Use of in vivo and in vitro assays for the characterization of mammalian excision repair and isolation of repair proteins

Elucidation of the molecular mechanism of mammalian nucleotide excision repair requires the availability of purified proteins, DNA substrates with defined lesions and suitable repair assays. Repair assays introduced in recent years vary from testing individual steps and successions of steps in vitro to systems that closely reflect the entire process in vivo. In the first part of this review, an in vivo microinjection system is discussed. The second part of the article reviews an in vitro system for study of repair synthesis promoted by cell extracts. Both systems can be utilized as assays during the purification of protein factors that complement repair-defective xeroderma pigmentosum cells. The effect of purified repair proteins from other organisms on mammalian repair is also considered.     

Habitat use and diet of sympatric Arctic charr (<Emphasis Type="Italic">Salvelinus alpinus</Emphasis>) and whitefish (<Emphasis Type="Italic">Coregonus lavaretus</Emphasis>) in five lakes in southern Norway: not only interspecific population dominance?

Stable coexistence of Arctic charr and whitefish does occur in a number of native lake fish communities in Scandinavia. Even so, whitefish introductions into Arctic charr lakes have resulted in serious decline and possibly local extinction of Arctic charr. In this article, we analyze the habitat use and diet of the two species in five Norwegian lakes differing in basin shape and environmental conditions. In two of the lakes, both species are native, and appear to live in a relatively stable coexistence. Here, whitefish mainly occupy the littoral and upper pelagic zone, while Arctic charr live in the deeper habitats. Diets are generally quite different in terms of the zooplankton species eaten. In the three other lakes, either whitefish or both species have been introduced. In the shallowest lake, habitat segregation is similar to that seen in the pristine lakes, although Arctic charr appears to be on the brink of extinction. In the remaining two lakes, however, Arctic charr dominates, and occurs in higher numbers than whitefish in all the habitats. Our observations indicate that coexistence of the two species in oligotrophic and relatively pristine lakes requires an extensive profundal zone to serve as a refugium for Arctic charr. If the littoral zone is rendered inaccessible or unprofitable for whitefish due to dominance of a third competitor or predator, or as a result of lake regulation, then Arctic charr may be the dominant species.     

ADAM12 is a four-leafed clover: the excised prodomain remains bound to the mature enzyme

The ADAMs (a disintegrin and metalloprotease) comprise a family of multidomain proteins with metalloprotease, cell adhesion, and signaling activities. Human ADAM12, which is implicated in diseases such as cancer, is expressed in two splice forms, the transmembrane ADAM12-L and the shorter and soluble ADAM12-S. ADAM12 is synthesized as a zymogen with the prodomain keeping the metalloprotease inactive through a cysteine-switch mechanism. Maturation and activation of the protease involves the cleavage of the prodomain in the trans-Golgi or possibly at the cell surface by a furin-peptidase. The aim of the present study was to determine the fate of the prodomain following furin cleavage. Here we demonstrate that, following cleavage of the human ADAM12-S prodomain in the trans-Golgi by a furin-peptidase, the prodomain remains non-covalently associated with the mature molecule. Accordingly, both the 68-kDa mature form of ADAM12-S and the 25-kDa prodomain could be detected using domain-specific antisera in immunoprecipitation and Western blot analyses of human serum ADAM12 and purified recombinant human ADAM12. Using electron microscopy after negative staining we have furthermore obtained the first visualization of a full-length ADAM molecule, human ADAM12-S, and report that it appears to be a compact clover composed of four globular domains, one of which is the prodomain. Finally, our data demonstrate that the presence of the metalloprotease domain appears to be sufficient for the prodomain to remain associated with the mature ADAM12-S. Thus, we conclude that the prodomain of human ADAM12-S is an integral domain of the mature molecule and as such might have specific biological functions in the extracellular space.     

Tight Junction Proteins in Human Schwann Cell Autotypic Junctions

Tight junctions (TJs) form physical barriers in various tissues and regulate paracellular transport of ions, water, and molecules. Myelinating Schwann cells form highly organized structures, including compact myelin, nodes of Ranvier, paranodal regions, Schmidt-Lanterman incisures, periaxonal cytoplasmic collars, and mesaxons. Autotypic TJs are formed in non-compacted myelin compartments between adjacent membrane lamellae of the same Schwann cell. Using indirect immunofluorescence and RT-PCR, we analyzed the expression of adherens junction (E-cadherin) and TJ [claudins, zonula occludens (ZO)-1, occludin] components in human peripheral nerve endoneurium, showing clear differences with published rodent profiles. Adult nerve paranodal regions contained E-cadherin, claudin-1, claudin-2, and ZO-1. Schmidt-Lanterman incisures contained E-cadherin, claudin-1, claudin-2, claudin-3, claudin-5, ZO-1, and occludin. Mesaxons contained E-cadherin, claudin-1, claudin-2, claudin-3, ZO-1, and occludin. None of the proteins studied were associated with nodal inter-Schwann cell junctions. Fetal nerve expression of claudin-1, claudin-3, ZO-1, and occludin was predominantly punctate, with a mesaxonal labeling pattern, but paranodal (ZO-1, claudin-3) and Schmidt-Lanterman incisure (claudins-1 and -3) expression profiles typical of compact myelin were visible by gestational week 37. The clear differences observed between human and published rodent nerve profiles emphasize the importance of human studies when translating the results of animal models to human diseases. (J Histochem Cytochem 57:523–529, 2009)     

Chromosome numbers of flowering plants from northern Norway and Svalbard

Chromosome numbers of 25 taxa of flowering plants from northern Norway and Svalbard are reported. Four species and one subspecies, Carex halophila F. Nyl. (2n = ca. 78), C. stenolepis Less. (2n = ca. 80), Salicorniapojarkovae N. Sem. (2n = 36), Alnus incano (L.) Moench ssp. kolaensis (Orlova) Love (2n = 28), and Saxifraga svalbardensis D. O. Øvstedal (2n = ca. 64), have not been studied karyologically before. Chromosome numbers of four additional species are reported for the first time from Norway; those of another six species for the first time from northern Norway. The hybrid Salix herbacea L. (2x) x 5. polaris Wahlenb. (6x) is proved tetraploid and may be fertile. A possible hybrid between Saxifraga hyperborea R. Br. and S. rivularis L. was revealed.     

Vitamin K-dependent carboxylase: Evidence for cofractionation of carboxylase and epoxidase activities,and for carboxylation of a high-molecular-weight microsomal protein

The vitamin K-dependent carboxylase from rat liver microsomes has been fractionated by submitting a crude preparation of this activity to chromatography on different column supports. A constant ratio of vitamin K epoxidation and vitamin K-dependent carboxylation was observed in all column fractions with good carboxylase activity, supporting the hypothesis that these two activities are carried out by the same enzyme complex. The preparation obtained (Complex B) is stable for several days when left on ice and has the same general properties as those observed in Triton X-100-solubilized microsomes. When antiserum raised against Complex B was incubated with Complex B, a twofold increase in carboxylase activity was observed. Benzidine staining showed that an appreciable pool of the antibody population was directed against hemeprotein(s). These data and spectral analyses indicated that a major contaminant of the preparation in cytochrome P-450. Although endogenous prothrombin precursors were absent in the crude starting preparation, a constant ratio of endogenous substrate carboxylation and carboxylation of a soluble substrate was observed during fractionation. A protein with a molecular weight of approximately 120,000 which copurified with Complex B was identified as substrate for the carboxylase.     

A Method for Collection of Bile in the Conscious Sheep

A method to collect bile directly from the hepatic duct is described for use in the sheep. The technique is a combination of the intestinal re-entrant cannulae and a catheter from the duodenal lumen to the hepatic duct. The cystic duct is ligated near its junction with the common bile duct. The catheter is fixed in the proximal visible end of the hepatic duct. One plastic cannula is fixed to the duodenum opposite to the opening of the common bile duct and the other is fixed in the same way about 15 cm posterior to the first one. The two plastic cannulae fixed together with a plastic tube serve as an extra-abdominal anastomosis. During the collection periods the bile duct catheter is passed through an opening in the wall of the connection tube into a collection bag that is fixed to the plastic cannulae. Between the collection periods the catheter ends in the lumen of the anastomosis.     

8-Hydroxy-5-deazaflavin-dependent electron transfer in the extreme halophile Halobacterium cutirubrum

Abstract Cell extracts of the extreme halophile Halobacterium cutirubrum were found to contain 8-hydroxy-5-deazaflavin as well as 8-OH-5-deazaflavin: NADPH oxidoreductase activity. The oxidoreductase was partially purified and showed maximum activity at pH 5.4, which is unusually low for halobacteria, and 5.3 M NaCl, close to the intracellular salt concentration. The results indicate the presence of an 8-OH-5-deazaflavin-dependent electron transfer system in a nonmethanogenic organism.     

Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities

Using immunological assays, we determined the relationship between the heparan sulfate proteoglycans produced by two different murine basement-membrane-producing tumors, i.e., the mouse Engelbreth-Holm-Swarm (EHS) tumor and the L2 rat yolk-sac tumor. Antibodies prepared against the heparan sulfate proteoglycans obtained from these two sources immunoprecipitated the same precursor protein with a molecular mass of 400,000 daltons from 35S-methionine pulse-labeled cells of both tumors. Immunohistochemistry showed the heparan sulfate proteoglycan to be distributed in the extracellular matrix and also in the native basement membrane of surrounding normal murine tissues. Blocking and ELISA assays demonstrated that the antibodies recognized both antigens. Using techniques involving the chemical and enzymatic degradation of 35S-sulfate-labeled glycosaminoglycans, the mouse EHS tumor cells were found to produce mainly heparan sulfate (75%) along with smaller amounts of chondroitin sulfate (19%), whereas the L2 rat yolk-sac tumor produced mainly chondroitin sulfate (76%) with smaller amounts of heparan sulfate (21%). We conclude that these two murine basement-membrane-producing tumors elaborate an immunologically and structurally similar type of high-molecular-weight heparan sulfate proteoglycan which subsequently becomes incorporated into basement-membrane-like material.