Effectory site in Escherichia coli inorganic pyrophosphatase is revealed upon mutation at the intertrimeric interface

Escherichia coli inorganic pyrophosphatase (E-PPase) is a homohexamer formed from two trimers related by a two-fold axis. The residue Asp26 participates in intertrimeric contacts. Kinetics of MgPPi hydrolysis by a mutant Asp26Ala E-PPase is found to not obey Michaelis-Menten equation but can be described within the scheme of activation of hydrolysis by a free PPi binding at an effectory subsite. Existence of such a subsite is confirmed by the finding that the free form of methylenediphosphonate activates MgPPi hydrolysis though its magnesium complex is a competitive inhibitor. The Asp26Ala variant is the first example of hexameric E-PPase demonstrated to have an activatory subsite.     

Immunomorphologic study of experimental enteric anc combined vaccination against cholera]

As a result of the enteral and combined subcutaneous-enteral immunization of adult rabbits with cholerogen toxoid and the specific fraction of chemical cholera vaccine in tablets immunological transformation occurred in the organism and a "protective" barrier consisting of antibody-containing cells was formed in the intestine on days 3-5. The combined method of immunization with cholera vaccine proved to be essentially as effective as enteral immunization in 2 administrations.     

Photobiological Properties of 13,15-N-(Carboxymethyl)- and 13,15-N-(2-Carboxyethyl)cycloimide Derivatives of Chlorin p6

Lipophilic derivatives of chlorin p6, 13,15-N-(carboxymethyl)cycloimide methyl ester (CIC1) and 13,15-N-(2-carboxyethyl)cycloimide methyl ester (CIC2), were shown to absorb light in 710 nm region and to be efficient IR photosensitizers. They exhibit similar phototoxicities on the cells of A549 human lung adenocarcinoma, which are 40- and 100-fold higher than those of chlorin p6 and the clinically used Photogem, respectively, and are not toxic in the absence of light irradiation. The confocal spectral imaging technique allowed us to demonstrate that the high phototoxicity of CIC1 and CIC2 is due to their ability to readily penetrate to cells and to be bound to the cell membranes and lipid-containing structures in the monomeric photoactive form. Under the irradiation, the membrane-bound CIC1 and CIC2 are characterized by high quantum yields of singlet oxygen generation (0.6 and 0.65, respectively) and the inability to produce hydroxyl radicals. A 1.5-M content of CIC1 and CIC2 in the incubation medium provides for their average cytoplasmic concentrations of 21 and 16.5 M, respectively. The incubation times to achieve 50% level of maximum accumulation for CIC1 and CIC2 in A549 cells are 30 ± 6 and 24 ± 12 min, and the times for 50% release of the dyes from the cells are 17 ± 4 and 50 ± 10 min, respectively. A diffuse distribution with the predominant accumulation in the membranes of the Golgi apparatus and mitochondria is characteristic of both CIC2 and CIC1, whereas, in addition, CIC1 is considerably accumulated in lipid droplets (cellular organelles responsible for the storage and metabolism of neutral lipids and sterol esters). Our results demonstrate that changes in the structure of the imide substituent could affect the intracellular localization and the rate of release of chlorin p6 cycloimide derivatives from cells while preserving their high photodynamic activity.     

A high-density SNP genomewide linkage scan for chronic lymphocytic leukemia-susceptibility loci

Chronic lymphocytic leukemia (CLL) and other B-cell lymphoproliferative disorders (LPDs) show clear evidence of familial aggregation, but the inherited basis is largely unknown. To identify a susceptibility gene for CLL, we conducted a genomewide linkage analysis of 115 pedigrees, using a high-density single-nucleotide polymorphism (SNP) array containing 11,560 markers. Multipoint linkage analyses were undertaken using both nonparametric (model-free) and parametric (model-based) methods. Our results confirm that the presence of high linkage disequilibrium (LD) between SNP markers can lead to inflated nonparametric linkage (NPL) and LOD scores. After the removal of high-LD SNPs, we obtained a maximum NPL of 3.14 (P=.0008) on chromosome 11p11. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under both dominant (HLOD 1.95) and recessive (HLOD 2.78) models. In addition, four other chromosomal positions (5q22-23, 6p22, 10q25, and 14q32) displayed HLOD scores >1.15 (which corresponds to a nominal P value <.01). None of the regions coincided with areas of common chromosomal abnormalities frequently observed for CLL. These findings strengthen the argument for an inherited predisposition to CLL and related B-cell LPDs.     

Potential use of radiation diagnosis of esophageal achalasia

The prime objective of the paper is to attempt to reevaluate the potentialities of current radiation diagnosis of esophageal achalasia. Moreover, its main idea is to assess the procedure of a traditional X-ray section of radiation study. In the authors' opinion, the main motivation of writing this paper was the emergence of new recent potentialities of radiation diagnosis, including its traditional X-ray section in gastroenterological pathology, as well as a considerable body of information on esophageal achalasia (144 cases). The tasks to be solved in the paper include the differential diagnosis of esophageal achalasia and cardioesophageal cancer, as well as the basic capacities of radiation diagnosis to help clinicians in characterizing the changes caused by medical and surgical treatments for this disease. The authors consider that the obtained volume of necessary information on this abnormality rather frequently encountered in esophagogastroenterological pathology can be increased by applying the current possibilities of radiation study and mainly its traditional X-ray section.     

A20 gene expression is regulated by TNF, Vitamin D and androgen in prostate cancer cells

A20 is a TNF-inducible primary response gene and its product, a zinc finger protein, has antiapoptotic function in several cancer cells. We studied A20 gene expression in the Vitamin D- and TNF-sensitive LNCaP cell line and in the Vitamin D- and TNF-resistant PC-3 cell line. The results of the quantitative real-time RT-PCR analyses demonstrated that the basal level of A20 mRNA production in PC-3 cells was considerably higher than in LNCaP cells that is associated with the resistance of PC-3 cells. TNF induced A20 gene expression in both cell lines, but with different effect. A20 mRNA expression was down-regulated by 10nM calcitriol within 3-9h after treatment and up-regulated by androgen reaching maximal values by 6h after stimulation in LNCaP cells. We conclude that A20 may be involved in the regulation of cell proliferation by TNF, Vitamin D, and androgen in prostate cancer.     

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.     

Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles.