Quantitative and qualitative immunohistochemical detection of myc and src oncogene proteins in normal, nodule, and neoplastic rat liver

This study examined the possibility of using an immunohistochemical technique to detect the expression of myc and src oncogene proteins (ops) in livers of male Sprague-Dawley rats after treatment with the carcinogen diethylnitrosamine (with or without phenobarbital promotion) or untreated. We found that the majority of nodules and tumors from these livers stained for myc and src ops, indicating that myc and src expression did occur in these structures. These results were expected, since myc and src expression has been previously observed by others using different techniques. However, in our study, myc and src op staining was also noted in normal liver areas from rats in any of the four treatment groups (DENA, DENA + PB, PB alone, or untreated). The staining pattern of normal liver was different for each oncogene probe but was consistent within the four groups. In most cases, oncogene expression of normal liver occurred at sites of abnormal (but non-neoplastic) hepatocytes. The method reported here used both a qualitative technique of op expression analysis and a quantitative method using a Zeiss computer-driven image analysis system.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

INTERCELLULAR COMMUNICATION AND TISSUE GROWTH : I. Cancerous Growth

Intercellular communication was examined with intracellular electrical techniques in primary and transplanted rat liver cancers. Normal liver cells communicate rather freely with each other through permeable junctional membranes. Cancer liver cells show no communication at all; their surface membrane is a strong barrier to diffusion all around the cell. Cancer cells induce alterations in membrane permeability in normal liver cells; communication among the latter is markedly reduced when cancer cells grow near them.     

Hepatocellular carcinoma versus carcinoma metastatic to the liver. Value of stains for carcinoembryonic antigen and naphthylamidase in fine needle aspiration biopsy material

Staining for amino acid naphthylamidase and carcinoembryonic antigen (CEA) was examined as an ancillary technique to improve the accuracy of differentiating hepatocellular carcinoma from metastatic carcinoma to the liver in fine needle aspiration (FNA) biopsy specimens. Twenty-four cases of FNA specimens from the liver, in which air-dried smears and/or cell blocks were available, were examined. Naphthylamidase-positive bile canalicular structures were present in 2 cases of hepatocellular carcinoma and absent in 8 cases of metastatic carcinoma studied. Ninety percent of the hepatocellular carcinomas were immunoreactive with the antibody to CEA, showing a predominantly bile canalicular pattern. Ninety percent of the cases of metastatic carcinoma were positive with the antibody to CEA, showing a diffuse cytoplasmic pattern. These findings indicate that both staining techniques may be useful in differentiating hepatocellular carcinoma from metastatic carcinoma. Since the naphthylamidase stain requires air-dried smears, which may not be available, whereas immunocytochemistry can be done on fixed material, the latter technique is more practical.     

Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry

The structure of rat liver vault ribonucleoprotein particles was examined using several different staining techniques in conjunction with EM and digestion with hydrolytic enzymes. Quantitative scanning transmission EM demonstrates that each vault particle has a total mass of 12.9 +/- 1 MD and contains two centers of mass, suggesting that each vault particle is a dimer. Freeze-etch reveals that each vault opens into delicate flower-like structures, in which eight rectangular petals are joined to a central ring, each by a thin hook. Vaults examined by negative stain and conventional transmission EM (CTEM) also reveal the flower-like structure. Trypsin treatment of vaults resulted exclusively in cleavage of the major vault protein (p104) and concurrently alters their structure as revealed by negative stain/CTEM, consistent with a localization of p104 to the flower petals. We propose a structural model that predicts the stoichiometry of vault proteins and RNA, defines vault dimer-monomer interactions, and describes two possible modes for unfolding of vaults into flowers. These highly dynamic structural variations are likely to play a role in vault function.     

Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels

Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental hepatic cryosurgery

In 28 dogs, a major portion of a lobe of the liver was frozen, either by contact with a cryoprobe or by spraying liquid nitrogen (?196 °C) over the exposed liver or by pouring liquid nitrogen into a plastic barrel placed on the lobe of the liver, which was wrapped in a Dacron velour cloth. With the probe technique, the amount of tissue that could he frozen was not as great as with direct application of liquid nitrogen to the liver. The spray technique allowed freezing of a larger area but with cracking of the liver substance with considerable mortality from bleeding. With the pour technique, even a larger area of liver could be frozen and bleeding was much less because of the protection afforded by the Dacron cloth. There were no toxic effects from the devitalized liver which was left to be resorbed. These techniques are applicable for treating localized lesions in the liver in humans, but the experiments demonstrate the difficulty of achieving great depth of freezing with any currently available technique of freezing tissue in situ. Clearly, advances in cryosurgical equipment are needed.     

Isolation of rat liver microsomes by gel filtration

A method was developed for the rapid isolation of liver microsomes by means of gel filtration. Such microsomes were compared to microsomes prepared by conventional centrifugation techniques. Both preparations were of similar composition as regards concentrations and activities of certain microsomal enzymes as well as contamination by other liver cell organelles. The main advantages over conventional techniques are the considerable decrease of time required for isolation of the microsomes, the improved removal of solutes like hemoglobin, the improved suspension stability of the preparation, and that the technique does not require facilities for ultracentrifugation.     

Localization of vitellogenin and serum albumin in hepatic parenchymal cells of normal and estradiol-treated immature chickens

The localization of albumin and vitellogenin was determined in liver sections from control and estradiol-treated chickens by two different immunocytochemical techniques: (1) The sandwich technique with rabbit anti-lipovitellin or rabbit anti-albumin IgG and fluorescent goat anti-rabbit IgG and (2) the mixed aggregation immunocytochemical technique with anti-lipovitellin IgG and fluorescent lipovitellin.The results show that the antibody against albumin bound only to all liver parenchymal cells. Furthermore, the fluorescence intensity was equally strong in the portal, intermediate and central zones of the lobules.The fluorescent stain for vittelogenin was not above background in livers of control chicks but was far above background in estradiol-treated chicks. As with albumin the fluorescent stain was distributed equally among the parenchymal cells.The results were quantitatively the same 2 and 4 days after estradiol treatment. The relative rates of synthesis and the concentrations of albumin and vitellogenin correlate well with values obtained for tissue sections by immunocytochemical techniques.     

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

Leptospirosis is an infectious disease of worldwide importance. The development of diagnostic techniques allows sick animals to be identified, reservoirs to be eliminated and the disease prevented and controlled. The present study aimed to compare different techniques for diagnosing leptospirosis in sheep. Samples of kidney, liver and blood were collected from 465 animals that originated from a slaughterhouse. The sera were analyzed by the Microscopic Agglutination Test (MAT), and kidney and liver samples of seropositive animals were analyzed using four techniques: bacteriological culture, the Warthin Starry (WS) technique, conventional PCR (cPCR), and quantitative PCR (qPCR). With the MAT, 21 animals were positive (4.5%) to serovars Hardjo (n=12), Hebdomadis (n=5), Sentot (n=2), Wolfii (n=1) and Shermani (n=1). Titers were 100 (n=10), 200 (n=2), 400 (n=6) and 1600 (n=3). No animal was positive by bacteriological culture; four animals were positive by the WS technique in kidney samples; six animals were positive by cPCR in kidney samples; and 11 animals were positive by qPCR, eight of which in kidney samples and three in liver. The bacterial quantification revealed a median of 4.3 bacteria/μL in liver samples and 36.6 bacteria/μL in kidney samples. qPCR presented the highest sensitivity among the techniques, followed by cPCR, the WS technique and bacteriological culture. These results indicate that sheep can carry leptospires of the Sejroe serogroup, and demonstrate the efficiency of quantitative PCR to detect Leptospira spp. in tissue samples.     

Innermembrane association of three mitochondrial β-oxidation enzymes revealed by immunoelectron microscopic technique

Summary Ultrastructural localization of three mitochondrial β-oxidation enzymes, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase in rat liver was studied by a post-embedding immunocytochemical technique. Rat liver was fixed by perfusion. Vibratome sections (100 μm thick) were embedded in Lowicryl K4M. Ultrathin sections were separately incubated with antibody to each enzyme, followed by protein A-gold complex. Gold particles representing the antigenic sites for all enzymes examined were confined exclusively to mitochondria of hepatocytes and other sinus-lining cells. Peroxisomes were consistently negative for the immunolabelling. In the mitochondria the gold particles were localized in the matrical side of inner membrane. The control experiments confirmed the specificity of the immunolabelling. The results firstly indicate that the mitochondrial β-oxidation enzymes are present in the matrix of mitochondria and associated with the inner membrane.     

Analysing the patterning of a sequence of discrete behavioural events

Existing techniques for analysing the organization of behavioural events are discussed. Cluster analysis, mutual replaceability, ‘melody’ detection and Markov analyses are examined in order to discover the validity and usefulness of the concepts searched for by each technique, and the mathematical and statistical background of the techniques is scrutinized. Some new techniques, designed to search for and detect possible important sequential organization patterns, are described in the light of the previous criticisms. Of these techniques, the Pre-post-state Histogram (PPSH)—a multi-order Markov graphical analysis—would appear to be the most important for future work, especially when used in conjunction with the other techniques described.     

Measurement of water transport during freezing in mammalian liver tissue: Part II--The use of differential scanning calorimetry.

There is currently a need for experimental techniques to assay the biophysical response (water transport or intracellular ice formation, IIF) during freezing in the cells of whole tissue slices. These data are important in understanding and optimizing biomedical applications of freezing, particularly in cryosurgery. This study presents a new technique using a Differential Scanning Calorimeter (DSC) to obtain dynamic and quantitative water transport data in whole tissue slices during freezing. Sprague-Dawley rat liver tissue was chosen as our model system. The DSC was used to monitor quantitatively the heat released by water transported from the unfrozen cell cytoplasm to the partially frozen vascular/extracellular space at 5 degrees C/min. This technique was previously described for use in a single cell suspension system (Devireddy, et al. 1998). A model of water transport was fit to the DSC data using a nonlinear regression curve-fitting technique, which assumes that the rat liver tissue behaves as a two-compartment Krogh cylinder model. The biophysical parameters of water transport for rat liver tissue at 5 degrees C/min were obtained as Lpg = 3.16 x 10(-13) m3/Ns (1.9 microns/min-atm), ELp = 265 kJ/mole (63.4 kcal/mole), respectively. These results compare favorably to water transport parameters in whole liver tissue reported in the first part of this study obtained using a freeze substitution (FS) microscopy technique (Pazhayannur and Bischof, 1997). The DSC technique is shown to be a fast, quantitative, and reproducible technique to measure dynamic water transport in tissue systems. However, there are several limitations to the DSC technique: (a) a priori knowledge that the biophysical response is in fact water transport, (b) the technique cannot be used due to machine limitations at cooling rates greater than 40 degrees C/min, and (c) the tissue geometric dimensions (the Krogh model dimensions) and the osmotically inactive cell volumes Vb, must be determined by low-temperature microscopy techniques.     

Time, Cost, and Efficacy Study of Identifying Group A Streptococci with Commercially Available Reagents

During the 12-month period primary throat, wound, and skin cultures, tentatively identified as B streptococci, were submitted by 10 different clinical laboratories for evaluation. A total of 692 beta-hemolytic streptococci were isolated from cultures submitted and examined in parallel by the fluorescent-antibody, precipitin, and bacitracin techniques. An evaluation of the specificity and sensitivity in conjunction with basic and personnel costs was determined for each method. The standard Lancefield precipitin method was established as the standard by which the bacitracin and flourescent antibody techniques were compared. With some variation depending on the commerical source of the disc, approximately 7% of the strains examined produced false reactions with the bacitracin disc. False-negative reactions were rarely noted by the group A fluorescent antibody technique (0.5%), but an appreciable number of other Lancefield groups (B, C, and G) were nonreactive with homologous conjugates.     

Mutagenesis by 4-nitrobenzofurazans and furoxans.

15 nitrobenzofurazans and 10 nitrobenzofuroxans synthesized primarily for testing as potential anti-rheumatic drugs were also tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. The method used involved placing each compound in a "well" cut out of a plate of selective medium previously seeded with the appropriate tester strain, and then adding a rat liver microsome/cofactor mixture to one of the two wells on each plate. This method is considerably cheaper and more convenient than the conventional agar overlay technique, but in the present series of experiments failed to detect 4 compounds which could be detected by the overlay technique. Using a combination of the two techniques, 21 of the 25 compounds tested were found to be mutagenic. All 10 benzofuroxans and 9 of the 15 benzofurazans were detected as direct-acting mutagens with at least one of the two tester strains. Two of the benzofurazans gave positive results only in the presence of rat liver microsomes, and hence are pro-mutagens. One of the 4 benzofurazans which gave negative results for mutagenicity in these tests was found to be an efficient inhibitor of a neutral protease activity found in rheumatic synovial fluid, and may therefore have some potential as an anti-rheumatic drug.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

Complex leaf-gathering skills of mountain gorillas (Gorilla g. beringei): Variability and standardization

The skills that mountain gorillas use to deal with the stings, tiny hooks, and spines protecting common plant leaves in their diet were examined for variation within and between animals. Many elements of uni- and bimanual performance were identified, often involving delicate precision and coordination, and varying idiosyncratically, erch individual having a different set of preferred elements. Many of these elements are functionally equivalent, and all but one weaned animals showed full processing capability; the history of the one exception suggests that early experience with the task may be important. Gorillas' idiosyncrasy in manual skill elements is entirely consistent with trial-and-error learning at this level. By contrast, each individual uses very few techniques (structured sequences of elements) for most processing, and these techniques are the same across the population. Where animals deviate from this generalization, they largely employ the simpler technique normally used for undefended leaves. Lateralization increases from start to finish, consistent with a logical structure in which each stage has a laterality bias and each stage is sequentially dependent on the last. Variations from their commonest, techniques occur in all animals (on average, about nine variant techniques were recorded from each animal). The repertoire of techniques increases significantly with age, whereas the repertoire of elements does not. This points to an initial reliance on a single logical structuring that is well established by weaning (about 3.5 years), with subsequent development of the ability to vary the technique used so as to take advantage of variations in the environment. Standardization of logical organization, despite variability between different animals in individual elements and behavioral laterality, suggests that the logical ordering of elements and the interrelationships of processing stages is copied by program-level imitation. © 1993 Wiley-Liss, Inc.     

Estrogen receptors in the wobbler mouse

Recent research has raised the interesting possibility that the neurological mutant mouse, wobbler (wr/wr), possesses an estrogen receptor deficit analogous to the androgen receptor deficiency found in androgen-resistant mice with testicular feminization. In the present report we examined estrogen-binding activity in cytosolic extracts of kidney, liver, and brain from wobbler mice, littermate control animals, and C57BL/6J mice, using DNA-cellulose chromatography. Estrogen binding components exhibiting properties of estrogen receptors were present in all tissues examined. Estrogen receptors adhered to DNA, displayed characteristic elution profiles from DNA-cellulose, and showed high affinity and limited capacity for estradiol, in contrast to non-receptor entities which bind estradiol. The qualitative elution patterns for estrogen receptors did not differ among groups within each tissue studied, and were similar to those reported previously in mouse kidney and brain. While estrogen receptors have been shown in mouse liver by other techniques, this is the first demonstration of putative estrogen receptors in mouse liver by DNA-cellulose chromatography. No consistent deficits in estrogen receptor concentration were found in wobblers compared to littermates. Thus, the data do not support the hypothesis that the wobbler mouse is an estrogen receptor-deficient mutant.     

Isolated hepatocytes - past, present and future

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca2+-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase.The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.     

Culture of fetal mouse liver in plastic chambers: a new technique for organ culture.

A new technique for organ culture which uses plastic culture chambers and the advantages of the cellophane-sheet technique is described with the results of a study of cultivations of fetal mouse liver. Two chambers, each containing cells, were placed in gas permeable roller tubes and rotated at 0.1 rpm in a CO2-air gassed incubator. The fetal mouse liver cells developed electron microscopic features similar to those of the in vivo adult liver by 9 days of cultivation. The albumin content and tyrosine aminotransferase (TAT) activity were detected in the cultivated liver. TAT activity was further induced by prednisolone. These results indicate that potential of this culture method for the study of physiological and pathological processes.