Christian P. Schultz

Study of chronic lymphocytic leukemia cells by FT-IR spectroscopy and cluster analysis

The peripheral mononuclear cells from 23 normal individuals and the purified B cells from 38 patients with chronic lymphocytic leukemia (CLL) were examined by Fourier transform infrared (FT-IR) spectroscopy. Differences were observed between the CLL and normal cells at the DNA, protein and lipid levels, with CLL cells having greater DNA and lower lipid contents than normal cells. In addition, the spectral character of the CLL and normal cells varied, demonstrating that there were also qualitative differences in the DNA and lipids. Statistical analysis, based on hierarchical clustering, separated normal from CLL cells completely and classified them into two subgroups for normal cells, while the CLL cells could be divided into three subgroups that were distinct from the normal cells. These differences were based on the lipid and DNA content and the overall spectral character of the cells.

Two‐dimensional mid‐IR and near‐IR correlation spectra of ribonuclease A: Using overtones and combination modes to monitor changes in secondary structure

We introduce near-IR spectroscopy as an ancillary tool for monitoring structural changes of proteins in aqueous solution using ribonuclease A (RNase A) as a model protein. The thermal unfolding of RNase A results in clear spectral changes in the near-IR and the mid-IR regions. In the near-IR the most pronounced changes are observed in the spectral region between 4820 and 4940 cm-1. The strong N-H combination band found at 4867 cm-1 in the spectrum of native RNase A shifts to 4878 cm-1 upon thermal unfolding. Hydrogen-deuterium exchange experiments that validate the N-H character of this mode can also be used to estimate the number of unexchanged amide protons after exposure to D2O. The transition profiles and temperatures derived from the temperature dependence of the N-H combination mode were found to be practically identical with those derived from the temperature dependence of the C = O amide I band in the mid-IR region, demonstrating that the near-IR region can be used as a conformation-sensitive monitor for the thermally induced unfolding of proteins in H2O solution. A 2-dimensional correlation analysis was applied to the mid-IR and near-IR spectra of RNase A to establish correlations between IR bands in both regions. The correlation analysis demonstrates that the thermal unfolding of RNase A is not a completely cooperative process; rather it begins with some changes in beta-sheet structure, followed by the loss of alpha-helical structures, and then ending with the unfolding of the remaining beta-sheets.

Comparison of infrared spectra of CLL cells with their ex vivo sensitivity (MTT assay) to chlorambucil and cladribine

The drug resistance of leukemic cells from 21 patients with chronic lymphocytic leukemia (CLL) to the alkylating agent chlorambucil (CLB) and the nucleoside analog cladribine or 2-chlorodeoxyadenosine (CdA) was investigated by infrared spectroscopy. Drug sensitivities, determined in vitro with the tetrazolium dye (MTT) assay, were correlated with the infrared spectra of the CLL cells, applying linear discriminant analysis (LDA). The 63 spectra (three from each of the 21 samples), obtained before drug exposure, were successfully partitioned into drug-sensitive and drug-resistant groups; the LDA-based ex vivo prediction of the sensitivity to CdA or CLB was 85.7% and 80.3%, respectively. Similar changes in the composition/structure of DNA were observed between the spectra of the drug-sensitive and drug-resistant CLL cells for both CdA and CLB. However, CdA-resistant CLL cells could also be differentiated from CdA-sensitive CLL cells by spectral changes associated with membrane lipids; these differences were much less pronounced between CLB-resistant and CLB-sensitive CLL cells. We demonstrate here for the first time that infrared spectroscopy can be used as a new tool for predicting ex vivo drug response (sensitivity/resistance).

Prognosis of chronic lymphocytic leukemia from infrared spectra of lymphocytes

Abstract Peripheral mononuclear cells obtained from blood of normal individuals and from patients with chronic lymphocytic leukemia (CLL) were investigated by infrared spectroscopy and multivariate statistical analysis. Not only are the spectra of CLL cells different from those of normal cells, but hierarchical clustering also separated the CLL cells into a number of subclusters, based on their different DNA content, a fact which may provide a useful diagnostic tool for staging (progression of the disease) and multiple clone detection. Moreover, there is evidence for a correlation between the increased amount of DNA in the CLL cells and the in-vivo doubling time of the lymphocytes in a given patient.

Infrared spectroscopic study of bryostatin 1-induced membrane alterations in a B-CLL cell line

Previous studies on intact cells have shown that bryostatin 1 (Bryo 1) induces significant alterations in the membranes of WSU-CLL cells (a drug-resistant B-CLL cell line), changes which may play an important role in the mechanism of reduced drug resistance of B-CLL cells to 2-chlorodeoxyadenosine (2-CdA). However, it is not clear whether the plasma membranes or the mitochondria, or both are involved; nor is it known which of these two targets is more important for regaining the cells former drug sensitivity. For the present study, we treated WSU-CLL cells with Bryo 1, isolated plasma membranes and mitochondria, and then subjected the purified fractions to infrared (IR) spectroscopic and chromatographic analyses. IR spectroscopy revealed a decreased glycosylation of both plasma membranes and mitochondria in Bryo 1-treated cells compared to untreated cells. The amount of lipid relative to protein was increased in both types of membranes, but considerably more enhanced in the plasma membrane fraction of the Bryo 1-treated cells than in mitochondria. Quantitative lipid analysis by thin layer chromatography also revealed that Bryo 1 treatment significantly increased the phospholipid content in plasma membranes, whereas the lipids in the mitochondria remained essentially unchanged. Changes in lipid composition were quite dramatic for plasma membranes where phosphatidylcholines were decreased by 50%, phosphatidylethanolamines doubled and sphingomyelins increased five-fold compared to the lipid composition in plasma membranes of untreated cells. In addition, the IR spectroscopic analysis provided evidence for an increased plasma membrane fluidity in Bryo 1-treated cells, whereas the fluidity of the mitochondria remained essentially unchanged; marker bands indicating mitochondrial DNA decreased upon Bryo 1 treatment. These results suggest that Bryo 1 increases the sensitivity of WSU-CLL cells to chemotherapeutic agents such as 2-CdA by action on two cell targets: (1) introduction of significant changes in plasma membrane permeability or fluidity through modifications in lipid content and composition as well as by reducing the surface glycosylation; (2) introduction of changes in lipid and DNA content of the mitochondria. Small alterations in the lipid composition of the mitochondria may provide the conditions for an altered proton gradient and transmembrane potential leading to apoptosis and decreased cell survival.

Infrared spectroscopic investigation of in vivo and ex vivo human nails

Abstract Mid (700–4000 cm −1 ) and near (4000–10000 cm −1 ) infrared spectra of viable and clipped human finger nails are presented. Mid-infrared depth profiles acquired by physically etching the nail plate and those acquired nondestructively using photoacoustic spectroscopy (PAS) are compared. The dorsal, intermediate, and ventral layers of the nail plate could be discerned spectroscopically. Near infrared (NIR) attenuated total reflectance (ATR), NIR-PAS and NIR diffuse reflectance spectra of the nail plate are compared. At high energies (> 7000 cm −1 ), ATR lacks sensitivity while the diffuse reflectance spectra contain significant contributions from deep within the finger. At lower energies ( −1 ) ATR can be used to probe the near surface of the nail while diffuse reflectance and PA spectra contain only minor contributions from the nail bed and primarily represent the deeper portion of the nail. The low energy near infrared region appears to be the most valuable region for viable nail plate diagnostic spectroscopy. This region is discussed in considerable detail.

A new subfamily of short bacterial adenylate kinases with the Mycobacterium tuberculosis enzyme as a model: A predictive and experimental study.

The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of “short” bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8°C). Based on various spectroscopic data, on the known three‐dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238–248.

Similarities between the sensitivity to 2-chlorodeoxyadenosine of lymphocytes from CLL patients and bryostatin 1-treated WSU-CLL cells: an infrared spectroscopic study

Infrared (IR) spectroscopy was used to compare the drug resistance mechanism of cells from chronic lymphocytic leukemia (CLL) patients with that of WSU-CLL cells. Bryostatin 1 (Bryo 1), a macrocyclic lactone and protein kinase C activator, was used to render WSU-CLL cells more susceptible to 2-chlorodeoxyadenosine (2-CdA). The IR spectroscopic analysis revealed some changes in protein and DNA content in Bryo 1-treated WSU-CLL cells, however, the most significant alterations were observed in the membrane lipids, which resemble those found between 2-CdA-sensitive and 2-CdA-resistant cells from CLL patients. In addition, Bryo 1 treatment induced WSU-CLL cells to become CD11c, CD25 and tartrate-resistant acid phosphatase-positive, specific markers for hairy cell leukemia, a disease exquisitely sensitive to 2-CdA. Our results suggest that 2-CdA-sensitive CLL cells have cellular characteristics resembling the hairy cell stage. The similarity between the membrane lipids in 2-CdA-sensitive CLL cells and the Bryo 1-treated WSU-CLL cell line supports the suggestion that membrane lipid alteration might be an important step in the drug resistance mechanism of CLL cells.

APPLICATION OF FT-IR MICROSPECTROSCOPY IN DIAGNOSING THYROID NEOPLASMS

Abstract Tissue biopsies and fine-needle aspirates (FNA) of patients with suspected benign or malignant thyroid neoplasms were investigated by infrared microspectroscopy and multivariate statistical methods. Unsupervised cluster analysis revealed four different spectral patterns for the aspirates analyzed, corresponding to colloid goiter, adenoma, carcinomas and negative diagnoses. Infrared microspectroscopic measurements of neoplastic cells on infrared transparent slides provide a potentially new tool for diagnostic screening of these FNA. Biopsy material obtained during surgical removal of gland tissue, was successfully used to generate statistically significant criteria for the distinction of neoplastic from normal tissue. Bivariate histogram plots demonstrate that two selected parameters, DNA and protein, are sufficient to separate control tissue from adenoma and carcinomas

Emerging Medical Applications of Infrared Technologies

Throughout the 70’s and 80’s, biologists discovered infrared spectroscopy; meanwhile, many complex biomolecules have yielded their structural secrets to the inquisitive IR spectroscopist. In the 90’s, infrared spectroscopy is now being challenged by the medical community, particularly so since other spectroscopic techniques such as magnetic resonance imaging and spectroscopy already are well entrenched in medical research and practice. The major endeavor for the biological infrared spectroscopists is now to apply the knowledge they have gained from studies on isolated biomolecules to the complex world of medicine. There is no doubt, however, that with increasing complexity of the molecular structures under investigation the role of infrared spectroscopy keeps changing, as different questions are being asked of this “new” and yet old spectroscopic technique.