Development of technologies aiding large-tissue engineering

Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.     

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.     

A kinetic and surface study of the thermal decomposition of cellulose powder in inert and oxidizing atmospheres

The thermal decomposition of fibrous cellulose powder from 275° to 340°C has been studied by thermogravimetry, scanning electron microscopy, krypton adsorption, and gas-chromatographic analysis of the gaseous products arising from pyrolysis in various oxidizing and inert atmospheres. The reaction kinetics fit a phase boundary model where the rate is controlled by the movement of an interface through a cylindrical particle and the principal kinetic parameters fit a compensation curve described previously for the decomposition of wood products. An explanation of the physical mechanism of pyrolysis is proposed which is consistent with the observed rate data and the structural changes observed by scanning electron microscopy.     

Dependence of coal liquefaction behaviour on coal characteristics. 6. Product distributions from coals treated in a continuous-flow reactor

Further experimentation with coals in a continuous-flow reactor system at 440 and 455°C confirms the significance of the previous classification of a set of coals by cluster analysis. The greatest yields of distillable products tend to be given by coals of high sulphur content in the middle of the range of the high-volatile bituminous rank classes. Yet the lowest yields of preasphaltene (toluene-insoluble material) tend to be given by coals of high rank and medium sulphur content. The group of low sulphur, low-rank coals (mostly Rocky Mountain province), in a solvent different from that used for the other groups, showed poor conversion and poor distillate yield at the higher temperature. It is concluded that this coal/solvent combination is particularly prone to retrogressive reactions, including coking.     

Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.     

Diffusion of fluid confined to nanotube with rectangular cross section

A dynamical model is proposed to study self-diffusion coefficient by confining the fluid in rectangular nanotube. The theoretical model is based on the consideration that the confinement affects the movement at atomic level. The model predicts that the diffusion parallel to walls of channel is different from that of diffusion perpendicular to the walls. Near the walls the dynamics of fluid has been found to slow down to an extent that below a certain value of ratio of width to the diameter of particle, the molecules behave as if these belong to solid. The results are contrasted with the result obtained from the model based on similar considerations for a fluid confined only in one direction. It is found that tendency of freezing near the wall increases due to confinement from second direction. Empirical relation which governs the behavior of diffusion coefficient as function of distance from the confining walls has also been proposed. The effect of confinement is more pronounced for denser fluids than for dilute fluid.     

Synthetic substrates for human factor VIIa and factor VIIa-tissue factor

A series of 100 tripeptide fluorogenic substrates has been synthesized. These substrates contain Arg in the P1 position, various amino acids in the P2 and P3 positions, and different 6-amino-1-naphthalenesulfonamides (ANSN) as the detecting group (P'). The 38 compounds possessing the highest initial rates of factor VIIa hydrolysis were evaluated for substrate kinetic parameters in the presence and absence of tissue factor (TF) and by factor Xa. Most of these substrates had a higher kcat/KM (keff) value for the factor VIIa-TF complex than for factor Xa. Substitution of different amino acids in the P2 position showed that substrates with bulkier amino acids such as Leu, Pro, and Val have higher values for KM and kcat than those with smaller amino acids (Gly or Ser). The highest second-order rate constants were found for substrates with Val or Pro in the P2 position. A decrease or increase in volume of the P2 substituent (Gly, Ser, or Leu) resulted in a decrease in this constant. Substrates with the highest keff values have Phe in the P3 position. As the hydrophobicity and volume of the amino acid in the P3 position decreased, the keff was reduced. The efficiency of substrates for hydrolysis by factor VIIa was enhanced by an increase of hydrophobicity in the P' structure. TF enhanced the amidolytic activity of the "family" of 38 substrates with ANSN in the P' position on an average of 58-fold.     

Changes of bone-perfusion in osteosynthesis of the femur with a marrow-nail (author's transl)

The perfusion of the bone in the hind leg after osteosynthesis (nailing of the bone-marrow) was studied. In 11 shepherd dogs (bastards) an osteotomy of the femur was done; it was treated with a marrow-nail without boring the marrow-cavity. With the "tracer-microsphere"-method the perfusion of femur, tibia and talus of both hind legs was measured. Measurements were performed before and after surgery, in 10 dogs 2 weeks and in 8 dogs 6 weeks after surgery. Immediately after the operation the perfusion was reduced considerably in all the examined bones of the operated leg. Two weeks later the perfusion was increased in all bones of both hind limbs. In the cancellous bone of the femur the perfusion reached the original preoperative values after 6 weeks; in cortical bone a further increase of the perfusion was noted. This increase was most marked in the cortical bone of the operated femur; it was less in the cortical bone of the other bones.     

Accelerated Cheddar Cheese Ripening by Added Microbial Enzymes

Good quality medium sharp Cheddar cheeses with 3-mo curing at 10 C were produced when the following enzyme combinations and concentrations were used: fungal protease 31000 (Miles), .005% + fungal lipase-MY (Meito) .00005 to .0002%; and fungal protease P-53 (Rohm & Haas), .0035% + fungal lipase-MY (Meito), .00005 to .0002%.Cheddar cheeses treated with microbial enzymes developed higher soluble protein and free volatile fatty acids and displayed better flavor and greater acceptability than control cheeses. Added microbial proteases contributed to the breakdown of casein, especially β-casein. Also, αs₁-I casein and free amino acids were high in cheeses treated with protease. Increased rate of proteolysis in enzyme-treated cheese had a direct relation to accelerated ripening.     

Prenatal diagnosis of partial monosomy 13q associated with occipital encephalocoele in a fetus

The pre- and postnatal findings of a fetus with a de novo del(13)(pter-->q21:) and an occipital encephalocoele are described. Maternal serum alpha-fetoprotein (AFP) screening at 19 weeks' gestation demonstrated a high level of 2.5 multiples of the median (MOM) and ultrasonography at 27 weeks' gestation showed severe intrauterine growth retardation, cardiomegaly, an occipital encephalocoele, and a calvarial defect. Genetic amniocentesis revealed a karyotype of 46,XX,del(13)(pter-->q21:). The proband postnatally displayed additional abnormalities such as microphthalmia, hypertelorism, large low-set ears, and micrognathia. We discuss the association of central nervous system (CNS) malformations with 13q deletions and emphasize that pregnancies with neural tube defects warrant cytogenetic analysis, especially when additional fetal abnormalities and neonatal dysmorphism are observed.