Surface Staining of Sawed Sections of Undecalcified Bone Containing Alloplastic Implants

A simple new method is described for the histological evaluation of bones containing alloplastic implants of ceramic and/or metallic materials. the undecalcified bone is embedded in acrylic resin and section at 50-200 μm using a sawing microtome. One surface of the preparation is stained up to 10 μm deep by floating the preparation on Giemsa stain. Other staining procedures are possible. Microscopic detail is Satisfactory for histological and morphometric evaluation.     

Purification and characterization of two tRNA-(guanine)-methyltransferases from rat liver

tRNA(guanine-1-)-methyltransferase (EC 2.1.1.31) and tRNA(N2-guanine)-methyltransferase I (EC 2.1.1.32) were isolated from rat liver. The (guanine-1-)-methyltransferase preparation is 6800-fold purified and is free from contaminating methyltransferases or ribonuclease. The molecular weight of (guanine-1-)-methyltransferase is 83 000. Of seven purified Escherichia coli tRNAs examined, only tRNAMetf was utilized as substrate by (guanine-1-)-methyltransferase. The methylation of tRNAMetf is maximally stimulated by 40 mM putrescine with a pH optimum of 8.0. Using E. coli K-12 tRNA, the Km for S-adenosylmethionine is 3 micrometer and Ki for S-adenosylhomocysteine is 0.11 micrometer for (guanine-1-)-methyltransferase. (N2-Guanine-)-methyltransferase is 6200-fold purified and is also free of interfering enzymes. It has a molecular weight of 69 000. E. coli tRNAPhe, tRNAVal and tRNAArg are substrates for this enzyme which introduces a methyl at the 2-amino group of the guanine at position 10 from the 5'-terminus of these tRNAs. The methylation of tRNAPhe is maximally stimulated by 100 micrometer spermidine with a pH optimum of 8.0. (N2-Guanine-)-methyltransferase has a Km for S-adenosylmethionine of 2 micrometer and a Ki for S-adenosylhomocysteine of 23 micrometer with E. coli K-12 tRNA as methyl acceptor.     

Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.     

Preparation and properties of rabbit-muscle glyceraldehyde-phosphate dehydrogenase with equal binding parameters for the third and fourth NAD+ molecules.

1. A method of preparing rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) is described which yields a preparation differing in important respects from those previously described and resembling the enzyme isolated from sturgeon muscle. 2. Direct binding measurements at 25 degrees C by equilibrium gel filtration fit dissociation constants for the first two molecules that are too low to be measured by this technique and 0.9 micrometer for the third and fourth molecules. The dissociation constant of the fourth molecule is much lower than that previously reported for the rabbit-muscle enzyme. 3. In contrast to previous results with the rabbit-muscle enzyme, the increase in absorbance at 360 nm between three and four molecules of NAD+ bound to the enzyme was, within experimental error, the same as that with each of the first three molecules. 4. Data on the quenching of the protein fluorescence by NAD+ at 15 degrees C at different enzyme concentrations closely fit dissociation constants of 0.028 micrometer for the first two molecules and 0.27 micrometer for the third and fourth molecules.     

A bone preservation protocol that enables evaluation of osseointegration of implants with micro- and nanotextured surfaces

Development of surface treatments has enabled secure attachment of dental implants in less than 1 month. Consequently, it is necessary to characterize accurately the osseointegration of the implant surface in the region of the bone-implant contact (BIC). We developed a method for sample preparation that preserves both bone and BIC to permit analysis of the contact interface. We prepared eight nanotextured implants and implanted them in rabbit tibias. After healing for 30 days, outcomes were analyzed using both our bone preservation protocol and routine decalcification followed by preparation of histological sections stained by hematoxylin and eosin (H & E). Pull-out tests for implant osseointegration were performed after healing. Non-implanted samples of rabbit mandible were used as a control for assessing organic and mineralized bone characteristics and bone structure. Our bone preservation protocol enabled evaluation of many of the same bone characteristics as histological sections stained with H & E. Our protocol enables analysis of implant samples, implant surfaces and osseointegration without risk of BIC damage.     

Scanning electron microscopy of plasma etched implant specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 microns histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Scanning Electron Microscopy of Plasma Etched Implant Specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 µl;m histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

A method for the isolation of intact, viable forespores from Bacillus species using high pressure

Large-scale preparation of Bacillus forespores was performed using nitrogen gas under high pressure to force an osmotically stable spheroplast suspension through a micrometer needle valve. Ninety-nine percent recovery of intact viable forespores at various stages of sporulation was achieved with a variety of Bacillus species. The isolated forespores are stable for up to 7 days when kept under nongerminating conditions.     

Tripeptide-specific aminopeptidase from Escherichia coli AJ005.

A tripeptidase, TP, from the ribosome-free fraction of Escherichia coli AJ005, a peptidase-deficient mutant of strain K-12, has been obtained using gel electrophoresis and chromatography on DEAE-Sephadex A-50, hydroxylapatite, and Sephadex G-200. Characterization studies on tripeptidase TP, freed of other detectable peptidases, indicate that this enzyme is capable of cleaving an amino-terminal leucine, lysine, methionine, or phenylalanine residue from certain tripeptides. Only one band of activity toward several tripeptides (and no activity toward dipeptides) was detected following gel electrophoresis of this preparation. Tripeptidase TP, the only strain AJ005 peptidase known to attack trilysine, was inactive toward all dipeptides, peptide amides, substituted peptides, esters, and tetrapeptides tested as substrates. Trilysine cleavage is optimal at about pH 8.5, as determined in Tris, borate, or phosphate buffers. Tripeptidase TP activity tested under a number of conditions was not inhibited by soybean trypsin inhibitor (3 mg/mL), phenylmethanesulfonyl fluoride (25 micrometer), or iodoacetate (9 mM). p-Mercuribenzoate (10 micrometer), divalent copper, cobalt, calcium (2.5 mM), zinc (25 micrometer), and mercury (10 micrometer) are inhibitory. Based on Sephadex G-200 chromatography tripeptidase TP has a particle weight of approximately 80 000 daltons. An apparent Km of 5.3 mM was determined for methionylglycylglycine cleavage.     

The assay of xylosyltransferase in cartilage extracts. A modified procedure for preparation of Smith-degraded proteoglycan.

A modification of the published method [Baker, Rodén & Stoolmiller (1972) J. Biol. Chem. 247, 3838--3847] for preparation of Smith-degraded proteoglycan is described. The new method is based on the finding that most of the chondroitin sulphate is cleaved from proteoglycan core protein by periodate oxidation. The borohydride reduction procedure was modified because the periodate-oxidized core protein is extensively degraded under the highly alkaline conditions previously used. The new method involves the separation of periodate-oxidized core protein from chondroitin sulphate by gel filtration on Sepharose 6B, and the reduction of the former in H3BO3/NaBH4 at pH 8.5 to produce the reduced species. Smith-degraded proteoglycan prepared by this method exhibited high acceptor activity for xylosyltransferase from embryonic-chick cartilage and had an apparent Km of 160 microgram/ml or 45 micrometer on a serine basis. In this assay system an apparent Km of 19 micrometer was obtained for UDP-xylose. The intermediate products periodate-oxidized core protein and reduced proteoglycen were inactive as xylosyltransferase acceptor substrates.     

Studies on NAD+ permeability in intact mitochondria from rabbit reticulocytes.

Functionally intact mitochondria from rabbit reticulocytes are characterized by a low NAD+ level after the preparation (0.29 nmoles NAD+ + NADH/mg protein). They are apparently impermeable for NADH and exhibit a slow net uptake of NAD+. From the increase of O2-uptake in state 3 and the increase of NADH concentration in state 4 of respiration after the addition of NAD+ we concluded that 3--10 min are necessary for the saturation with NAD+ at 23 degrees C. 2mM NAD+ extramitochondrially are not sufficient to saturate the mitochondria with NADH and probably NAD+, too. Because of the net uptake of NAD+ we assume that reticulocyte mitochondria lose NAD+ during their preparation. If they are incubated with the physiological concentration of 300 micrometer NAD+, which was found in reticulocytes, a value of 1.9 nmoles NAD+ + NADH mg protein was calculated. At an extramitochondrial NAD+ concentration of 300 micrometer, reticulocyte mitochondria exhibit an almost maximal O2-uptake in the presence of oxaloacetate or alpha-ketoglutarate. It is concluded that the mitochondria in intact reticulocytes contain the "normal" complement of NAD+ + NADH.     

Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy

Electron microscopy of biofilms and the localization of extracellular polymers at high resolution require the adaptation of conventional electron microscopic preparation and imaging techniques. A method developed for in situ fixation and embedding of biofilms, imaging of unstained thick sections with electron spectroscopic imaging and the application of lectin or antibody-based marker systems allowed interpretation of extracellular polymer distribution at micrometer scale. By this way, it is possible to discriminate in situ between extracellular polymers produced by different organisms.     

Histological analysis of the aortic nerve in the rat raised in space (Rapid communication on Neurolab Project).

To study development of the aortic nerve baroreflex under conditions of microgravity, we examined the cross section of the left aortic nerve (LAN), which is the afferent of the baroreflex, in the neonate rats aged 25 days raised in microgravity on the space shuttle Columbia (flight:FLT group) for 16 days. In this paper, we report a part of the result obtained from the data of the myelinated fibers of LAN analyzed with an electron microscope. Two kind of ground control groups were compared to the FLT group; one was asynchronous ground control (AGC) group where the rats were housed in the same cage as that on the shuttle, and the other was vivarium(VIV) group where the rats were housed in a commercial cage. The LANs in each group were extirpated the from rats perfused with a fixative and embedded for histological analysis. We observed the transverse sections of LAN and took pictures of several areas (magnified to x 2K to x 200K). No irregular myelination was found in all fibers of FLT group when they were compared with two control groups. The thickness of myelin of the maximally myelinated fibers were 0.55 +/- 0.17 micrometer in FLT(n=5), 0.45 +/- 0.10 micrometer in AGC(n=5), and O.47 +/- 0.06 micrometer meter in VIV(n=5). There was no significant difference among three groups (unpared t-test). The results suggest that there is no effect of space environment on the myelin formation of each nerve fiber in the aortic nerve.     

A new method to correlate histology with biomechanical properties in urethral tissue. An in-vitro study using light microscopy and scanning acoustic microscopy

When surveying the classical biomechanical theory of flow and resistance, the passive elastic properties of the urethra seems to be important for the transport of urine though the urethra. The aim of this study was to show that scanning acoustic microscopy (SAM) is a suitable methodology for investigating elastic properties of the urethra, and that it can be used to correlate elastic properties to histological areas. One 40 kg female pig and one 2 kg male rabbit comprised the material. A SAM2000 was used at a working frequency of 1000 MHz. Sections of nominal 3 micrometer thickness fixed urethral tissue were prepared for SAM and stained for light microscopy. The histological layers of the urethra were evident in the SAM image, and showed highly variable values of elastic properties. The layers seen with SAM correlated well with those seen with light microscopy. In conclusion, we have provided the first images of the microelastic properties of the urethra and correlated them to histology.     

Stability and local toxicity evaluation of a liposomal prilocaine formulation

This study reports a physicochemical stability evaluation of a previously reported liposomal prilocaine (PLC(LUV)) formulation (Cereda et al. J. Pharm. Pharmaceut. Sci. 7:235, 2004) before and after steam sterilization as well as its local toxicity evaluation. Prilocaine (PLC) was encapsulated into extruded unilamellar liposomes (LUVs) composed by egg phosphatidylcholine:cholesterol:alfa-tocopherol (4:3:0.07, mole %). Laser light-scattering analysis (p > 0.05) and thiobarbituric acid reaction (p > 0.05) were used to evaluate the liposomes physical (size) and chemical (oxidation) stability, respectively. The prilocaine chemical stability was followed by (1)H-nuclear magnetic resonance. These tests detected no differences on the physicochemical stability of PLC or PLC(LUV), sterilized or not, up to 30 days after preparation (p > 0.05). Finally, the paw edema test and histological analysis of rat oral mucosa were used to assess the possible inflammatory effects of PLC(LUV). PLC(LUV) did not evoke rat paw edema (p > 0.05), and no significant differences were found in histological analysis, when compared to the control groups (p > 0.05). The present work shows that PLC(LUV) is stable for a 30-day period and did not induce significant inflammatory effects both in the paw edema test and in histological analysis, giving supporting evidence for its safety and possible clinical use in dentistry.     

Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture.

1. Adenosine, a potent vasodilator, is transported very efficiently by pig aortic endothelium in monolayer culture (approx. 50pmol/min per 10(6) cells at 2 micrometer). Uptake proceeds by diffusion at high (millimolar) substrate concentrations, and by two discrete transport processes (Km approx. 3 micrometer and 250 micrometer) at lower concentrations. Over 90% of the adenosine taken up at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides (mainly ATP). 2. The high-affinity process is selectively inhibited by dipyridamole and by nitrobenzylthioinosine. Adenine preferentially inhibits the lower-affinity process, papapaverine inhibits both transport processes, and inosine has no significant effect. 3. Pig aortic smooth-muscle cells in culture show no high-affinity transport system for adenosine; uptake is much slower at low concentrations than that by endothelium (approx. 5pmol/min per 10(6) cells at 2 micrometer). Over 80% of the incorporated adenosine at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides. 4. The uptake of adenosine by smooth-muscle cells is powerfully inhibited by adenine, but dipyridamole is much less potent than in endothelium. 5. We conclude that endothelial cells are mainly responsible for the removal of circulating adenosine.     

Stability and Local Toxicity Evaluation of a Liposomal Prilocaine Formulation

This study reports a physicochemical stability evaluation of a previously reported liposomal prilocaine (PLC_LUV) formulation () before and after steam sterilization as well as its local toxicity evaluation. Prilocaine (PLC) was encapsulated into extruded unilamellar liposomes (LUVs) composed by egg phosphatidylcholine:cholesterol:alfa-tocopherol (4:3:0.07, mole?%). Laser light-scattering analysis (p?>?0.05) and thiobarbituric acid reaction (p?>?0.05) were used to evaluate the liposomes physical (size) and chemical (oxidation) stability, respectively. The prilocaine chemical stability was followed by ¹H-nuclear magnetic resonance. These tests detected no differences on the physicochemical stability of PLC or PLC_LUV, sterilized or not, up to 30 days after preparation (p?>?0.05). Finally, the paw edema test and histological analysis of rat oral mucosa were used to assess the possible inflammatory effects of PLC_LUV. PLC_LUV did not evoke rat paw edema (p?>?0.05), and no significant differences were found in histological analysis, when compared to the control groups (p?>?0.05). The present work shows that PLC_LUV is stable for a 30-day period and did not induce significant inflammatory effects both in the paw edema test and in histological analysis, giving supporting evidence for its safety and possible clinical use in dentistry.     

Calcium ions and the regulation of NAD+-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues.

The effects of Ca2+ on the activity of isocitrate dehydrogenase (NAD+) in extracts of rat heart mitochondria were explored in the presence of MgCl2 by using EGTA buffers. In the absence of ADP, Ca2+ (about 30 micrometer) resulted in a slight increase in apparent Km for threo-Ds-isocitrate; in the presence of ADP, Ca2+ (about 25 micrometer) greatly lowered the apparent Km for threo-Ds-isocitrate from 227 micrometer to 53 micrometer without changing the maximum velocity. At 100 micrometer-threo-Ds-isocitrate and 1 mM-ADP, there was an 8-fold activation by Ca2+, with a Km for Ca2+ of 1.2 micrometer. This activation was also observed with Sr2+ (Km 3.1 micrometer), but not with Mn2+ (at concentrations below 2.5 micrometer). Similar effects of Ca2+ were also observed on isocitrate dehydrogenase (NAD+) activity in extracts of mitochondria from liver, kidney, brown adipose tissue and white adipose tissue of the rat. The possible regulatory role of changes in the intramitochondrial concentration of Ca2+ is discussed.     

Method of determination of specific length of histological specimens with the aid of square-net eyeglass]

The author has analytically expressed the relationship between the inaccessible for measurement total length of non-randomly oriented stretched objects and the sum of lengths of their projections on the longitudinal axis of the preparation (or another line). The sum of their projection lengths can be measured by means of a square-net ocular. The specific length of objects on a histological section is proportional to the number of their intersections with the transversal lines of the net and the value of its division and inversly proportional to the cosine of the average meaning of angles between the longitudinal axis of the preparation and the objects under study. Verification of the validity of the formula deduced was carried on in the histological section model prepared by the author. The proposed method ensures determination of the specific length of stretched histological preparations with a no more than 3% error.     

Hepatic zonation of drug metabolizing enzymes. Studies on hepatocytes isolated from the periportal or perivenous region of the liver acinus

Models of hepatic intraacinar zonation have been proposed previously; in most models, direct visualization of the acinar destruction is not possible while intact hepatocyte recovery-viability often presents a problem for subsequent metabolic studies. In the present studies, the liver is isolated in situ and perfused with Krebs-Henseleit buffer, pH 7.4. A 1.5-mL intrahepatic volume of a 7 mM digitonin solution is then injected at a flow rate of 6 mL/min for 15 s via the portal vein or via the vena cava for selective destruction of the periportal (PP) or perivenous (PV) region of the acinus. To avoid diffusion of the detergent throughout the acinus, the liver is then immediately perfused with oxygenated Hanks buffer in the direction opposite to that of digitonin injection. The preparation can then be used for histological evaluation, for studies on isolated-perfused liver, or for isolation of hepatocytes. Direct visualization of the acinar destruction can be achieved by coloring the permeabilized cells with 0.2 mM trypan blue; the liver is then fixed in situ by a 10-min perfusion with paraformaldehyde and histological evaluation is achieved by eosine staining of liver slices. Following isolation of hepatocytes by collagenase perfusion, a highly significant PV localization was found for the synthesis of glutamine, the N-demethylation of aminopyrine, and the glucuronidation of p-nitrophenol, whereas a highly significant PP zonation was found for alanine aminotransferase. By contrast, no specific acinar zonation was found for the enzymes 7-ethoxycoumarin O-deethylase and aniline p-hydroxylase. Total cytochrome P-450 was 0.42 +/- 0.006 and 0.4 +/- 0.03 nmol/10(6) hepatocytes in PV and PP, respectively (nonsignificant).(ABSTRACT TRUNCATED AT 250 WORDS)