Comparison of apoptosis and terminal differentiation: the mammalian aging process.

Apoptosis is the ordered chain of events that lead to cell destruction. Terminal differentiation (denucleation) is the process in which cells lose their nuclei but remain functional. Our group examined cell death in three tissues using two different fixatives and a postfixation procedure, involving young (5 months) and old (2 years) guinea pigs. The data reveal that B-DNA and Z-DNA content decreases, whereas single-stranded (ss-) DNA increases, in older tissues undergoing apoptosis (skin and cornea) and terminal differentiation (ocular lens). We speculate that some of the factors that contribute to the aging process might also be responsible for the enhanced amount of damaged DNA in older tissues undergoing cell death. (J Histochem 49:929-930, 2001)

Novel use of bovine zeta-crystallin as a conformational DNA probe to characterize a phase transition zone and terminally differentiating fiber cells in the adult canine ocular lens.

Using a novel immunocytochemical staining method, we aimed to characterize the phase transition zone (PTZ) (approximatly 100 microm) in adult ocular lenses and the process of terminal differentiation (denucleation) within normal fiber cells. The binding to DNA of zeta-(zeta) crystallin (Z-DNA-binding protein) and anti-double-stranded (ds-)-B-DNA antibody probes was found to decline gradually throughout denucleating fibers, with a precipitous decrease occurring at about 100 microm (PTZ). Nuclei of superficial fiber cells (in front of the PTZ) showed the highest DNA probe-binding values, followed by middle fibers (MF) and deep fibers (DF). With the use of zeta-crystallin, anti-ds-B-DNA antibody, and anti-single stranded (ss-) DNA antibody probes, it was possible to reveal a loss of reactivity of fiber cell ds-DNA. Ss-DNA antibody binding was seen initially in the MF and reached its highest intensity level in the DF. The pattern of zeta-crystallin probe-DNA reactivity correlates with the loss of anti-B-DNA antibody staining and decreased eosin-protein staining. These data suggest that a reorganization of DNA and intracellular protein supramolecular order in normal adult lenses occurs at a depth of about 100 microm (PTZ).

Damage-resistant DNA synthesis in Fanconi anemia cells treated with a DNA cross-linking agent.

Fanconi anemia (FA) is a recessive disorder associated with diverse congenital anomalies, progressive bone marrow failure, and a marked predisposition to develop cancer. At the cellular level, FA is characterized by a prolonged G(2) phase in proliferating cells and a marked hypersensitivity to both the cytotoxic and the clastogenic effects of agents which produce DNA interstrand cross-links. Treatment with these agents leads to even further prolongation of the G(2) phase in FA cells. We now show that FA cells, from four different complementation groups, fail to decrease their rates of replicative DNA synthesis, as do normal cells, following treatment with a DNA cross-linking agent. This may be responsible for the prolongation of the G2 phase seen in these cells, and suggests that the fundamental defect in response of FA cells to DNA cross-linking agents may be in the S phase, rather than the G(2) phase, of the cell cycle.

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.

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.

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.

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.

Echogenicity of hepatic versus portal vein walls revisited with histologic correlation.

The portal vein wall typically is hyperechoic over a wide range of beam-vessel angles, whereas the hepatic vein wall is hyperechoic only when the incident beam and the vessel are perpendicular. This has been attributed to marked discrepancies in mural thickness, collagen content, or perivascular fat between portal and hepatic veins. We evaluated histologically the walls of portal and hepatic veins using three cadaveric livers. For vessels with luminal diameter above 2 to 3 mm, hepatic vein and portal vein wall thicknesses were similar such that portal vein walls were not more than 50% thicker than those of hepatic veins of comparable size. Hepatic vein walls were mostly composed of parallel, tightly packed collagen fibers. In contrast, portal vein walls were composed of loosely arrayed, nonparallel connective tissue fibers which were separated by multiple intervening spaces and only a minority of which were collagenous. Perivascular fat was not identified adjacent to intrahepatic vessels beyond the liver hilus. The marked differences in echogenicity between portal vein and hepatic vein walls typically observed at ultrasonography thus cannot be attributed to differences in mural thickness, collagen content, or perivascular fat between these vessels. Rather, the distinct composition of the hepatic vein wall renders it a specular reflector, which is hyperechoic only when the angle between the ultrasound beam and the vessel wall is close to 90 degrees, whereas the composition of the portal vein wall enables it to appear hyperechoic at a wide range of beam-vessel angles.

Localization of B-DNA and Z-DNA in terminally differentiating fiber cells in the adult lens.

We examined histochemically and immunohistochemically the distribution of B- and Z-DNA in the epithelium and terminally differentiating dog lens fiber cells. On the basis of anti-DNA antibody reactivity, qualitative and quantitative data on B- and Z-DNA in cells were determined. Anti-B-DNA immunoreactivity gradually declined throughout nucleated fibers, with a precipitous decrease at approximately 90 microns. Anti-Z-DNA antibody binding decreased with a sudden loss of immunoreactivity at approximately 90 microns. The pattern of anti-B- and Z-DNA staining correlates with the loss of alpha-crystallin immunoreactivity, the major lens crystallin, and decreased eosin staining of proteins. Germinative zone cell nuclei showed the highest DNA probe binding values, followed by the superficial fibers, central zone, middle fibers, and deep fibers. The presence of single-stranded (ss)DNA in deeper fibers was detected by anti-ss-DNA antibodies. This is indicative of DNA degradation. These observations suggest that a dramatic reorganization of lens fiber cells' supramolecular order occurs at approximately 90 microns, the phase transition zone.

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.

Xeroderma pigmentosum.

Xeroderma pigmentosum is a rare, recessively transmitted disease associated with increased sensitivity to ultraviolet radiation in wavelengths found in sunlight, development of cancers in sun-exposed areas of the body in much larger numbers and much earlier in life than in normal individuals, and in some patients, neurologic deficiencies unrelated to sun exposure. Extensive cellular, biochemical, and molecular genetic studies in numerous laboratories have revealed that cells derived from patients with this disease have defective repair of ultraviolet-light-induced damage in cellular DNA, and that extensive genetic heterogeneity and numerous distinct genes are involved in the genetics of this disease and the etiopathogenesis of its associated changes. A number of these genes and gene products are now being, or have been, cloned, and their gene products characterized.