Lipofuscin Pigment Research Articles

Induction of angiogenic cytokine expression in cultured RPE by ingestion of oxidized photoreceptor outer segments.

Normal aging is associated with accumulation of lipofuscin pigment in the retinal pigment epithelium (RPE). This may occur as a result of phagocytosis and incomplete degradation of oxidized photoreceptor outer segments (POS). This study was undertaken to determine whether phagocytosis of UV-irradiated POS (artificial lipofuscin) would increase expression in the RPE of various chemotactic and angiogenic cytokines. ARPE-19 cells were exposed to latex beads (0.76 micro m), naïve bovine POS, and UV-irradiated POS (Ox-POS; 2 x 10(7)/mL), and supernatants were collected at 18 and 36 hours. The supernatants were assayed for IL-8, monocyte chemotactic protein-(MCP)-1, and TNF-alpha by ELISA. Protein synthesis and NFkappaB activity were inhibited by actinomycin D and SN50, respectively. Phagocytosis and generation of intracellular reactive oxygen species were assessed by flow cytometry. Confocal and electron microscopy studies were also performed to verify phagocytosis and cellular integrity. IL-8 and MCP-1 levels were decreased in the naïve POS group (IL-8: 473.76 +/- 66.9 pg/mL, P = 0.0005; MCP-1: 550.1 +/- 21.8 pg/mL, P = 0.0001), but were increased in the Ox-POS group (IL-8: 1348.8 +/- 164.9 pg/mL; MCP-1: 1772.28 +/- 65.19 pg/mL) compared with the control (IL-8: 741.09 +/- 39.8 pg/mL; MCP-1: 1413.47 +/- 38.4 pg/mL) and latex bead groups (data not shown). TNF-alpha levels were not affected. At 12 hours (but not at 6 hours), ROS were increased in the Ox-POS group. The cytokine increases observed were dependent on de novo protein synthesis and were NF-kappaB dependent. Ingestion by RPE of oxidized bovine POS stimulates expression of the chemotactic and angiogenic factors IL-8 and MCP-1 that have the capability to promote angiogenesis directly, or indirectly through the accumulation of immune cells such as macrophages, which themselves may release angiogenic promoters and degrade Bruch's membrane. This may be of significance in the development of exudative AMD.

Ultrastructural Study of the Pancreas in AIDS

Frequent histologic changes (90%) in the pancreas suggesting protein-energy malnutrition were found in a previous necropsy study of pancreas morphology in patients with AIDS. However, additional studies were required to clarify subcellular changes. To ultrastructurally analyze pancreas changes in AIDS patients through transmission electron microscopy. Pancreas specimens for necropsy were obtained from nine consecutive AIDS patients and four normal controls. A semiquantitative histologic and ultrastructural analysis of exocrine pancreas was carried out with the following findings: preserved pancreas structure with little autolysis, marked decrease in zymogen granules (100%), increased lipofuscin pigment (80%), augmented and dilated rough endoplasmic reticulum (100%), and increased number and size of mitochondria. The Golgi complex could be identified only in two cases. In all cases, acinar nuclei were decreased in size, with peripherally condensed chromatin and undulated membrane suggesting early apoptosis. In addition, immunohistochemical evaluation of the pancreas was carried out to detect opportunistic agents. Decreased zymogen granules, acinar atrophy, increased lipofuscin pigment, and rarefying Golgi complex represent the morphologic substrate of protein-energy malnutrition in AIDS patients. The combination of rough endoplasmic reticulum and mitochondria changes could be due to the need for supplying vital plasma proteins rather than exportation protein synthesis associated, or not, with the deleterious effects of inflammatory cytokines and/or therapy for disease.

The nucleus raphe interpositus in spinocerebellar ataxia type 3 (Machado–Joseph disease)

The nucleus raphe interpositus (RIP) plays an important role in the premotor network for saccades. Its omnipause neurons gate the activity of the burst neurons for vertical saccades lying within the rostral interstitial nucleus of the medial longitudinal fascicle and that for horizontal saccades residing in the caudal subnucleus of the pontine reticular formation. In the present study we investigated the RIP in five patients with clinically diagnosed and genetically confirmed spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease. Polyethylene glycol-embedded 100 μm serial sections stained for lipofuscin pigment and Nissl material as well as paraffin-embedded Nissl stained thin sections revealed the hitherto overlooked involvement of this pontine nucleus in the degenerative process underlying SCA3, whereby in four of our SCA3 patients the RIP underwent a conspicuous loss of presumed omnipause neurons. As observed in other affected brain structures, the RIP of all our SCA3 patients displayed reactive astrocytes and activated microglial cells, while some of the few of its surviving neurons harbored an ataxin-3-immunopositive intranuclear inclusion body. The findings of the present pathoanatomical study suggest that (1) neurodegeneration in the brain stem of terminal SCA3 patients is more widespread than previously thought and is not confined to cranial nerve nuclei involved in the generation of saccades but likewise involves the premotor network for saccades and (2) damage to the RIP may contribute to slowing of horizontal saccades in SCA3 patients but is not associated with saccadic oscillations as occasionally speculated.

Degeneration of the external cuneate nucleus in spinocerebellar ataxia type 3 (Machado–Joseph disease)

Owing to its anatomical connections, the external cuneate nucleus (ECU) plays a crucial role in processing proprioceptive input from the upper trunk and upper limbs. Here, we studied this dorsal column nucleus post-mortem in five individuals with clinically diagnosed and genetically confirmed spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, who had manifested upper trunk and upper limb ataxia. Polyethylene glycol-embedded 100-μm sections stained for lipofuscin pigment and Nissl material, as well as paraffin-embedded Nissl-stained thin sections, revealed serious neuronal loss in the ECU of all five SCA3 patients. As observed in other affected central nervous system structures, the ECU of these individuals displayed an astrogliosis, and some of the few surviving neurons harbored one or even two ataxin-3-immunopositive intranuclear inclusion bodies. The findings of the present study suggest that (1) the ECU is among the consistent targets of the degenerative process underlying SCA3 and (2) interruption of the proprioceptive pathway at the level of the ECU contributes significantly to upper limb and trunk ataxia in SCA3 patients.

Oral (gavage) two-generation (one litter per generation) reproduction study of pentachlorophenol (penta) in rats.

The potential for pentachlorophenol (penta) to induce general and reproductive/developmental toxicity was evaluated in Crl Sprague-Dawley rats, employing a two-generation reproduction toxicity study. Penta was administered by gavage at doses of 0, 10, 30, and 60 mg/kg/day. In both generations, the parental animals (30/sex/group) were intubated daily for 10 weeks before cohabitation and continuing through cohabitation, gestation, and lactation periods. Intubation of the F1 generation was begun 28 days postpartum. Animals were evaluated daily for mortality and general toxicity (clinical observations, body weights and gains, feed consumption). Organ weights were recorded and histopathological evaluations were made. Specific indices of reproductive function evaluated included estrous cycles, mating and fertility, parturition, lactation, viability, and growth and development of offspring, including sexual maturation, sperm parameters, and numbers of ovarian primordial follicles. All deaths in the parental rats were unrelated to penta. Expected metabolic effects of penta, sporadic increased liver weights associated with hepatocellular centrilobular hypertrophy and vacuolation and lipofuscin pigmentation, were evident in the 10-, 30-, and 60-mg/kg/day dose group P1 and F1 animals. Toxicity, in the form of liver pathology (single cell necrosis), reduced body weights and associated reductions in organ weights, and reduced feed consumption were noted in both generations at the 30- and 60-mg/kg/day doses. Developmental toxicity associated with these doses included reduced pup weights and viability. The 60-mg/kg/day dose also resulted in delayed sexual maturation, decreased spermatid counts, small prostates and testes, decreased implantations, reduced fertility, and increased resorptions of embryos. Based on these results, it was concluded that 30 mg/kg/day is the lowest-observable-adverse-effect level (LOAEL) and 10 mg/kg/day is the no-observable-adverse-effect level (NOAEL) for both reproductive and general toxicity. These findings are consistent with results from previously conducted studies wherein reproductive/developmental toxicity was observed only at doses that also induced general toxicity. It differs from previous findings in that the NOAEL for general toxicity is two to three times higher for the more pure product than for products produced and tested previously. In addition, the results did not indicate bioaccumulation of penta. Thus, penta did not selectively affect reproduction or development of the offspring of rats at a dose of 10 mg/kg/day, a dose that is 7000 to 20,000 times higher than human exposure.

Pigments in aging: an overview.

Although during the normal aging process there are numerous pigmentary changes, the best recognized are those of melanin and lipofuscin. Melanin may increase (e.g., age spots, senile lentigo, or melanosis coli) or decrease (e.g., graying of hair or ocular melanin) with age, while lipofuscin (also called age pigment) always increases with age. In fact, the time-dependent accumulation of lipofuscin in lysosomes of postmitotic cells and some stable cells is the most consistent and phylogenetically constant morphologic change of aging. This pigment displays a typical autofluorescence (Ex: approximately 440; Em: approximately 600 nm), sudanophilia, argyrophilia, PAS positiveness, and acid fastness. Advances on its biogenesis, composition, evolution, and lysosomal degradation have been hampered by the persistent confusion between lipofuscin and the large family of ceroid pigments found in a variety of pathological conditions, as evidenced by the frequent use of the hybrid term lipofuscin/ceroid by investigators mainly working with in vitro systems of disputable relevance to in vivo lipofuscinogenesis. While lipofuscin and ceroid pigments may share some of their physicochemical properties at one moment or another in their evolutions, these pigments have different tissue distribution, rates of accumulation, origin of their precursors, and lectin binding affinities. Although it is widely believed that lipofuscin is a marker of oxidative stress, and that it can be, therefore, modified by antioxidants and prooxidants, these assumptions are mainly based on in vitro experiments and are not generally supported by in vivo studies. Another common misconception is the belief that lipofuscin can be extracted from tissues by lipid solvents and measured spectrofluorometrically. These and other disturbing problems are reviewed and discussed in this presentation.

Neurocalcin-α immunoreactivity in the enteric nervous system of young and aged rats

The distribution of the calcium binding protein neurocalcin a has been examined in the entericnervous system of young adult (3 months) and aged (24+ months) male rats by immunofluorescence. Neurocalcin-immunoreactive (NC-ir) neurons were observed in the submucous and myenteric plexuses throughout the gastrointestinal tract from the oesophagus to the distal large intestine. NC-ir nerve terminals were also seen on NC-ir and NC-negative neurons. Semiquantitative estimates revealed fewer NC-ir neurons in the submucous plexus than in the myenteric plexus. The gre atest occurrence of NC-ir neurons was in the small and large intestine. NC-ir axons were seen in the mucosa and also in between the ganglia of the myenteric plexus. In the aged rats, there were no discernible changes in the numbers of NC-ir neurons in th e oesophagus and stomach, with an increase in the pylorus and slight decreasesin the small and large intestines. No decrease in NC-ir was observed in the distal large intestine. NC-ir neurons never contained lipofuscin age pigment and many enteric neuro ns devoid of NC-ir contained age pigment. Like other previously investigated calcium-binding proteins in enteric neurons, the distribution of NC shows much variability from one part of the intestine to another. The observed slight decreases in the number of NC-ir enteric neurons in aged rats may compromise the regulation of calcium in these neurons.

Morphometry of age pigment (lipofuscin) and of ceroid pigment deposits associated with vitamin E deficiency

Consistent amounts of lipofuscin and of ceroid pigment associated with vitamin E deficiency are reported to represent morphological correlates of aging and increased oxidative stress. A reliable quantification of these yellow autofluorescent deposits is of critical biological significance, thus we carried out a computer-assisted morphometric study on the accumulation of lipofuscin in physiological aging and of ceroid pigment in vitamin E deficiency, respectively. The total area and the size distribution of lipofuscin or ceroid pigment deposits were measured in CA3 hippocampal pyramidal neurons of 6-, 12-, 18- and 25-month-old rats, as well as in vitamin E deficient animals of 18 months of age. An increase in the mean total area of lipofuscin and ceroid pigment was found in aging and in vitamin E deficiency. In both conditions, the proportion of large discrete deposits also increased. The similarity of changes observed in old and adult vitamin E deficient animals suggests that the underlying processes initiated by the absence of α-tocopherol from the diet of adult rats and in physiological aging may share some common mechanisms.

Differential lectin histochemical studies on lipofuscin (age-pigment) and on selected ceroid pigments

The persistent indiscriminate use of the term lipofuscin for the pigments encountered in pathological conditions, and which should be most properly termed ceroid pigments, is still creating unnecessary conceptual and nomenclature problems, and a great deal of confusion. While both the age-dependent lipofuscin and the pathologically formed ceroid pigments have somewhat similar physical and histochemical properties, sufficient differences to properly identify these two types of pigments are presented in this communication. In addition, because little is known on the saccharide components of lipofuscin and ceroid pigments in situ, we have in recent years explored the lectin binding characteristics of lipofuscin in human and rats, as well as in diverse ceroid pigments experimentally induced in rats. Our lectin histochemical results showed qualitative and quantitative differences in the saccharide composition between human cerebral neurolipofuscin and the intra and extracellular ceroid pigment of human atheromas, as well as, between rat lipofuscin and the ceroid pigments induced in these animals.

Lipofuscins and sclerotial differentiation in phytopathogenic fungi.

Lipofuscins of lipidic and proteinaceous origin were identified by their excitation and emission spectra in phytopathogenic fungal representatives of different sclerotial differentiation types. Lipofuscin pigments in Sclerotium rolfsii, Rhizoctonia solani, Sclerotinia minor and Sclerotinia sclerotiorum showed similar excitation and emission maxima (ex-em 330-450, 330-450, 330-470 and 330-470 nm, respectively). Sclerotial differentiation of these fungi was proceeded by a 4.2, 2.5, 2.7, 2.5 and 6, 2.9, 3.8, 3.1 fold increase of lipofuscin accumulation (per lipid and protein content), per respective fungus, as compared to their undifferentiated stage. Lipofuscin levels were higher in older than in younger mycelia and this phenomenon was more profound in S. rolfsii. Since lipofuscins are considered as indicators of oxidative stress, these data are in accordance with the hypothesis that suggests oxidative stress to be a common underlying factor in sclerotial differentiation of sclerotia-forming filamentous phytopathogenic fungi.

Quantifying dense bodies and lipofuscin during aging: a morphologist's perspective

Secondary lysosomes, residual or dense bodies containing lipofuscin or age pigment accumulate in post-mitotic and inter-mitotic cells during aging. The consensus is that the accumulation of this auto-fluorescent material is an index of cellular senescence. Biochemical and morphological studies have independently demonstrated marked age-related increases in the cell and tissue contents of lipofuscin. Most morphological studies on aging have been qualitative, have included only two or three age groups and have not yielded data that are easily correlated with biochemical analyses. One of the best documented age-related changes in hepatocytes and cardiac myocytes is the accumulation of dense bodies and lipofuscin inclusions. Independent stereologic studies reported two- to eightfold age-related increases in the dense body volume fraction of rat hepatocytes. Furthermore, we reported a fourfold increase in the dense body volume fraction of cardiac myocytes in rats between 6 and 30 months of age. These and other studies confirm the use of quantitative morphology to estimate the increases in dense body and lipofuscin inclusions as indices of age. Whether or not the accumulated lipofuscin compromises cell functions in senescent animals has not been adequately addressed. On the one hand, there is little evidence that several-fold increases in this subcellular compartment impair the functional capacities of either hepatocytes or cardiac myocytes. On the other hand, the age-related accumulation of immunoprecipitable, but catalytically inactive, lysosomal enzymes in both liver and heart muscle may be a reflection of increased lipofuscin deposits in the dense bodies.

Degenerative Disorders of the Central Nervous System

Degenerative Disorders of the Central Nervous System

The autofluorescent age pigment lipofuscin: key to age, growth and productivity of the Antarctic amphipod Waldeckia obesa (Chevreux, 1905).

Peracarid crustaceans are among the most important taxa in terms of biodiversity and carbon-flow within the Weddell Sea benthos; however, very few data on their age, growth and productivity are available. This study uses the pigment lipofuscin as an age marker in the scavenging amphipod Waldeckia obesa (Chevreux, 1905) from the eastern Weddell Sea. Resin brain sections of 159 trap-caught specimens (1.2 to 7.7 mm coxal plate length L(cox) equal to 5 to 31 mm total length) were recorded digitally by confocal microscopy, and images were analysed. A modal progression analysis of the lipofuscin concentration-frequency distribution revealed five regularly spaced modes presumed to reflect consecutive annual age classes. Single females outside the range of mode V occurred, indicating maximum age of up to 8 years in females. No regular modes were obvious from the comparable length-frequency distribution of 386 individuals. Average yearly pigment accumulation was linear, and accumulation rates did not differ between sexes. The estimates of the growth parameters L(infinity) and k of the von Bertalanffy growth function were 7.47 mm L(cox) and 0.50 per year in females, respectively, and 6.92 mm L(cox) and 0.60 per year in males, respectively. Mortality, estimated from catch curves, amounted to 0.27 per year in females and 0.43 per year in males. P/B ratio, calculated from the mass specific growth rate method, was 0.38 per year for the pooled population (0.25 per year in females, 0.31 per year in males, 2.26 per year in juveniles). The results are discussed with regard to advantages and drawbacks of the methodology, and are compared with results from warmer water habitats.

Age determination in the Antarctic shrimp Notocrangon antarcticus (Crustacea: Decapoda), using the autofluorescent pigment lipofuscin

Determination of basic population parameters in long-lived Crustacea is hindered by a lack of appropriate methods for age determination. This study uses the pigment lipofuscin as an age marker in the common Antarctic decapod Notocrangon antarcticus (Pfeffer, 1887) from the eastern Weddell Sea. Resin brain sections of the lateral somacluster of the olfactory lobe of 189 specimens were digitally recorded by confocal microscopy and images were subsequently analyzed. A modal progression analysis of the lipofuscin concentration–frequency distribution revealed a total of eight regularly spaced modes presumed to reflect consecutive annual age classes. All eight modes contained females, and the first four contained males. No regular modes were obvious in the comparable length-frequency distribution. The average yearly pigment accumulation was nearly linear and estimated as 0.02% area fraction year−1, which is considerably lower than rates published for species from lower latitudes. This is probably explained by the effect of low water temperature on metabolism and lipofuscin accumulation rate. The growth parameters CL∞ and k from the von Bertalanffy growth function were 22.3 mm and 0.79 year−1 for females, respectively, and 16.9 mm and 0.64 year−1 for males, respectively. Mortality, estimated from catch curves, amounted to 0.44 year−1 in females and 0.92 year−1 in males, whereas P/B, calculated from the mass specific growth rate method, was slightly lower. The results indicate that the lipofuscin-inferred population parameters are an improvement over what can be learned about N. antarcticus with traditional methods.

Age-related Macular Degeneration: THE LIPOFUSCIN COMPONENTN-RETINYL-N-RETINYLIDENE ETHANOLAMINE DETACHES PROAPOPTOTIC PROTEINS FROM MITOCHONDRIA AND INDUCES APOPTOSIS IN MAMMALIAN RETINAL PIGMENT EPITHELIAL CELLS

10-20% of individuals over the age of 65 suffer from age-related macular degeneration (AMD), the leading cause of severe visual impairment in humans living in developed countries. The pathogenesis of this complex disease is poorly understood, and no efficient therapy or prevention exists to date. A precondition for AMD appears to be the accumulation of the age pigment lipofuscin in lysosomes of retinal pigment epithelial (RPE) cells. In AMD, these cells seem to die by apoptosis with subsequent death of photoreceptor cells, and light may accelerate the disease process. Intracellular factors leading to cell death are not known. Here we show that the lipophilic cation N-retinyl-N-retinylidene ethanolamine (A2E), a lipofuscin component, induces apoptosis in RPE and other cells at concentrations found in human retina. Apoptosis is accompanied by the appearance of the proapoptotic proteins cytochrome c and apoptosis-inducing factor in the cytoplasm and the nucleus. Biochemical examinations show that A2E specifically targets cytochrome oxidase (COX). With both isolated mitochondria and purified COX, A2E inhibits oxygen consumption synergistically with light. Inhibition is reversed by the addition of cytochrome c or cardiolipin, a negatively charged phospholipid that facilitates the binding of cytochrome c to membranes. Succinate dehydrogenase activity is not altered by A2E. We suggest that A2E can act as a proapoptotic molecule via a mitochondria-related mechanism, possibly through site-specific targeting of this cation to COX. Loss of RPE cell viability through inhibition of mitochondrial function might constitute a pivotal step toward the progressive degeneration of the central retina.

Different patterns of structural luteolysis in the human corpus luteum of menstruation.

Structural luteolysis is a complex process responsible for the elimination of the corpus luteum (CL). The aim of this study was to analyse the luteolytic process of the CL of menstruation. For this, we have morphologically studied 654 ovaries from 340 cycling women. Apoptotic cells were observed almost exclusively during the perimenstrual period and were extremely scarce at advanced stages of involution. Steroidogenic luteal cells surviving to the perimenstrual apoptotic wave underwent characteristic degenerative changes, consisting of intense cytoplasmic vacuolation, expression of macrophage markers and accumulation of lipofuscin pigment, and they persisted for long periods of time. Accumulation of corpora albicantia (CA) was observed in only 25% of a subset of 168 women, whereas 28% showed involuting CL without hyalinization, consisting of clusters of pigment-filled cells, and 46.4% showed ovaries with a mixture of CA and involuting CL without hyalinization or involuting CL with intermediate features. Evolution of the CL towards CA seemed to be related to the presence of a large, blood-filled cavity. The data from this study suggested that different patterns of structural luteolysis exist during CL involution, and that the final fate of the involuting CL is dependent on the presence of a large, central, blood-filled cavity.

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts.

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.

Effect of Toxaphene on Isolated Hepatocytes of the Yellowtail Flounder, Pleuronectes ferrugineus Storer

The histochemical and enzyme cytochemical effects of Toxaphene were investigated using isolated hepatocytes in suspension culture from laboratory-bred juvenile, female yellowtail flounder (Pleuronectes ferrugineus). Hepatocytes were kept in suspension culture for 4 days and exposed for 3 days to a control medium, to a medium with hexane (the solvent of Toxaphene), or to a medium with Toxaphene in two different concentrations (1 and 10 μg/ml). Subsequently, the cultivated cells were examined histochemically (Sudan black B, oil red O, Schmorl's reaction) and enzyme cytochemically (acid phosphatase, NADPH-ferrohemoprotein reductase). Toxaphene decreased the viability of the isolated cells significantly, as compared to the control suspensions. Toxaphene also increased the storage of total and neutral lipids (as demonstrated by Sudan black B and oil red O, respectively) in a dose-dependent manner. In addition, Toxaphene increased the enzymatic activity of acid phosphatase, and increased the storage of lipofuscin pigment (as demonstrated by the Schmorl's reaction) within the hepatocytes, suggesting an increase in the number and/or size of the lysosomes. Hexane did not have a significant toxic effect on the isolated hepatocytes. It is concluded that Toxaphene is potentially toxic to fish in a marine environment and that this in vitro system may provide a model for assessing the direct effect of various toxicants on fish hepatocytes.

Modification of bovine serum albumin structure following reaction with 4,5(E)-epoxy-2(E)-heptenal.

Bovine serum albumin (BSA) was incubated for different periods of time and in the presence of several concentrations of 4, 5(E)-epoxy-2(E)-heptenal, at pH 7.4 and 37 degrees C, in an effort to analyze the changes produced in its structure as a consequence of its reaction with this product of lipid oxidation. The epoxyalkenal modified the primary structure of BSA as determined by lysine losses and formation of oxidative stress product epsilon-N-pyrrolylnorleucine (Pnl), which depended on the concentration of the aldehyde and the incubation time. These changes also modified secondary and tertiary structures of the protein, which were determined by studying protein denaturation and polymerization. In addition, all these modifications were parallel to the development of color and fluorescence, which were produced as a consequence of the formation and polymerization of pyrrole amino acid residues. The above results indicated that epoxyalkenals modify the protein structure and develop color and fluorescence. A failure in the degradation of these modified proteins might induce their accumulation and, thus, participation in lipofuscin or age pigments formation.

Photoreceptor renewal and the pigment epithelium of the retina--congratulations to a pioneer in retinal research: Richard W. Young

In 1999, a pioneer in retinal cell biology celebrates his seventieth birthday: Richard W. Young, Professor of Anatomy at the Dept. of Anatomy and Jules Stein Eye Institute, University of Southern California, Los Angeles, California, USA. Against the current dogma of visual cells as static structures he demonstrated that they undergo continual renewal of their light-sensitive outer segments. Entire membranes and/or single molecules are being replaced, and the tips of outer segments are shed (disk-shedding), and phagocytized and degrade by pigment epithelial (PE) cells. About 100 disks are made per rod within 24 hours, and about 30,000 disk membranes from overlying rods are degraded by one PE cell thus rendering the PE one of the most active phagocytic systems of the body. It is not surprising, therefore, that the age pigment lipofuscin accumulates within PE cells, which is mainly composed of undigestible outer segment material. It is generally concluded that lipofuscin can contribute to the pathogenesis of age related macular degeneration (AMD). Early on Young has postulated that light exposure may accelerate AMD and some forms of retinitis pigmentosa (RP). Today we know that indeed in several animal models of RP light exposure can significantly enhance the disease progression. With a similar insight and intuition he described apoptosis of the retina thus preceding the "apoptotic wave" in eye research. Apoptosis now is considered the final common death pathway of many retinal diseases including degenerations and dystrophies. With his work young has created may scientific children, who directly or indirectly were inspired by his pioneering work.