Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.     

Isolation and immunological characterization of the four non-identical subunits of the soluble NAD-linked hydrogenase from Alcaligenes eutrophus H16

The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000. Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS). Against each of the 4 subunits, polyclonal antibodies were produced. From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography. By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides. Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits. The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical. A. eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000. Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a native subunit of the NAD-linked hydrogenase isolated from a mutant of Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits. From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity. The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits. In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000. The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels. It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels. Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c. The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule. As shown by ESR studies, the iron is organized in a [4Fe-4S] cluster but is partially present also in the 3Fe-form. No nickel signal could be detected. The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.     

Three-dimensional structure of a regular surface layer from Pseudomonas acidovorans

The regular surface layer of Pseudomonas acidovorans was investigated by computer processing of a series of tilted view electron micrographs, and a reconstruction of the three-dimensional structure was obtained. The pattern is tetragonal and consists of massive identical subunits, block-like in face-view, which interlock loosely in a simple cobblestone pattern. The square unit cell has a lattice constant of 11 nm. The surface layer pattern of P. acidovorans appears to be more dependent on the underlying membrane for maintaining its integrity than those so far studied in other bacteria.     

A neural cocktail-party processor

Sensory segmentation is an outstanding unsolved problem of theoretical, practical and technical importance. The basic idea of a solution is described in the form of a model. The response of neurons within the sensory field is temporally unstable. Segmentation is expressed by synchronization within segments and desynchronization between segments. Correlations are generated by an autonomous pattern formation process. Neuronal coupling is the result both of peripheral evidence (similarity of local quality) and of central evidence (common membership in a stored pattern). The model is consistent with known anatomy and physiology. However, a new physiological function, synaptic modulation, has to be postulated. The present paper restricts explicit treatment to the peripheral evidence represented by amplitude modulations globally present in all components of a sound spectrum. Generalization to arbitrary sensory qualities will be the subject of a later paper. The model is an application and illustration of the Correlation Theory of brain function.This work has been supported by Grant I/37-821 of the Stiftung Volkswagenwerk.     

Antigenic reactivity: Molecular delineation of monohaptenic anaphylactogens

Anaphylactogenic, monohaptenic conjugates carrying a 2,4-dinitro-5-carboxyphenyl substituent as haptenic group and either one of two types of auxiliary groups essential for their anaphylactogenicity were studied. The “hydrocarbon auxiliary groups” require the presence of a hydrocarbon structure such as aliphatic chains of discrete length or planar rings such as provided by phenyl- or nicotinoyl residues and become particularly effective in conjunction with a carboxylate group. The benzylpenicilloyl group is an effective auxiliary structure, but the thiazolidine ring as such is not. It appears that the distance between haptenic and hydrocarbon auxiliary groups can be quite large. The “carbohydrate auxiliary group” becomes effective via a different mechanism. It requires disaccharide residues or two closely connected monosaccharides. Single monosaccharides are ineffective. A concept of interest with regard to drug allergy is the possibility that attachment of a single haptenic molecule to a glycoprotein constitutes an anaphylactogen.     

Immunosuppression as a high-risk factor in the development of condyloma acuminatum and squamous neoplasia of the cervix

The iatrogenic immunosuppression in renal transplant recipients has been associated with an increased incidence of malignancy in these patients. Among 132 female transplant recipients at risk for the development of squamous lesions of the cervix, 11 (8.5%) developed cervical condylomas. Six (4.5%) of the 11 patients developed cervical neoplasia. The average age of the patients at the time of initial diagnosis was 32.2 years. The lag time from transplantation to the diagnosis of the condyloma was 22.4 months, and the lag time from transplantation to the diagnosis of cervical neoplasia was 38.0 months. The increased incidence of condylomas as well as of intraepithelial neoplasia of the cervix in this group of patients with an established higher risk of malignancy supports the hypothesis that condylomas may represent a precursor lesion of cervical cancer. Immunosuppression should be included among the high-risk factors in the development of cervical neoplasia.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.     

6-Hydroxytoxol esters and a seco-dammarane derivative from Abrotanella forsterioides

Abrotanella forsterioides afrorded euparin, 6-hydroxytremetone and three 6-hydroxytoxol esters, one of them not being isolated previously. Furthermore a seco-triterpene was isolated. The tribal position of the genus Abrotanella in the Compositae is still an unsolved problem. Morphological investigations suggest that this genus should be transferred from the tribe Anthemideae to the Senecioneae [1,2]. So far two species have been studied chemically; one atforded ent-kaurane derivatives, while both contained euparin and hydroxytremetone [3].