Fine needle aspiration cytology of renal infarcts. Cytomorphologic findings and potential diagnostic pitfalls in two cases

The fine needle aspiration (FNA) cytologic features of two cases of renal infarction are presented. Both patients did not have a classic clinical history for infarction, and the radiologic findings were suggestive of neoplasia. In one of our cases, necrotic glomeruli and tubules were present, which suggested the correct diagnosis of renal infarction and permitted appropriate early treatment. In the other case, groups of atypical renal tubular cells undergoing repair secondary to the infarction was misinterpreted as renal cell carcinoma. This case illustrates that renal infarction, like pulmonary infarction, can be a potential pitfall for a false-positive cytologic diagnosis of malignancy in FNA biopsy. A conservative approach is warranted when there is scanty cellularity and atypical cells having features of a repairlike reaction are present.     

Distribution of flourescently labeled α-actinin in living and fixed fibroblasts

The distribution of flourescently labeled α-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-α-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of α-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected α-actinin. Endogenous tropomyosin in the injected cells was found to be distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected α-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected α-actinin was incorporated into the foci of the networks. Thus, the microinjected fluorescent derivative of α-actinin appears to be incorporated into the functional pools of α-actinin within the living cell and to be utilized by the cell with fidelity.     

Plasma membrane ectoglycosyltransferase activity of L1210 murine leukemic cells

L1210 murine leukemia cells after treatment with Cl. perfringens neuraminidase at pH 7.0 incorporated six times more N-acetylneuraminic acid-[C¹⁴] than control cells when incubated for 30 minutes with cytidine 5′-monophosphate N-acetylneuraminic-[C¹⁴] acid and three times more galactose-[C¹⁴] when incubated with uridine diphosphate galactose-[C¹⁴]. These sugars were incorporated in a 10% trichloracetic acid insoluble fraction and more than 75% of the incorporated N-acetylneuraminic acid- [C¹⁴] could be removed by further treatment of these cells with neuraminidase. The incorporation of N-acetylneuraminic acid- [C¹⁴] as a function of time was divided into two rates: a rapid one, active during the first 30 minutes followed by a slower one, similar to the rate observed with untreated cells. The addition of Ba⁺⁺ and Ca⁺⁺ ions at 8.3 mM increased the incorporation of N-acetylneuraminic acid- [C¹⁴] by 25% while 8.3 mM EDTA decreased activity by 58% . The addition of Zn⁺⁺ or Hg⁺⁺ at similar concentrations abolished the incorporation almost completely. The optimal pH for the incorporation of N-acetylneuraminic acid- [C¹⁴] by these neuraminidase treated cells was 6.5. These data suggest that ectoglycosyltransferases are present on the outer surface of the plasma membrane of L1210 cells and are able to catalyze the addition of radiolabeled nucleotide sugars onto macromolecular acceptors (cell surface glycoproteins and glycolipids) prepared by prior incubation of the cells with neuraminidase. Use of these procedures for labeling outer cell surfaces may also prove to be valuable for the study of plasma membrane glycoprotein and glycolipid structure, synthesis, and turnover.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Inhibition of human HT-29 colon carcinoma cell adhesion by a 4-fluoro-glucosamine analogue

Cell surface glycoconjugates play an important role in cellular recognition and adhesion. Modification of these structures in tumour cells could affect tumour cell growth and behaviour, including metastasis. 2-Acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro--D-glycopyranose (4-F-GlcNAc) was synthesized as a potential inhibitor and/or modifier of tumour cell glycoconjugates. The effect of this sugar analogue on the adhesive properties of human colon carcinoma HT-29 cells was evaluated. Treatment of HT-29 cells with 4-F-GlcNAc led to reduced cell surface expression of terminal lactosamine, sialyl-Le^x and sialyl-Le^a, as determined by Western blotting and flow cytometry. The aberrant expression of these oligosaccharide structures on the HT-29 cell surface resulted in: (1) decreased E-selectin mediated adhesion of human colon cells to human umbilical cord endothelial cells (HUVEC); (2) impaired adhesion of HT-29 cells to -galactoside binding lectin, galectin-1; and (3) reduced ability to form homotypic aggregates. After exposure to 4-F-GlcNAc, lysosomal associated membrane proteins (lamp) 1 and 2, and carcinoembryonic antigen (CEA) detected in HT-29 cells were of lower molecular weight, probably due to impaired glycosylation. These results strongly suggest that modification of tumour cell surface molecules can alter tumour cell adhesion and that tumour cell surface oligosaccharides may be suitable targets for therapeutic exploitation.Abbreviations 4-F-GlcNAc 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro--glucopyranose - GlcNAc N-acetylglucosamine - s-Le^x sialyl-Lewis^x - s-Le^a sialyl-Lewis^a - lamp-1 and lamp-2 Lysosomal Associated Membrane Protein 1 and 2 - CEA carcinoembryonic antigen - DMEM Dulbecco's Modified Eagle Medium - PBS Phosphate Buffered Saline (2.7 mM KCl, 1.5 mM KH₂PO₄, 137 mM NaCl, 6.5 mM Na₂HPO₄, pH 7.3) - BSA Bovine Serum Albumin - PMSF Phenylmethylsulfonylfluoride - TBS Tris Buffered Saline (10 mM Tris, 20 mM NaCl, pH 7.3) - TCA Trichloroacetic Acid - DSA Datura stramonium agglutinin     

Role of galaptin in ovarian carcinoma adhesion to extracellular matrix in vitro

Immunohistochemical studies indicated that galaptin is a major protein of ovarian carcinoma cells present in patient effusions and it is distributed throughout the cytoplasm. Enzyme-linked immunoadsorbent assay (ELISA) and immunoprecipitation experiments demonstrated that galaptin is also a major protein of the A121 ovarian carcinoma cell line, constituting less than or equal to 1% of extractable protein bound by DEAE Sephacel. Western blot analyses revealed that the galaptin present in ovarian carcinoma consists of a 14.5 KD subunit. Ovarian carcinoma and mesothelial cells isolated from patient effusions display surface receptors for galaptin with an apparently greater density of receptors present on the carcinoma cells. A121 cells also display surface receptors for galaptin: binding sites/cell = 3 X 10(8) and Ka = 1.2 X 10(9) M-1. The presence of galaptin in bovine corneal endothelial cells (BCEC) and BCEC-derived extracellular matrix (ECM) was demonstrated by ELISA. Of the total ECM-bound galaptin, about 75% appears to be insoluble in phosphate-buffered saline (PBS) lactose. ECM was also found to contain abudnant receptors for galaptin. Treatment of ECM with lactose increased the apparent galaptin receptor density:binding sites/cm2 = 7 X 10(13) and Ka = 2.6 X 10(9) M-1. Pretreatment of A121 cells with galaptin inhibited adhesion to ECM. The addition of exogenous galaptin to ECM had variable effect on cell adhesion. The data presented here suggest that early adhesion events may be carbohydrate-specific involving interaction between ECM-bound galaptin and cell surface galaptin receptors.     

Synthesis and Biological Effects of 5′-Deoxy-5′-(Cyclo-Propylmethylthio)Adenosine

Abstract

A novel synthesis of the nucleoside analog, 5′-deoxy-5′-(cyclopropylmethylthio)adenosine (CPMTA, 1) has been developed. CPMTA is a closely related structural analog of 5′-deoxy-5′-(isobutylthio)-adenosine (SIBA, 2), which has been widely studied and shown to exert a multitude of biological effects. The in vitro and in vivo antitumor (L1210 leukemia) activity of CPMTA has been found to be comparable to that of SIBA, whereas its in vitro antiviral (HSV and VSV) activity is diminished. These agents are being developed as inhibitors of methylation and/or polyamine synthesis.     

Length-dependent aggregation of uninterrupted polyalanine peptides

Polyalanine (polyA) is the third-most prevalent homopeptide repeat in eukaryotes, behind polyglutamine and polyasparagine. Abnormal expansion of the polyA repeat is linked to at least nine human diseases, and the disease mechanism likely involves enhanced length-dependent aggregation. Because of the simplicity of its side chain, polyA has been a favorite target of computational studies, and because of their tendency to fold into α-helix, peptides containing polyA-rich domains have been a popular experimental subject. However, experimental studies on uninterrupted polyA are very limited. We synthesized polyA peptides containing uninterrupted sequences of 7 to 25 alanines (A7 to A25) and characterized their length-dependent conformation and aggregation properties. The peptides were primarily disordered, with a modest component of α-helix that increased with increasing length. From measurements of mean distance spanned by the polyA segment, we concluded that physiological buffers are neutral solvents for shorter polyA peptides and poor solvents for longer peptides. At moderate concentration and near-physiological temperature, polyA assembled into soluble oligomers, with a sharp transition in oligomer physical properties between A19 and A25. With A19, oligomers were large, contained only a small fraction of the total peptide mass, and slowly grew into loose clusters, while A25 rapidly and completely assembled into small stable oligomers of ~7 nm radius. At high temperatures, A19 assembled into fibrils, but A25 precipitated as dense, micrometer-sized particles. A comparison of these results to those obtained with polyglutamine peptides of similar design sheds light on the role of the side chain in regulating conformation and aggregation.