Expression and subcellular localization of a 35-kDa carbonic anhydrase IV in a human pancreatic ductal cell line (Capan-1).

The high intraluminal concentrations of HCO(3)(-) in the human pancreatic ducts have suggested the existence of a membrane protein supplying the Cl(-)/HCO(3)(-) exchanger. Membrane-bound carbonic anhydrase IV (CA IV) is one of the potential candidates for this protein. The difficulties in isolating human pancreatic ducts have led the authors to study the molecular mechanisms of HCO(3)(-) secretion in cancerous cell lines. In this work, we have characterized the CA IV expressed in Capan-1 cells. A 35-kDa CA IV was detected in cell homogenates and purified plasma membranes. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase-C indicated that this CA IV was not anchored by a glycosylphosphatidylinositol (GPI). In contrast, its detection on purified plasma membranes by an antibody specifically directed against the carboxyl terminus of human immature GPI-anchored CA IV indicated that it was anchored by a C-terminal hydrophobic segment. Immunoelectron microscopy and double-labeling immunofluorescence revealed that this CA IV was present on apical plasma membranes, and in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules, suggesting its transport via the classical biosynthesis/secretory pathway. The expression in Capan-1 cells of a 35-kDa CA IV anchored in the apical plasma membrane through a hydrophobic segment, as is the case in the healthy human pancreas, should make the study of its role in pancreatic HCO(3)(-) secretion easier.     

Crystal structure determination and refinement of pike 4.10 parvalbumin (minor component from Esox lucius)

The crystal and molecular structure of the minor component of pike parvalbumins has been determined at 1.93 A resolution by molecular replacement (1 A = 0.1 nm). The crystals are orthorhombic, space group P2(1)2(1)2 with a = 59.62 A, b = 59.83 A and c = 26.35 A. A location of the secondary cation binding site is proposed for this parvalbumin of the beta phylogenetic series.     

A chromatin core particle obtained by selective cleavage of histones by clostripain.

Rat liver chromatin core particles digested with clostripain yield a structurally well-defined nucleoprotein particle with an octameric core made up of fragmented histone species (designated H'2A, H'2B, H'3 and H'4, respectively) after selective loss of a sequence segment located in the N-terminal region of each core histone. Sequential Edman degradation and carboxypeptidase digestion unambiguously establish that histones H2A, H2B, H3 and H4 are selectively cleaved at the carboxyl side of Arg 11, Lys 20, Arg 26 and Arg 19 respectively and that the C-terminal sequences remain unaffected. Despite the loss of the highly basic N-terminal regions, including approximately 17% of the total amino acids, the characteristic structural organization of the nucleosome core particle appears to be fully retained in the proteolyzed core particle, as judged by physicochemical and biochemical evidence. Binding of spermidine to native and proteolyzed core particles shows that DNA accessibility differs markedly in both structures. As expected the proteolyzed particle, which has lost all the in vivo acetylation sites, is not enzymatically acetylated, in contrast to the native particle. However, proteolyzed histones act as substrates of the acetyltransferase in the absence of DNA, as a consequence of the occurrence of potential acetylation sites in the core histones thus rendered accessible. The possible role of the histone N-terminal regions on chromatin structure and function is discussed in the light of the present observations with the new core particle obtained by clostripain proteolysis.     

A STUDY OF GLUCO-CORTICOSTEROID-INDUCED PYKNOSIS IN THE THYMUS AND LYMPH NODE OF THE ADRENALECTOMIZED RAT

Pyknotic nuclei, observed in the thymus of steroid-treated rats, are dense, homogeneous, intensely basophilic and Feulgen positive. Under the electron microscope, the image is that of a complete segregation of the chromatin from the nuclear sap producing a margin or crescent of condensed chromatin. Approximately 30% of all small thymocytes appeared to undergo this type of degeneration within 3–4 hr after administration of the synthetic corticosteroid, dexamethasone. At this time, pyknotic thymocytes were observed in clusters, probably as a result of the activity of dense reticular cells and macrophages. Topographical and experimental data suggest the existence of a select population of steroid-sensitive thymic cells. Furthermore, on the basis of thymidine-³H incorporation studies, it appears that the steroid-sensitive population of thymocytes does not correspond to "aged" cells. In addition, many plasma cells became pyknotic after the same steroid treatment, indicating an unexpected similarity between their nuclei and those of lymphocytes. Finally, steroid failed to induce pyknosis of thymocytes in a variety of in vitro experiments, suggesting that the in vivo effect of steroid is of an indirect nature. The results are discussed in terms of (a) the nature of the nuclear changes characterizing pyknosis, (b) the hypothetical mechanism whereby steroids trigger such changes, and (c) the population of cells susceptible to steroid-induced pyknosis.     

Plasmid-mediated resistance to fosfomycin in Staphylococcus epidermidis

Staphylococcus epidermidis strain BM2641, isolated from a patient, was resistant to penicillin G, methicillin, aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin B-type (MLS) antibiotics, and to high levels of fosmycin. Resistance to forsfomycin and/or to MLS was lost at low frequencies either spontaneously or after curing with novobiocin. The plasmid DNA from BM2641 and its cured derivatives was purified, analyzed by agarose gel electrophoresis and transferred to a nitrocellulose sheet. Comparative analysis of the resistance phenotypes with the plasmid content of the strains indicated that fosfomycin and MLS resistance were encoded by plasmids pIP1842 (2.5 kb) and pIP1843 (2.6 kb), respectively. Southern hybridization with a probe specific for gene fosA of Serratia marcescens showed that the fosfomycin resistance determinant in Staphylococcus is not homologous to that of Gram-negative bacteria.     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

Cellular Quantification of Tyrosine Hydroxylase in the Rat Brain by Immunoautoradiography

Abstract: We developed a rapid and sensitive radioimmunohistochemical method for the quantification of tyrosine hydroxylase (TH) at both the anatomical and cellular level. Coronal tissue sections from fresh-frozen rat brains were incubated in the presence of a TH monoclonal antibody. The reaction was revealed with a ³⁵S-labeled secondary antibody. TH content was quantified in catecholaminergic brain areas by measuring optical density on autoradiographic films or silver grain density on autoradiographic emulsion-coated sections. Regional TH concentrations determined in the locus ceruleus (LC), substantia nigra pars compacta (SNC), and ventral tegmental area (VTA) were significantly increased by 45% after reserpine treatment in the LC but unchanged in the SNC and VTA. Microscopic examination of TH radioimmunolabeling showed a heavy accumulation of silver grains over catecholaminergic cell bodies. In the LC, grain density per cell was heterogeneous and higher in the ventral than in the dorsal part of the structure. After reserpine treatment, TH levels were significantly increased (57%) in the neurons of the LC but not in those of the SNC or VTA. The data support the validity of this radioimmunohistochemical method as a tool for quantifying TH protein at the cellular level and they confirm that TH protein content is differentially regulated in noradrenergic and dopaminergic neurons in response to reserpine.