Hon-Reen Kuo

Binding properties of bovine ocular lens zeta-crystallin to right-handed B-DNA, left-handed Z-DNA, and single-stranded DNA.

Bovine zeta‐crystallin has the ability to bind with different DNAs. Initially, this protein was named regulatory factor 36 (Kang et al., 1985), but it has been shown to be an ocular lens zeta‐crystallin (Jörnvall et al., 1993), which is considered an enzyme‐crystallin (Rodakanaki et al., 1989). The enzyme‐linked immunosorbent assay (ELISA) was used to quantitate the binding of bovine zeta‐crystallin to purified high molecular weight double‐stranded (ds‐) and single‐stranded (ss‐) DNA (bovine and synthetic DNA). ELISA quantitation was achieved by the addition of anti‐zeta‐crystallin antibodies to the DNA—zeta‐crystallin complex, using a novel immunochemical avidin—biotin method. Zeta‐crystallin shows much greater binding intensity for ss‐DNA and for ds‐Z‐DNA than for ds‐B‐DNA. It also reacts slightly more with ds‐Z‐DNA than ss‐DNA. Therefore, we speculate that zeta‐crystallin may act as a transcriptional enhancer (outer lens cortex), possibly binding to Z‐DNA regulatory elements within lens crystallin genes. It may also act to protect DNA from endogenous DNase activity and as a DNA unwinding (destabilizing) protein also involved with transcription, occurring in normal adult bovine lens nucleated secondary fiber cells.

Terminal differentiation and left-handed Z-DNA: a review.

Nucleic acids control the expression of genes, and different conformations of DNA structure may regulate cell death. Left‐handed Z‐DNA, which is speculated to function as a transcriptional enhancer, may be directly influenced by the destructive effects of terminal differentiation. The nicking‐denaturation of double‐stranded Z‐DNA could possibly initiate and enhance terminal differentiation within specific tissues.

Identification of early apoptosis in Feulgen‐stained cultured cells in situ by computerized image analysis

A method is presented in which Feulgen-stained apoptotic cells are identified among cultured cells in situ using computerized image analysis of their nuclei. Images of either control (untreated) human lymphoblastoid cell nuclei or of similar cells treated with 3mM methyl methanesulfonate for 1 h, which induces apoptosis in 100% of the cells, were converted to a standard size (80 x 80 pixels) and Fourier transforms and boundary images based on 21 proportional mean gray-level thresholds obtained. The perimeter and the mean fractal dimension of the latter and 39 selected coefficients of the former were then obtained from which data were chosen heuristically and subjected to multivariate linear discrimination analysis. Eighty-two percent of a teaching set of 50 nuclei and 69% of a test set of 29 nuclei were correctly identified as apoptotic versus nonapoptotic by the computer, compared with 57% identified correctly by a panel of 7 pathologists. This study shows the feasibility of using this type of analysis to directly identify apoptotic cells in culture, and probably also in tissues, by direct observation using computerized imaging technology.

Effect of Antihypertensive Therapy on Renal Injury in Type 2 Diabetic Rats With Hypertension

In a previous study, we demonstrated that doxazosin (DZN), an alpha(1)-adrenergic blocker, prevented proteinuria in streptozotocin diabetic rats. In this study, we investigated whether DZN would lower established proteinuria by improving glomerular sclerosis in spontaneously hypertensive corpulent rats with type 2 diabetes mellitus. DZN treatment was compared with treatment with angiotensin-converting enzyme inhibitor, lisinopril (LIS) alone, and DZN in combination with LIS. Combination therapy was used to examine any additive effect of either drug alone in the reduction of proteinuria and glomerular sclerosis. Both male and female rats age 6 months with established proteinuria were used. The rats were allocated randomly to 1 of 4 groups: untreated, DZN treated, LIS treated, or a combination of DZN and LIS treatment. Drug treatment was continued for 16 weeks. The results show that (1) either drug alone or in combination significantly lowered systolic blood pressure; (2) DZN, LIS, or combination therapy reduced albuminuria at 16 weeks of treatment from baseline by 38.61+/-5.77%, 30.70+/-4. 21%, and 42.17+/-4.77% (mean+/-SE), respectively. No difference in albuminuria was observed among the 3 groups of rats; (3) the fractional mesangial area, which was 20.55+/-3.77% in untreated rats, was significantly reduced to 11.18+/-1.32% in DZN-treated rats, with a further reduction to 8.72+/-0.64% in LIS-treated rats and to 3.48+/-0.35% in rats treated with DZN+LIS; and (4) DZN but not LIS significantly improved plasma glucose levels in spontaneously hypertensive corpulent rats (untreated 21.06+/-0.97 mmol/L versus DZN treated 15.81+/-0.93 mmol/L or DZN+LIS treated 17.38+/-1.10 mmol/L; P<0.025 to 0.005). Thus, the data suggest that 16-week treatment with either DZN or LIS improves established proteinuria and glomerular sclerosis, but combination therapy is superior to either DZN or LIS alone in preventing glomerular sclerosis in type 2 diabetic rats with hypertension.

Xeroderma Pigmentosum and Related Disorders

Inherited diseases with cutaneous manifestations currently believed to be associated with defective DNA repair and/or chromosomal instability, of which xeroderma pigmentosum is the prototype, are all very rare. The more important of these, discussed here, are listed in Table 81-1. Diseases above the open space are all (mainly) autosomal recessive disorders; those below it, autosomal dominant. Of the recessive diseases, there are four major entities: (1) xeroderma pigmentosum, which may occur together with a distinctive autosomal recessive disorder, Cockayne disease, and the cells of which may show some very similar changes, with failure of complementation, with those of cells from some cases with yet a third disorder, trichothiodystrophy; (2) Fanconi anemia, an important variant of which, lacking only the congenital anomalies characteristic of Fanconi anemia, is the Diamond-Blackfan syndrome (also known as the Estren-Dameshek variant of Fanconi anemia); (3) Ataxia-Telangiectasia, which shares distinctive cell culture but not clinical characteristics with two other disorders, the Nijmegen breakage syndrome and the Berlin breakage syndrome, which complement each other in complementation assays but are otherwise identical; and (4) Bloom syndrome. There is also less-well-documented evidence for defective DNA repair and/or chromosomal instability in a number of other disorders, such as progeria of various subtypes and dyskeratosis congenita.

Novel DNA Staining Method and Processing Technique for the Quantification of Undamaged Double-stranded DNA in Epidermal Tissue Sections by PicoGreen Probe Staining and Microspectrophotometry

Histotechnological processing of DNA can cause damage to and loss of DNA and can change its structure. DNA probes have severe tissue-staining limitations. New DNA probes and improved histotechnology are needed to enhance the characterization of fixed tissue-bound DNA. Our team developed a novel DNA staining technique and histotechnological processing procedure that improves tissue-bound DNA retention and the qualification and quantification of intact double-stranded (ds)-B-DNA. We used the ultrasensitive PicoGreen ds-DNA probe for the histochemical characterization of ds-DNA. Fifteen fixatives were examined to determine which were best for preventing DNA denaturation and retaining original DNA content and structures. Our use of a microwave–vacuum oven reduced heating temperatures, shortened heating and processing times, and enhanced fixation. We achieved better qualitative and quantitative results by using superior tissue-acquisition techniques (e.g., reduced prefixation times) and improved histotechnology. We also compared our novel approach with archival tissues, delayed fixation, less sophisticated and conventional histological processing techniques, and by experimenting with preservation of tissue-bound ds-Z-DNA. Results demonstrate that our histotechnological procedure and nucleic acid staining method significantly improve the retention of intact, undamaged ds-DNA which, in turn, allows the investigator to more precisely quantify the content and structures of unaltered and undamaged tissue-bound ds-B-DNA. (J Histochem Cytochem 55: 999–1014, 2007)

Localization and quantification of intact, undamaged right-handed double-stranded B-DNA, and denatured single-stranded DNA in normal human epidermis and its effects on apoptosis and terminal differentiation (denucleation).

Quantification of two types of nucleic acids [double-stranded (ds-) and single-stranded (ss-) DNA] was performed to understand the distribution of DNA within the epidermal strata and to examine the effects of DNA structure on gene expression, viz., apoptosis and terminal differentiation. In addition, we examined the precise starting point of cell death within the epidermis (suprabasal layer); examined how DNA structure affects gene expression of melanocytes; and characterized the “transitional cells” located between the stratum granulosum and stratum corneum, viz., epidermal phase transition zone (EPTZ). Ultrasensitive anti-DNA antibody probes (ds-DNA, ss-DNA), the Feulgen reaction, histological stains (morphological characterization) and the terminal deoxyribonucleotidyl transferase (TUNEL) assay (apoptosis) were used to characterize cell death in normal human epidermis. This study characterized, for the first time, the deterioration of right-handed ds-B-DNA and the increase in denatured ss-DNA during epidermal maturation. For the first time, this approach also allowed for the quantitative and qualitative characterization of DNA content and structure in all epidermal strata, using anti-ds-B-DNA and anti-ss-DNA antibodies. In order to improve the retention and quality of DNA, a novel histotechnological processing procedure was used. The results indicate that the largest decline in DNA occurred within the stratum granulosum, followed by the EPTZ, and the stratum spinosum. Not all epidermal nuclei lost DNA, indicating two differentiating keratinocyte pathways, viz., apoptotic and non-apoptotic. Both pathways united in the stratum granulosum. These results suggest that keratinocyte terminal differentiation and apoptosis are distinct cellular events, cell death begins earlier than expected, and molecular epidermal events take place in a gradual and orderly manner within keratinocytes. During maturation, ds-B-DNA decreases as ss-DNA increases. Therefore, during differentiation of keratinocytes, both DNA content and DNA structure are altered.

Introduction of macromolecules into synaptosomes using electroporation

Synaptic terminals are sites of high metabolic activity and thus are particularly vulnerable to oxidative stress. Oxidative damage to proteins can be toxic to neurons and may cause irreversible cell damage and neurodegeneration. A neuroprotective mechanism used by cells to combat oxidative damage is to selectively degrade damaged proteins. Therefore, it is of interest to study the mechanism of degradation of oxidatively damaged proteins in synaptosomes. One way of oxidizing synaptosomal proteins in vitro is by incubating intact synaptosomes in the presence of an oxidizing agent. A problem with this approach is that it may also cause oxidative damage to the machinery required to recognize and degrade oxidized proteins. We have, therefore, introduced a fluorescent macromolecule into synaptosomes to assess the feasibility of using this technique to study how oxidized proteins are degraded and removed from synaptic terminals. Synaptosomes were subjected to electroporation in the presence of FITC labelled-dextran with an average molecular weight of 70000 (FD-70) and non-specific binding was determined by running parallel experiments in lysed synaptosomes. Following extensive washing, synaptosomes were assayed for the presence of intra-synaptosomal FD-70 by measuring fluorescence in a microplate fluorescence reader. Significant differences in fluorescence were found between intact and lysed synaptosomes with maximal uptake at 100 V/ 1500 microF (approx. 36 pmol/mg protein). To determine if membrane transport was compromised by electroporation, uptake of 3H-arginine was compared in control and electroporated synaptosomes. While untreated electroporated synaptosomes showed a loss of 22% in the ability to transport arginine, preincubation in the presence of 1 mM ATP resulted in a complete restoration of arginine transport. These results show that electroporation is a potentially useful technique for introducing a specific oxidized protein, into synaptic terminals so its metabolic fate can be examined.

Use of anti-single-stranded DNA antibodies to localize and quantify denatured DNA during cell death.

The present discussion is a brief overview which examines the most widely utilized methods for the characterization of cell death. We believe that the development of novel molecular biological techniques to identify cell death in tissue sections has not progressed in a rapid manner. Our group believes that the use of experimentally induced anti-single-stranded (ss-) DNA monoclonal antibodies (MAb; Stollar, 1992) is a specific tool which can be used to characterize cell death, with many advantages over the more frequently utilized procedures. Both singleand double-stranded DNA breaks occur under a normal range of non-apoptotic situations. DNA fragmentation does not always accompany apoptosis. Therefore, the use of positive and negative controls is critical to proper evaluation of cell death. The development of denatured single-stranded breaks in the doublestranded (ds-) DNA molecule is an important event in the genesis of cell death. A variety of techniques can be used to characterize denatured ss-DNA, e.g. denatured DNA can be quantified by the OliGreen ss-DNA quantitation kit (Molecular Probes, Inc., Eugene, OR, U.S.A.). Sophisticated methodologies, employ flow cytometry to identify and quantify normal cells or cells undergoing apoptosis and necrosis, based on changes in light scattering, DNA stainability, DNA sensitivity to denaturation or changed plasma membrane transport of Hoechst 33342 (Li et al., 1995). Two other techniques used for examining DNA strand breaks occurring in apoptotic cells can label individually fixed, permeabilized cells or tissue sections by

The quantification and distribution of nasal sebaceous glands using image analysis

The distribution of sebaceous glands in nasal skin is of interest because the presence of these adnexal structures significantly influences the outcome of healing. Using whole nasal skins dissected from cadavers, we prepared tissue sections from the nasal bridge to the nasal tip, both from the midline and lateral aspects. The sebaceous glands in these sections were analyzed for the following parameters: (1) size of the glands, (2) width of luminal cross-sections, and (3) depth of the glands. These parameters were studied using a Leitz Quantimet 500 Plus image analyzer and software to quantify the results. We found that the superior or proximal nasal skin contains fewer, smaller, more superficially located sebaceous glands. The inferior or distal nasal skin contains increased numbers of sebaceous glands which are markedly larger in size. The glands in the distal nose have larger lumina, are situated both superficially and deep in the dermis, and also occupy a greater percentage of the dermis. We identified an anatomical breakpoint on the nasal skin, marking the transition from superficial, small sebaceous glands to superficial-and-deep, enlarged glands. The columella was found to be similar to the proximal nasal skin.