Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.     

The parasitism of the itch mite Sarcoptes scabiei (Acariformes: Sarcoptidae)]

The life cycle of the itch mite Sarcoptes scabiei (L.), an intracutaneous parasite of man and animals, has been studied. The paper concerns morphological adaptations, embryonal and postembryonal development, life cycle pattern, scabious passage as a reproductive formation, invasive stages, feeding, reproduction and topical relationships with the host, distribution and survival in the environment.     

Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula

The coordinate appearance of the bispecific NAD(P)H-nitrate reductase (NR; EC 1.6.6.2) and nitrite reductase (NiR; EC 1.7.7.1) was investigated in leaves and roots from European white birch seedlings (Betula pendula Roth). Induction by nitrate and light of both enzymes was analyzed by in vitro assays and by measuring NR- and NiR-encoding mRNA pools with homologous cDNAs as probes. When birch seedlings were grown on a medium containing ammonium as the sole nitrogen source, low constitutive expression of NR and NiR was observed in leaves, whereas only NiR was significantly expressed in roots. Upon transfer of the seedlings to a nitrate-containing medium, mRNA pools and activities of NR and NiR dramatically increased in leaves and roots, with a more rapid induction in leaves. Peak accumulations of mRNA pools preceded the maximum activities of NR and NiR, suggesting that the appearance of both activities can be mainly attributed to an increased expression of NR and NiR genes. Expression of NR was strictly light-dependent in leaves and roots and was repressed by ammonium in roots but not in leaves. In contrast with NR, constitutive expression of NiR was not affected by light, and even a slight induction following the addition of nitrate was found in the dark in roots but not in leaves. No effect of ammonium on NiR expression was detectable in both organs. In leaves as well as in roots, NiR was induced more rapidly than NR, which appears to be a safety measure to prevent nitrite accumulation.     

Nanosecond fluorescence of tryptophans in cytochrome P-450scc (CYP11A1): effect of substrate binding.

Fluorescence of eight tryptophan residues in cytochrome P-450scc with bound endogenous cholesterol could be fitted with a two component model: a single exponential and a "top-hat" distribution of lifetimes as the second component. The short-lived component (tau 1 about 700 ps) does not change significantly upon binding of substrate (22R-hydroxycholesterol). The parameters of the long-lived component (central lifetime tau m about 3.4 ns) change upon binding of carbon monoxide and substrate. 22R-hydroxycholesterol binding broadens the distribution of the long-lived component; that is the heterogeneity of the Trp environment is increased when this substrate displaces the endogenous cholesterol.     

A substance released by metamorphosing larvae and young polyps ofHydractinia echinata induces metamorphosis in conspecific larvae

Summary A metamorphosis-inducing factor was isolated from medium conditioned by either metamorphosing larvae or 3-day postmetamorphic primary polyps. The factor has a molecular weight 8 kDa and is heatlabile. It does not induce metamorphosis of isolated posterior fragments and is therefore not identical to the internal signal described by Schwoerer-Böhning et al. (1990). The biological significance of the substance is currently unclear, therefore its inducing activity may be a side effect.     

Identification of a central regulator of stationary-phase gene expression in Escherichia coli

During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30-50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation-induced csi::lacZ fusions using the lambda placMu system. One operon fusion (csi2::lacZ) has been studied in detail. csi2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi2::lacZ did not synthesize at least 16 proteins present in an isogenic csi2+ strain. Cells containing csi2::lacZ or csi2::Tn10 did not produce glycogen, did not develop thermotolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi2::lacZ defines a regulatory gene of central importanc e for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi2 demonstrated that the csi2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose 'rpoS' ('sigma S') as appropriate designations.     

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

Zymosterol is located in the plasma membrane of cultured human fibroblasts

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. 1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. 2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. 3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool.     

Rapid Cellular Regulation of D-Glucose Transport in Cultured Neural Cells

Previous studies have revealed two different kinds of regulation of glucose utilization in cell lines derived from the nervous system (Keller et al., 1981). We found glucose metabolism of C-6 glioma cells to be limited and regulated by membrane transport. In contrast, glucose utilization of C-1300 neuroblastoma (N2A) cells was limited by the known regulatory enzymes of the Embden-Meyerhof pathway. Under the given experimental conditions the "membrane-limited" C-6 glioma cells were characterized by periodically changing glucose transport rates and very low intracellular glucose concentrations, which remained constant in spite of widely differing transport rates. These findings suggest the close functional coupling between transport and phosphorylation required for the regulation of glucose transport by cellular metabolic needs.