Rat Brain DNA Research Articles

The Pattern of DNA and Histone H3 Methylation in Rat Brain in Response to Severe Hypobaric Hypoxia and Hypoxic Postconditioning

Methylation of histone H3 Lys4 (meН3К4) and Lys9 (meН3К9), as well as DNA methylation (meDNA), was investigated in rat hippocampal and neocortex neurons in an original model of severe hypobaric hypoxia (SHH) and hypoxic postconditioning (PostC). It was shown that, in hippocampal field CA1, a day after SHH, the content of meH3K4 increased, while the level of meDNA decreased. Later, the amount of meH3K9 decreased and the meDNA content increased. PostC increased meH3K4, normalized the level of meH3K9, and decreased the level of meDNA in the hippocampal field CA1 of rats that had survived SHH. In the neocortex, significant changes were detected only 1–2 days after SHH, consisting in the stimulation of histone H3 methylation and decreased meDNA. Thus, a complex pattern of changes in the methylation of H3 histone and DNA was observed in both the hippocampus and neocortex in response to SHH. However, the protective effect of PostC was accompanied by the correction of only hippocampal reactions, while methylation in the neocortex returned to the initial level regardless of the PostC.

Effect of intensive endurance training on rat brain and hepatic 8-oxoguanine DNA glycosylase and 8-hydroxy-2-deoxyguanosine levels

Aims: The purpose of this study was to investigate the effect of intensive endurance training on 8-oxoguanine DNA glycosylase (OGG1) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in liver and brain of rats. Materials & Methods: 16 adult Wistar albino rats were randomly divided into sedentary control and endurance exercise training groups. Animals ran on treadmill for 6 weeks, 6 days a week, at at a speed of 10 m/min (85 percent of maximal oxygen consumption). The content of OGG1 and 8-OHdG were measured using sandwich ELISA assay. Data analyzed using Student’s T-test at P≤0.05 level. Findings: our results showed that intensive endurance training has no significant effect on 8-OHdG contents in liver (t14=1.09, p=0.29) and brain (t14=0.93, p=0.36) of rats. However, contents of OGG1 in liver (t14=5.84, p=0.001) and brain (t14=4.09, p=0.001) of rats significantly increases following intensive endurance training. Finally, there were no significantly differences between changes in contents of 8-OHdG (t14=0.44, p=0.66) and OGG1 (t14=1.72, p=0.10) in liver and brain of rats following endurance training. Conclusion: intensive endurance training maintains 8-OHdG genomic damage in baseline level in liver and brain of rats by increasing contents of OGG1.

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3′-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11–13 nt away in the top strand and 9–10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5′-CN11–13↓N9–10G-3′/3′-GN9–10↓N11–13C-5′, with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells.

Effects of static magnetic field exposure on antioxidative enzymes activity and DNA in rat brain

The present study was undertaken in order to investigate the effects of static magnetic field (SMF) exposure on the antioxidative enzymes activity, malondialdehyde (MDA) concentration and DNA oxidation in male rat brain. The exposure of rats to SMF (128 mT, 1 h/day during 30 consecutive days) decreased the glutathione peroxidase (GPx; -39%, p < 0.05), CuZn superoxide dismutase (CuZn-SOD; -35%, p < 0.05) and catalase (-59%, p < 0.05) activities in frontal cortex. The same treatment decreased the CuZn-SOD (-51%, p < 0.05) and Mn-SOD (-13%, p < 0.05) activities in hippocampus. However, the glutathione levels remained unchanged in the both brain structures. In the hippocampus, SMF exposure increased MDA concentration (+32%, p < 0.05). Interestingly, exposed-rats to SMF displayed a significant increase of metallothioneins level in frontal cortex (+100%, p < 0.05), while the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) concentration remained unaffected, indicating the absence of DNA oxidation. Our results indicated that sub-chronic exposure to SMF induced oxidative stress in rat hippocampus and frontal cortex. Metallothionein induction protected probably DNA against oxidative damage.

A novel rat genomic simple repeat DNA with RNA-homology shows triplex (H-DNA)-like structure and tissue-specific RNA expression

Mammalian genome contains a wide variety of repetitive DNA sequences of relatively unknown function. We report a novel 227 bp simple repeat DNA (3.3 DNA) with a d {(GA) 7A (AG) 7} dinucleotide mirror repeat from the rat ( Rattus norvegicus) genome. 3.3 DNA showed 75–85% homology with several eukaryotic mRNAs due to (GA/CU) n dinucleotide repeats by nBlast search and a dispersed distribution in the rat genome by Southern blot hybridization with [ 32P]3.3 DNA. The d {(GA) 7A (AG) 7} mirror repeat formed a triplex (H-DNA)-like structure in vitro. Two large RNAs of 9.1 and 7.5 kb were detected by [ 32P]3.3 DNA in rat brain by Northern blot hybridization indicating expression of such simple sequence repeats at RNA level in vivo. Further, several cDNAs were isolated from a rat cDNA library by [ 32P]3.3 DNA probe. Three such cDNAs showed tissue-specific RNA expression in rat. pRT 4.1 cDNA showed strong expression of a 2.39 kb RNA in brain and spleen, pRT 5.5 cDNA showed strong expression of a 2.8 kb RNA in brain and a 3.9 kb RNA in lungs, and pRT 11.4 cDNA showed weak expression of a 2.4 kb RNA in lungs. Thus, genomic simple sequence repeats containing d (GA/CT) n dinucleotides are transcriptionally expressed and regulated in rat tissues. Such d (GA/CT) n dinucleotide repeats may form structural elements (e.g., triplex) which may be sites for functional regulation of genomic coding sequences as well as RNAs. This may be a general function of such transcriptionally active simple sequence repeats widely dispersed in mammalian genome.

Oxidative damage and breakage of DNA in rat brain after transient MCA occlusion

As thrombolytic therapy for treatment of ischemic stroke was propagated, much attention has been paid to reperfusion brain injury. Oxidative stress is one of the most important factors that exacerbate tissue damage by reperfusion. Thus, we investigated the extent of oxidative damage in rat brain after transient middle cerebral artery (MCA) occlusion by immunohistochemical analysis for 8-hydroxy-2′-deoxyguanosine (8-OHdG), which is one of the best markers of oxidative damage. Furthermore, in order to investigate its role in neuronal cell death, we performed terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) study, and compared the results with that of 8-OHdG immunohistochemistry. There was no immunoreactive 8-OHdG in sham-operated brain, but it became present in neurons of MCA territory at 3 h of reperfusion after 90-min ischemia. At 48 h after reperfusion, cerebral tissue of MCA territory was severely destroyed, and many cells in that area revealed TUNEL positivity. Some neurons in MCA territory showed mild immunoreactivity for 8-OHdG at that time, but it was strongest in neurons in the outer area of MCA territory. Those cells did not show TUNEL positivity, suggesting that 8-OHdG production is not necessarily followed by early cell death. Here, it was demonstrated that oxidative DNA damage occurs in more extended area than that where cell death is recognized. Although this damage does not cause early cell death, this might result in more prolonged cell dysfunction and eventual neuronal loss. Anti-oxidant therapy might be required for treatment of stroke in the future.

PCR assay of DNA damage and repair at the gene level in brain and spleen of γ-irradiated young and old rats

The PCR amplification of fragments of transcribed (β-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from γ-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The β-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of γ-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene β-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of γ-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old γ-irradiated animals for both of the repair phases.

Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia.

The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.

Formation of 8-oxodeoxyguanosine in brain DNA of rats exposed to acrylonitrile.

Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.

Characterization of the 5′ region of the rat brain glycine transporter GLYT2 gene: identification of a novel isoform

We have identified two alternative 5′ ends for the GLYT2 glycine transporter in rat brain DNA by using rapid amplification of cDNA ends (RACE) analysis. Study of the genomic DNA revealed that the isoform diversity is generated by alternative use of exons Ia or Ib, respectively. Upon translation, the mRNA corresponding to the novel isoform GLYT2b would yield a protein five amino acids longer than the previously characterized isoform GLYT2a. Both forms display similar regional distribution and kinetics characteristics. However, whereas GLYT2a is able to actively accumulate glycine into transfected COS cells, GLYT2b seems only to exchange (or release) glycine.

Age-dependent increase of indigenous DNA adducts in rat brain is associated with a lipid peroxidation product.

Indigenous DNA adducts (I-compounds) are considered to be a biomarker of aging tissues. Thus far, few studies have been conducted to investigate the accumulation patterns of I-compounds in the brain during aging. Particularly, identities of age-dependent I-compounds have largely remained unknown. In the current study, we have determined the amounts of I-compounds in the brains of male Fischer 344 rats at ages 1, 6, 12, 18, and 24 months using a 32P-postlabeling technique. The results indicate that I-compounds increase in the rat brain age dependently from 6 to 24 months of age. Total I-adduct levels (central and upper cutouts) increase 3.5-fold from 6 to 24 months. Contrary to the results of other investigators, brains of 1-month-old rats contain the highest level of I-compounds, which may be due to the hypermetabolic status during the infant period. In an effort to characterize I-compounds, different deoxynucleosides were coincubated with malondialdehyde (MDA). The results show that only deoxyguanosine (dGMP)-MDA adducts overlap with I-compounds of the rat brain DNA adducts map. A total of five dGMP-MDA adducts have been identified as responsible for I-compounds in brain tissues. It is known that brain tissue contains high levels of lipids that are susceptible to oxygen free radicals and that MDA is the most abundant and genotoxic product of lipid peroxidation. The present study provides supporting evidence that lipid peroxidation and its product (MDA) may play an important role in endogenous brain DNA modification, which may partly contribute to cerebral aging and age-related degenerative disorders of the brain. The accumulation of I-compounds with aging may serve as an index of indirect oxidative damage to DNA as evidenced by the presence of MDA-DNA adducts.

Age-dependent increases of DNA adducts (I-compounds) in human and rat brain DNA.

Brain DNA from 20 humans ranging in age from neonatal to 100 years was analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay for bulky covalently modified nucleotides. A reproducible pattern of three 32P-labeled spots was obtained by thin-layer chromatography followed by autoradiography. Two of these spots increased with age (Mann-Whitney U-test; P < 0.001; comparison of ages < or = 60 years and ages > 60 years). Thus, these spots met the definition of I-compounds. Rat brain DNA exhibited the same two I-spots, whose intensities also increased with animal age (1, 4, and 10 months). In humans, considerable individual variation of brain I-compound levels was observed, especially at ages > 60 years, presumably reflecting environmental, life-style, or genetic factors. This variation was not noted for brain DNA of laboratory rats. Thus, human brain DNA undergoes progressive covalent modifications with aging.

Environmental stimuli modulate the circadian rhythm of [ 3H-methyl]thymidine incorporation into brain DNA of male rats

A circadian rhythm of DNA synthesis is present in most organs of adult mammals, with peak levels (acrophase) generally in the rest period. We have recently reported that in the rat brain the acrophase of the rhythm of thymidine incorporation into DNA occurs on the contrary during the active period. To determine whether the brain waking activity was exerting a modulatory action we measured the circadian rhythm of incorporation in the brain and kidney of young adult male rats housed in conditions of sensory and social enrichment or impoverishment for four days. Biochemical and autoradiographic data show that the brain rhythm persists in the enriched condition, but is abolished in the impoverished condition. On the other hand, the rhythm of incorporation in the kidney is maintained in both conditions. These results suggest that the permanence of the brain oscillation is selectively dependent on the complexity of the sensory and social stimulation.

Multiple mRNAs of rat brain alpha-crystallin B chain result from alternative transcriptional initiation.

Two major classes of mRNAs for the alpha-crystallin B chain (or alpha(B)crystallin), about 0.9 and 1.2 kilobases in length, are expressed in rat brain. To examine the structures of these mRNAs, we isolated cDNA clones from rat brain and genomic DNA from rat liver. Characterization of these clones as well as Northern blot analysis indicated that the various mRNAs differed in the lengths of their 5' leader sequences. RNase protection assays revealed that the gene for alpha-crystallin B chain contains multiple start sites. The transcriptional start sites of the longer mRNAs are preceded by a putative CAAT box and that of the shorter mRNA by a putative TATA box. The shorter mRNA encodes the alpha-crystallin B chain protein, whereas the longer mRNA contained three extra small open reading frames upstream of the AUG start codon for the protein. The shorter mRNA is abundant in lens, heart, muscle, and kidney, while the longer mRNAs are constitutively expressed at low levels in a wide variety of tissues. The shorter mRNA was increased by treatment with phorbol 12-myristate 13-acetate in rat C6 glioma cells. Since there is only a single copy of the alpha-crystallin B chain gene, our results indicate that the two classes of mRNAs are generated by alternative transcriptional initiation from different promoters and their expressions are regulated differentially.

Molecular cloning, sequencing and expression of an ?2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet alpha 2-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney alpha 2-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3'-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5'-untranslated region. Southern-blot analysis with a human platelet alpha 2-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain alpha 2-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A+) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the alpha 2-adrenergic antagonist [3H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the alpha 2A subclass. We conclude that the rat brain cDNA encodes a new alpha 2-adrenergic receptor subtype that may be brain-specific.

Differential cloning of genomic DNA: cloning of DNA with an altered primary structure by in-gel competitive reassociation.

A procedure was developed for cloning (anonymous) DNA sequences whose primary structures differ between two DNA samples. The procedure is based upon in-gel competitive DNA reassociation after electrophoresis of a mixture of restriction enzyme-digested target DNA (from which clones are to be isolated) and a large excess of unclonable reference DNA (competitor DNA). Inclusion of polyethylene glycol in the reassociation buffer greatly improved the in-gel reassociation efficiency, which was critical for the practical use of the procedure. Using this technique, we obtained several clones from rat brain (target) DNA, which may have been derived from tissue (brain)-specific altered DNA structures. The details of this procedure and its possible applications are discussed.

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.

A tissue-specific change in repetitive DNA in rats.

From a genomic library constructed from an EcoRI digest of Wistar rat brain DNA, we isolated a clone (BL-1) that gave a 0.6-kilobase restriction fragment only in brain (or lens) DNA upon Southern hybridization. The tissue-specific fragment was present in Wistar and other strains (Sprague-Dawley and Donryu) of rats regardless of their sex and age. Sequencing of the clone indicated that it is closely related to a part of the LINE 3 sequence, one of the highly repetitive sequences present throughout mammalian genomes. Polymerase chain reaction using primer sequences in the BL-1 clone indicated that it is derived from an amplified (rear-ranged) sequence, although other explanations are possible. These results suggest that there are tissue-specific changes in DNA primary structure during mammalian developmental processes.

Distribution of O6-ethylguannine in DNA exposed to ethylnitrosourea in vitro as visualized by electron microscopy using a monoclonal antibody

Application of a monoclonal antibody (Mab ER-6; Rajewsky et al., 1980) specific for O 6-ethyl-2′-deoxyguanosine ( O 6-EtdGuo), in conjunction with a protein-free spreading procedure for double-stranded DNA molecules and transmission electron microscopy, permits the visualization of antibody molecules complexed to O 6-EtdGuo residues formed in DNA upon reaction with hte carcinogen N-ethl- N-nitrosourea (EtdGuo) Nehls et al., 1984). To obtain information on the distribution of O 6-eEtdGuo in native DNA exposed to EtNU in vitro, samplesof purified rat brain DNA were briefly incubated with EtNU at concentrations differing by a factor of 8 (0.5 and 4 mg of EtNU/ml, respectively). As determined in DNA hydrolysates by competitive radioimmunoassay, the resulting DNA preparations contained O 6-EtdGuo at O 6-EtdGuo/2′-deoxyguanosine molar ratios of 15.1 x 10 −5 and 116 x 10 −5, respectively. Interspace distances between Mab-binding sites in both sets of ethylated DNA were determined by electron microscopy both individual DNA fragments of different size, and in computer-generated, long-thread DNA constructs. Comparative statistical analyses by a newly developed MOLRANDO computer program show a non-random distribution pattern of Mab-binding O 6-EtdGuo redisues.

Stimulation of rat kidney, spleen and brain DNA-(cytosine-5)-methyltransferases by divalent cobalt ions

Rat kidney, spleen, brain, and liver DNA-methylases were partially purified by chromatography on DEAE—Trisacryl columns and their catalytic properties were studied. Crude extracts contain one or several inhibitors which are thermostable and resistant to acidic or alkaline treatments and which can be eliminated by dialysis, or by chromatography on DEAE—Trisacryl. These are most probably divalent ions, such as, Pb 2+, Zn 2+, Cu 2+, Fe 2+, Mg 2+, Mn 2+ or Ca 2+, which inhibit the DNA-methylase activity. However, Co 2+, at concentrations ranging from 0.05 mM to 1 mM, has an efficient stimulatory action on spleen, kidney or brain DNA-methylase activity. The spleen DNA-methylase activity on chicken erythrocyte DNA could be increased 10-fold, by a 0.2 mM concentration of Co 2+, but no stimulation was found with liver DNA-methylase. The fact that significant differences exist between the DNA-methylases from the different organs in their behavior towards Co 2+ could indicate that these enzymes are different.