Effects of Ni(2+), Co(2+), and Mn(2+) on desensitized butyrylcholinesterase prepared from human serum

Human serum butyrylcholinesterase (BChE) has been converted into a stable but less active desensitized form when heated at 45°C for 24 h. The desensitized BChE follows Michaelis-Menten kinetics, whereas native enzyme exhibits slightly negative cooperativity with respect to butyrylthiocholine binding. In this study, we investigated the effects of Ni²⁺, Co²⁺, and Mn²⁺ on the desensitized BChE. It is found that all three ions were noncompetitive inhibitors of the desensitized BChE, and K _i values have been determined as 7.816±1.060 mM, 48.722±4.635 mM, and 84.795±5.249 mM for Ni²⁺, Co²⁺, and Mn²⁺, respectively. In our previous study, these ions were linear mixed-type inhibitors of the native BChE. This finding confirms that desensitized BChE changes to a different conformation than native BChE. From the comparison of K _i values of the trace elements, it can be said that Ni²⁺ is a more effective inhibitor of the desensitized BChE than Co²⁺ and Mn²⁺.     

Inhibitory effect of galanin on dopamine induced increased oxytocin secretion in rat neurohypophyseal tissue cultures

The regulation of oxytocin (OT) release by galanin (GAL) at the neurohypophyseal (NH) nerve terminal is not adequately understood. The effect of GAL on the secretion of OT was studied in 13- to 14-day cultures of isolated rat NH tissue. By this time, the hormone content of the medium had become constant. The OT content of the supernatant medium was determined by RIA after a 1- or 2-h incubation. A significantly decreased content of OT was found following incubation with 10(-6)-10(-8) M doses of GAL. Dopamine (DA) and the DA-active drugs apomorphine (APM) and Pro-Lys-Gly (PLG) (10(-6) M in each medium) increased the OT synthesis of NH tissue cultures. This elevation of OT secretion could be blocked by the administration of GAL together with DA, APM or PLG. The DA-blocking effect of GAL was prevented by previous treatment with the GAL receptor antagonist galantid (M15). The results indicate that OT release from the NH is directly influenced by the GAL-ergic system. The GAL-ergic control of OT secretion from NH tissue in rats can occur at the level of the posterior pituitary.     

De novo translocation (2;18)(q21;q22) in a child with severe epilepsy, developmental delay and mild dysmorphism

De novo translocation (2;18)(q21;q22) in a patient with severe epilepsy developmental delay and mild dysmorphism: We report on a patient presenting with severe epilepsy, hypotonia, developmental delay, blepharophimosis, low-set ears, camptodactyly and tapering fingers, and cutaneous syndactyly of toes II and III of the right foot. The MRI showed some loss of volume of the white matter and delayed myelination, no other specific anomalies were present. Chromosome analysis revealed a translocation involving chromosomes 2 and 18, which was characterized further by FISH using band-specific probes. The possibility of a submicroscopic deletion is discussed and the patient is compared with patients reported in the literature with either 2q21 or 18q22 deletion.     

Molecular models in nickel carcinogenesis

Nickel compounds are known human carcinogens, but the exact molecular mechanisms of nickel carcinogenesis are not known. Due to their abundance, histones are likely targets for Ni(II) ions among nuclear macromolecules. This paper reviews our recent studies of peptide and protein models of Ni(II) binding to histones. The results allowed us to propose several mechanisms of Ni(II)-inflicted damage, including nucleobase oxidation and sequence-specific histone hydrolysis. Quantitative estimations of Ni(II) speciation, based on these studies, support the likelihood of Ni(II) binding to histones in vivo, and the protective role of high levels of glutathione. These calculations indicate the importance of histidine in the intracellular Ni(II) speciation.     

De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B₁4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing ¹⁵NH₄Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.     

Function and Expression of CD44 during Spreading, Migration, and Invasion of Murine Carcinoma Cells

The cell surface glycoprotein CD44 is proposed as a main participant in cell adhesion and migration. We studied the function, expression, and distribution of CD44 in the invasive and metastatic F3II murine carcinoma cell line during adhesion, spreading, migration, and invasion. A mAb anti-CD44 (KM 201) dramatically blocked F3II cell adhesion on both plastic and hyaluronic acid coatings, as well as spreading on uncoated plastic surfaces (P< 0.01). KM201 mAb significantly inhibited F3II cell migration and invasion in Transwell chambers. Immunocytochemistry of spreading cells revealed that CD44 distributed in bands on the cell surface, particularly in the tip of leading edges and in the perinuclear zones of the cell membrane. CD44 antigen was never detected in filopodia or lamellipodia nor in focal adhesion-like structures, but was also detectable as strong interlamellar bands. Fully spread cells showed a decreased CD44 signal compared to cells in early stages of spreading. This decrease correlated with a reduced expression of CD44 as detected by Western blot. We also investigated the signals that may regulate CD44 expression in F3II cells. Treatment of F3II cells, with phorbol myristate acetate (PMA) or phosphatidic acid (PA, the product of PLD-dependent hydrolysis of phosphatidylcholine), significantly enhanced CD44 expression. Conversely, the treatment of F3II cells with H7, a specific PKC inhibitor, or propranolol, which blocks PA conversion to DAG, significantly decreased CD44 expression levels. These results suggest the involvement of PKC and PLD pathways in CD44 expression. These results demonstrate that CD44 plays an important role during F3II cells adhesion, spreading, migration, and invasion. In addition we provide information linking the PLD- and PKC-dependent pathways with the regulation of CD44 expression.     

A Protease-Resistant Novel Hemagglutinin Purified from Type A Clostridium botulinum

Botulinum neurotoxin is a food poisoning agent produced by Clostridium botulinum. The neurotoxin is a 150-kDa protein that causes flaccid muscle paralysis by blocking neurotransmitter release at neuromuscular junctions. The neurotoxin is produced along with a group of neurotoxin associated proteins (NAPs), which protect it from the low pH and proteases of the gastrointestinal (GI) tract. We have isolated, for the first time, one of the major components of NAPs in a pure form. The isolated protein is a 33-kDa single polypeptide (Hn-33) that exhibits hemagglutination activity. Specific polyclonal antibodies against the Hn-33 are able to block the hemagglutination activity of the neurotoxin complex, which indicates that perhaps Hn-33 is the only strong hemagglutinating protein in the complex. The Hn-33 was found be resistant to trypsin and other protease digestion, a feature that could play a role in the protection of the neurotoxin in the GI tract during its toxicoinfection.     

Construction of a xylanase A variant capable of polymerization

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created.