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Recent results indicate that the longevity of both invertebrates and vertebrates can be altered through genetic manipulation and pharmacological intervention. Most of these interventions involve alterations of one or more of the following: insulin/IGF-I signaling pathway, caloric intake, stress resistance and nuclear structure. How longevity regulation relates to aging per se is less clear, but longevity increases are usually accompanied by extended periods of good health. How these results will translate to primate aging and longevity remains to be shown.

High energy phosphate levels fall rapidly during cardiac ischemia and recover slowly (more than one week) during reperfusion. The slow recovery of ATP may reflect a lack of purine metabolic precursors and/or increased activity of purine catabolic enzymes such as 5′-nucleotidase (5′-NT, EC 3.1.3.5) and adenosine deaminase (ADA, EC 3.5.4.4). The activity of enzymes involved in both the catabolism of ATP precursors (5-NT and ADA) and the restoration of ATP from slow synthetic pathways [adenosine kinase (AK, EC 2.7.1.20), adenine phosphoribosyl transferase (APRT, EC 2.4.2.7) and hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8)] may directly affect the rate of ATP recovery. Strategies to enhance recovery will depend on the relative activity of these enzymes following ischemia. Their activity in different species and their response to ischemia are not well characterized. Hence, rapid assay methods for these enzymes would facilitate detailed time course studies of their activities in postischemic myocardium. We modified a single ion-exchange column chromatographic method using DEAE-Sephadex to determine the products of incubation of 5′-NT, AK, APRT and HPRT with their respective substrates. The uniformity of the final product measurement procedure for all assays permits the activities of the four enzymes to be rapidly determined in a single tissue sample and facilitates the study of a large number of samples. This technique should also be useful for enzymes of the pyrimidine metabolic pathway.

In an article published in the EMBO reports Special Issue on Time and Ageing, Aubrey D.N.J. de Grey criticizes biogerontologists for what he sees as their generally pessimistic view of the possibilities for intervening in the ageing process (de Grey, 2005). In his view, “resistance [of biogerontologists] to debate on how to postpone ageing is delaying progress and costing lives.” de Grey believes he has formulated a research plan that, in his words, will “stop people from getting frailer and more prone to life‐threatening diseases as they get older, and moreover [will] restore the already frail to youthful vitality”. Similar to vintage cars maintained by careful mechanics, individuals will then retain this happy state “indefinitely … even at ages many times what we reach today”. In de Grey's opinion, “the failure of most biogerontologists to maintain an open mind concerning the scientific options … [has] the result that much longer healthy lives are being denied those who will die before ’real anti‐aging medicine' arrives.” In the words of the great American journalist H.L. Mencken, “for every complex problem, there is a simple solution, and it is wrong.” de Grey's research programme, which he terms ’strategies for engineered negligible senescence' (SENS), involves a combination of preventative and therapeutic interventions (de Grey, 2003). To solve the problem of apoptosis in senescent cells, one simply uses “senescence marker‐tagged toxins”. To cure cancer, one just calls on “total telomerase deletion plus cell therapy”. To prop up the failing immune system, one can turn on “IL‐7 mediated thymopoiesis”. To reverse mitochondrial mutations, one need only use “allotopic [mitochondrial]‐coded proteins” of the type favoured by algae. Cell replacement can be accomplished by “stem cell therapy and growth factors”, whereas retooling the endocrine system relies on “genetically engineered muscle”. Cleavage of glycosylation crosslinks will involve periodic exposure …

Over 15 years of research on correlations between superoxide dismutase (SOD) activity and aging or life span have failed to provide a consistent picture of the role of SOD in aging. While genetic manipulations that increase CuZn-SOD activity have only a slight, if any, effect on maximum life span in several species, they do increase resistance to oxidative stress. However, increasing both CuZn-SOD and catalase does significantly increase maximum life span. Decreased SOD expression in a variety of species increases their vulnerability to oxidative stress, and in the case of genetically altered CuZn-SOD, leads to premature death of motor neurons in humans. Little is known about the regulation of expression of SOD and other antioxidant defense enzymes in eukaryotes. The research summarized below collectively suggest that SOD plays an important role in longevity and degenerative disease, but much remains to be learned before manipulation of SOD expression can be considered for effective intervention in either process.

The National Institute on Aging Interventions Testing Program (ITP) was established to evaluate agents that are purported to increase lifespan and delay the appearance of age-related disease in genetically heterogeneous mice. Up to five compounds are added to the study each year and each compound is tested at three test sites (The Jackson Laboratory, TJL; University of Michigan, UM; and University of Texas Health Science Center, San Antonio, UT). Mice in the first cohort were exposed to one of four agents: aspirin, nitroflurbiprofen (NFP), 4-OH-α-phenyl-N-tert-butyl nitrone (4-OH-PBN), or nordihydroguiaretic acid (NDGA). Sample size was sufficient to detect a 10% difference in lifespan in either sex, with 80% power, using data from two of the three sites. Pooling data from all three sites, a log-rank test showed that both NDGA (p = 0.0006) and aspirin (p = 0.01) led to increased lifespan of male mice. Comparison of the proportion of live mice at the age of 90% mortality was used as a surrogate for measurement of maximum lifespan; neither NDGA (p = 0.12) nor aspirin (p = 0.16) had a significant effect in this test. Measures of blood levels of NDGA or aspirin and its salicylic acid metabolite suggest that the observed lack of effects of NDGA or aspirin on lifespan in females could be related to gender differences in drug disposition or metabolism. Further studies are warranted to find whether NDGA or aspirin, over a range of doses, might prove to postpone death and various age-related outcomes reproducibly in mice.

The central theme of the 3rd International Conference on Functional Genomics of Ageing was tissue regeneration as a remedial strategy to address age-related cellular damage and the pathology that ensues. The conference included sessions on maintaining genome integrity and the potential of stem cells to restore function to damaged tissues. In addition to several human syndromes that appear to reflect accelerated ageing, there are now a number of mouse models that prematurely display phenotypes associated with ageing. The intent of this summary presented at the end of the conference was to: (1) discuss various human syndromes and mouse models of accelerated ageing; (2) evaluate whether the phenotypes displayed might result from an elevated rate of cell death coupled with an inability to adequately maintain cell number in various tissues with increasing age; and (3) discuss whether similar events may be occurring during normal ageing, albeit much more slowly.

The National Institute on Aging (NIA) started a program in 1993 to identify genes involved in the regulation of longevity in a variety of species, including yeast, nematodes, fruit flies, and mice. The initial success of this program has attracted the interest of many investigators working with these organisms. Of primary interest are single-gene mutants that have identified genes and processes involved in longevity regulation across species. These processes include the insulin-like signaling pathway, stress resistance, and most recently, chromosome and nuclear architecture. Mutations in genes that regulate these processes indirectly are also being identified in this program. The ultimate goal of this program is to extend these results to humans to identify the major biological risk factors for age-related decline of function in human physiological systems.

During the past ten years considerable progress has been made in discovering genes that regulate longevity by identifying single gene mutations that lead to increased longevity. The initial success in nematodes was quickly followed by comparable success in fruit flies and mice. In contrast, mutations that cause a decrease in longevity have been largely discounted as unlikely to be informative about aging mechanisms. However, the recent creation of several mutant mouse models that develop a variety of aging-like phenotypes and die prematurely, suggests that such models may be useful in understanding aging mechanisms, particularly as they relate to progressive tissue and organ dysfunction. A possible common feature of these models may be an imbalance between loss of cells by apoptosis and subsequent cell replacement, leading gradually to a net loss of cells in multiple tissues.

In summary, the progressive loss of post-mitotic tissue during aging may be due to the death of damaged post-mitotic cells which cannot be replaced, or possibly their malfunction. The damage may derive from either intrinsic or extrinsic sources, and may consist of damage to proteins as well as DNA. The loss of neuronal cells is particularly problematic, since they are least readily replaced (if at all). Mitotic cells may also respond to intrinsic or extrinsic damage by cell death, but this generally is not problematic in mitotic tissues. In contrast, however, damage or repeated cell division can cause mitotic cells to express a senescent phenotype, which may compromise the function and integrity of the surrounding tissue. Finally, imperfect repair of DNA damage may cause the accumulation of potentially oncogenic mutations, which can cause mitotic, but not post-mitotic, cells to express a hyperplastic or neoplastic phenotype.

HE first experimental evidence that genetics plays a role in aging was provided by Luckinbill and colleagues (1) and Rose (2), who produced long-lived fruit fly lines by repeatedly selecting for progeny from the oldest females. Although it was also known that some human families have an unusual number of individuals who live a long time, only in the last 10 years has it been possible to elucidate some of the mechanisms by which this may occur. Richard Miller (3) provided a lot of clarity to this question in his 1998 Kleemeier lecture titled: ‘‘Are There Genes for Aging?’’ Whereas it is fairly obvious that the broad range of life spans among animals must be genetically determined, it is less clear why the longevity of individuals within a species is so variable, even in an apparently genetically homogeneous population. Environmental factors presum

When Lockshin and Zakeri discussed the relevance of apoptosis to aging, the common view was that apoptosis had primarily a negative impact on aging by destroying essential and often irreplaceable cells. That view has now changed to one that acknowledges that there are two general ways in which apoptosis can play a role in aging: (1) elimination of damaged and presumably dysfunctional cells (e.g., fibroblasts, hepatocytes) which can then be replaced by cell proliferation, thereby maintaining homeostasis and elimination of essential postmitotic cells (e.g., neurons) which cannot be replaced, thereby leading to pathology. Evidence exists in two systems (fibroblasts and thymocytes/lymphocytes) that there are age-related decreases in the potential for apoptosis, although the molecular bases for these decreases appear to differ (Table II). Fibroblasts (and neurons?) lose the ability to downregulate bcl-2 in response to an apoptotic signal; thus, apoptosis is blocked even though an initiating signal has been received. In contrast, thymocytes/lymphocytes lack the ability to initiate the signal due to downregulation of the cell surface receptor Fas. There is limited information available for other tissue types, and nothing is known about why and how these age-related changes occur. An interesting observation, but not necessarily a critical one, is that the frequency of upregulation of the bcl-2 gene due to chromosome translocation increases with age. The role of apoptosis in regulating cell number is also a promising area of research. The studies on liver damage and neoplastic lesions suggest an extremely important role for apoptosis in controlling cancer. This may be particularly important in the prostate, where hypertrophy and cancer are a virtual certainty with ever-increasing age. It is not known whether the ability to undergo apoptosis declines in the prostate with increasing age, but it appears likely that it does. One problem in answering questions about the actual regulation of apoptosis is the lack of a quantitative assay. Apoptosis appears to be either "on" or "off" in cells, while the basic cell-killing machinery may often be present, but in an inactive form. Most assays for apoptosis are microscopic rather than kinetic, and the rate-limiting step may be at the level of the initiating signal. Thus, if CR, which extends the life span of rodents, does upregulate apoptosis, it is not clear how to quantify the magnitude of this effect or what should be quantified. The best one can do is to measure the frequency of occurrence of apoptotic bodies. This is essentially a pool size assay which provides little knowledge about how rapidly cells are leaving and entering the pool. Nevertheless, the results currently available do suggest that apoptosis is a process which may be important in aging, at least in some tissues, and the mechanism of its regulation needs to be understood. Although a variety of tumor suppressor gene and oncogene products are known to be involved in signal transduction associated with apoptosis, it remains to be shown which of these, if any, are actually involved in "on-off" switches for apoptosis and which might regulate the intrinsic rate of apoptosis. As Driscoll has already pointed out: "regulation and execution of cell death is an absolutely critical process that interfaces with nearly every aspect of life. Future investigation of the links of cell death to cellular aging and the aging of organisms should be an exciting enterprise."

Publisher Summary This chapter provides an overview of superoxide dismutase (SOD1), oxygen radical metabolism, and amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) belongs to a common subtype of the motor neuron diseases, which are a heterogeneous group of disorders that produce muscle weakness and atrophy by their effects on the anterior horn cells of the spinal cord. The most important immediate and obvious implication of the finding of various SOD1 mutations in familiar ALS (FALS) patients is its potential therapeutic benefit. If toxicity caused by oxygen-free radicals is the primary pathogenic mechanism for motor neuron death in FALS and perhaps sporadic ALS, diminishing this toxicity might stop or retard the course of this disease. Clinical trials with either SOD itself or compounds that penetrate the central nervous system and decrease levels of free radicals should be feasible. Such drugs are currently being tested for treatment of Parkinsonism. In a more general sense, this finding is strong, perhaps compelling, evidence in favor of a very significant role for ROS and perhaps other radicals in degenerative processes associated with human aging and in diseases associated with aging. Conversely, a study of the molecular biology of the mutated forms of SODl should reveal a fundamental understanding of how protein structure and function are interrelated.

There is general agreement that diet has important implications for health and susceptibility to disease, but there is little direct evidence that dietary factors alter the progress of aging per se. One difficulty is that it is not easy to distinguish changes due to “true aging” from changes associated with age-related disease. There are numerous theories of aging (Warner et al. 1987), many of which are not mutually exclusive, but share common elements. However, it seems clear that the longevity of an individual within a population depends on his/her genetic constitution and environmental experience. Thus, one can ask how dietary factors might modulate agerelated changes in gene expression and/or macromolecular damage due to environmental insults, both of which presumably determine the rate of aging of any particular individual of a species.

Human longevity depends strongly on health, but there is considerable uncertainty about the potential increase in human longevity that could result from diverse curative and preventive interventions (Manton et al., 1992). The basis for some of the most profound intervention strategies in the future lies in the realm of rapidly developing molecular biological technologies. The purpose of this chapter is to review some molecular biological approaches that may be useful in studying the aging process and intervening to delay or prevent age-dependent degenerative processes that lead to disease.

Our goal is to test mesenchymal stem cell therapy in a mouse model of Hutchinson-Gilford Progeria Syndrome (HGPS), a lethal childhood disorder for which no definitive therapy is available. HGPS patients have failure to thrive, lipodystrophy, skeletal dysplasia, sclerotic skin, alopecia, and occlusive vascular disease that results in premature death from myocardial infarction or stroke in early teens. In HGPS, the accumulation of incompletely processed nuclear architectural protein lamin A (a farnesylated version of prelamin A, termed progerin) results in dysfunction of multiple organ systems, particularly those of mesenchymal origin. Consistent with this, progerin has been shown to interfere with the proliferation and differentiation of mesenchymal stem cells (MSCs). Furthermore, MSC depletion is thought to account for the specific segmental nature of human progeria. The identification of HGPS-specific MSC dysfunction, and emerging evidence about the plasticity and paracrine effects of MSCs have prompted us to test the hypothesis that MSCs might be coaxed into becoming cells able to correct widespread pathology in HGPS. To determine whether MSCs can be used to rescue progeria phenotype, MSCs from wild-type donors were tested. First, donor MSCs (0.5 × 106 per recipient) were stably transfected to express the red fluoroform DsRed2, which permitted donor cell tracking in tissues post mortem. Next, to achieve wide biodistribution DsRed2+ MSCs were injected intra-arterially into 2-week-old ZMPSTE24−/− mutant mice. Like HGPS cells, ZMPSTE24−/− mutant mice accumulate progerin and the mice exhibit many progeria-like signs, including alopecia, micrognathia, dental abnormalities, osteolytic lesions in bones, osteoporosis, retarded growth, muscle weakness, and shortened lifespan. Therefore, survival, growth (measured by weight gain), and muscle weakness (measured by grip strength) were analyzed. MSC-treated (n = 13) versus untreated (n = 23) ZMPSTE24−/− mice had significantly increased survival (65% versus 100%), weight gain after 6 weeks of life, and grip strength (judged by the ability to hang onto an upside–down grid). These data are consistent with the hypothesis that the transfer of wild-type MSCs reduces the severity of the lamin A defect. To investigate whether the benefits associated with MSC infusion were attributable to persistent tissue engraftment or to a transient paracrine effect of donor MSCs, all mice were electively harvested at 20 weeks of age. Because dermal lipodystrophy, cardiac arteriosclerosis, and skeletal myopathy are prominent pathological findings in both human and murine progeria, post-mortem analyses focused on donor cell engraftment and progerin expression in corresponding organs of pathology (skin, liver, and skeletal muscle). Donor cells were identified in skin, liver, and skeletal muscle specimens with a frequency of 1%–5%, indicating that donor MSCs can repopulate tissue niches presumed to be dysfunctional in progeria. Experiments are ongoing to evaluate tissue levels of prelamin A and mature lamin A, abnormalities in nuclear shape, and pertubations in intracellular signaling pathway genes known to be dysregulated in human progeria. Collectively, these data demonstrate the proof of principle of MSC transfer for phenotypic reversion of lamin A defect in murine progeria. This may offer a valuable approach for treatment of human HGPS with human MSCs, which are already in clinical use.

Females of the human species live longer than males, and the longevity differential is probably not entirely explained by reasons which are presently obvious. Genotypic sex has long been suspected to affect longevity to the advantage of the female. Several recent findings about the X and Y chromosomes must be reckoned with in considering determinants of longevity which derive from genotypic sex. The advantages of having two X chromosomes are apparent, notwithstanding X-chromosome inactivation. Not only can some cells compensate for biosynthetic deficiencies of others, but also cell selection according to which X chromosome is active can occur during development according to cell viability and proliferative capacity. It has recently been observed that at least some genes on inactive X chromosomes are reactivated late in life. Details of the reactivation process must be studied to determine its significance and the effects of the process on late life survival. The recent mapping of the catalytic polypeptide of DNA-polymerase-alpha to the X chromosome calls attention to a new property of the genotype which could affect the basic ability of cells to proliferate. It is likely that this enzyme, perhaps in concert with DNA-polymerase-delta, is required for DNA replication, suggesting that two alleles for this enzyme and cell selection within the female phenotypic mosaic for DNA replication may provide a sex-linked determinant of cell proliferation which could be advantageous in late life. Much remains to be learned about the Y chromosome, although there are early results consistent with a determinant of longevity on that chromosome which operates to the male disadvantage and probably does not involve sex hormones. The genotype may be a significant determinant of longevity in humans even if it does not appear to be so in non-human animals, because causes of death are different. Determinants of longevity are based on susceptibility or vulnerability to the causes and diseases of mortality, and these differ in different species.

Uracil-DNA glycosylase activities from etiolated Zea mays seedling nuclei and mitochondria were partially purified and characterized. Nuclei and mitochondria were separated using sucrose differential and step gradient centrifugation. Experiments with osmotically shocked organelles indicated that enzyme activity from mitochondria was soluble, whereas nuclear enzyme activity was only partially soluble under the conditions tested. Purification using DEAE-cellulose and Affigel Blue column chromatography yielded distinct elution profiles from both columns for each of the organellar enzyme activities. Final purification was 490- and 850- fold for the nuclear and mitochondrial uracil-DNA glycosylase, respectively. Characterization studies demonstrated significant differences between the nuclear and mitochondrial uracil-DNA glycosylase with respect to K(m), temperature, and pH activity optimum, the effect of salts, and substrate preference. Molecular weight as determined by gel filtration was 18,000 for enzymes from both sources. Both were also sensitive to the sulfhydryl group-blocking agent N-ethylmaleimide. A number of uracil analogs were tested for their ability to inhibit nuclear and mitochondrial uracil-DNA glycosylase activities. 5-Azauracil, uracil, 6-aminouracil, 6-azauracil, 5-aminouracil, and 5-fluorouracil all inhibited both activities to variable degrees.

A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent K(m) was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl(2), or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA-containing organelles of eucaryotic cells.

Uracil is incorporated into newly synthesized DNA by mutants of Escherichia coli with reduced levels of dUTPase (dUTP nucleotidohydrolase; EC 3.6.1.23). Excision-repair of the incorporated uracil results in the generation of labeled DNA fragments that appear after brief pulses with [ 3 H]thymidine [Tye, B-K., Nyman, P.-D., Lehman, I. R., Hochhauser, S. & Weiss, B. (1977) Proc. Natl. Acad. Sci. USA 74, 154-157]. Uracil is also incorporated into the newly synthesized DNA of strains of E. coli that contain normal levels of dUTPase. DNA fragments generated by the postreplication excision-repair of uracil may therefore contribute to the pool of nascent DNA (Okazaki) fragments that normally appear in wild-type strains. Discontinuous DNA replication has been examined in the absence of uracil excision by comparing Okazaki fragments in strains that are defective in DNA polymerase I ( polA - ) and polA - strains that are also defective in uracil N -glycosidase, an enzyme required for the excision-repair of uracil in DNA ( polA - ung - ). Little or no difference was detected in the level of Okazaki fragments in the polA - strain as compared with the polA - ung - strain. Thus, the uracil-induced cleavage of DNA cannot be the sole mechanism for the generation of Okazaki fragments. Mutants that are defective both in dUTPase and in uracil N -glycosidase incorporate uracil into their DNA with a high frequency (up to 1 per 100 nucleotides). These uracil residues, once incorporated, persist in the DNA without an adverse affect on the growth of the cells.

The incorporation of uracil into and excision from DNA were studied in vitro using lysates on cellophane discs made from Escherichia coli strains with defects in the enzymes dUTPase (dut) and uracil-DNA glycosylase (ung). Results with dut ung lysates indicate that dUTP is competitively incorporated with dTTP at the replication fork. Such incorporation is not due to DNA polymerase I. There is a mild discrimination (2.5-fold) against incorporation of dUTP versus dTTP. These data, together with in vivo uracil incorporation data (Tye et al., 1978) permit a rough estimate of the pool of dUTP in vivo (~0.5% of the dTTP pool). These in vitro data indicate that uracil-DNA glycosylase is the initial step in at least 90% of uracil excision events. However, in a strain defective in uracil-DNA glycosylase (ung-1), uracil-containing DNA is still more subject to single-strand scission than non-uracil-containing DNA, albeit at a rate at least tenfold less than in an ung+ strain. A number of qualitative statements may also be made about different steps in uracil incorporation and subsequent excision and repair events. When high levels of dUTP are added in vitro, a dut ung+ strain has a higher steady-state level of uracil in newly synthesized DNA than does an isogenic dut+ ung strain. Thus the dUTPase in these lysates has a higher capacity to be overloaded than does the excision system (i.e. uracil DNA glycosylase). However, the DNA sealing system (presumably DNA polymerase I and DNA ligase) apparently can handle all single-strand interruptions being introduced by uracil excision at the maximal rate, at least so that DNA synthesis can continue.

The incorporation of uracil into DNA in an in vitro repair reaction catalyzed by native Escherichia coli DNA polymerase I renders the newly synthesized uracil-containing portion of the resultant polymer susceptible to an endonucleolytic-like cleavage by the polymerase itself. This response is related to the presence of the 5' → 3' nuclease activity of this enzyme, since subtilisin-treated polymerase which lacks this nuclease activity also fails to attack uracil-containing DNA in this way.

A mutant of bacteriophage T4 unable to induce exonuclease A has been isolated. The mutation responsible for this defect maps between genes 39 and 56, in a region of the chromosome devoid of other known markers. Four deletion mutants lacking part of the genome located between genes 39 and 56 also fail to induce exonuclease A. The ability of all of these mutants to replicate suggests that exonuclease A is not essential for replication of phage T4.

A method was devised for identifying nonlethal mutants of T4 bacteriophage which lack the capacity to induce degradation of the deoxyribonucleic acid (DNA) of their host, Escherichia coli. If a culture is infected in a medium containing hydroxyurea (HU), a compound that blocks de novo deoxyribonucleotide biosynthesis by interacting with ribonucleotide reductase, mutant phage that cannot establish the alternate pathway of deoxyribonucleotide production from bacterial DNA will fail to produce progeny. The progeny of 100 phages that survived heavy mutagenesis with hydroxylamine were tested for their ability to multiply in the presence of HU. Four of the cultures lacked this capacity. Cells infected with one of these mutants, designated T4 nd 28, accumulated double-stranded fragments of host DNA with a molecular weight of approximately 2 × 10 8 daltons. This mutant failed to induce T4 endonuclease II, an enzyme known to produce single-strand breaks in double-stranded cytosine-containing DNA. The properties of nd 28 give strong support to an earlier suggestion that T4 endonuclease II participates in host DNA degradation. The nd 28 mutation mapped between T4 genes 32 and 63 and was very close to the latter gene. It is, thus, in the region of the T4 map that is occupied by genes for a number of other enzymes, including deoxycytidylate deaminase, thymidylate synthetase, dihydrofolate reductase, and ribonucleotide reductase, that are nonessential to phage production in rich media.

Nuclear disruption after infection of Escherichia coli with a bacteriophage T4 mutant deficient in the ability to induce endonuclease II indicates that either (i) the endonuclease II-catalyzed reaction is not the first step in host deoxyribonucleic acid (DNA) breakdown or (ii) nuclear disruption is independent of nucleolytic cleavage of the host chromosome. M-band analysis demonstrates that the host DNA remains membrane-bound after infection with either an endonuclease II-deficient mutant or T4 phage ghosts.

Advances in understanding aging processes and their consequences are leading to the development of therapies to slow or reverse adverse changes formerly considered to be "normal" aging and processes that underlie multiple age-related conditions. Estimating the effectiveness of candidate aging therapies, whose effects on human aging may require many years to determine, is a particular challenge. Strategies for identifying candidate interventions can be developed through multiple approaches, including the screening of molecular targets and pathways in vitro and in animal models, informed as well by evidence from human genetic and epidemiologic data. A number of recently established programs and networks can serve as resources for such research. For all these research approaches, from in vitro molecular studies to clinical trials, contributions of cell and molecular biology are crucial and offer the prospect of therapeutic advances that address fundamental biological processes as well as the clinically important challenges of aging.

T4 bacteriophage DNA containing as much as 30% of its thymine replaced by uracil can be synthesised in Escherichia coli deficient in both dUTPase and uracil--DNA glycosidase. This uracil-containing DNA is competent for RNA transcription, and can be packaged into phage which are viable, if the host cells are deficient in uracil--DNA glycosidase activity. If the host cells are not deficient in this glycosidase activity the infecting phage DNA is rapidly attacked, resulting in more than 50% acid-solubilisation of the DNA. The infected cells are inefficiently killed, presumably because of very limited, if any, expression of the phage DNA. These results indicate that this replacement of thymine by uracil in DNA does not seriously impair the biological functionality of T4 DNA, provided the DNA is not subjected to the breakdown (repair) pathway initiated by uracil--DNA glycosidase.

Some mutants of bacteriophage T4 selected for their inability to replicate in the presence of hydroxyurea are defective in the degradation of host DNA. Nine such mutants which all map in the same locus (the "denA" locus) were all deficient in the induction of T4 endonuclease II. We conclude that this enzyme is involved in the breakdown of cellular DNA.

Bacteriophages T5 and BF23 have been the two most extensively investigated of the T5 group, which also includes bacteriophages PB, BG3, and 29 alpha. Unusual features of T5, BF23, and PB include the existence of interrupted phosphodiester bonds (“nicks”) at specific locations in only one of the strands of their duplex DNA, a two-step mechanism for transfer of their DNA into host cells, and large terminal repetitions in their DNA.

A number of mutant strains of Escherichia coli have been examined for their sensitivity to nitrous acid and in some instances to methylmethanesulfonate. All ung- mutants tested are abnormally sensitive to nitrous acid. Since the ung mutation is phenotypically expressed as a defect in uracil DNA glycosidase, this observation supports the contention that treatment of cells with nitrous acid causes deamination of cytosine to uracil. In addition the observed sentitivity indicates that the ung gene is involved in the repair of uracil in DNA. Studies with other mutants suggest that both exonuclease III and DNA polymerase I of E. coli are involved in the repair of nitrous acid damage in vivo.

In this chapter, we review evidence that certain metabolic systems established during the process of infection of Escherichia coli with bacteriophage T4 are associated with the cell envelope of the host. Information pertaining to this subject is incomplete and often controversial. Nevertheless, we are encouraged to review what is known on the premise that the T4 phage system, as has been demonstrated so often in the past, is uniquely endowed with favorable properties for exploring important questions in biology including the structure and functions of cell membranes. Our format is intended to be descriptive rather than comprehensive, and the literature cited is illustrative rather than exhaustive.

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.

The characteristics of the nicks (single-strand breaks) introduced into damaged DNA by Escherichia coli endonucleases III, IV, and VI and by phage T4 UV endonuclease have been investigated with E. coli DNA polymerase I (DNA nucleotidyltransferase). Nicks introduced into depurinated DNA by endonuclease IV or VI provide good primer termini for the polymerase, whereas nicks introduced into depurinated DNA by endonuclease III or into irradiated DNA by T4 UV endonuclease do not. This result suggests that endonuclease IV nicks depurinated DNA on the 5' side of the apurinic site, as does endonuclease VI, whereas endonuclease III has a different incision mechanism. T4 UV endonuclease also possesses apurinic endonuclease activity that generates nicks in depurinated DNA with low priming activity for the polymerase. The priming activity of DNA nicked with endonuclease III or T4 UV endonuclease can be enhanced by an additional incubation with endonuclease VI and, to a lesser extent, by incubation with endonuclease IV. These results indicate that endonuclease III and T4 UV endonuclease (acting upon depurinated and irradiated DNA, respectively) generate nicks containing apurinic/apyrimidinic sites at their 3' termini and that such sites are not rapidly excised by the 3' leads to 5' activity of DNA polymerase I. However, endonuclease IV or VI apparently can remove such terminal apurinic/apyrimidinic sites as well as cleave on the 5' side of the unnicked sites. These results suggest roles for endonucleases III, IV, and VI in the repair of apurinic/apyrimidinic sites as well as pyrimidine dimer sites in DNA. Our results with T4 UV endonuclease suggest that the incision of irradiated DNA by T4 UV endonuclease involves both cleavage of the glycosylic bond at the 5' half of the pyrimidine dimer and cleavage of the phosphodiester bond originally linking the two nucleotides of the dimer. They also imply that the glycosylic bond is cleaved before the phosphodiester bond.

With the discovery some six years ago of uracil DNA glycosylase by Lindahl,1 it became possible to define a new mode of excision repair, “base excision repair,”2 which was hypothesized to occur by (i) removal of a DNA base by hydrolysis of the glycosylic bond, (ii) cleavage of a phosphodiester bond adjacent to the resulting apurinic/ apyrimidinic (AP) site by an “AP endonuclease,” and (iii) subsequent excision of the sugar and resynthesis by DNA polymerase. In the past few years it has been the goal of our laboratory to study the base-excision processes in depth in order to determine exactly the sequence and mechanisms of the enzymatic events involved, and the relation of this repair mode to nucleotide excision repair processes. This article summarizes some of the recent studies from our laboratory toward this goal. Space considerations prevent a review of related studies from other laboratories and such an omission should not be taken to indicate that our results are particularly unique. Indeed we encourage the reader to consult articles on similar endeavors that are found elsewhere in this volume as well as to consult Lindahl’s recent comprehensive review.3

The theory of aging which proposes that DNA repair capacity declines with age and/or DNA damage accumulates with age, resulting in increasingly aberrant gene expression, is attractive but still unproven. Most results that do not support the theory are at least neutral. We propose that much of the ambiguity produced by work so far is due to (a) the difficulty of controlling all relevant variables, including particularly the proliferation state of the cells used and the location and nature of the damage being repaired, and (b) the existence of multiple and overlapping DNA repair pathways. A better knowledge of the critical, rate-limiting events in DNA repair in vivo, and the application of more sophisticated approaches for studying these events, may ultimately resolve the ambiguities. The causal relationship between DNA damage and cancer seems more secure. Declining DNA repair capacity with age would be expected to accelerate the increased incidence of cancer with age, but it is not known how important this is compared to other genetic and environmental variables. Results with genetic diseases generally support this view. Diseases characterized by DNA repair deficiencies are accompanied by phenomena that are also characteristic of normal aging, but a causal relationship has not been established. However, some of these diseases do constitute a strong risk factor for cancer. Even less convincing is a causal link between DNA repair deficiencies and aging in the so-called premature aging syndromes.

Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.

Ribonucleoside diphosphate (RDP) reductase activity can be readily assayed in ether-treated Escherichia coli cells. The rate of cytidine 5′-diphosphate (CDP) reduction observed in ether-treated cells by using saturating substrate concentrations is about 25% of the rate of de novo deoxyribonucleotide synthesis required to account for in vivo deoxyribonucleic acid synthesis. Optimal activity is observed in the presence of magnesium ions and a positive effector. Adenosine 5′-triphosphate (ATP), deoxy ATP (dATP), and deoxythimidine triphosphate serve as positive effectors, and dATP also serves as a negative effector. These effects on the activity in ether-treated cells resemble those observed in vitro with highly purified enzyme. When the RDP reductase activity in these cells is assayed by using high specific activity 3H-CDP as substrate, even at nonsaturating substrate concentrations, the sensitivity of the assay is sufficient to make it useful for the assay of the low levels of reductase activity in cells not derepressed by thymine starvation or in cells containing mutationally altered RDP reductase. This assay is much easier to perform than the usual in vitro assay, since thioredoxin, thioredoxin reductase, and enzyme subunits B1 or B2 need not be first purified and added to the reaction mixtures.