candida glabrata accounted for 14% of all Candida isolates and for 31% of urinary Candida isolates. The culture site was urine for 68% of C. glabrata isolates compared to 30% of all Candida isolates (p < 0.001, chi 2). To study the association between C. glabrata and isolation from the urine, we analyzed all available C. glabrata urinary isolates over a 3-month period (23 isolates from 20 patients) using electrophoretic karyotyping, random amplified polymorphic DNA analysis, and fluconazole susceptibility testing. Random amplified polymorphic DNA generated eight types, although electrophoretic karyotyping generated 17 types. Combining the two methods resulted in 19 types indicating that urinary C. glabrata strains at our hospital are genetically diverse and the association between C. glabrata and urinary tract isolation does not appear to be due to horizontal transmission of a single or small number of strains. In vitro susceptibility tests showed that C. glabrata isolates from patients receiving fluconazole had significantly higher minimum inhibitory concentrations to fluconazole than those not receiving fluconazole (p < 0.05). Despite a limited number of patients and isolates, our data suggest that selection of less susceptible organisms by the presence of antifungal agents may be an important contributor to increased urinary isolation of C. glabrata from patients in our hospital.