Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form

The crystal structure of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-GK) in a dimeric form was determined at a resolution of 2.4 A. This is the first crystal structure of a hyperthermophilic glycerol kinase. The overall structure of the Tk-GK dimer is very similar to that of the Escherichia coli glycerol kinase (Ec-GK) dimer. However, two dimers of Ec-GK can associate into a tetramer with a twofold axis, whereas those of Tk-GK cannot. This may be the reason why Tk-GK is not inhibited by fructose 1,6-bisphosphate, because the fructose 1,6-bisphosphate binding site is produced only when a tetrameric structure is formed. Differential scanning calorimetry analyses indicate that Tk-GK is a highly thermostable protein with a melting temperature (T(m)) of 105.4 degrees C for the major transition. This value is higher than that of Ec-GK by 34.1 degrees C. Comparison of the crystal structures of Tk-GK and Ec-GK indicate that there is a marked difference in the number of ion pairs in the alpha16 helix. Four ion pairs, termed IP1-IP4, are formed in this helix in the Tk-GK structure. To examine whether these ion pairs contribute to the stabilization of Tk-GK, four Tk-GK and four Ec-GK derivatives with reciprocal mutations at the IP1-IP4 sites were constructed. The determination of their stabilities indicates that the removal of each ion pair does not affect the stability of Tk-GK significantly, whereas the mutations designed to introduce one of these ion pairs stabilize or destabilize Ec-GK considerably. These results suggest that the ion pairs in the alpha16 helix contribute to the stabilization of Tk-GK in a cooperative manner.     

Identification of the putative protein phosphatase gene PTC1 as a virulence-related gene using a silkworm model of Candida albicans infection

Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Δ, yvh1Δ, sit4Δ, and ptc1Δ) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Δ, yvh1Δ, and sit4Δ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Δ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.     

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.     

Correlations of calcium accumulations in arteries, veins, cartilages, ligaments, and bones in single humans

To show the relationships of calcium accumulation in the thoracic aorta to the other tissues, calcium contents were determined with a microwave-induced plasma-atomic emission spectrometer on arteries, veins, cartilages, ligaments, and bones. These tissues were resected from 18 individuals, consisting of 11 men and 7 women who died in the age range 59–91 yr. As thoracic and abdominal aortas are routinely used for radiographic examination of arterial calcification, they appear to be standard tissues of the calcium accumulation. The calcium accumulations were determined in the femoral artery, the superior and inferior venae cavae, the internal jugular vein, cartilages of the articular disk of the temporomandibular joint and the intervertebral disk, both the ligaments of the anterior cruciate ligament and the ligamentum capitis femoris, and the calcaneus, in contrast with the thoracic aorta. As calcium increased in the thoracic aorta, it increased in the femoral artery, the articular disk of the temporomandibular joint, the intervertebral disk, both ligaments of the anterior cruciate ligament, and the ligamentum capitis femoris, but it did not increase in veins, such as the superior and inferior venae cavae and the internal jugular vein. In contrast, it decreased in the calcaneus.     

In vivo conformation of mitochondrial DNA revealed by pulsed-field gel electrophoresis in the true slime mold, Physarum polycephalum.

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.     

Clinical significance of serum bone Gla protein and urinary gamma-Gla as biochemical markers in primary hyperparathyroidism

The serum bone Gla-protein (BGP) and urinary gamma-carboxyglutamic acid (gamma-Gla) levels were determined in patients with primary hyperparathyroidism (PHP). The mean serum BGP and urinary gamma-Gla levels were 18.6 +/- 2.34 ng/ml and 65.5 +/- 4.62 nmoles/mgCr, respectively, for the 11 patients with the skeletal type of PHP, 5.13 +/- 0.85 ng/ml and 45.2 +/- 1.33 nmoles/mgCr for the 4 with the chemical type, and 7.91 +/- 2.43 ng/ml and 43.2 +/- 3.47 nmoles/mgCr for the 5 with the renal type. Thus, patients with skeletal-type PHP had significantly higher serum BGP and urinary gamma-Gla levels than those with the other type of PHP. Serum BGP levels had significant positive correlations with serum Ca (r = 0.64, P less than 0.005), serum A1-p (r = 0.77, P less than 0.001) and serum PTH (r = 0.45, P less than 0.005). Urinary gamma-Gla levels also had significant positive correlations with serum Ca (r = 0.50, P less than 0.05), serum A1-p (r = 0.67, P less than 0.005), serum 1,25(OH)2D (r = 0.62, P less than 0.02), and serum BGP (r = 0.72, P less than 0.001). Mineral content in the left radius had significant negative correlations with serum BGP levels (r = -0.73, P less than 0.001) and urinary gamma-Gla levels (r = -0.59, P less than 0.01). As these data show, serum BGP and urinary gamma-Gla levels clearly reflect the abnormal bone metabolism and can therefore be useful biochemical markers in PHP.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Mutations affecting retina development in Medaka

In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation.     

Age-dependent decreases of phosphorus and magnesium in human achilles’ tendons

To elucidate changes of human tendons with aging, the authors studied age-related changes of elements in human Achilles’ tendons by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. It was found that the content of calcium increased progressively with aging in the Achilles’ tendons, whereas the contents of phosphorus and magnesium decreased gradually with aging. The previous investigations demonstrated that the content of calcium and phosphorus increased progressively with aging in most, but not all, human tissues, except for the bones. In ligaments, such as the anterior cruciate ligament and the ligament of the head of the femur, which are histologically similar to the Achilles’ tendon, it was previously found that both the contents of calcium and phosphorus increased with aging in the ligaments. It should be noted that the content of phosphorus in the Achilles’ tendons decreased during the aging process. In addition, it was found that there was a very high direct correlation between phosphorus and magnesium contents in the tendons, but not between calcium and phosphorus contents.