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Distraction osteogenesis for lengthening of the hard palate: Part II. Histological study of the hard and soft palate after distraction.

To correct velopharyngeal incompetence, a new treatment concept was proposed in Distraction Osteogenesis for Lengthening of the Hard Palate: Part I (using lengthening of the hard and soft palate by distraction osteogenesis). Cephalometry and computed tomography showed successful elongation of the posterior hard palate with gradual calcification. Here the sequential use of fluorochrome markers (oxytetracycline, xylenol orange, DCAF [2,4-bis-N-N'-dicarboxymethyl aminomethyl fluorescein], and alizarin complexone) during the distraction and retention period is reported together with the histologic investigations using light and laser scan microscopy without prior demineralization. The experimental gap showed de novo osteogenesis in all dogs. The new bone was always in continuity with the original anterior and posterior palatal bone margins. It either bridged the experimental gap fully or left a small central zone of fibrous tissue, in which eventual ossification occurred. Several distinct zones could be distinguished: A small central zone was found with parallel strains of collagen fibers, oriented longitudinally in the direction of the distraction. Next to this zone a layer of undifferentiated mesenchymal precursor cells was seen in direct contact to newly formed bone. The next zone was coarse woven bone, showing a transition to mature lamellar bone through remodeling. No evidence of endochondral bone formation was found, i.e., all dogs showed exclusively intramembranous bone formation. The soft tissues showed no signs of alteration: in particular, there was no necrosis or scar formation. The soft tissues were not thinned but appeared to have followed the longitudinal displacement. In conclusion, gradual distraction osteogenesis of the hard palate could be a possible method for lengthening the palate to treat velopharyngeal incompetence.

Animals↗

The incidence and characteristics of some paraneoplastic syndromes of hepatocellular carcinoma in Nigerian patients.

OBJECTIVE: To determine whether paraneoplastic syndromes of hepatocellular carcinoma such as erythrocytosis, hypoglycaemia, hypercholesterolaemia and hypercalcaemia are found in Nigerian patients with this tumour, and, if present, to determine their incidence and characteristics. DESIGN: Consecutive patients with a diagnosis of hepatocellular carcinoma seen over a 10-year period at Ile-Ife, Nigeria, underwent haematocrit (100 patients), plasma glucose (65), cholesterol (65) and calcium studies (30). Shortage of laboratory reagents did not allow all the 100 patients to be tested for plasma glucose, cholesterol and calcium. METHODS: Haematocrit was determined by the microhaematocrit method, and the glucose oxidase-peroxidase, Liebermann-Burchard and O-cresolphthalein complexone methods were employed for the estimation of plasma glucose, cholesterol and calcium respectively. RESULTS: The haematocrit values ranged from 19 to 63% with a mean of 34.51% (SD 7.72). Only 1 out of 100 patients (1%) had erythrocytosis (haematocrit 63%). Sixty-five patients had their plasma glucose determined. The range of values for fasting plasma glucose (FPG) was 1-8.4 mmol/l with a mean of 4.3 (SD 1.6). Hypoglycaemia occurred in 18 patients (27.7%)(14 males, 4 females). The mean liver span for patients with FPG > 5.0 mmol/l was 16.87 cm (SD 3.03), while the value for those with FPG < 3.0 mmol/l was 22.0 cm (SD 6.14, P < 0.05). Plasma cholesterol was determined in 65 patients and the range of values was 2.1 -18.1 mmol/l with a mean of 5.65 (SD 2.42). Hypercholesterolaemia occurred in 16 patients (24.6%) (15 males, 1 female). The range of plasma calcium obtained in 30 patients was 1.6-3.15 mmol/l with a mean of 2.36 (SD 0.32). Three patients (10%) (all males) had hypercalcaemia. CONCLUSION: While erythrocytosis has a low incidence, hypoglycaemia and hypercholesterolaemia are important paraneoplastic syndromes of hepatocellular carcinoma in Nigerian patients. The study confirms that the larger the liver mass, the greater the tendency towards hypoglycaemia. Also, the paraneoplastic syndromes studied occurred predominantly in hepatocellular carcinomas with cirrhosis.

Calcium↗

Isolation and biological characterization of staphyloferrin B, a compound with siderophore activity from staphylococci.

A highly hydrophilic compound with siderophore activity has been isolated from the supernatant of Staphylococcus hyicus DSM 20459 grown under iron-restricted conditions. The metabolite, named staphyloferrin B, is strictly iron-regulated and produced by a large variety of staphylococci strains. In vivo iron transport measurements and the growth-promoting activity in a bioassay establish staphyloferrin B as the second siderophore for staphylococci besides the previously described staphyloferrin A. The structure elucidation revealed 2,3-diaminopropionic acid, citrate, ethylenediamine and 2-ketoglutaric acid as structural components of the compound. Thus, staphyloferrin B is a structurally new siderophore of the complexone type.

Biological Assay↗

[Synthesis and structure-function study of artificial nucleases on the basis of hemin conjugates with peptide fragments of cell differentiation factor HLDF].

A series of octa-hexapeptide fragments of HLDF and their conjugates with hemin were obtained by solid phase peptide synthesis. A relationship between the structure and the nuclease activity of the compounds was established. The effect of various factors (medium pH, the presence of metal ions, complexons, reducers, and buffer composition) on DNA destruction with hemin peptides was studied. Preliminary information confirming an oxidative mechanism of this process was obtained. The cleavage of plasmid DNA under the action of hemin peptides was studied by the methods of electron microscopy, gel electrophoresis, and atomic force microscopy.

DNA↗

Measurement of serum calcium concentration after administration of gadoversetamide in dogs.

PURPOSE: To measure serum calcium concentration with three different analytic methods after administration of gadoversetamide and three other gadolinium chelates in dogs. MATERIALS AND METHODS: Six dogs were injected with 0.1-, 0.3-, 1.0-, and 3.0-mmol/kg doses of gadoversetamide; 1.0-mmol/kg doses of gadodiamide, gadopentetate, and gadoteridol; and a 6-mL/kg dose of saline. Baseline blood samples were collected before injection; 5, 15, 30, and 60 minutes after each treatment; and 2, 4, 6, 12, and 24 hours after each treatment. Serum calcium levels were measured with inductively coupled plasma mass spectrometry, an arzenazo III dye assay, and an orthocresolphalthalin (OCP) complexone system. Analysis of variance coupled with the Dunnett procedure was used to compare serum calcium concentrations at different time points after injection with baseline values. RESULTS: Administration of gadoversetamide caused no decrease in serum calcium levels, as measured with inductively coupled plasma mass spectrometry or arzenazo III analytic techniques. In contrast, OCP assays showed a significant (P <.05) decrease in serum calcium values after administration of gadoversetamide. The decrease in serum calcium values peaked immediately after injection, and serum calcium values quickly returned to baseline. Injection of gadodiamide caused a significant (P <.05) decrease in the value of serum calcium when analyzed with the OCP technique but not when analyzed with inductively coupled plasma mass spectrometry or arzenazo III assays. In contrast, administration of gadopentetate or gadoteridol produced no significant change in serum calcium values, regardless of analytic method. CONCLUSION: Gadoversetamide and gadodiamide caused a transient artifact in measurement of serum calcium levels with an OCP assay but not with an arzenazo III technique or inductively coupled plasma mass spectrometry. The presence of gadopentetate or gadoteridol in the blood did not affect measurement of serum calcium levels.

Analysis of Variance↗

Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme catabolism and presumably is involved in cellular iron homeostasis. It is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. We examined induction of HO-1 mRNA in skin fibroblasts and investigated the mechanism by which it occurs. Hypoxia did not appear to act via induction of oxygen free radicals: induction of HO-1 was not sensitive to the free radical scavenger GSH or other antioxidants. Moreover, hypoxia did not increase steady-state levels of free radicals generated by fibroblasts. In contrast, HO-1 induction by the oxidants, H(2)O(2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) was significantly attenuated in the presence of free radical scavengers. This correlated with increased levels of free radical production in fibroblasts treated with these oxidants. Iron depletion by desferrioxamine mesylate, a specific iron complexon, completely inhibited hypoxic stimulation of HO-1 but did not attenuate the effect of H(2)O(2) and CCCP on HO-1 mRNA. Addition of Fe(2+), Fe(3+), or holo-transferrin to fibroblasts increased levels of HO-1 mRNA. Treatment of cells with hypoxia, but not H(2)O(2) or an exogenous source of iron, significantly increased the half-life of HO-1 mRNA. The data suggest hypoxia regulates HO-1 gene expression by a specific posttranscriptional mechanism: stabilization of mRNA. Hypoxia has previously been shown to increase fibroblast collagen synthesis and is thought to play a role in pathogenesis of systemic sclerosis (SSc). Skin fibroblasts isolated from patients with SSc demonstrated significantly stronger induction of HO-1 by hypoxia than did fibroblasts from normal controls. We hypothesize that exposure of SSc fibroblasts to hypoxic conditions leads to in vivo selective proliferation of cells that adapt to hypoxia.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗

Renal mercury content in HgCl2-induced acute renal failure in furosemide/saline-protected and nonprotected rats.

Renal mercury content, urinary mercury excretion and renal function were studied in rats with acute renal failure (ARF) induced by subcutaneous injection of 2, 3, 6, or 10 mg/kg HgCl2. Similarly poisoned rats were protected against ARF by continuous intravenous infusion of furosemide and saline. Excellent protection was obtained in rats receiving 2,3, and 6 mg/kg HgCl2, whilst some animals developed moderate azotemia after 10 mg/kg HgCl2. Renal mercury content 48 h after HgCl2 injection did not differ appreciably between protected and nonprotected groups of rats and showed no relation to the dose of HgCl2 injected or to the degree of renal failure. Urinary Hg excretion was variable during the first 24 h after HgCl2 injection and tended to be higher with higher dosage unless the animals became anuric early on. Hg excretion during the second 24 h was independent of dosage, but was comparatively high in functionally well protected rats and low in oliguric animals with severe renal failure. Attempts at detoxication with the potent chelating agent complexon I after 6 mg/kg HgCl2 failed completely: Renal mercury content was similar to that in the other groups of rats and every single rat so treated developed severe anuric renal failure. Although dose-dependent functional injury after HgCl2 may be related to the amount of Hg reaching the kidney during the initial phase, we have to conclude that HgCl2 toxicity is unrelated to the amount of Hg found in renal tissue at 48 h. Furthermore, furosemide/saline protection does not act through increasing urinary Hg excretion or decreasing the amount of toxin accumulating in renal tissue.

Acute Kidney Injury↗

Polychromatic labeling of otoconia for the investigation of calcium turnover.

The calcium ion turnover into otoconia of adult guinea pigs was investigated by the use of different fluorochromes, i.e. tetracycline (TC), calcein (Cal) and alizarin complexone (AC). The administration of all fluorochromes induced yellow (TC), green (Cal) and red (AC) fluorescence on the outer surface of otoconia, respectively. Sequential polychromatic labeling with AC followed by TC induced only red fluorescence after 1 week administration of TC, combined fluorescence after 2 weeks administration of TC and only yellow fluorescence after more than 3 weeks of administration of TC. The otoconia labeled with both AC and TC showed red fluorescence on the side faces of otoconia and yellow fluorescence on the terminal faces. These results may indicate the existence of a dynamic exchange of calcium ions in the otoconia and this exchange is mainly restricted to the terminal faces. The sequential polychromatic labeling may thus be of great use for the further investigation of the calcium dynamics of otoconia.

Acoustic Maculae↗

Physicochemical background for ambiguity of clinical recommendations in treating phosphate nephrolithiasis.

Efficiency of clinical recommendations for curing and prevention of phosphate nephrolithiasis founded on physicochemical investigations is unexpectedly low and apparently did not exceed 50%. We tried to consider possible physicochemical causes of this fact. For this purpose the influence of changes of urine pH and of calcium, phosphate and complexon concentrations on the probability of different precipitation phases of solid calcium phosphate has been reviewed. The importance of tendencies of changing the urine dynamic physicochemical parameters such as the precipitation induction period and the amplitude of urine pH variations in time has been shown. It was established that the probability of the existence of Ca3(PO4)2, Ca8H2(PO4)6.5H2O, CaHPO4.2 H2O and MgNH4PO4.6 H2O in urine depends on the time of its transition from the supersoluble state and is determined in the final analysis by the relationship of formation rates and solid phase dissolution rates. This was verified by experiments and by data obtained in the clinical laboratory.

Humans↗

A study of the regional distribution of bone formed around hydroxyapatite implants in the tibiae of streptozotocin-induced diabetic rats using multiple fluorescent labeling and confocal laser scanning microscopy.

The present study was designed to compare the amount and regional distribution of bone formation around hydroxyapatite (HA) implants in normal (control) rats with that of animals with diabetes mellitus (DM), induced by streptozotocin 2 weeks prior to implant placement. Calcein (CAL), alizarin complexone (AL), and tetracycline (TC) were injected on the 7th, 14th, and 21st days after implantation, respectively, and the rats were sacrificed on the 28th day after implantation. Seventy-microns undecalcified sections of the HA-bone interface in both groups were then prepared for confocal laser scanning microscopy (CLSM) observation. In both groups, bone formation developed from the HA surface to the endosteum, periosteum, or bone marrow. In the control group, around the HA close to the endosteum and periosteum, the new bone showed an extensive lamination pattern of three color layers (CAL, AL, and TC), but in the DM group the labeling density of TC on the 21st day was low. In contrast, on the lateral part of the HA surface (away from the endosteum and periosteum), there was considerably less bone formation in the control group, and in the DM group it was almost completely suppressed. These findings indicate that bone formation around the HA was initiated from the HA surface in the control group, while in the DM group, bone formation along the lateral part of the HA away from the endosteum and periosteum was almost completely suppressed. Furthermore, it is also suggested that in the new bone along the HA close to the endosteum and periosteum, only calcification on the 21st day was depressed.

Animals↗

A sequential injection system for the spectrophotometric determination of calcium, magnesium and alkalinity in water samples.

A sequential injection methodology for the spectrophotometric determination of calcium, magnesium and alkalinity in water samples is proposed. A single manifold is used for the determination of the three analytes, and the same protocol sequence allows the sequential determination of calcium and magnesium (the sum corresponds to the water hardness). The determination of both metals is based on their reaction with cresolphtalein complexone; mutual interference is minimized by using 8-hydroxyquinoline for the determination of calcium and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for the determination of magnesium. Alkalinity determination is based on a reaction with acetic acid, and corresponding color change of Bromcresol Green. Working ranges of 0.5 - 5 mg dm(-3) for Ca, 0.5 - 10 mg dm(-3) for Mg, and 10 - 100 mg HCO3- dm(-3), for alkalinity have been achieved. The results for water samples were comparable to those of the reference methods and to a certified reference water sample. RSDs lower than 5% were obtained, a low reagent consumption and a reduced volume of effluent have been accomplished. The determination rate for calcium and magnesium is 80 h(-1), corresponding to 40 h(-1) per element, while 65 determinations of alkalinity per hour could be carried out.

Alkalies↗

[Process of regeneration of cementum observed by various fluorescent-labeling methods].

The purpose of this study was to evaluate the effect of various fluorescent-labeling methods on longitudinal information associated with the process of regeneration of cementum after periodontal therapy. Mandibular bilateral premolars and molars in three monkeys were used. Prior to the labeling, experimental periodontitis was surgically produced at the mesial site of each tooth. In four weeks, scaling and root planing were carried out, and time marking was performed by injection of 3 kinds of fluorescent-labeling materials, tetracycline hydrochloride (30 mg/kg), calcein (8 mg/kg), and alizarin complexon (20 mg/kg), intramuscularly. The animals were sacrificed 4 days after the last injection and serial sections without decalcification were prepared. They were examined under a fluorescence microscope and further observed by contact microradiography (CMR) and staining with toluidine blue. According to the observations made by fluorescence microscopy, marked regeneration of the cementum was revealed by each of the 3 labeling agents. Also, the presence of regenerated cementum was supported by the observations by CMR and after toluidine blue staining. In addition, the regeneration of new cementum was shown to start in about 2 weeks after scaling and root planing. Judging from the results of this experiment, the various fluorescent-labeling methods seem to be effective for observing the process of regeneration of cementum.

Animals↗

Peptide antibiotics, beta-lactams, and related compounds.

In the field of natural peptides, beta-lactams, and related compounds, recent exciting developments are discussed. The increasing interest in this class of bioactive amino-acid derived structures has been attributed to the use of new directed screens (enzyme inhibition assays, beta-lactam detection, immunomodulator studies), new and improved applications (antibiotic, transplantation, and cancer chemotherapy), and advances in functional studies (DNA binding peptides, nucleotide complexones, cell wall and protein processing inhibitors). Peptides offer unique access to modifications and analog production by in vivo (directed biosynthesis) and in vitro procedures (enzymatic synthesis) due to their general linear precursors permitting point replacements. Of special interest are recent developments in the genetics of these compounds (cyclic peptides and beta-lactams), which will find applications in production methods in the near future.

Amino Acid Sequence↗

[Enhancement of 67Ga tumor-to-blood ratios by chelating agent (author's transl)].

Chelating agent, such as CaEDTA, CaDTPA, D-penicillamine, DMSA, desferoxamine, NTA, cysteine ethyl ester, BAL, alpha-MPG, phthalein complexone, were tested as a possible contrast enhancing agent for tumor imaging with 67Ga-citrate. The intravenous administration of a chelating agent to Ehrlich's tumor bearing mice, one hour after the injection of 67Ga-citrate, accelerated the blood clearance with only a very slight change of activity in the target, increasing the tumor-to-blood ratio, and consequently achieving a better visualization. Among the tested chelating agents, D-penicillamine showed the highest target-to-nontarget ratio at a shorter time: a good tumor-to-blood ratio, performed after 24 hr with non-treated animals, was achieved in only 1-3 hr with post-treated animals. Thus, D-penicillamine hold a considerable promise as a contrast enhancing agent for future clinical use.

Animals↗

Characterization of an in vitro model of calcification in retinal pigmented epithelial cells.

Little is known about the relationship at the molecular and cellular levels between vascular calcification and elastic fibers essential for elasticity. To gain a better understanding of the physiological function of elastin in vascular calcification, we developed a calcification model on cultured bovine retinal-pigmented-epithelial cells (RPEs) that do not express endogenous tropoelastin. The addition of inorganic phosphate (NaH2PO4; Pi) induced calcium deposition in RPEs. The Pi-induced calcification, as assessed by the o-cresolphthalein complexone method, Goldenbergs method, and von Kossa staining, was completely inhibited by treatment with clodronate (DMDP) and phosphonoformic acid (PFA) and was weakly suppressed by treatment with levamisole. Moreover, the osteopontin mRNA expression was upregulated in the Pi-induced calcification of RPEs. These reactions in RPEs were characteristically consistent with those already established in cultured bovine aortic smooth muscle cells (BASMCs). Furthermore, bacterially expressed tropoelastin inhibited calcium deposition in RPEs as well as in BASMCs. Finally, Pi-induced calcification was partially suppressed after the addition of tropoelastin due to elastic fiber formation. In conclusion, we suggest that this calcification model in RPEs is useful for analyzing the relation between elastic fibers and vascular calcification, and that tropoelastin and elastic fibers may contribute to the inhibition of vascular calcification.

Animals↗

Tropoelastin inhibits vascular calcification via 67-kDa elastin binding protein in cultured bovine aortic smooth muscle cells.

In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.

Animals↗

Review of calcium methodologies.

A review of calcium methodologies for serum has been described. The analytical systems developed over the past century have been classified as to type beginning with gravimetry and extending to isotope dilution-mass spectrometry by covering all of the commonly used technics which have evolved during that period. Screening and referee procedures are discussed along with comparative sensitivities encountered between atomic absorption spectrophotometry and molecular absorption spectrophotometry. A procedure involving a simple direct reaction for serum calcium using cresolphthalein complexone is recommended in which high blanks are minimized by repressing the ionization of the color reagent on lowering the dielectric constant characteristics of the mixture with dimethylsulfoxide. Reaction characteristics, errors which can be encountered, normal ranges and an interpretative resume are included in its discussion.

Blood Chemical Analysis↗

Evaluation of the revised serum calcium procedure used with the Du Pont automatic clinical analyzer.

We evaluated a revised serum calcium method (o-cresolphthalein complexone) used with the Du Pont aca. Our results correlated well with those obtained by atomic absorption spectrometry. Within-run and day-to-day coefficients of variation were 0.5 and 1.5%, respectively. Color produced and calcium concentration are linearly related and the relation is not affected by hemolysis, lipemia, or icterus.

Autoanalysis↗