Specific tumor markers in diagnostic cytology. Immunoperoxidase studies of carcinoembryonic antigen, lysozyme and other tissue antigens in effusions, washes and aspirates

The immunoperoxidase technique was used to identify specific tumor markers in exfoliated cells in fine needle aspirates and body fluids. Carcinoembryonic antigen (CEA) and lysozyme staining was evaluated in cytocentrifuge preparations from 42 malignant effusions and aspirates and 16 benign effusions. Reactive mesothelial cells were negative for CEA and lysozyme or showed faint peripheral cytoplasmic staining. Malignant cells from 50% of the adenocarcinomas studied were positive for CEA. All tumors studied were negative for lysozyme. These staining patterns are helpful in the differential diagnosis of reactive mesothelial and adenocarcinoma cells, a frequent diagnostic dilemma. Moreover, demonstration of specific tumor antigens (e.g., prostatic acid phosphatase, calcitonin and immunoglobulin) helped define the origin of metastatic malignancy in selected cases. Estrogen receptor activity was also identified in tumor cells using this technique. Immunoperoxidase was helpful in the evaluation of malignant cytologic specimens from patients with more than one tumor. Interpretation of staining patterns is discussed, with reference to the limitations of the technique. Immunoperoxidase methods maintain cytologic detail, are readily adaptable to diagnostic cytology and increase the specificity of cytologic diagnosis.     

The Loss of In Vivo Activity of Recombinant Human Erythropoietin by Active Oxygen Species

The effects of active oxygen species on the in vivo activity of recombinant human erythropoietin (EPO) treated by Fenton system, xanthine (X) plus xanthine oxidase (XO) system and hydrogen peroxide (H₂O₂) has been studied by means of counting the increase in number of hemolyser-resistant cells (HRCs) in EPO-injected mice. The results showed that both Fenton and X plus XO systems caused a significant reduction of the activity in proportion to the concentration of generated active oxygen species. Meanwhile, the treatment of EPO with H₂O₂ alone resulted in a relatively slight reduction of the activity. Electrophoretic studies on the structure of EPO revealed that its main protein band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) disappeared in proportion with the extent of exposure to active oxygen generating systems. Both Fenton and X plus XO systems caused a significant loss of fluorescence in the pyridylamino (PA-) sugar chain in proportion to the concentration of generated active oxygen species, and no degradation products in the sugar chain part of the PA-sugar chain were detected. This showed that aromatic groups in EPO were sensitive to attack by active oxygen species. These results provide evidence that hydroxyl radical and other active oxygen species have a potential to react with EPO, leading to a reduction of its in vivo activity.     

Production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene

Soybean (Glycine max. Merrill. cv. Fayette) cotyledonary nodes were transformed with bean pod mottle virus (BPMV) coat protein precursor (CP-P) gene via Agrobacterium-mediated transformation. The transformation rate was low, and only five primary transformants derived from five different cotyledons were obtained from 400 original cotyledons. Southern blot hybridization verified the integration of the BPMV CP-P gene. Inheritance and expression of this gene in R₁ plants were also demonstrated. About 30% of R₂ plants derived from one transgenic line showed complete resistance to BPMV infection, as assessed by symptomatology and ELISA, suggesting that homozygous, but not hemizygous, plants exhibit the resistant phenotype.Abbreviations BAP 6-benzyladenine phosphate - BPMV bean pod mottle virus - CP-P coat protein-precursor - CTAB hexadecyltrimethylammonium bromide - DAS-ELISA double antibody sandwich-enzyme-linked immunosorbent assay - IBA indole-butyric acid - kbp kilobase pairs - MES 2-(N-Morpholino)ethanesulfonic acid - NOS nopaline synthase - NPTII neomycin phosphotransferase II - NTP nucleoside triphosphate - PBS phosphate-buffered saline - PCR polymerase chain reaction - PVP polyvinyl pyrrolidone - VPg viral genome-linked protein     

Separation of the components of pectolytic complex produced byTubercularia vulgaris in solid state culture

Summary Tubercularia vulgaris produces extracellular pectolytic activity in solid state culture using citrus pulp pellets as a carbon source. Five peaks containing hydrolase activities were separated by ion exchange chromatography (DEAE-Sephacel). Two enzymatic forms, PG II and PG III, were purified about 8 and 10 fold, respectively. The molecular weight of PG II was similar to that of PG III (24.000 Da). The two enzymes had similar endo activities on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing groups release.     

Nature of the intrinsic protein kinases involved in phosphorylation of non-histone proteins in intact prostatic nuclei: further identification of androgen-sensitive protein kinase reactions

Nuclei isolated from rat ventral prostate contain a number of messenger-dependent and -independent protein kinases. Studies were undertaken to determine the relative contribution of these protein kinases in phosphorylation of non-histone proteins (NHPs) in isolated nuclei. The data suggest that messenger-dependent protein kinases such as those dependent on cAMP or Ca²⁺/calmodulin or Ca^2–/phospholipid may be present in very small amounts in intact isolated nuclei, and thus appear not to be significantly involved in phosphorylation of endogenous NHPs. Messenger-independent nuclear associated protein kinases PK-N1 and PK-N2 are known to catalyze the phosphorylation of NHPs in vitro (Goueli SA, et al., Eur J Biochem 113: 45–51, 1980). Of these, the intrinsic heparin-sensitive PK-N2 as compared with heparin-insensitive PK-N1 appeared to be the predominant protein kinase engaged in phosphorylation of NHPs in intact nuclei. About 78–88% of NHP phosphorylation in intact nuclei was inhibited by heparin suggesting that the remaining 12–22% phosphorylation of NHPs was catalyzed via the heparin-insensitive protein kinase(s). Further, the data provide additional evidence that heparin-sensitive PK-N2 is the one that is most responsive to androgenic status in the animal.Abbreviations NHP Non-Histone Protein - PMSF Phenylmethylsulfonyl Fluoride - DTT Dithiothreitol - SDS Sodium Dodecyl Sulfate     

Monitoring of naturally acquired and artificially induced immunity toAmblyomma variegatum andRhipicephalus appendiculatus ticks under field and laboratory conditions

The ability of rabbits, goats and cattle to acquire immunity to the ixodid ticksAmblyomma variegatum andRhipicephalus appendiculatus was studied under laboratory and field conditions. Rabbits were successfully immunized with crude salivary gland extract (SGE) and midgut extract (ME) obtained from flat or partly fed femaleR. appendiculatus ticks. The lowest numbers of larvae were produced by females fed on rabbits immunized with unfed midgut extract. Similar reductions in larval production could be induced after three infestations of rabbits with adultR. appendiculatus. Also, successive feedings of nymphs ofR. appendiculatus on rabbits resulted in significantly reduced engorgement weights. Skin testing with SGE induced delayed-type hypersensitivity reactions, which could be correlated with immunity toR. appendiculatus in rabbits. Moreover, circulating antibodies were detected in rabbits with an ELISA using SGE ofR. appendiculatus.Immunity toA. variegatum nymphs could be induced in rabbits by repeated infestations, but this failed in goats. Immunization of goats with midgut extract from adultA. variegatum did not protect against subsequent nymphal challenge, but strong skin reactions were noticed when adults ticks fed on immunized goats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of SGE and ME fromA. variegatum revealed the presence of 48 protein bands in SGE and 29 bands in midgut extract. Western blotting employing serum from a rabbit immune toR. appendiculatus recognized a number of bands in SGE fromR. appendiculatus, but also in SGE ofA. variegatum.Immunity acquired by cattle to ixodid tick infestations under field conditions was monitored by skin testing with SGE and western blot analysis. In general, cattle with the lowest tick numbers manifested the strongest delayed-type hypersensitivity responses. Finally, western blot analysis employing sera from tick-infested and tick-naive cattle could not be related to actual immune status.     

Interaction of Doxorubicin with phospholipid monolayer and liposomes

The effect of Doxorubicin which is (an anthracycline antibiotic with a broad spectrum of antitumor activity) on the monolayer and bilayer in the form of large Multilamellar Vesicles (MLV's) of Dipalmitoyl phosphatidylcholine (DPPC) were studied by means of monolayer techniques (surface pressure, penetration kinetics, and association constant) and light scattering technique. The monolayer technique showed that addition of DXR to a lipid film composed of (DPPC/CHOL/PEG-PE) at a molar ratio of (100:0:0) produced a less condensed Monolayer. In the (π-A) curves, DXR induced shift towards larger area/molecule, where the area/molecule was shifted from 61 to 89 A², and 116 A² in the presence of 20 and 40 nM DXR, respectively. The three curves collapsed at a pressure π = 45 mN/m. In penetration kinetics experiment (Δπ-t), the change in pressure with time was 8 and 14 mN/m for a DXR concentration of 20 and 40 nM, respectively, and the increase in surface pressure presented a plateau over a period of 30 min. The measured association constant (K) was found to be 5 × 10⁵/M. In the light scattering experiment, there was a shift of the transition temperature (T_m) of (MLV's) of the same composition of the monolayer towards a smaller value from 40.5° to 34.5°C. Incorporation of CHOL and PEG-PE as DPPC/CHOL/PEG-PE at a molar ratio of (100:20:0), (100:0:4) and (100:20:4) greatly counteracted the effect of DXR and made the lipid membrane more condense and rigid. Moreover, the penetration of DXR into the membrane was greatly reduced. There was a very small shift for the (π-A) and (Δπ-t) curves, and the association constant of the drug for these different lipid compositions was greatly reduced down to 2.5 × 10⁵/M and the transition temperature (T_m) was increased up to (42.5°C) in the presence of 40 nM DXR. Our results suggest that DXR has a great effect on the phospholipid membrane, and that addition of CHOL or PEG-PE to the phospholipid membrane causes stabilization for the membrane, and reduces the interaction with Doxorubicin.     

DEVELOPMENTAL INSTABILITY IN WILD CHROMOSOMAL HYBRIDS OF THE HOUSE MOUSE

In wild populations of the house mouse from Tunisia, fluctuating asymmetry and character size of tooth traits were compared between chromosomal races (2n = 40, all acrocentric standard karyotype, and 2n = 22, with nine fixed Robertsonian fusions) and their natural hybrids. Developmental stability was impaired in hybrids compared to both parental groups. Because genetic divergence measured by allozyme markers was low, genomic incompatibilities were not expected between the chromosomal races. This suggests that differentiation of gene systems specifically involved in development may have occurred between the chromosomal races. Support for the latter was found in the study of character size which showed that the 2n = 22 mice had smaller teeth than either the hybrid or the standard mice. The study of Tunisian chromosomal races thus shows that chromosomal evolution may lead to important changes in coadapted gene systems without involving extensive genic differentiation.     

Detection of oligodendrocytes in tissue sections using PCR synthesis of digoxigenin-labeled probes.

Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.     

Structure of the exceptionally large nonrepetitive carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-82164.

The structures of the oligosaccharides obtained after acetic acid hydrolysis and alkaline deacylation of the rough-type lipopolysaccharide (LPS) from Pectinatus frisingensis strain VTT E-82164 were analysed using NMR spectroscopy, MS and chemical methods. The LPS contains two major structural variants, differing by a decasaccharide fragment, and some minor variants lacking the terminal glucose residue. The largest structure of the carbohydrate backbone of the LPS that could be deduced from experimental results consists of 25 monosaccharides (including the previously found Ara4NP residue in lipid A) arranged in a well-defined nonrepetitive structure: We presume that the shorter variant with R1 = H represents the core-lipid A part of the LPS, and the additional fragment is present instead of the O-specific polysaccharide. Structures of this type have not been previously described. Analysis of the deacylation products obtained from the LPS of the smooth strain, VTT E-79100T, showed that it contains a very similar core but with one different glycosidic linkage.