Decrease in specific activity of heart and muscle aldolase in old mice

1. 1. Aldolase was purified from mouse heart and muscle cells by column chromatography. The enzyme from 3- and 30-month old mouse tissues was assayed under different temperature and pH conditions in order to determine the effect of these parameters on the activity of aldolase from the two age groups.2. 2. The results showed that the specific activity of aldolase decreases as a function of mouse senescence. The specific activity of young mouse muscle aldolase is times that of the enzyme from old mouse muscle. This trend was also observed with heart cell aldolase.3. 3. These results indicate that the heart and muscle cells of old mice accumulate inactive molecules of aldolase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23068/1/0000642.pd

Alkaline opening of imidazole ring of 7-methylguanosine. 2. Further studies on reaction mechanisms and products

High performance liquid chromatography (HPLC) was used to follow the kinetics of the alkaline induced opening of the imidazole ring of 7-methylguanosine (7-meGuo). The kinetics show an initial rapid formation of a major transient intermediate and some minor products that were chromato-graphically separable into seven peaks. This phase of the reaction is followed by the formation of a dominant pyrimidine derivative whose liquid chromatography retention time in a 6% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent is 6 min; during the rest of the reaction time this dominant species was progressively converted to a co-dominant species that has a 4.5-min column retention. Mass spectroscopy confirmed the existence of two species of ring opened 7-methylguanine (7-meGua), one formylated and another deformylated. Schiff's reaction demonstrated that the species in the second HPLC peak is the formylated one. The ring opened 7-methylguanine (rom7Gua) released by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species. These results demonstrate that the enzyme excises formylated rom7Gua from DNA Analysis of rom7Guo by NMR showed that there are two signals assignable to methyl protons and two to formyl protons. These chemical shifts were interpreted as being due to the opening of the imidazole ring at two sites and to the formation of formylated and deformylated rom7Gua.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23913/1/0000156.pd

Zimbabwe National Cancer Registry: summary data, 1986-1989

A summary data of the national registry of cancer in Zimbabwe in the period 1986 to 1989.The Zimbabwe National Cancer Registry began operation in 1986. Between 1986-1989, a total of 8 276 cases were identified. Among men of African descent, oesophageal (11,2 pc) and liver cancer (11,0 pc) were most common. Cervical cancer was by far the most common among women of African descent (34,5 pc). Among both males and females of non-African descent, skin cancers (other than melanoma) accounted for one-third of cancers followed by prostate cancer (7,7 pc) in males and breast cancer (18,5 pc) in females. These findings arc comparable to earlier reports of the epidemiology of cancer in Zimbabwe

Alkaline opening of imidazole ring of 7-methylguanosine. 1. Analysis of the resulting pyrimidine derivatives

Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo). High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom7Gua) in equal amounts. Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks. The species in one peak appears to be composed of formylated and that in the other of deformylated rom7Gua. The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened [8-14C]guanosine was analyzed by HPLC. The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH4H2PO4 (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent. Subsequent to Dowex 50 chromatography in an ammonium formate solvent, about 90% of the material was formylated. When stored at 24[deg]C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species. These results indicate that the two species of rom7Gua are in equilibrium. The rom7Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23912/1/0000155.pd

Changes in structural integrity of heart DNA from aging mice

The state of the structural integrity of the DNA from mouse myocardial cells has been investigated by utilizing both CsCl density gradient sedimentation and digestion by S1 endonuclease from . The DNA from myocardial cells of young mice sedimented in a narrow peak at the expected density of 1.701 g/cm3, while the DNA from the heart cells of senescent mice became broadly distributed in CsCl gradients, banding even more multimodally in alkaline sucrose gradients. This mode of sedimentation indicates that old mouse DNA becomes partially fragmented. When the native DNA of myocardial cells from 6, 20 and 30 month old mice was treated with single-strand specific S1 endonuclease, it was the DNA from the senescent mice that showed a progressive increase in sensitivity to digestion by the enzyme. The results indicate that the heart DNA of aging mice develops single-stranded gaps in addition to a breakdown into differently sized fragments.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21749/1/0000143.pd

Caffeine enhancement of digestion of DNA by nuclease S1

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNa when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (pH 7.0) or sodium phosphate buffer (pH 6.8).Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibity of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis.The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A---T base-pairs in DNA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21852/1/0000256.pd

Age-associated structural alterations in senescent mouse brain DNA

The maintenance of structural integrity in the DNA of aging mice has been examined with the aim in view of determining whether changes in genome structure constitute the molecular basis of aging. Cell lysate DNA from brains of differently aged mice was subjected to alkaline sucrose gradient sedimentation. The results show that brain DNA from young mice sediments monodispersely while that from senescent mice exhibits polydisperse sedimentation patterns, banding in four peaks corresponding to number-average molecular weights of 1.4 [middle dot] 108, 70 [middle dot] 106, 15 [middle dot] 106 and 3 [middle dot] 106.When treated with nuclease S1, it was the 30 month mouse DNA whose sedimentation shifted to the top of the gradient indicating a reduction in its molecular weight as a result of nuclease digestion. The apparent increase in single strand breaks implies that the rate of breakage in old mouse brain DNA is faster than that of repair replication. The conclusion is drawn that senescence could result from an accumulation of defects in the genome.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22999/1/0000567.pd

Some properties of RAT liver mitochondrial RNA polymerase

A rapid method suitable for purifying large amounts of mitochondria from rat liver using isopycnic zonal centrifugation is described. The RNA polymerase isolated from the purified mitochrondria was found associated with one peak when resolved by DEAE Sephadex chromatography. The enzyme was next fractionated on a phosphocellulose column followed by glycerol gradient centrifugation. A 600-fold purification was achieved when the enzyme was finally filtered through agarose gel. This final enzyme fraction consisted of one polypeptide chain as shown by polyacrylamide gel electrophoresis profiles. The enzyme has a greater preference for poly [d(A-T)] templates than for rat liver mitochondrial DNA. Inhibition of the enzyme activity required high concentrations of the inhibitors. The resistance of the enzyme to α -amanitin indicated that there was no contamination from nuclear RNA polymerase II. The conclusion is drawn that the mitochondrial RNA polymerase activity is associated with a single polypeptide.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45331/1/11010_2005_Article_BF01731777.pd

The Fpg/Nei Family of DNA Glycosylases

During the initial stages of the base excision DNA repair (BER) pathway, DNA glycosylases are responsible for locating and removing the majority of endogenous oxidative base lesions. The bifunctional formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of the Fpg/Nei family, one of the two families of glycosylases that recognize oxidized DNA bases, the other being the HhH/GPD (or Nth) superfamily. Structural and biochemical developments over the past decades have led to novel insights into the mechanism of damage recognition by the Fpg/Nei family of enzymes. Despite the overall structural similarity among members of this family, these enzymes exhibit distinct features that make them unique. This review summarizes the current structural knowledge of the Fpg/Nei family members, emphasizes their substrate specificities, and describes how these enzymes search for lesions

Specialization of an Exonuclease III family enzyme in the repair of 3′ DNA lesions during base excision repair in the human pathogen Neisseria meningitidis

We have previously demonstrated that the two Exonuclease III (Xth) family members present within the obligate human pathogen Neisseria meningitidis, NApe and NExo, are important for survival under conditions of oxidative stress. Of these, only NApe possesses AP endonuclease activity, while the primary function of NExo remained unclear. We now reveal further functional specialization at the level of 3′-PO4 processing for NExo. We demonstrate that the bi-functional meningococcal glycosylases Nth and MutM can perform strand incisions at abasic sites in addition to NApe. However, no such functional redundancy exists for the 3′-phosphatase activity of NExo, and the cytotoxicity of 3′-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes. A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3′-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level. This specialization of two Xth enzymes for the 3′-end processing and strand-incision reactions has not previously been observed and provides a new paradigm within the prokaryotic world for separation of these critical functions during base excision repair