sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast

In the fission yeast Schizosaccharomyces pombe, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein. Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast. In this paper we describe one such component, encoded by the sud1(+) gene. sud1(+) shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast. Cells lacking sud1(+) accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators. Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Deltasud1 cells, but is not the cause of the defect in Cdc18 proteolysis. Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo. Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1. Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles.     

The primary structure of rabbit serum amyloid A protein isolated from acute phase serum

Serum amyloid A (SAA) protein, a sensitive acute phase protein and the precursor of protein AA in secondary amyloid, was purified from pooled acute phase rabbit serum using two different methods: isolation of protein SAA directly by octyl-Sepharose chromatography of total serum, and dissociation and isolation of apoSAA from acute phase high density lipoprotein (HDL). The protein SAA fraction obtained was further purified using gel filtration and ion exchange chromatography. Rabbit protein SAA has 104 amino acid residues, like human SAA, and has a partially blocked N terminus. The highly conserved region from position 33 to position 63 found in SAA from all species studied was confirmed also in rabbit SAA. No microheterogeneities were observed. The amino acid sequence showed extensive N-terminal homology with the rabbit amyloid A protein, except for the microheterogeneity in position 12 in protein AA. It also showed identical amino acid sequence with that deduced from the rabbit cDNA clone pSAA 55. Complete homologies were found with clone SAA 2, except for positions 22 and 78, clone SA8-1, except for positions 22 and 79 and clone SA7-3, except for position 22. This pSAA 55/SA7-3/SA8-1/SAA2-like protein was the only SAA isotype found both in total serum and in the HDL fraction. Isotypes corresponding to other SAA-like genes could not be found in this pool of acute phase rabbit sera.     

Exercise training for intermittent claudication: does it adversely affect biochemical markers of the exercise-induced inflammatory response?

OBJECTIVES: To identify a stable biochemical marker of disease severity in patients with intermittent claudication and to use these findings to assess the effect of therapeutic exercise training. DESIGN: Case-control study: prospective randomised-controlled trial of exercise training. MATERIALS AND METHODS: Plasma fibrinogen, serum amyloid A protein (SAA), C-reactive protein (CRP) and urinary albumin-creatinine ratio (ACR) were measured in 67 claudicants and 15 controls. Twenty-two patients were randomised to supervised exercise training and 17 randomised to observation. Subjects were reviewed at 3, 6 and 12 months. RESULTS: The median (interquartile range) baseline fibrinogen was 3.7 g/l (3.3-4.25) in claudicants and 3.5 g/l (2.9-3.95) in controls (p = 0.045); CRP was 4.7 mg/l (2.2-9.0) and 2.1 mg/l (1.0-2.8), respectively (p < 0.0001); SAA was 72 mg/l (35-132) and 30 mg/l (20-89) (p = 0.0009). Claudicants showed an increased urinary ACR following treadmill exercise (Wilcoxon, p < 0.0001) with no change in controls. Exercise training reduced SAA at 6 months, CRP at 3 months and progressively attenuated the post-exercise increase in ACR. No similar changes were found in controls. CONCLUSIONS: Repetitive low-grade inflammatory events in claudicants lead to elevation of serum acute-phase proteins. Exercise training is associated with symptomatic improvement and reduction inflammatory markers. The concern that exercise has adverse systemic effects therefore seems to be unjustified.     

Social senses: G-protein-coupled receptor signaling pathways in Dictyostelium discoideum

Incubation of phenol-induced cells of the yeast Candida maltosa SBUG 700 with mono- and dichlorophenols resulted in the formation of metabolites of the substrates and of further metabolites not related to the degradation pathway of the substrates. These additional compounds, identified as 4-hydroxyphenylacetic acid (4-HPA), phenylacetic acid (PA), indolylacetic acid (IA) and indolylethanol (i.e.) by means of HPLC and GC/MS, were not excreted in incubation experiments with glucose. The excretion of these metabolites of aromatic amino acid metabolism is not caused by toxic effects of the phenol derivatives, but seems to be a result of carbon and nitrogen starvation of yeast cells.     

A chromatographic method for the preparative separation of phosphohistidines

The driving force behind this new chromatographic technique was to develop a method for purifying preparative quantities of phosphohistidines in a single step that provided good resolution wit eluants that could be easily removed. The current method can provide milligram quantities of each phosphohistidine; however, the later 1H NMR analysis of the dried, individually purified phosphohistidines showed that histidine was present along with each of the individual phosphohistidines. The stability of each phosphohistidine during storage does not appear to be a problem because the amounts of histidine contamination of freshly freeze-dried samples were compared with those of samples stored at -80 degrees C under nitrogen for 2 weeks and were found to be similar (data not shown). Possibly, the lyophilization and preparation of solutions for NMR analysis resulted in a certain amount of hydrolysis of phosphohistidine, particularly with the less stable 1- and 1,3-forms (5, 6). It was noted that when the lyophilized samples were initially dissolved in D2O for NMR analysis, the pH was between 6 and 7; this may have resulted in some hydrolysis of the phosphohistidines. This hydrolysis can be reduced by the addition of 50 mM potassium hydroxide to the pooled fractions collected from the chromatography, so that the alkalinity of the samples is maintained throughout the subsequent processes. The data obtained for the assignment of individual phosphohistidines by 1H and 31P NMR analysis seem consistent with those obtained by other independent studies (6, 10). The NMR analysis was a powerful tool in assigning the identity of each purified phosphohistidine, although caution should be used when considering free phosphohistidines as references for NMR detection of phosphohistidines in native proteins. Lecroisey et al. (10) showed that there were differences between the chemical shifts observed for free phosphohistidine compared to those for phosphohistidine in dipeptides. However, for the purposes of phosphoamino acid analysis, these purified phosphohistidines are used by this group as references in the detection of phosphohistidine in proteins.     

k-tree method for high-speed spatial normalization

The general approach to spatial normalization using a deformation field is presented. Current high degree-of-freedom deformation methods are extremely time-consuming (10-40 hr), and a k-tree method is proposed to greatly reduce this time. A general k-tree method for analysis of source and target images and synthesis of deformation fields is described. The k-tree method simplifies scale control and feature extraction and matching, making it highly efficient. A two-dimensional (2-D), or quadtree, application program was developed for preliminary testing. The k-tree method was evaluated with 2-D images to test rotating ability, nonhomologous region matching, inner and outer brain-structure independence, and feasibility with human brain images. The results of these tests indicate that a three-dimensional (3-D), or octree, method is feasible. Preliminary work with an octree application program indicates that a processing time of under 10 min for 256(3) image arrays is attainable on a Sun Ultra30 workstation.     

Effect of strength training on EMG of human skeletal muscle

An 8-year-old white girl with a history of vertigo, nausea, and vomiting developed a progressive hearing loss, bilateral retinal arteriolar narrowing in each eye, vasoproliferation, and subsequent intravitreal hemorrhage. An attempt at peripheral retinal ablation with cryotherapy in the left eye resulted in retinal detachment. Spontaneous retinal detachment occurred in the right eye and was successfully repaired. Repeated intermittent hemorrhages occurred despite intraocular diathermy. Three years after onset, visual acuity was R.E.: 6/21 (20/66) and L.E.: light perception. She remains totally deaf. A 20-year-old white woman developed severe bilateral sensorineural hearing loss with poorly functioning labyrinths, followed by midperipheral retinal arteriolar occlusions and vasoproliferation on the optic nerve head. Progressive retinal neovascularization was followed by rubeosis iridis and repeated episodes of intravitreal bleeding. Six years after onset, visula acuity was R.E.: hand motions, and L.E.: 6/3 (20/100). She remains totally deaf. Both patients were of normal gestation, development, and mentality, without evidence of other systemic disease. The cause of this disease was not found.