Role of postreplicative DNA mismatch repair in the cytotoxic action of thioguanine.

It is proposed here that the delayed cytotoxicity of thioguanine involves the postreplicative DNA mismatch repair system. After incorporation into DNA, the thioguanine is chemically methylated by S-adenosylmethionine to form S6-methylthioguanine. During DNA replication, the S6-methylthioguanine directs incorporation of either thymine or cytosine into the growing DNA strand, and the resultant S6-methylthioguanine-thymine pairs are recognized by the postreplicative mismatch repair system. Azathioprine, an immunosuppressant used in organ transplantation, is partly converted to thioguanine. Because the carcinogenicity of N-nitrosamines depends on formation of O6-alkylguanine in DNA, the formation of the analog S6-methylthioguanine during azathioprine treatment may partly explain the high incidence of cancer after transplantation.

Susceptibility of feline spinal cord energy metabolism to severe incomplete ischemia.

Feline spinal cords were subjected to 10 to 30 minutes of severe incomplete ischemia (average reduction in blood flow of 92%) with and without 90 minutes of recirculation, and the L-2 segment was analyzed for high-energy phosphates and certain glycolytic metabolites. Spinal cord tissue lactic acid levels were stepwise elevated, and adenosine triphosphate (ATP), phosphocreatine (P-creatine), and glucose were progressively consumed by increasing durations of ischemia. However, upon restoration of blood flow, there was extensive recovery of energy metabolites and normalized lactic acid, demonstrating resumption of mitochondrial oxidative metabolism. These data indicate that the spinal cord can tolerate at least 30 minutes of severely reduced blood flow before recovery of energy metabolism is significantly impaired upon restitution of blood flow.

Pretreatment with alpha tocopherol enhances neurologic recovery after experimental spinal cord compression injury.

Lipid hydrolysis with subsequent production of eicosanoids and lipid peroxidation are two of the earliest potentially pathochemical events induced in spinal cord tissue by mechanical trauma. Although these membrane lipid disturbances are thought to contribute to the paralysis that occur subsequent to spinal cord injury, such a correlation has not been demonstrated directly. Consequently, the purpose of this study was to test the capacity of alpha tocopherol, the major lipid antioxidant in cellular membranes and a compound that limits the injury-induced lipid hydrolysis and peroxidation in spinal cord tissue, to promote functional recovery in a static loading model of spinal cord injury. After laminectomy, the L2 spinal cord of cats was compressed with 180 g for 5 min. For 5 days before injury and for 5 days postinjury, treated cats received orally 1000 IUD-alpha tocopherol acetate daily. Control cats were similarly injured but untreated. All cats were blindly evaluated weekly for 4 weeks for their neurologic recovery based on an 11 point behavioral scale that assessed walking, running, and stair climbing. By the second postinjury week, alpha tocopherol-pretreated cats demonstrated significantly better recovery than untreated controls. By 4 weeks, treated cats had recovered 72% of their preinjury function as compared with 20% for untreated controls, i.e., a 3.5-fold difference. These results strongly suggest that lipid peroxidation and/or hydrolysis is primarily involved in the genesis of posttraumatic paralysis and that alpha tocopherol exerts its protection of injured spinal cord tissue, at least in part, by its antioxidant and/or antilipolytic activity.

Spinal cord energy metabolism in normal and postlaminectomy cats.

The purpose of this study was to determine 1) normal concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), phosphocreatine (P-creatine), glucose, lactate, and pyruvate in upper (L-2) and lower (L-5) feline lumbar spinal cord, and 2) whether previously reported laminectomy-induced reduction in spinal cord blood flow (SCBF) resulted in disturbance of spinal cord energy metabolism. Concentrations of ATP, P-creatine, pyruvate, and glucose were significantly higher at L-5 than at L-2, probably as the result of larger amounts of gray matter at L-5 than L-2. Significant increases in ADP and AMP levels were the only metabolite changes noted 15 minutes following laminectomy. The authors speculate that the increase in ADP and AMP is due to a laminectomy-induced stimulation of ATP utilization. However, lack of change in other metabolites implies an efficient energy homeostasis. These results indicate that although laminectomy can reduce SCBF, the degree of this reduction is insufficient to adversely affect spinal cord energy metabolism. Thus, tissue from beneath or near the laminectomy site is viable and essentially normal.

Effect of methylprednisolone in compression trauma to the feline spinal cord.

The purpose of this study was to determine the effect of methylprednisolone sodium succinate on clincal recovery and tissue preservation following compression trauma of feline spinal cord. Cats were anesthetized with pentobarbital and injured by placing a 170-gm weight on the spinal cord for 5 minutes. One hour after injury, the animals were given intravenous steroid (15 mg/kg/day) for 2 days in three devided doses, 15 mg/kg/day for 1 day intramuscularly, 7.5 mg/kg/day intramuscularly for 3 days, and 3.75 mg/kg/day intramuscularly for 3 days, for a total of 9 days. In a control group, the animals were injured but untreated. At 60 days after injury, the animals were sacrifieced by perfusion fixation with 10% formalin. The spinal cord was removed and evaluated for a number of morphometric parameters, including percentage of spinal cord cross-sectional area containing the cavity (%area) and percentage of spinal cord volume occupied by the cavity (%volume). A clinical recovery score (recovery index) was devised to evaluate neurological recovery. Steroid-treated cats showed significantly greater recovery than the untreated controls (p less than 0.001). Moreover, the spinal cord of treated cats displayed greater tissue preservation as measured by %area (p leass than 0.005) and %volume (p less than 0.004). Correlation coefficients comparing the recovery index with morphometric parameters revealed a negative correlation between cavity size and recovery. These data provide evidence for a beneficial effect of methylprednisolone in promoting recovery and preserving spinal cord tissue following blunt injury to the feline spinal cord.

Evaluation of an intensive methylprednisolone sodium succinate dosing regimen in experimental spinal cord injury.

Beginning 30 minutes after compression trauma of the upper lumbar (L-2) spinal cord, cats were treated with either a high-dose regimen of methylprednisolone (MP) administered as the sodium salt of the 21-succinate ester (Solu-Medrol sterile powder) or the MP vehicle. Animals were randomly assigned to either treatment group (10 cats per group), and all personnel were blind as to which animals received vehicle or drug. The intensive 48-hour dosing regimen was designed to maintain therapeutic tissue levels of MP and consisted of an initial 30 mg/kg intravenous bolus of MP; 2 and 6 hours later additional 15 mg/kg MP doses were administered by intravenous bolus. Immediately following the bolus given at 6 hours, a continuous MP infusion of 2.5 mg/kg/hr was started. The infusion was stopped abruptly at 48 hours with no dose tapering. Animals in the vehicle group received an equivalent volume of MP vehicle. The total MP dose administered over 48 hours was 165 mg/kg. Animals were evaluated weekly for neurological recovery based upon a 12-point functional scale which assessed general mobility, running, and stair-climbing. Mean recovery scores at 1 month after injury (+/- standard error of the mean) were: vehicle group (seven cats) 3.7 +/- 0.9, and MP group (10 cats) 8.7 +/- 0.2; (p less than 0.001). Histological evaluation of the spinal cords revealed a strong negative correlation between neurological recovery and size of the spinal cord cavity at 1 month (r = -0.88). Three of 10 animals in the vehicle group became ill and had to be dropped from the study, whereas all of the 10 MP-treated animals survived in excellent health. The results demonstrate the therapeutic effectiveness and low incidence of side effects associated with an intensive MP dose regimen for treatment of experimental spinal cord injury.

Spinal cord energy metabolism following compression trauma to the feline spinal cord.

The purpose of this study was to determine the spinal cord metabolic state for 24 hours after compression trauma to the feline spinal cord. Cats were anesthetized with pentobarbital and injured by placing a 190-gm weight on the spinal cord for 5 minutes. Biochemical analysis of the injured segment revealed a significant depletion in the levels of adenosine triphosphate (ATP), phosphocreatine (P-creatine), and total adenylates for the entire 24-hour recovery period. Glucose levels initially declined, but by 1 hour had normalized, and at 8 and 24 hours were significantly supranormal. The lactate/pyruvate ratio and tissue lactate concentrations increased four and five and half times, respectively, for the first 4 hours after injury. Between 8 and 24 hours, lactate levels remained elevated, whereas the lactate/pyruvate ratio declined to contol levels as the result of a significant rise in the tissue pyruvate concentration. This sequence of metabolic changes suggested that metabolism was probably not homogeneous throughout the injured segment, and that tissue metabolic rate was depressed for the initial 4 hours after trauma then increased in metabolically active tissue for the remainder of the 24-hour recovery period. This model of spinal cord trauma results in a severe, prolonged ischemia and metabolic injury to the affected tissue. Whether these metabolic changes results from or cause the tissue damage and irreversible paraplegia associated with this type of spinal cord injury remains to be determined.

Microvascular perfusion and metabolism in injured spinal cord after methylprednisolone treatment.

The purpose of this study was to determine the effect of treatment with the synthetic glucocorticoid, methylprednisolone, on the microvasulature and metabolism of the traumatized spinal cord. Spinal cords of cats were compressed with a 170-gm weight for 5 minutes and were treated with either high-dose methylprednisolone (HDMP, 15 mg/kg/24 hrs) or megadose methylprednisolone (MDMP,60 mg/kg/24 hrs). Animals were sacrificed at 2, 8, or 24 hours following injury. Treatment with HDMP resulted in substantial preservation of injured spinal cord microvascular perfusion at 8 hours as compared with injured untreated cats. Compression trauma caused a partial derangement of energy metabolism and a shift toward anaerobic glycolysis in both treated and untreated groups for the entire 24-hour postinjury period. Tissue levels of adenosine triphosphate, phosphocreatine, and total adenylates in the HDMP-treated cats sacrificed at 8 hours after trauma were significantly elevated over untreated controls, but those in the 2- and 24-hour groups were not. Concentration of energy intermediates in MDMP-treated cat were either equal to or below those of injured untreated animals al all three postinjury time period. The postinjury metabolite pattern and concentrations seen in this study possibly result from differing levels of blood flow and neuronal activity in the injured untreated, HDMP-, and MDMP-treated spinal cords. Better tissue perfusion in the HDMP-treated cats might be expected to result in an improved tissue energy state in these animals. However, intensive high-dose glucocorticoid treatment has been demonstrated to augment spinal cord monosynaptic and polysynaptic reflex transmission and primary afferent excitability. Furthermore, acute single intravenous dose studies have shown this direct neuronal action to be dose-related. Thus, additional high-energy phosphate molecules that may be reformed as a result of HDMP treatment were perhaps used as the energy source for any increased neuronal activity caused by steroid administration. The beneficial effects of glucocorticoid treatment in experimental spinal cord trauma might derive from preserved cellular structural integrity. This could result in increased levels of neuronal activity, energy utilization, and production in treated as compared with untreated tissue.

Effects of treatment with U-74006F on neurological outcome following experimental spinal cord injury.

The compound U-74006F is one of a series of 21-aminosteroids that lack glucocorticoid or mineralocorticoid activity. These potent inhibitors of lipid peroxidation have been specifically developed for the acute treatment of central nervous system trauma and ischemia. This study evaluated the dose-response characteristics and capability of U-74006F to promote functional recovery in cats subjected to compression trauma of the upper lumbar (L-2) spinal cord. Thirty minutes following injury, randomized and investigator-blinded treatment was initiated with the intravenous administration of either vehicle (citrate-buffered saline) or one of eight doses of U-74006F. Initial doses of U-74006F ranged from 0.01 to 30 mg/kg. Subsequent doses consisted of intravenous bolus injections followed by a continuous 42-hour intravenous infusion. Over the 48-hour treatment period, cats received total U-74006F doses ranging from 0.048 to 160 mg/kg. The animals were evaluated weekly for neurological recovery based upon an 11-point behavioral scale. With the exception of two cats in one group, the animals receiving accumulated doses of U-74006F (ranging from 1.6 to 160.0 mg/kg/48 hrs) exhibited nearly 75% of normal neurological function by 4 weeks after injury. Lower total doses of 0.16 and 0.48 mg/kg/48 hrs were associated with approximately 50% return of normal function, which was not significantly better than the recovery in the vehicle-treated control group. The lowest total dose tested (0.048 mg/kg/48 hrs) gave results indistinguishable from those in vehicle-treated cats, which had recovered only 20% of their preinjury neurological function by 4 weeks. These findings demonstrate that over a 100-fold range of doses, U-74006F has a remarkable capacity to promote functional recovery in spinal cord-injured cats.

Thymine-DNA glycosylase and G to A transition mutations at CpG sites.

About 23% of mutations in hereditary human diseases and 24% of mutations in p53 in human cancers are G to A transitions at sites of cytosine methylation suggesting that these sites are either foci for DNA damage, or foci for damage that is poorly repaired. Thymine produced at these sites by the hydrolytic deamination of 5-methylcytosine is removed by thymine-DNA glycosylase. Thymine-DNA glycosylase will also remove 3,N(4)-ethenocytosine and uracil from DNA. The action of this enzyme is limited by its very low k(cat) and by tight binding to the apurinic site produced when the thymine is removed. These properties of the enzyme suggest that the inefficiency of the base excision repair pathway that it initiates may be the underlying cause of the prevalence of these mutations.

Evaluation of the revised NIOSH lifting equation. A cross-sectional epidemiologic study.

STUDY DESIGN: A cross-sectional study of the 1-year prevalence of low back pain was conducted in workers employed in manual lifting jobs. OBJECTIVES: To provide epidemiologic data to determine the correlation between the prevalence of low back pain and exposure to manual lifting stressors, measured with the lifting index component of the revised lifting equation from the National Institute for Occupational Safety and Health (NIOSH). SUMMARY OF BACKGROUND DATA: The NIOSH lifting equation has been proposed as a practical, yet valid tool for assessing the risks of low back pain caused by manual lifting. To date, however, there have been few studies in which the effectiveness of the equation to identify jobs with elevated rates of low back pain has been evaluated. METHODS: Fifty jobs from four industrial sites were evaluated with the NIOSH lifting equation. A symptom and occupational history questionnaire was administered to 204 people employed in lifting jobs and 80 people employed in nonlifting jobs. Regression analysis was used to determine whether there was a correlation between the lifting index and reported low back pain. RESULTS: As the lifting index increased from 1.0 to 3.0, the odds of low back pain increased, with a peak and statistically significant odds ratio occurring in the 2 < lifting index < or = 3 category (odds ratio = 2.45). For jobs with a lifting index higher than 3.0, however, the odds ratio was lower (odds ratio = 1.45). CONCLUSIONS: Although low back pain is a common disorder, the lifting index appears be a useful indicator for determining the risk of low back pain caused by manual lifting.

Accuracy of measurements for the revised NIOSH lifting equation. National Institute for Occupational Safety and Health.

Twenty-seven non-ergonomists who participated in a one-day training session on the use of the NIOSH lifting equation (NLE) were subsequently tested on a simulated lifting task eight weeks later to determine their accuracy in measuring the variables. Analysis of the results indicate that (1) inter-observer variability was small, especially for the most important factor (i.e. horizontal distance); (2) individuals can be trained to make measurements with sufficient accuracy to provide consistent recommended weight limit and lifting index values; and (3) measurement of the coupling and asymmetric variables were the least accurate.

Ergonomic job design to accommodate and prevent musculoskeletal disabilities.

Work-related musculoskeletal disorders (MSDs) account for a major portion of the cost of work-related injury and illness in the United States. Many of these injuries and illnesses lead to temporary or permanent disability. It is generally accepted that the incidence of MSDs increases when the demands of the job exceed the capabilities of the worker. As the workforce ages and physical capabilities decline, it is anticipated that many more Americans will request disability-related leave resulting from musculoskeletal disorders because they are unable to meet the demands of the job. To prevent these disabilities and to accommodate a wider range of people in the workforce, physical job demands may have to be reduced so that a larger portion of the population will be capable of working. Providing engineering controls or alternative work arrangements allows for accommodation of workers with a wide range of capabilities and can assist in rehabilitation and early return to work following injury.

Interventions to reduce low-back injury risk among youth who perform feed handling and scooping tasks on farms.

Children and adolescents who perform farm chores are exposed to cumulative trauma injury risks, particularly to the low back. For example, they may routinely handle heavy materials and need to adopt awkward postures during farm chores. Two potential interventions aimed at reducing low-back injury risk were examined in the laboratory: the use of a rugged cart and proper orienting of feed bags to ease feed transport; and the use of a modified feed bin, intended to allow easier access to product scooped from the bin at different levels. A lumbar motion monitor device was used to quantify trunk movement and determine injury risk level. Fourteen male and female youth who regularly perform these farm chores participated in the study. The cart significantly reduced low-back injury risk by nearly 10%, compared with manual feed bag lifting and carrying. The modified feed bin did not significantly reduce low-back injury risk, compared with traditional scooping. Regardless of the method used, however, scooping feed from the top of the bin reduced lower back disorder risk by 50% or more compared to the two lower levels. This study showed that relatively simple and low-cost solutions can be applied to farm environments to help protect the low backs of youth who perform farm chores.

New procedure for assessing sequential manual lifting jobs using the revised NIOSH lifting equation.

A sequential manual lifting job is defined as a job where workers rotate between a series of manual lifting rotation slots or elements at specified time intervals during the course of a work shift. The original NIOSH lifting equation lacked a method for assessing the physical demands of these types of jobs. This paper presents the sequential lifting index (SLI), a new conceptual method for assessing the physical demands for sequential manual lifting jobs. The new method is similar to the composite lifting index (CLI) method that was provided by NIOSH for assessing multi-task jobs. The SLI method expands upon the methods originally provided by NIOSH by providing a simple method for estimating the relative magnitude of physical stress for sequential manual lifting jobs. It should also be useful in assisting safety and health specialists to prioritize or rank hazardous jobs within a plant.

Interaction of the restriction endonuclease EcoRV with the deoxyguanosine and deoxycytidine bases in its recognition sequence.

The interaction of the EcoRV restriction endonuclease with the dG and dC bases in its recognition sequence (GATATC) has been studied using base analogues. These modified dG and dC bases each have a single potential protein contact removed. The analogues have been incorporated into the self-complementary dodecamer d(pGACGATATCGTC) at the appropriate positions (underlined). Many of the analogues caused no change in the Tm of the duplex or else lowered the Tm by a small amount such that a duplex was still formed at temperatures suitable for enzyme assay. However, the dG analogue 2-aminopurine-1-beta-D-2'-deoxyriboside destabilized the duplex to such an extent that the 12'-mer could not be used for enzyme assays. To overcome this, a longer self-complementary 18'-mer was used with this modified base. The circular dichroism spectra of the modified base containing 12'-mers (and the 18'-mer in the case of 2-aminopurine) were very similar to the parent sequences lacking modified bases. This demonstrates the formation of B-DNA structures in all cases and similar overall conformations. The Km and kcat values for the various modified oligomers have been determined, and these data have been used to assess the roles that functional groups on the dG and dC bases play in the recognition and hydrolysis of GATATC sequences by the endonuclease. The results obtained here have been compared to the crystal structures of the EcoRV complexed with a GATATC sequence, and this has allowed a critical evaluation of the base analogue approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect.

A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed. The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate. The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases. Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis. Both of the hexameric products are single stranded under the assay conditions. The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect. This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.

Cytotoxic mechanism of 6-thioguanine: hMutSalpha, the human mismatch binding heterodimer, binds to DNA containing S6-methylthioguanine.

It has been suggested that the cytotoxicity of 6-thioguanine depends upon (1) incorporation of 6-thioguanine into DNA, (2) methylation by S-adenosylmethionine (SAM) of the thio group to give S6-methylthioguanine, (3) miscoding during DNA replication to give [SMeG] x T base pairs, and (4) recognition of these base pairs by proteins of the postreplicative mismatch repair system. Here we have investigated systematically the ability of proteins present in human cell extracts to bind to DNA containing S6-methylthioguanine. We found that [SMeG] x T base mismatches were bound by the mismatch binding complex, hMutS alpha, and that the level of binding was dependent upon the base 5' to the S6-methylthioguanine in the order G > C = A > T. Extracts from cells that lack either hMSH2 (LoVo cells) or GTBP (HCT-15 cells), two components of the hMutS alpha complex, were unable to bind the [SMeG] x T base pair. We also found that hMutS alpha was able to bind to [SMeG] x C base pairs when the S6-methylthioguanine was in the sequence 5'-Cp[SMeG]. This suggests that miscoding by S6-methylthioguanine residues in DNA during DNA synthesis may not be an absolutely required step in the mechanism of cytotoxicity. Also, since CpG sequences are so important in gene regulation, this result may be of considerable significance.

Kinetics of the action of thymine DNA glycosylase.

The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G.T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5'-flanking base pair to G.T mismatches influences the rate of removal of thymine: kcat values with C.G, T.A, G.C, and A.T as the 5'-base pair were 0.91, 0.023, 0. 0046, and 0.0013 min-1, respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S6-methylthioguanine, but, unlike G.T mismatches, a 5'-C.G does not have a striking effect on the rate: kcat values for removal of thymine from SMeG.T with C.G, T. A, G.C, and A.T as the 5'-base pair were 0.026, 0.018, 0.0017, and 0. 0010 min-1, respectively. Thymine removal is fastest when it is from a G.T mismatch with a 5'-flanking C.G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the 5'-flanking C.G pair. Disrupting either of these sets of contacts (i.e. replacing the 5'-flanking C.G base pair with a T.A or replacing the G.T mismatch with SMeG.T) has essentially the same effect on rate as disrupting both sets (i.e. replacing CpG.T with TpSMeG.T), and so these contacts are probably cooperative. The glycosylase removes uracil from G.U, C.U, and T.U base pairs faster than it removes thymine from G.T. It can even remove uracil from A.U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.

Job hazards for musculoskeletal disorders for youth working on farms.

A series of eight focus groups were convened to: (1) identify tasks and activities performed by youth on farms with the potential for causing non-traumatic work-related Musculoskeletal Disorders (MSDs); (2) determine the participants perceptions about risks of MSDs for youths performing those tasks; and (3) determine other factors that might relate to MSDs for youth working on farms, such as possible interventions for prevention. Seventy-two farm family members, 40 adults and 32 farm youth aged 8-18, participated in focus groups. Ten questions were posed to each of the eight groups about what tasks youth perform on the farm, how the work is assigned, and what risk factors are associated with the work. There was general agreement among the adults that maturity rather than age is the dominant factor for determining what tasks are performed by youth workers on the farm. Youth, on the other hand, believed that task urgency dictated what jobs were assigned to youth workers. Most adults indicated that lifting objects, forking, or shoveling was responsible for most of the serious non-traumatic injuries. Bending over while working, sitting in an awkward position looking back at equipment from a tractor, sitting in a cramped position, looking down at a combine header, and long hours of work were also identified as potential problems. Youth described muscle aches and strains of the legs, arms, shoulder, back or neck as everyday occurrences. According to the youth, "If it's not broken, you're fine". Only basic training is provided and most respondents believed that youth learned best through observation. There was general agreement that physician recommended guidelines for assigning youth to tasks would be ignored unless they carried the force of the law.