Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate.

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.     

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.     

Body Position Influences Which Neural Structures Are Recruited by Lumbar Transcutaneous Spinal Cord Stimulation

Transcutaneous stimulation of the human lumbosacral spinal cord is used to evoke spinal reflexes and to neuromodulate altered sensorimotor function following spinal cord injury. Both applications require the reliable stimulation of afferent posterior root fibers. Yet under certain circumstances, efferent anterior root fibers can be co-activated. We hypothesized that body position influences the preferential stimulation of sensory or motor fibers. Stimulus-triggered responses to transcutaneous spinal cord stimulation were recorded using surface-electromyography from quadriceps, hamstrings, tibialis anterior, and triceps surae muscles in 10 individuals with intact nervous systems in the supine, standing and prone positions. Single and paired (30-ms inter-stimulus intervals) biphasic stimulation pulses were applied through surface electrodes placed on the skin between the T11 and T12 inter-spinous processes referenced to electrodes on the abdomen. The paired stimulation was applied to evaluate the origin of the evoked electromyographic response; trans-synaptic responses would be suppressed whereas direct efferent responses would almost retain their amplitude. We found that responses to the second stimulus were decreased to 14%±5% of the amplitude of the response to the initial pulse in the supine position across muscles, to 30%±5% in the standing, and to only 80%±5% in the prone position. Response thresholds were lowest during standing and highest in the prone position and response amplitudes were largest in the supine and smallest in the prone position. The responses obtained in the supine and standing positions likely resulted from selective stimulation of sensory fibers while concomitant motor-fiber stimulation occurred in the prone position. We assume that changes of root-fiber paths within the generated electric field when in the prone position increase the stimulation thresholds of posterior above those of anterior root fibers. Thus, we recommend conducting spinal reflex or neuromodulation studies with subjects lying supine or in an upright position, as in standing or stepping.     

A low-resolution 3D model of the tetrameric alcohol dehydrogenase from Sulfolobus solfataricus

We describe the computation of a model of the thermophilic NAD-dependent homotetrameric alcohol dehydrogenase from the archaeon Sulfolobus solfataricus (SsADH). Modeling is based on the knowledge that each monomer contains two Zn ions with catalytic and structural function, respectively. In the database of known structures, proteins with similar functions are either dimers containing two zinc ions per monomer or tetramers with one zinc ion per monomer. In any case, the sequence identity of the target to the possible templates is low. A threading procedure is therefore developed which includes constraints taking into account residue conservation both at the zinc ion binding and at the monomer-monomer interaction sites in the tetrameric unit. The model is consistent with previously reported data. Furthermore, cross-linking experiments are described which support the computed tetrameric model.     

Acute Cardiovascular Events after Herpes Zoster: A Self-Controlled Case Series Analysis in Vaccinated and Unvaccinated Older Residents of the United States

Background

Herpes zoster is common and can have serious consequences. Additionally, emerging data suggest an increased risk of acute cardiovascular events following herpes zoster. However, to our knowledge, existing association studies compare outcomes between individuals and are therefore vulnerable to between-person confounding. In this study, we used a within-person study design to quantify any short-term increased risk of acute cardiovascular events (stroke and myocardial infarction [MI]) after zoster and to assess whether zoster vaccination modifies this association.

Methods and Findings

The self-controlled case series method was used to estimate rates of stroke and acute MI in defined periods after herpes zoster compared to other time periods, within individuals. Participants were fully eligible Medicare beneficiaries aged ≥65 y with a herpes zoster diagnosis and either an ischemic stroke (n = 42,954) or MI (n = 24,237) between 1 January 2006 and 31 December 2011. Age-adjusted incidence ratios (IRs) for stroke and MI during predefined periods up to 12 mo after zoster relative to unexposed time periods were calculated using conditional Poisson regression. We observed a marked increase in the rate of acute cardiovascular events in the first week after zoster diagnosis: a 2.4-fold increased ischemic stroke rate (IR 2.37, 95% CI 2.17–2.59) and a 1.7-fold increased MI rate (IR 1.68, 95% CI 1.47–1.92), followed by a gradual resolution over 6 mo. Zoster vaccination did not appear to modify the association with MI (interaction p-value = 0.44). We also found no evidence for a difference in the IR for ischemic stroke between vaccinated (IR 1.14, 95% CI 0.75–1.74) and unvaccinated (IR 1.78, 95% CI 1.68–1.88) individuals during the first 4 wk after zoster diagnosis (interaction p-value = 0.28). The relatively few vaccinated individuals limited the study’s power to assess the role of vaccination.

Conclusions

Stroke and MI rates are transiently increased after exposure to herpes zoster. We found no evidence for a role of zoster vaccination in these associations. These findings enhance our understanding of the temporality and magnitude of the association between zoster and acute cardiovascular events.