Simulation of chemical metabolism for fate and hazard assessment. III. New developments of the bioconcentration factor base-line model.

The new development of the bioconcentration factor (BCF) base-line model of Dimitrov et al. [SAR QSAR Environ. Res. 6 (2005), pp. 531-554] is presented. The model applicability domain was expanded by enlarging the training set of the model up to 705 chemicals. The list of chemical-dependent mitigating factors was expanded by including water solubility of chemicals. The original empirical term for estimating ionization of chemicals was mechanistically analysed using two different approaches. In the first one, the ionization potential of chemicals was estimated based on the acid dissociation constant (pK(a) ). This term was found to be less adequate for inclusion in the ultimate BCF model, due to overestimating ionization of chemicals. The second approach, estimating the ionization as a ratio between distribution and partition coefficients (log P and log D), was found to be more successful. The new ionization term allows modelling of chemicals with both acidic and basic functionalities and chemicals undergoing different degrees of ionization. The significance of the different mitigating factors which can reduce the maximum bioconcentration potential of the chemicals was re-formulated and model parameters re-evaluated.

[Femoral lengthening. A critical review of ten-year results]. / Kritické zpracování prodluzování femuru za posledních deset let.

PURPOSE OF THE STUDY: To evaluate the results of femoral lengthening in the patients treated from 2000 to 2009 in whom complete radiographic data were available and the lengthening procedure involved mainly the use of a Mephisto fixator. MATERIAL AND METHODS: A total of 28 femoral lengthening procedures were carried out in 26 patients,16 girls and 10 boys. The external fixator Mephisto was used in 24 cases; fixators Prospon and Orthofix in one and three cases, respectively. Fifteen patients were treated for congenital short femur, the rest had secondary femoral shortening due to following pathologies: distal femoral epiphysiolysis in five children, proximal femoral osteomyelitis in one child, avascular necrosis of the femoral head in one, diaphyseal femur fracture in one, enchondromatosis of the distal femur with growth plate destruction in one, and contralateral femur overgrowth following a fracture in one child. The average age at the beginning of treatment was 11 (range, 4-16) years. Complications were classified as mild, serious and critical. The results were statistically analysed using several statistical tests. RESULTS: The average parameter values for the group included: total femoral lengthening, 40.2.mm (SD±11.1); osteotomy index (OI), 41 % (SD±9.8); lengthening percentage (LP), 10.9 % (SD±3.8); lengthening index (LI), 14.5 (SD±3.5) days/cm; hea- ling index (HI), 52.6 (SD±20.1) days/cm; and consolidation index (CI), 93.3 (SD±40.0) days/cm. Mild complications were recorded in 11 (39.2 %), and serious and critical in eight patients (28.6 %). Fourteen patients (53.8 %) were free of any complications. Two complications were concurrently found in five patients (17.9 %). There was a statistically significant difference in the LP values related to the number of complications (p=0.019). No significant relationship was recorded on comparison of the HI value with the patient's age at the time of surgery (p=0.836) and patient's gender (p=0.546) (Mann- Whitney test). The relationship of the OI value to the HI value was non-significant (p=0.492), as was the relationship between the osteotomy technique (oscillating or Gigli saw osteotomy) and the occurrence of complications (p=1.000) (Fisher's exact test). Correlation between the LI and HI values was significant (p<0.001). DISCUSSION: The results of healing after lengthening, as assessed by the healing and the consolidation index, were in agreement with other authors' data. The lower number of complications, particularly fractures of bone regenerate, can be explained by the facts that, in our study, the lengthening percentage was lower and that the post-operative care was strictly observed, including dynamic axial loading which stimulates bone consolidation at the lengthened section, with adherence to the proof of three developed cortices. CONCLUSIONS: Our results did not confirm the assumption that slower lengthening will have a favourable effect on the healing index. Key words: femoral lengthening, external fixator, complications.

Bifidobacterium bombi sp. nov., from the bumblebee digestive tract.

Gram-positive-staining, anaerobic, non-spore-forming, lactate- and acetate-producing bacterial strains were isolated from the digestive tracts of different bumblebee species (Bombus lucorum, Bombus pascuorum and Bombus lapidarius). All of the isolates produced fructose-6-phosphate phosphoketolase activity. A representative strain, BluCI/TPT, was characterized further. Cells of strain BluCI/TPT showed occasional bifurcation and irregular constrictions. The bacterium utilized a wide range of carbohydrates. Glucose was fermented to acetate and lactate. The DNA base composition was 47.2 mol% G+C. Complete 16S rRNA and partial hsp60 gene sequences were obtained and phylogenetic relationships were determined. Strain BluCI/TPT and related isolates were located in the actinobacterial cluster and were closely related to the genera Bifidobacterium, Scardovia, Aeriscardovia and Parascardovia. The results presented support the proposal of a novel species to accommodate strain BluCI/TPT, with the name Bifidobacterium bombi sp. nov.; the type strain is BluCI/TPT (=DSM 19703T=ATCC BAA-1567T).

A rapid, reproducible method for coating Rotorod Sampler collector rods with silicone grease.

BACKGROUND: The Rotorod Sampler (Sampling Technologies, St. Louis Park, MN) is a rotating-arm impactor that recovers airborne particles on two rapidly moving plastic collector rods. For decades, the standard method for applying silicone grease to collector rods has been with one's finger. Although this method can yield excellent results when performed by practiced investigators, a relatively high skill level is required, and significant intrapreparer variability has been reported in the medical and technical literature. OBJECTIVE: The purpose of this investigation was to develop and evaluate a new method for coating Rotorod collector rods with silicone grease. METHODS: Collector rods were coated with silicone grease by dipping them into a solution consisting of silicone grease and hexane. Pollen recovery by these dipped collector rods was compared with pollen counts obtained with hand-greased collector rods. RESULTS: Twenty-three paired samples were obtained during five sampling periods. Pollen recovery by the hand-greased and dipped collector rods was similar (P = 0.410). Dipped collector rods generally offered a lower standard deviation than hand-greased collector rods, however, differences were not statistically significant (F = 1.782, P = 0.087). Dipped collector rods were also superior to hand-greased collector rods in several qualitative categories such as grease uniformity, time required for microscopic analysis, and visual quality. CONCLUSIONS: Dipped collector rods offered a time-efficient means to obtain atmospheric samples with excellent visual quality. The resulting pollen counts were similar to data obtained via the standard, manual method. Allergists are encouraged to consider using this new method in their office practices and for drug studies.

Dynamic structure of proteins in solid state. 1H and 13C NMR relaxation study.

Temperature dependencies of 1H non-selective NMR T1 and T2 relaxation times measured at two resonance frequencies and natural abundance 13C NMR relaxation times T1 and T1r measured at room temperature have been studied in a set of dry and wet solid proteins - Bacterial RNase, lysozyme and Bovine serum albumin (BSA). The proton and carbon data were interpreted in terms of a model supposing three kinds of internal motions in a protein. These are rotation of the methyl protons around the axis of symmetry of the methyl group, and fast and slow oscillations of all atoms. The correlation times of these motions in solid state are found around 10(-11), 10(-9) and 10(-6)s, respectively. All kinds of motion are characterized by the inhomogeneous distribution of the correlation times. The protein dehydration affects only the slow internal motion. The amplitude of the slow motion obtained from the carbon data is substantially less than that obtained from the proton data. This difference can be explained by taking into account different relative inter- and intra- chemical group contributions to the proton and carbon second moments. The comparison of the solid state and solution proton relaxation data showed that the internal protein dynamics in these states is different: the slow motion seems to be few orders of magnitude faster in solution.

The functional size of ferrochelatase determined in situ by radiation inactivation.

Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.

Immunochemical studies of ferrochelatase protein: characterization of the normal and mutant protein in bovine and human protoporphyria.

Protoporphyria is a hereditary disorder characterized by a marked decrease in the activity of ferrochelatase, the terminal enzyme in the heme biosynthetic pathway. We have prepared specific polyvalent antibodies against bovine ferrochelatase in rabbits. The specificity of the antibody preparation against ferrochelatase was demonstrated by western blot analysis and immunoprecipitation of ferrochelatase activity. The antibody also cross-reacted weakly with ferrochelatase from human mitochondria. To quantify immunoreactive ferrochelatase in tissue samples, a kinetic-based enzyme-linked immunosorbent assay (k-ELISA) was developed. Ferrochelatase activity and the level of immunoreactive protein were measured in hepatic mitochondria isolated from six normal and nine protoporphyric (homozygous) cattle. Ferrochelatase activity was less than 10% of normal in mitochondria from protoporphyric animals; the amount of immunoreactive material was equivalent to that from normal animals. Similar studies were performed with samples from three normal and two protoporphyric (heterozygous) humans. Ferrochelatase activity was decreased in protoporphyric samples (about 17% of normal, but there was no concomitant decrease in immunoreactive material. These data demonstrate that a normal amount of ferrochelatase protein is present and suggest that bovine and human protoporphyria result from point mutations in the gene encoding ferrochelatase.

Comparison of bile porphyrin concentrations in cattle and human beings with protoporphyria.

Blood and bile porphyrin concentrations were measured in cattle with protoporphyria and compared with those in human beings with the disease. Whereas the mean RBC porphyrin concentration in cattle was 18-fold greater than in human beings, the mean bile porphyrin concentration was only 78% greater. Sequential measurements over a 30-hour period in 1 animal with a bile fistula indicated that the ratio of total porphyrin to total bile acid in bile varied minimally. When the animal was given an IV infusion of taurocholate, the biliary excretion rate of porphyrin increased in parallel with that of bile acid, because of enhancement of bile flow. Thus, in cattle with protophorphyria, the concentration of porphyrin in bile is low compared with that of porphyrin in RBC, in contrast with findings in human beings, and adequate amounts of bile acids are secreted to maintain efficient protoporphyrin excretion. This explains, in part, why hepatobiliary disease has not been observed in cattle with protoporphyria, but has been seen in human beings with the disease.

Porphyria and porphyrin metabolism.

Porphyrins, their reduced congeners (porphyrinogens), and their precursors are accumulated and excreted in excessive amounts in the porphyrias because of defects in the enzymes of heme biosynthesis. The nature of these defects is being defined using biochemical and molecular biological techniques. The principal clinical manifestations in the porphyrias, photocutaneous lesions and neurological dysfunction, are linked to the biochemical abnormalities, and appropriate therapeutic interventions have accordingly been developed. The exogenous administration of metalloporphyrins and porphyrin derivatives, unlike the harmful effects of porphyrins in the porphyrias, may be of use in some clinical conditions, such as the treatment of hyperbilirubinemic states and the detection and therapy of certain cancers.

Modulation of hepatic ferrochelatase activity by dietary manipulation of mitochondrial phospholipid fatty acyl groups.

Ferrochelatase is an enzyme bound to the inner mitochondrial membrane, which is important in heme biosynthesis. Activity of purified ferrochelatase is affected by the presence of certain fatty acids. In the present study, we examined whether the activity of ferrochelatase is altered by dietary manipulation of the composition of mitochondrial membrane phospholipid fatty acyl groups. Rats were fed diets containing triolein, safflower or menhaden oil as 5% (w/w) of the diet. After 3 weeks, the animals were killed and liver mitochondria were isolated. Phospholipid fatty acid composition and ferrochelatase activity were assayed in the isolated mitochondria. Marked differences were seen. The proportion of oleic acid was highest in the triolein oil-fed group, that of linoleic and arachidonic acid was highest in the safflower oil-fed group and the proportion of eicosapentaenoic acid was highest in the menhaden oil-fed group. Ferrochelatase activity was greatest in the triolein oil-fed group and lowest in the menhaden oil-fed group regardless of whether the mitochondria were intact, sonicated or sonicated and treated with Tween 20. Mixing of mitochondria from menhaden oil-fed rats with triolein oil resulted in a significant increase in ferrochelatase activity. Membrane fluidity and activities of the mitochondrial membrane enzymes succinic dehydrogenase and cytochrome oxidase did not differ among the groups. We conclude that dietary manipulation of mitochondrial membrane phospholipid fatty acyl group composition can directly modulate hepatic ferrochelatase activity. This has potential application in the treatment of protoporphyria, the genetic disorder in which ferrochelatase activity is deficient.

Hepatic and bile porphyrins in patients with protoporphyria and liver failure.

The livers of patients who have protoporphyria and hepatic failure contain large amounts of pigment crystals. Two such patients underwent liver transplantation, providing the opportunity to identify the pigment crystals. Portions of liver were digested enzymically, sedimented through a sucrose gradient, treated with 1% sodium dodecylsulfate, and centrifuged to purify the crystals. Spectrophotometric and high-performance liquid chromatography analysis demonstrated them to be composed of protoporphyrin. Bile samples were obtained from the 2 patients, 4 other patients who did not have liver disease, and 10 control subjects. The porphyrin concentrations in bile from the 6 patients were significantly increased above controls (range 254-7884 micrograms/dl compared with 11-109 micrograms/dl). The ratio of protoporphyrin to bile acid in bile distinguished the 2 patients with advanced liver disease (3105 and 2756 micrograms/mmol) from the 4 patients without liver disease (range 61-926 micrograms/mmol). Thus, analysis of bile from patients with protoporphyria may help in evaluating their hepatobiliary status.

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents.

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase.

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.

Dysphagia: its treatment in the profoundly retarded patient with cerebral palsy.

Oral motor and swallowing patterns of six profoundly retarded cerebral palsied patients were examined with videofluoroscopy. All subjects had delayed swallow reflexes and lingual dysfunction. Two subjects aspirated 10% or more of the bolus. Based upon patterns observed in x-ray evaluations, treatment programs were designed to remediate deviant areas. These programs consisted of dietary modifications, oral motor treatment, and thermal stimulation. Therapy was implemented seven days a week and a minimum of three times per day. X-rays were repeated at two four-month intervals following initiation of treatment procedures. Results showed significant gains in pharyngeal transit times, amount of material aspirated, amount of residue in the valleculae and pyriform sinuses, and number of swallows required to clear the oropharynx. Thermal stimulation was withdrawn on three of the subjects and after four months, when the x-rays were repeated. Withdrawal patients regressed in pharyngeal transit times but continued to make gains in other areas. Treatment patients showed minimal regression and substantial continued gains. Results suggest that the profoundly retarded cerebral palsied patient is capable of making gains in swallowing function based upon a passive treatment paradigm. The swallowing mechanism was felt to operate more quickly, more efficiently, and with fewer swallows at the end of the 18-month study.