Seymour Zigman

An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation

Abstract Superoxide dismutase activity was assayed in terms of its ability to inhibit the radical-mediated chain-propagating autoxidation of epinephrine. The enzyme assay based on adrenochrome absorption at 480 nm has been improved by measuring the absorption change at 320 nm. This alternative procedure was found to be 6 to 10 times more sensitive and more consistent than that measured at 480 nm.

Studies on the structural proteins of the human lens.

In the aging and cataractous human lens, soluble protein decreases and insoluble protein increases in concentration. This change is more marked in the cataractous than in the aging normal lens. Of the soluble lens proteins, γ-crystallin decreases and α-crystallin increases in both aging and cataract formation, and this change is more pronounced in the cataract. In the human, as in bovine lenses, α-crystallin and albuminoid appear to be more closely related than any of the other fractions. Senile normal lenses and senile cataracts resemble each other as seen in their amino acid analyses, E11 values and sulfhydryl content. In the human lens, albuminoid appears to derive from α-crystalling mostly (as in bovine lens), not from γ-erystallin as in the rat and dogfish.

Human RAD2 Homolog 1 5′- to 3′-Exo/Endonuclease Can Efficiently Excise a Displaced DNA Fragment Containing a 5′-Terminal Abasic Lesion by Endonuclease Activity

Repair of abasic lesions, one of the most common types of damage found in DNA, is crucial to an organisms well-being. Studies in vitro indicate that after apurinic-apyrimidinic endonuclease cleaves immediately upstream of a baseless site, removal of the 5′-terminal sugar-phosphate residue is achieved by deoxyribophosphodiesterase activity, an enzyme-mediated β-elimination reaction, or by endonucleolytic cleavage downstream of the baseless sugar. Synthesis and ligation complete repair. Eukaryotic RAD2 homolog 1 (RTH1) nuclease, by genetic and biochemical evidence, is involved in repair of modified DNA. Efficient endonucleolytic cleavage by RTH1 nuclease has been demonstrated for annealed primers that have unannealed 5′-tails. In vivo, such substrate structures could result from repair-related strand displacement synthesis. Using 5′-tailed substrates, we examined the ability of human RTH1 nuclease to efficiently remove 5′-terminal abasic residues. A series of upstream primers were used to increasingly displace an otherwise annealed downstream primer containing a 5′-terminal deoxyribose-5-phosphate. Until displacement of the first annealed nucleotide, substrates resisted cleavage. With further displacement, efficient cleavage occurred at the 3′-end of the tail. Therefore, in combination with strand displacement activity, RTH1 nucleases may serve as an important alternative to other pathways in repair of abasic sites in DNA.

Cataract Induction in Mice Exposed to Near UV Light

When young mice were exposed to 40-watt black light lamps for 12 h a day, microanatomical and biochemical changes were observed in their lenses after 35 weeks. From this time on, many undifferentiat-Mice ed cells with pyknotic nuclei were found in the lens cortex. Accumulation of soluble crystalline was depressed, especially γ-crystallin. High levels of insoluble protein were found in lenses of UV-irradiated mice. High molecular weight aggregates increased in this insoluble phase. From 60 weeks on, cortical opacities were seen. Thus, cataracts have now been shown to result from biochemical and anatomical changes in lenses of mice exposed to low levels of near UV light for extended time periods.

Replication Protein A Stimulates Long Patch DNA Base Excision Repair

Two pathways for completion of DNA base excision repair (BER) have recently emerged. In one, called short patch BER, only the damaged nucleotide is replaced, whereas in the second, known as long patch BER, the monobasic lesion is removed along with additional downstream nucleotides. Flap endonuclease 1, which preferentially cleaves unannealed 5′-flap structures in DNA, has been shown to play a crucial role in the long patch mode of repair. This nuclease will efficiently release 5′-terminal abasic lesions as part of an intact oligonucleotide when cleavage is combined with strand displacement synthesis. Further gap filling and ligation complete repair. We reconstituted the final steps of long patch base excision repair in vitro using calf DNA polymerase ε to provide strand displacement synthesis, human flap endonuclease 1, and human DNA ligase I. Replication protein A is an important constituent of the DNA replication machinery. It also has been shown to interact with an early component of base excision repair: uracil glycosylase. Here we show that human replication protein A greatly stimulates long patch base excision repair.

The Role of Sunlight in Human Cataract Formation

This review summarizes and integrates new findings concerning the role of near-ultraviolet radiation, as is present in sunlight and common artificial light sources, in stimulating human and animal cataract formation. Epidemiological and basic research studies are summarized and critical statements concerning them are offered. Although certain questions still remain unanswered, the evidence that near-UV radiation does stimulate cataract formation is very strong. Avoidance of excessive exposure to near-ultraviolet light and the use of protective lenses that filter it out are suggested to prevent the enhancement of human cataract formation by near-UV light.

Ocular protein alterations by near UV light

Abstract Human lenses in vitro contain sufficient levels of near UV sensitive substances to cause darkening of the lens from exposure to near UV light at levels not exceeding those present in sunlight. Ascorbic acid, when supplied to the medium inhibits such darkening. Exposure of the aqueous humor to near UV light leads to pigmentation and chemical alterations of aqueous humor proteins resulting from the binding of photo-oxidized small aromatic molecules normally present. The electrophoretic mobilities of near UV irradiated gamma crystallins are grossly altered both by the binding of low molecular weight photoproducts to them and also by direct action on aromatic amino acids comprising the proteins themselves. Denaturation enhances the near UV alterations in these proteins.

OCULAR LIGHT DAMAGE

This review describes papers recently published on the damage to and protection of ocular tissues from environmental short-wavelength radiation. The focus is on radiant energy in the 295-450 nm range, as the ozone layer filters out wavelengths shorter than 295 nm. It is useful mechanistically to consider the effects of UV-B (280-3 15 nm)* and UV-A (3 15400 nm) wavelengths separately, but the natural environment always contains a mixture of near-UV wavelengths from 295 to 400 nm. Generally, it is 97% UV-A and 3% UV-B. From direct sun, at an intermediate latitude the eye would receive -300 kJ/m2 of UV-A and 10 kJ/m2 UV-B in 30 min. Near-UV energy is strongly absorbed in the anterior segment of the eye (cornea, lens). The exception is that in aphakic eyes (z.e. after lens removal), the posterior segment can also receive sufficient near-UV energy to be damaged by it. As the lens is an efficient UV filter, the greatest damage to the retina in intact eyes is from blue light. The blue retinal photoreceptors absorb blue light for vision, but they can also be damaged by intense exposure to it. Blue lightand near-UV radiation-induced cellular damage is often enhanced by natural photosensitizers present in all living cells and body fluids (i.e. riboflavin). Natural antioxidants also protect ocular tissues from light damage. Thus, near-UV and blue energy can damage the ocular tissues both by direct and photosensitized reactions. Antioxidants can be used to protect against these toxic reactions.

Coloration of human lenses by near ultraviolet photo-oxidized tryptophan ☆

Abstract When human lenses in vitro and solutions of extracted crystallins were irradiated for 48 hr with near u.v. light in the presence of tryptophan their absorption of visible light (at 440 nm) increased and they became more intensely yellow-brown. As a result the absorption of u.v. light and the blue visible fluorescence of all lens crystallin fractions increased at least several fold. The observations suggest a role of environmental near u.v. light in the intensification of human lens color with aging. Both beneficial and harmful effects may result from such lens color darkening.

NEAR UV LIGHT AND CATARACTS

Abstract. Sunlight and many types of artificial lighting contain near‐UV light (300–400 nm). These wavelengths can enter the eye and are maximally absorbed in the lens due to its chemical composition. The lenses of certain animals develop cataracts from exposure to this light, and changes similar to those that occur in human lenses with certain types of cataracts and with aging are inducible in isolated human lenses. These changes seem to be associated with chemical alterations in the essential amino acid tryptophan either as a part of proteins or in free form. Such changes in tryptophan would result in lens cell toxicity, in increased pigmentation of the lens, and in large aggregates of proteins. The latter two changes would result in losses in the ability of the lens to transmit visible light needed for vision, and the abnormal state called cataract would result. Much more work is needed to prove that near‐UV light can accelerate cataractous changes in the lenses of living humans. Studies at the basic chemical level are needed, but population studies would be most essential for the final proof. Many preventive measures could become available, including the use of special types of spectacles and dietary additives.