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PubMed2004

Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting.

The accumulation of PrP(Sc), an abnormal and disease-associated form of the normal prion protein (PrP(c)), within the central nervous system (CNS) is a key pathological feature of Creutzfeldt-Jakob disease (CJD). Following limited proteolytic digestion of PrP(Sc), the detection of PrP(res) within lymphoid tissues is a unique characteristic of variant CJD in comparison with other human prion diseases, raising fears of an increased risk of iatrogenic spread. Because levels of PrP(res) in lymphoid tissues are lower than those found in CNS tissue, there is concern that other peripheral tissues may harbour infectivity at levels that current detection systems cannot demonstrate PrP(res). We have modified the paraffin-embedded tissue blot (PET blot), a technique combining immunohistochemistry (IHC), histoblot and Western blotting, for the detection of PrP(res) in paraffin sections in peripheral tissues in variant CJD. Five cases of variant CJD were examined, using a panel of anti-PrP antibodies. In each of these five cases, spleen, tonsil, lymph nodes and dorsal root ganglia showed an increase in the sensitivity and specificity of labelling using the PET blot when compared with optimized PrP(res) IHC methods. Control cases showed no evidence of PrP accumulation in either peripheral or CNS tissues. Autopsy and biopsy brain material from sporadic CJD cases also showed an increased sensitivity of PrP(res) detection with the PET blot, confirming its value as an important diagnostic and research tool in human prion diseases.

Adolescent↗
PubMed1992

Identification and isolation of common and tissue-specific geranylgeranylated gamma subunits of guanine-nucleotide-binding regulatory proteins in various tissues.

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.

Amino Acid Sequence↗
PubMed1997

Influence of changes in pancreatic tissue morphology and capillary blood flow on antibiotic tissue concentrations in the pancreas during the progression of acute pancreatitis.

BACKGROUND: The ability of an antibiotic to reach bactericidal concentrations in tissue depends on numerous factors including tissue composition and regional perfusion. Although necrotising pancreatitis is characterised by progression of pancreatic necrosis over at least 96 hours and microcirculatory alterations, the impact of these changes on the concentration of antibiotics in the pancreas has not yet been investigated. AIM: To determine and compare pancreatic tissue concentrations of imipenem and cefotaxime at different stages of acute necrotising pancreatitis in an animal model that has been shown to mimic closely the pathomorphological and bacteriological features of severe human pancreatitis. METHOD: Acute necrotising pancreatitis was induced in rats by a standardised intraductal infusion of glycodeoxycholic acid and intravenous cerulein. Six hours (n = 16) and 48 hours (n = 16) after induction of pancreatitis, the animals were randomised for intravenous therapy with either imipenem or cefotaxime. Fifteen minutes after injection of the antibiotic, the animals were killed. Blood and the head of the pancreas were collected for determining imipenem or cefotaxime in serum and tissue; the splenic portion of the pancreas was prepared for histological examination. In an additional set of identically treated animals, pancreatic capillary blood flow (PCBF) was assessed by intravital microscopy before induction of acute necrotising pancreatitis and at the time of antibiotic therapy. RESULTS: Imipenem accumulates in the pancreas in the initial phase of acute necrotising pancreatitis characterised by pronounced oedema and decreased PCBF, and tends to decrease with resolution of the oedema and the progression of acinar cell necrosis in the later course of the disease. Concentrations of cefotaxime are low in oedematous pancreatic tissue early after induction of acute necrotising pancreatitis and increase with the resolution of oedema and normalisation of PCBF. CONCLUSIONS: Concentrations of antibiotics in the pancreas vary in acute necrotising pancreatitis, depending on changes in pancreatic tissue morphology and capillary blood flow. This suggests that antibiotic tissue concentrations may not be consistent from one agent to another and that efficacy of antibiotics in acute pancreatitis cannot be estimated solely on the basis of their pharmacological and microbiological properties.

Acute Disease↗
PubMed2002

Tissue counter analysis of histologic sections of melanoma: influence of mask size and shape, feature selection, statistical methods and tissue preparation.

BACKGROUND: Tissue counter analysis is an image analysis tool designed for the detection of structures in complex images at the macroscopic or microscopic scale. As a basic principle, small square or circular measuring masks are randomly placed across the image and image analysis parameters are obtained for each mask. Based on learning sets, statistical classification procedures are generated which facilitate an automated classification of new data sets. OBJECTIVE: To evaluate the influence of the size and shape of the measuring masks as well as the importance of feature selection, statistical procedures and technical preparation of slides on the performance of tissue counter analysis in microscopic images. As main quality measure of the final classification procedure, the percentage of elements that were correctly classified was used. STUDY DESIGN: HE-stained slides of 25 primary cutaneous melanomas were evaluated by tissue counter analysis for the recognition of melanoma elements (section area occupied by tumour cells) in contrast to other tissue elements and background elements. Circular and square measuring masks, various subsets of image analysis features and classification and regression trees compared with linear discriminant analysis as statistical alternatives were used. The percentage of elements that were correctly classified by the various classification procedures was assessed. In order to evaluate the applicability to slides obtained from different laboratories, the best procedure was automatically applied in a test set of another 50 cases of primary melanoma derived from the same laboratory as the learning set and two test sets of 20 cases each derived from two different laboratories, and the measurements of melanoma area in these cases were compared with conventional assessment of vertical tumour thickness. RESULTS: Square measuring masks were slightly superior to circular masks, and larger masks (64 or 128 pixels in diameter) were superior to smaller masks (8 to 32 pixels in diameter). As far as the subsets of image analysis features were concerned, colour features were superior to densitometric and Haralick texture features. Statistical moments of the grey level distribution were of least significance. CART (classification and regression tree) analysis turned out to be superior to linear discriminant analysis. In the best setting, 95% of melanoma tissue elements were correctly recognized. Automated measurement of melanoma area in the independent test sets yielded a correlation of r=0.846 with vertical tumour thickness (p<0.001), similar to the relationship reported for manual measurements. The test sets obtained from different laboratories yielded comparable results. CONCLUSIONS: Large, square measuring masks, colour features and CART analysis provide a useful setting for the automated measurement of melanoma tissue in tissue counter analysis, which can also be used for slides derived from different laboratories.

Adult↗
PubMedDate not supplied

Imprint cytology of parathyroid tissue in relation to other tissues of the neck and mediastinum.

OBJECTIVE: To retest the hypothesis that imprint cytology may be used to reliably diagnose parathyroid tissue and, if so, to ascertain whether accuracy in this technique may be easily attained. STUDY DESIGN: Imprint preparations from 15 parathyroid, 10 thyroid, 8 lymphoreticular and 2 adipose tissue specimens were assessed blindly by two pathologists, one of whom (pathologist B) had only limited experience with endocrine tissue imprint cytology. RESULTS: Both assessors consistently distinguished parathyroid and thyroid preparations from lymphoreticular and adipose tissue preparations. While there was occasional difficulty in distinguishing between parathyroid and thyroid preparations, this was usually attributable to the scanty nature of the preparations. No single cytologic feature allowed a distinction between parathyroid and thyroid tissue. However, by considering several relatively diagnostic features collectively, pathologist B showed an increase in specificity and sensitivity rates for distinguishing parathyroid from thyroid imprints from 82% to 100% and 57% to 83%, respectively. CONCLUSION: The high accuracy rates and rapid [table: see text] learning curve shown by imprint cytology in distinguishing between different neck or mediastinal tissue types, together with its time- and cost-cutting potential, support a role for the technique in the intraoperative diagnosis of parathyroid tissue.

Cytodiagnosis↗
PubMed1999

Comparison of the inhibitory effects of ApoB100 and tissue factor pathway inhibitor on tissue factor and the influence of lipoprotein oxidation.

The procoagulant activity of tissue factor is regulated by circulating inhibitors such as tissue factor pathway inhibitor (TFPI) and LDL. These 2 inhibitors also readily associate making the distinction between their activities difficult. We have examined the relative contributions of intact and C-terminal truncated TFPI and ApoB100. By following the inhibitory potential of the preparations, over a period of 120 minutes, it was demonstrated that TFPI and LDL-resembling particles inhibited tissue factor at different rates. TFPI was found to be a short, fast-acting inhibitor, whereas the action of LDL-resembling particles was more prolonged but slower. The oxidation of LDL has been closely associated with the development of cardiovascular disease, including atherosclerosis and thrombosis. Positively charged amino acids, particularly lysine residues, are prone to alterations via the formation of adducts by lipid peroxidation products. These residues are important in the inhibition of tissue factor activity by ApoB100. They also play an important role in the inhibitory Kunitz domains of TFPI. We have shown that the decline in the ability of LDL to inhibit tissue factor was as a result of modifications in LDL arising from oxidation. By examining the effects of oxidation on full-length and C-terminal truncated TFPI bound to LDL-resembling particles, we found that TFPI is only affected when in close association with ApoB100. C-terminal truncated TFPI was not affected significantly by oxidation. Finally, chemical modification of lysine and arginine residues reduced the overall inhibition of tissue factor by TFPI. We propose that TFPI and LDL act separately to inhibit tissue factor in vivo. However, the oxidation of LDL can alter both the endogenous activity of ApoB100 and reduce that of closely associated TFPI, compromising normal hemostasis.

Apolipoprotein B-100↗
PubMed1998

Role of tissue-specific blood flow and tissue recruitment in insulin-mediated glucose uptake of human skeletal muscle.

BACKGROUND: Conflicting evidence exists concerning whether insulin-induced vasodilation plays a mechanistic role in the regulation of limb glucose uptake. It can be predicted that if insulin augments blood flow by causing tissue recruitment, this mechanism would enhance limb glucose uptake. METHODS AND RESULTS: Twenty healthy subjects were studied with the forearm perfusion technique in combination with the euglycemic insulin clamp technique. Ten subjects were studied at physiological insulin concentrations (approximately 400 pmol/L) and the other 10 at supraphysiological insulin concentrations (approximately 5600 pmol/L). Four additional subjects underwent a saline control study. Pulse injections of a nonmetabolizable extracellular marker (1-[3H]-L-glucose) were administered into the brachial artery, and its washout curves were measured in one ipsilateral deep forearm vein and used to estimate the extracellular volume of distribution and hence the amount of muscle tissue drained by the deep forearm vein. Both during saline infusion and at physiological levels of hyperinsulinemia we observed no changes in blood flow and/or muscle tissue drained by the deep forearm vein. However, supraphysiological hyperinsulinemia accelerated total forearm blood flow (45.0+/-1.8 versus 36.5+/-1.3 mL x min(-1) x kg(-1), P<0.01) and increased the amount of muscle tissue drained by the deep forearm vein (305+/-46 versus 229+/-32 g, P<0.05). The amount of tissue newly recruited by insulin was strongly correlated to the concomitant increase in tissue glucose uptake (r=0.789, P<0.01). CONCLUSIONS: Acceleration of forearm blood flow mediated by supraphysiological hyperinsulinemia is accompanied by tissue recruitment, which may be a relevant determinant of forearm (muscle) glucose uptake.

Adult↗
PubMedDate not supplied

Tissue engineered skeletal muscle: preparation of highly dense, highly oriented hybrid muscular tissues.

We prepared highly dense, highly oriented hybrid muscular tissues that are composed of C2C12 cells (skeletal muscle myoblast cell line) and type I collagen. A cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into capillary tube molds of two different sizes (inner diameters; 0.90 and 0.53 mm, respectively). One end of each mold was sealed. Upon centrifugation (1000 rpm, 5 min) and subsequent thermal gelation, a rod-shaped gel was obtained. It was cultured in an agarose gel-coated dish for 7 days (first for 3 days in a growth medium and then for 4 days in a differentiation medium), during which time it shrank to become a highly dense tissue. Small-diameter rod-shaped, highly dense cellular assemblages with multinucleated myotubes were formed and only few necrotic cells at the core of the tissue were observed. On the other hand, a ring-shaped tissue prepared using a specially devised agarose gel mold was subjected to cyclic stretching at 60 rpm, resulting in the formation of a highly dense, highly oriented hybrid muscular tissue in which both densely accumulated cells and collagen fiber bundles tended to be aligned in the direction of stretching. The hybrid muscular tissues that were prepared using via sequential procedures of a centrifugal cell packing method and a mechanical stress-loading method became closer to native muscular tissues in terms of cell density and orientation.

Animals↗
PubMed2001

Autofluorescence microscopy of fresh cervical-tissue sections reveals alterations in tissue biochemistry with dysplasia.

Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 +/- 39) relative to normal tissue (85 +/- 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 +/- 34 [dysplasia] vs 151 +/- 44 [normal]) and 460 nm excitation (93 +/- 35 [dysplasia] vs 137 +/- 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17-40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

Adult↗
PubMed1994

Absorption and incorporation into tissue lipids of 3H-arachidonic- and 14C-linoleic acid: effects of ethanol in jejunal tissue cultures and in vivo.

The effects of ethanol in the rat on the absorption and incorporation of 3H-arachidonic (20: 4, n-6) and 14C-linoleic acid (18:2, n-6) into tissue lipids was examined in jejunal tissue cultures in vivo. The pattern of incorporation earlier seen in the small intestine in vivo, ie. a preferential incorporation of 3H-20: 4 in comparison to 14C-18: 2 into phospholipids (PL), particularly phosphatidylethanolamine (PE) and phosphatidylinositol (PI) was seen in the jejunal tissue cultures. 10 mM ethanol slightly decreased the incorporation of 3H-20: 4 into PE and PI, but did not change the partitioning of labelled fatty acids into phospholipids and triacylglycerols (TG). Ethanol (100 mM) decreased the incorporation of both 3H and 14C into both PL and TG and caused a moderate increase in the TG/PL radioactivity ratio. Rats were also infused intraduodenally with either 10% ethanol or saline and given 3H-20: 4 and 14C-18: 2 in Intralipid. Radioactivity of tissue lipids were analysed after 1, 2 and 3 h. Ethanol did not significantly influence the absorption or the retention in small intestine, liver or heart of 3H or 14C, or the time course for the 3H- and 14C lipid radioactivity in serum. The distribution of 3H and 14C between total nonpolar lipids and individual PLs was also similar in the two groups, 3H-20: 4 thus exhibiting the same preferential incorporation into PLs, compared to 14C-18: 2, in both groups. Significant amounts of 3H and 14C appeared in phosphatidylethanol (PEth) of the small intestine, heart and liver, when the tissues from the ethanol-infused animals were stored frozen before extraction. When the tissues were extracted immediately after the experiment, the proportions of 3H and 14C migrating as PEth did not exceed 0.5%. Although sufficient amounts of ethanol and the phospholipase D activity necessary for PEth formation were thus present in the tissues examined, little PEth formation thus occurred during the ethanol infusion. In the ethanol treated group about 1% of the 3H and 14C radioactivity of the small intestine was in ethyl ester. Intraduodenal infusion of 10% ethanol in vivo thus has little acute effects on the metabolism of absorbed 18: 2 and 20: 4 in the intestinal mucosal cells. Formation of some ethyl ester occurs, however.

Animals↗
PubMedDate not supplied

Cerebral blood flow response to the tissue temperature in tumour and brain tissues.

The response of regional-cerebral blood flow (rCBF) to change in the tissue temperature was studied using normal and tumour-bearing monkeys. The local brain was selectively heated by the external microwave irradiation, while the body was kept hypothermic (30.1 +/- 0.1 degrees C, mean +/- standard error) by immersion in a cold water bath. The rCBF in brain and/or tumour tissues was sequentially measured by inhalation hydrogen clearance method. In the normal animal study (n = 7), rCBF changed in response to the tissue temperatures over a range of 29.4-40.7 degrees C with a constant rate 15.2% per degree Celsius change. Similarly, rCBF in the tumour-bearing animals (n = 7) changed proportionately with change in the tissue temperatures over a range of 28.4-42.5 degrees C in tumour and 27.6-41.8 degrees C in brain tissue. The rate in rCBF change per degree Celsius was 6.5% for tumour, which was significantly smaller than that for brain tissue (13.5%) (P less than 0.01). These results indicated that rCBF can be controlled by the defined application of selective heating with temperatures ranging from shallow hypothermia to modest hyperthermia. Vascular response to temperatures in the tumour and brain tissues may play a significant role in the application of heat to brain tumour treatment.

Animals↗
PubMed1977

[Study of distribution of 169Yb, 69Ga and 111In in tumor tissues by macroautoradiography; comparison between viable and necrotic tumor tissues].

The localization of 169Yb, 67Ga and 111In in tumor tissues was determined macroautoradiographically. 169Yb-citrate and 111In-citrate were injected intravenously to the rats subcutaneously transplanted Yoshida sarcoma and were injected intraperitoneally to the mice subcutaneously transplanted Ehrlich tumor. These animals were sacrificed 3, 24 and 48 hours after injection. These tumor tissues were frozen in n-hexane (-70 degrees C) cooled with dry ice-acetone. After this, these frozen tumor tissues were cut into serial thin sections (10 micron) in the cryostat (-20 degrees C). One of the slice of these sections was then placed on X-ray film and this film was developed after exposure of several days. On the other hand, next slice of these sections were then stained using the hematoxylin and eosin. From the observations of these autoradiogram and H-E stained slice, the following results were obtained. Concentration of 169Yb, 67Ga and 111In was predominant in viable tumor tissue rather than in necrotic tumor tissue, regardless of time after the administration. 67Ga and 111In were distributed uniformly in viable tumor tissue, but deposition of 169Yb was observed more avidly in viabl tumor tissue neighboring to necrotic tumor.

Animals↗
PubMed1975

Quantitative studies of the renal corpuscles iii: the influence of post mortem delay before taking renal tissue samples and of the duration of tissue fixation.

Comparative quantitative studies of glomeruli in biopsies of the guenon monkey kidney were made to give a numerical measure of the effect of varying periods of fixation of the biopsy tissue in Carnoy's fluid (1-2-24 hrs.), and of the effect of progressive post mortem delay before renal tissue samples were taken (2-4-6-12 hrs.). Additional aims were to determine possible quantitative differences between subcapsular, intermediate and juxtamedullary glomeruli, and to determine whether the glomerular area should be determined with reference to the inner aspect of Bowman's capsule (total area) or to tangents laid at the top of the capillary loops (corrected total area). The quantitative methods used were total and differential counts of glomerular nuclei by light microscopy and point counting for determination of glomerular and mesangial areas. Statistical evaluation was made by a two-way analysis of variance. The differential counts of nuclei were not affected by duration of fixation, nor by post mortem delay up to 12 hrs. before tissue samples were taken. The mesangial area expressed as a percentage of total area was significantly lower two hours post mortem, while total nuclear concentration was significantly lower at time 0 and two hours post mortem than at four, six and 12 hours post mortem. Total nuclear concentration increased as duration of fixation was increased, probably a reflection of tissue shrinking. Differences in nuclear concentration were also noted at different levels of the renal cortex. These differences were only apparent when the glomerular area was determined with reference to the inner aspect of Bowman's capsule, and disappeared when the corrected total area was used. It is concluded that duration of tissue fixation and post mortem delay before tissue samples were taken both affect method sensitivity, as reflected by some, not all, of the parameters measured. The numerical differences in the parameters were not large, however, and it will be the specific study which will determine whether variable fixation times and post mortem delay in obtaining tissue samples is acceptable. As most renal biopsies contain both subcapsular and juxtamedullary glomeruli there is probably no advantage in using the corrected glomerular area instead of the total glomerular area limited by Bowman's capsule.

Animals↗
PubMed2001

Tissue-implant response following soft tissue implantation of poly-L-lysine coated UHMW-polyethylene into adult male rats.

It is well demonstrated in the literature that polyethylene materials used in orthopedic applications are highly compatible and non-immunogenic with host tissues. However, recent studies in our laboratory have demonstrated the need for further study of these devices in vivo to further elucidate the mechanisms involved in biocompatibility. The purpose of this investigation was to study the tissue-implant response resulting from the implantation of ultra high molecular weight polyethylene (PE) coated with poly-L-lysine at 90 days post-implantation. In this investigation, implants of PE coated with poly-L-lysine and saline were implanted into 8 adult male rats subcutaneously (S/C) and intraperitoneally (I/P). At 90 days post-implantation, the animals were euthanized; and the PE devices, the fibrous tissue, and vital organs were harvested. Evaluation of routinely stained sections (5 microns, Hematoxylin & Eosin) of the fibrous tissue capsule surrounding the PE implants revealed the following: 1) all the devices had fibrous connective tissue capsules of slightly varying degrees of thickness surrounding them present at the time of sacrifice, 2) the thickness of the fibrous tissue capsule was dependent on the site of implantation and type of coating, 3) the thickness of the fibrous tissue capsules around the subcutaneous devices differed markedly than those retrieved from the peritoneal cavity, and 4) there were statistically significant differences (T-test, p < 0.05) in the numbers and types of cellular components with respect to implantation site and type of coating.

Animals↗
PubMed1975

Retinoic acid-binding protein in rat tissue. Partial purification and comparison to rat tissue retinol-binding protein.

When the 100,000 X g supernatant fractions of several rat organs are incubated with all-trans-[3H]retinoic acid, a binding component for retinoic acid with a sedimentation coefficient of 2 S can be detected by sucrose gradient centrifugation. This tissue binding protein for retinoic acid is distinct from the tissue binding protein for retinol which has been previously described. The tissue retinoic acid-binding protein has been partially purified from rat testis and this partially purified protein would appear to have a molecular weight of 14,500 as determined by gel filtration and high binding specificity for all-trans-retinoic acid. Binding of [3H]retinoic acid is not diminished by a 200-fold molar excess of retinal, retinol, or oleic acid but is reduced by a 200-fold excess of unlabeled retinoic acid. Tissue retinoic acid-binding protein can be detected in extracts of brain, eye, ovary, testis, and uterus but is apparently absent in heart muscle, small intestine, kidney, liver, lung, gastrocnemious muscle, serum, and spleen. This distribution is different than that observed for the tissue retinol-binding protein. Tissue retinol-binding protein was also purified extensively from rat testis. The partially purified protein has an apparent molecular weight of 14,000 and high binding specificity for all-trans-[3H]retinol as only unlabeled all-trans-retinol but not retinal, retinoic acid, retinyl acetate, retinyl palmitate, or oleic acid could diminish binding of the 3H ligand under the conditions employed. The partially purified protein has a fluorescence excitation spectrum with lambda max at 350 nm. In contrast, the retinol-binding protein isolated from rat serum and described by others has a fluorescence excitation spectrum with lambda max at 334 nm and an apparent molecular weight of 19,000. When partially purified tissue retinol-binding protein is extracted with heptane, the heptane extract has a fluorescence excitation spectrum similar to that of all-trans-retinol.

Animals↗
PubMed2001

[Construction of a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma tissue].

OBJECTIVE: To construct a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma (HCC) tissue. METHODS: Using the suppression subtractive hybridization (SSH), a novel technique has been described recently. cDNA fragments of missing or low expressing tumor suppressor genes in HCC tissue were isolated using paracancerous normal liver tissue and HCC tissue as targets. Then these cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of E.coli by high voltage electroperforation. One hundred positive bacteria clones were randomly picked and identified using enzyme restriction method. RESULTS: The amplified library contained more than 4,000 positive bacteria clones. Random analysis of 100 clones with enzyme restriction method showed that all clones contained 200-600 bp inserts. CONCLUSIONS: A subtracted cDNA library of differentially expressed genes in human normal liver tissue and HCC tissue is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new and specific missing or low expressing tumor suppressor genes of HCC.

Carcinoma, Hepatocellular↗
PubMed2004

[Expression of HPV16-E6 and E7 oncoproteins in squamous cell carcinoma tissues of esophageal cancer and non-cancer tissues].

BACKGROUND & OBJECTIVE: Evidences indicate that high-risk type human papillomavirus (HPV) are closely associated with the carcinogenesis, progression and transformation of several kinds of human tumors. This study was designed to determine the expression of HPV16- E6 and E7 oncoproteins in normal tissues, dysplasia tissues, and carcinoma tissues of patients with esophageal cancer and to investigate the biological significance of high-risk type HPV in the esophageal squamous cell carcinogenesis. METHODS: HPV16-E6 and E7 oncoproteins were determined using immunohistochemical staining in normal mucosa tissues (70 cases), dysplasia tissues (43 cases), and carcinoma tissues (18 cases). RESULTS: The positive rates of HPV16-E6 in the tissues of normal mucosa, dysplasia, and carcinoma of esophagus patients were 59.3%,88.4%,and 83.3%,respectively; the positive rates of HPV16-E7 protein were 62.1%, 90.7%, and 88.9%, respectively. The positive rates of HPV16-E6 and E7 in dysplasia and carcinoma of esophagus were significantly higher than those in normal mucosa (P< 0.05). Double expression of HPV16-E6 and E7 in normal mucosa was 25.7%, while in dysplasia and carcinoma were 88.3% and 83.3%,respectively. CONCLUSION: HPV16-E6 and E7 are highly associated with esophageal squamous cell carcinogenesis. And cooperation of HPV16-E6 and E7 may play an important role in genesis of esophageal squamous carcinoma.

Carcinoma, Squamous Cell↗
PubMed2005

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.

Coloring Agents↗