Infrared spectroscopic analysis of tumor pathology

Infrared spectra of normal and malignant breast tissues were measured in the 600 cm(-1) to 4000 cm(-1) region. The measured spectroscopic features which are the spectroscopic fingerprints of the tissues contain the vital information about the malignant and normal tissues. Fourier Transform Infrared (FTIR) data on 25 cases of infiterating ductal carcinoma of breast with different grades of malignancy from patients of different age groups were analyzed. The samples were taken from the tumor sections of the tissue removed during surgery. Infrared spectra demonstrate significant spectral differences between the normal and the cancerous breast tissues. In particular changes in frequency and intensity in the spectra of protein, nucleic acid and glycogen vibrational modes as well as the band intensity ratios for lipid/proteins, protein/nucleic acids, protein/glycogen were observed. This allows to make a qualitative and semi quantitative evaluation of the changes in proliferation activity from normal to diseased tissue. It was evident that the sample to sample or patient to patient variations were small and the spectral differences between normal and diseased tissues were reproducible. The findings establish a framework for additional studies, which may enable us to establish a relation of the diseased state with its infrared spectra.     

Physico-chemical and kinetic properties of hexokinase isoenzymes from human normal and tumor tissues]

Individual hexokinase isoenzymes (isoHK) are isolated from normal and malignant human stomach mucosa. IsoHK from tumour tissue are found to have KM for glucose 10 times as low as isoHK from normal tissue. Molecular weights of individual isoHK from normal and tumour tissues are similar (at the range of 112,000-125,000). The treatment of protein preparation with 8M urea in the presence of 1% sodium docecyl sulphate resulted in the appearance of a single band with molecular weight of 58,000-60,000 for all the isoHK under polyacrylamide gel electrophoresis. Intensive bands with molecular weight of 60,000 and 96,000 and a number of minor bands were observed under polyacrylamide gel disc elect-ophoresis in the absence of urea. 2-Mercaptoethanol did not affect the results of disc electrophoresis. It is concluded that the molecule of human hexokinase consists of two subunits with molecular weight of 60,000.     

In vivo investigation of progressive alterations in rat mammary gland tumors by near-infrared spectroscopy

We have investigated mammary gland tissues of female rats treated with 7,12-dimethylbenz[a]anthracene in sesame oil by a near infrared (NIR) spectroscopy finding that the DNA and water contents in the cancerous tissues were larger than those in the normal tissues but that the lipid content in the former was less than that in the latter. With protein contents, however, little difference was observed between the two. Thus, we used a lipid band around 1725 nm (the first overtone of n-alkane) and a protein band around 2054 nm (a combination band of amide A and amide II of polypeptides) for a quantitative evaluation of malignant changes in the mammary gland tissues. The lipid/protein band intensity ratios were calculated from the spectra of the mammary glands in the control animals and those of the noncancerous and cancerous sites in the treated animals. The lipid/protein ratios in the control animals, in the noncancerous sites, and in the cancerous sites were 1.452 +/- 0.221 (n = 5), 0.728 +/- 0.069 (n = 5), and 0.362 +/- 0.060 (n = 5), respectively. These values were significantly different from each other (P < 0.001). The lipid changes observed by near-infrared (NIR) spectroscopy were confirmed by the results obtained from chemical methods for the evaluation of lipid levels in the same samples. Thus, our NIR spectroscopic method would be able not only to discriminate between cancerous and normal tissues but also to distinguish animals with cancers from normal animals. In addition, as the cancer grew, the lipid band intensity decreased, this band was shifted to higher wavelengths, and collagen peaks appeared in the tissues. These findings were supported by histological examinations of the cancerous and normal tissues. The present study indicates that NIR spectroscopy has high specificity and sensitivity in discriminating cancerous tissues from normal mammary glands in animals and it may offer potential for noninvasive, in vivo diagnosis of female breast cancer in the near future.     

Evaluation of the suitability of ex vivo handled ovarian tissues for optical diagnosis by Raman microspectroscopy

A pilot Raman microspectroscopy study of formalin-fixed, paraffin-embedded, and deparaffinized sections from the same ovarian normal and malignant tissues was carried out. This approach was considered in order to evaluate the suitability of these ex vivo tissue handling procedures in discrimination as well as biochemical characterization. The spectra of formalin-fixed normal and malignant tissues exhibited no contamination due to formalin, which is indicated by the absence of strong formalin peaks; spectral features also show significant differences for normal and malignant tissues. The differences between spectral profiles of deparaffinized normal and malignant tissues are subtle and spectra show few residual sharp peaks of paraffin. Complete dominance of paraffin swamping signals from tissues was observed in the spectra of paraffin-embedded tissues. Principal components analysis (PCA), which was employed for discrimination of tissue type, provided good discrimination for formalin-fixed and paraffin-embedded tissue spectra. PCA of deparaffinized tissues resulted in a poor classification with significant overlap among the clusters. Thus, this study indicates that formalin fixation is the most suitable among the three procedures employed in the study. Significant differences between spectral profiles of normal and malignant formalin-fixed tissues can not only be exploited for discrimination but can also provide information on biochemical characteristics of the tissues. Deparaffinized tissues provide poor discrimination and information on tissue biochemistry is lost. Paraffin-embedded tissues may provide good discrimination, but predominance of paraffin in the spectra could jeopardize biochemical characterization. Prospectively, as a result of the better availability of paraffin-embedded tissues and problems associated with frozen sectioning of formalin-fixed tissues, the results of this study using paraffin-embedded tissues are very encouraging.     

Vibrational spectroscopy studies of formalin-fixed cervix tissues

Optical histopathology is fast emerging as a potential tool in cancer diagnosis. Fresh tissues in saline are ideal samples for optical histopathology. However, evaluation of suitability of ex vivo handled tissues is necessitated because of severe constraints in sample procurement, handling, and other associated problems with fresh tissues. Among these methods, formalin-fixed samples are shown to be suitable for optical histopathology. However, it is necessary to further evaluate this method from the point of view discriminating tissues with minute biochemical variations. A pilot Raman and Fourier transform infrared (FTIR) microspectroscopic studies of formalin-fixed tissues normal, malignant, and after-2-fractions of radiotherapy from the same malignant cervix subjects were carried out, with an aim to explore the feasibility of discriminating these tissues, especially the tissues after-2-fractions of radiotherapy from other two groups. Raman and FTIR spectra exhibit large differences for normal and malignant tissues and subtle differences are seen between malignant and after-2-fractions of radiotherapy tissues. Spectral data were analyzed by principal component analysis (PCA) and it provided good discrimination of normal and malignant tissues. PCA of data of three tissues, normal, malignant, and 2-fractions after radiotherapy, gave two clusters corresponding to normal and malignant + after-2-fractions of radiotherapy tissues. A second step of PCA was required to achieve discrimination between malignant and after-2-fractions of radiotherapy tissues. Hence, this study not only further supports the use of formalin-fixed tissues in optical histopathology, especially from Raman spectroscopy point of view, it also indicates feasibility of discriminating tissues with minute biochemical differences such as malignant and after-2-fractions of radiotherapy.     

Oxytocin,oxytocin-associated neurophysin and the oxytocin receptor in the human prostate

Oxytocin has been implicated in the regulation of prostate growth. However, the cellular localisation of oxytocin in the normal and diseased human prostate is not known. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were detected by immunohistochemistry in tissues from patients undergoing routine prostatectomy and in normal human prostate epithelial and stromal cell lines. Western blot analysis detected a single band at 14 kDa with neurophysin antiserum and a 66-kDa band with oxytocin receptor antiserum in epithelial and stromal cell lines. Similar sized bands were also detected in extracts of hyperplastic and adenocarcinomic prostate tissues. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were present in stromal and epithelial cell lines and in tissue from patients with benign prostatic hyperplasia. The peptides were localised predominantly to the epithelial cells, although discrete areas of stromal staining were also observed. There was a significant difference in the intensity of oxytocin-staining between tissue displaying benign prostatic hyperplasia and invasive carcinoma, with less immunoreactivity being present in the malignant epithelial cells. Thus, oxytocin and its neurophysin and receptor are present in epithelial and stromal cells of the human prostate. Oxytocin expression is reduced with tumour progression and may provide a marker for invasive disease.This work was supported by a Project Grant (007756) from the Wellcome Trust and from Lottery Health Research     

Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection.Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues.An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders.     

FTIR spectroscopic analyses of human placental membranes

We investigated the differences in the Fourier transform infrared (FTIR) spectra of normal and abnormal human placentas. Normal placentas, placentas with infant intrauterine growth restriction (IUGR), and placentas from mothers with diabetes mellitus (DM) were used, none of which had been treated before measurement. The tissues were divided into three parts: the upper one-third portion (P1), the middle portion (P2), and the lower one-third portion (P3). Placental tissues were also investigated histochemically. The differences of the main second-derivative FTIR spectra among P1, P2, and P3 in normal placentas were observed in bands appearing between 1080 and 1090 cm(-1). Bands in P2 were observed at 1083 cm(-1), which was significantly higher than that in P3 (p < 0.05). The spectrum of P2 tissue in placentas with infant IUGR had a peak at 1081 cm(-1), which was significantly different from those of P1 and P3 (p < 0.05). In placentas with DM, the P2 band was shifted to a peak at 1088 cm(-1). These data were well correlated with the histochemical sugar-chain staining pattern of the P2 portion of the placenta. Our data suggested that this IR technique is applicable to the clinical diagnosis of diseases in the gynecological field.     

A1 reduction in intact cyanobacterial photosystem I particles studied by time-resolved step-scan Fourier transform infrared difference spectroscopy and isotope labeling

Time-resolved step-scan Fourier transform infrared (FTIR) difference spectroscopy, with 5 mus time resolution, has been used to produce P700(+)A(1)(-)/P700A(1) FTIR difference spectra in intact photosystem I particles from Synechococcus sp. 7002 and Synechocystis sp. 6803 at 77 K. Corresponding spectra were also obtained for fully deuterated photosystem I particles from Synechococcus sp. 7002 as well as fully (15)N- and (13)C-labeled photosystem I particles from Synechocystis sp. 6803. Static P700(+)/P700 FTIR difference spectra at 77 K were also obtained for all of the unlabeled and labeled photosystem I particles. From the time-resolved and static FTIR difference spectra, A(1)(-)/A(1) FTIR difference spectra were constructed. The A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled trimeric photosystem I particles from both cyanobacterial strains are very similar. There are some mode frequency differences in spectra obtained for monomeric and trimeric PS I particles. However, the spectra can be interpreted in an identical manner, with the proposed band assignments being compatible with all of the data obtained for labeled and unlabeled photosystem I particles. In A(1)(-)/A(1) FTIR difference spectra obtained for unlabeled photosystem I particles, negative bands are observed at 1559 and 1549-1546 cm(-)(1). These bands are assigned to amide II protein vibrations, as they downshift approximately 86 cm(-)(1) upon deuteration and approximately 13 cm(-)(1) upon (15)N labeling. Difference band features at 1674-1677(+) and 1666(-) cm(-)(1) display isotope-induced shifts that are consistent with these bands being due to amide I protein vibrations. The observed amide modes suggest alteration of the protein backbone (possibly in the vicinity of A(1)) upon A(1) reduction. A difference band at 1754(+)/1748(-) cm(-)(1) is observed in unlabeled spectra from both strains. The frequency of this difference band, as well as the observed isotope-induced shifts, indicate that this difference band is due to a 13(3) ester carbonyl group of chlorophyll a species, most likely the A(0) chlorophyll a molecule that is in close proximity to A(1). Thus A(1) reduction perturbs A(0), probably via a long-range electrostatic interaction. A negative band is observed at 1693 cm(-)(1). The isotope shifts associated with this band are consistent with this band being due to the 13(1) keto carbonyl group of chlorophyll a, again, most likely the 13(1) keto carbonyl group of the A(0) chlorophyll a that is close to A(1). Semiquinone anion bands are resolved at approximately 1495(+) and approximately 1414(+) cm(-)(1) in the A(1)(-)/A(1) FTIR difference spectra for photosystem I particles from both cyanobacterial strains. The isotope-induced shifts of these bands could suggest that the 1495(+) and 1414(+) cm(-)(1) bands are due to C-O and C-C modes of A(1)(-), respectively.     

Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation.

The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So(2)) in vivo. We implemented a system for in vivo noninvasive measurements of So(2). A laser light was focused onto areas of 15-30 microm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So(2) was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm(-1) range (oxidation state marker band nu(4)), as well as from the nu(19) and nu(10) bands (1,500- to 1,650-cm(-1) range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary path length and Hb concentration did not affect So(2) estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So(2) estimations and So(2) measured by CO-oximetry was highly significant for nu(4), nu(10), and nu(19) bands. The method allowed So(2) determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So(2) determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.     

Replication kinetics of X chromosomes in fibroblasts and lymphocytes

Summary The kinetics of replication for early and late replicating X chromosomes in karyotypically normal fibroblasts and lymphocytes was studied using terminal bromodeoxyuridine (BrdU) treatment followed by Hoechst/light/Giemsa staining. Although the order of band appearance differs between the two tissues, the programme (order and interval between band appearances) for early replicating bands (dark R-bands) is identical in the two homologues. This is probably also the case for later replicating bands (dark G-bands) though the criteria for derermining mean band appearance times are less reliable for these bands when terminal BrdU treatment is used. This means that the late X has a delayed start but thereafter proceeds at the same pace as its early counterpart.     

Characteristics and variations of B-type DNA conformations in solution: a quantitative analysis of Raman band intensities of eight DNAs

Raman spectra were obtained from four bacterial DNAs varying in GC content and four periodic DNA polymers in 0.1 M NaCl at 25 degrees C. A curve fitting procedure was employed to quantify and compare Raman band characteristics (peak location, height, and width) from 400 to 1600 cm-1. This procedure enabled us to determine the minimum number of Raman bands in regions with overlapping peaks. Quantitative comparison of the Raman bands of the eight DNAs provided several new results. All of the DNAs examined required bands near 809 (+/- 7) and 835 (+/- 5) cm-1 to accurately reproduce the experimental spectra. Since bands at these frequencies are associated with A-family and B-family conformations, respectively, this result indicates that all DNAs in solution have a mixture of conformations on the time scale of the Raman scattering process. Band characteristics in the 800-850-cm-1 region exhibited some dependence on CG content and base pair sequence. As previously noted by Thomas and Peticolas [Thomas, G. A., & Peticolas, W. L. (1983) J. Am. Chem. Soc. 105, 993], the poly[d(A)].poly[d(T)] spectra were qualitatively distinct in this region. The A-family band is clearly observed at 816 cm-1. The intensity of this band and that of the B-family band at 841 cm-1 were similar, however, to intensities in the natural DNA spectra. Three bands at 811, 823, and 841 cm-1 were required to reproduce the 800-850-cm-1 region of the poly[d(A-T)].poly[d(A-T)] spectra. This may indicate the presence of three backbone conformations in this DNA polymer. Analysis of intensity vs. GC content for 42 Raman bands confirmed previous assignments of base and backbone vibrations and provided additional information on a number of bands.     

Neuromedin B and its receptor are mitogens in both normal and malignant epithelial cells lining the colon

Bombesin-like peptides are uniformly thought to act as mitogens in cancer. Yet by studying human tissues, we have recently shown that bombesin and its mammalian homologue gastrin-releasing peptide act as morphogens, promoting tumor differentiation when aberrantly upregulated in colon cancer. In contrast, little is known about the bombesin-like peptide neuromedin B (NMB) and its receptor (NMB-R) in the human gastrointestinal tract. We therefore studied their presence and function in normal and malignant human colonic epithelia. Anti-NMB monoclonal antibodies were made against keyhole limpet hemocyanin (KLH)-conjugated human NMB, whereas anti-NMB-R antibodies were raised in rabbits against KLH-conjugated peptides corresponding to the third intracellular loop and COOH-terminal tail of the receptor protein. NMB antibody recognized two bands at approximately 1.2 kDa and approximately 1.5 kDa. NMB-R antibodies recognized a band at 80 kDa (predicted 43 kDa); whereas treatment with the deglycosylating agent peptide-N-glycosidase generated bands at 65, 47, and 43 kDa. By immunohistochemistry, both NMB and NMB-R were expressed in normal and cancerous colonic epithelial tissues. In cancer, the amount of NMB was similar to that expressed by proliferating epithelial cells located within the crypt. In contrast, NMB-R expression was increased in cancer, with higher levels detected in better differentiated tumor cells. To assess NMB function, proliferation was determined in the nonmalignant human colonic epithelial cell line NCM-460 and in the colon cancer cell lines Caco-2 and HT-29. Exogenously added NMB was 50-100% more efficacious than gastrin-releasing peptide in causing tumor cell proliferation, whereas only NMB increased NCM-460 cell proliferation. These findings indicate that NMB and its receptor are coexpressed by proliferating cells in which they act in an autocrine fashion with similar and modest potency in both normal and malignant colonic epithelial cells.     

Laser Raman investigations of intact single muscle fibers. Protein conformations

Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.     

Study of articular cartilage and the synovial membrane using IR spectroscopy

The infrared spectra of normal knee joint cartilage, normal and rheumatoid arthritis-affected human synovial membrane and the same normal bovine tissues were obtained over the region of 400--4000 cm-1. A comparative analysis of the spectra of these tissues and those containing hyaluronate, protein-chondroitin-keratan sulfate aggregates of cartilage proteoglycans and heparin made it possible to identify greater absorption bands of these biopolymers in the tissue spectra. The interpretation of the results obtained is presented.     

Discrimination of normal, benign, and malignant breast tissues by Raman spectroscopy

Breast cancers are the leading cancers among females. Diagnosis by fine needle aspiration cytology (FNAC) is the gold standard. The widely practiced screening method, mammography, suffers from high false positive results and repeated exposure to harmful ionizing radiation. As with all other cancers survival rates are shown to heavily depend on stage of the cancers (Stage 0, 95%; Stage IV, 75%). Hence development of more reliable screening and diagnosis methodology is of considerable interest in breast cancer management. Raman spectra of normal, benign, and malignant breast tissue show significant differences. Spectral differences between normal and diseased breast tissues are more pronounced than between the two pathological conditions, malignant and benign tissues. Based on spectral profiles, the presence of lipids (1078, 1267, 1301, 1440, 1654, 1746 cm(-1)) is indicated in normal tissue and proteins (stronger amide I, red shifted DeltaCH2, broad and strong amide III, 1002, 1033, 1530, 1556 cm(-1)) are found in benign and malignant tissues. The major differences between benign and malignant tissue spectra are malignant tissues seem to have an excess of lipids (1082, 1301, 1440 cm(-1)) and presence of excess proteins (amide I, amide III, red shifted DeltaCH2, 1033, 1002 cm(-1)) is indicated in benign spectra. The multivariate statistical tool, principal components analysis (PCA) is employed for developing discrimination methods. A score of factor 1 provided a reasonable classification of all three tissue types. The analysis is further fine-tuned by employing Mahalanobis distance and spectral residuals as discriminating parameters. This approach is tested both retrospectively and prospectively. The limit test, which provides the most unambiguous discrimination, is also considered and this approach clearly discriminated all three tissue types. These results further support the efficacy of Raman spectroscopic methods in discriminating normal and diseased breast tissues.     

Isoforms of wild type proteins often appear as low molecular weight bands on SDS‐PAGE

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS‐PAGE), is a technique commonly used to detect specific proteins. SDS‐PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography – mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non‐malignant HEK293 and cancerous MDA‐MB231 (MB231) cells separated using SDS‐PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS‐PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer‐therapy studies.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

Complex mitochondrial DNA in animal thyroids. A comparative study.

1. The frequency of circular dimers and catenanes was determined in thyroid mitochondrial DNA (mtDNA) from rabbits, mice, pigs, sheep and cattle. 2. The mtDNA from freshly removed thyroids was isolated by buoyant density centrifugation in ethidium bromide/CsCl gradients after DNAase treatment of the mitochondrial pellet. Typically, more than 90% of the recovered mtDNA was found in the lower band, indicating a low rate of nicking during isolation. A sample of the total mtDNA (upper and lower bands) was examined by electron microscopy after preparation by the aqueous protein film technique. 3. The frequency of circular dimers generally ranged from 0.1 to 0.3%. However, in an mtDNA sample from cow thyroid, the frequency of circular dimers was 0.6% (0.9% if circular dimers occuring in catenanes are included(, differing significantly from the frequency of these forms in bull thyroid, 0.1%. A small but significant variability also occurred in the frequency of catenanes ranging from 2 to 8% in the different groups; this variation is within the limits usually observed in normal tissues. 4. These observations indicate that thyroids, like other normal tissues examined so far, have a low content of circular dimers. A high frequency of these forms seems to be the trademark of some genetically and physiologically abnormal cells such as certain established cell lines, virus-transformed cells and malignant or otherwise pathological tissues.     

Synthesis and conformational studies of ribooligonucleotides which contain an alternating C-G sequence and show unusual circular dichroism spectra

The poly [r(C-G)] duplex shows an unusually large negative CD band in the long wavelength region. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized by a phosphotriester method and their properties were examined by UV, CD, 1H and 31P NMR spectroscopy. These ribooligomers form self-duplexes at low temperature, the CD spectra of which show negative bands at around 290 nm and positive bands at around 265 nm. The results of 1H nuclear Overhauser effect experiments, 1H chemical shift-temperature profiles of base protons, and the sharp singlet observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. The CD-temperature profiles and 31P NMR spectra support this conclusion. These results indicate that RNA duplexes with an alternating C-G sequence can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.