Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements.

The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.     

Monoclonal antibody uptake in B-cell lymphomas: Experimental studies in nude mouse xenografts

Accumulation of radiolabelled monoclonal antibodies (mAb) in human B-lymphoma xenografts was found to result in two distinct patterns. The basic elements leading to these patterns were elucidated by autoradiographic and immunohistological analysis applied to the nude mouse xenografts BJAB and OCI.LY1. With BJAB, accumulation occurred exclusively in peripheral cell layers of the lymphoma nodule, while central areas were not accessible irrespective of mAb dose. This feature was the consequence of an inefficient transport across intratumoral vessels together with peripheral mAb supply through a subcapsular pseudosinus. With OCI.LY1, intratumoral vessels showed generalized leakiness. Furthermore, interstitial transport was operative to a fair extent, such that in early images multiple sites of mAb extravasation were obvious, which coalesced during the course of prolonged uptake. The pattern of peripheral mAb uptake resulted in a low overall tumour uptake, while multifocal uptake yielded substantial accumulation values.     

Coated-vesicle formation in vitro: conflicting results using different assays

Cell-free systems provide essential tools for elucidating the molecular mechanisms underlying complex cellular processes such as vesicular transport. The biochemical utility of these model systems is strengthened by assays that allow rapid, quantitative detection of the events being studied. Two model systems have recently been developed to reconstitute coated-vesicle budding, and two different biochemical assays are used to detect this event. Striking differences in the biochemical requirements for 'coated-vesicle budding' are detected by these two assays, suggesting that two distinct events are being measured. These findings have wide implications for the use of cell-free assay systems in cell biology.     

Effects of a protein synthesis inhibitor on the hormonally mediated regression and death of motoneurons in the tobacco hornworm,Manduca sexta

The larval–pupal transformation of Manduca sexta is accompanied by the loss of the abdominal prolegs. The proleg muscles degenerate, the dendritic arbors of proleg motoneurons regress, and a subset of the proleg motoneurons dies. The regression and death of proleg motoneurons are triggered by the prepupal peak of ecdysteroids in the hemolymph. To investigate the possible involvement of protein synthesis in these events, we gave insects repeated injections of the protein synthesis inhibitor, cycloheximide (CHX), during the prepupal peak. Examination of insects 3–5 days following CHX treatment showed that CHX inhibited the death of proleg motoneurons and the production of pupal cuticle in a dose-dependent fashion. When insects were allowed to survive for 10 days after the final CHX injection, motoneuron death and pupal cuticle production sometimes occurred belatedly, apparently in response to the ecdysteroid rise that normally triggers adult development. CHX treatments that inhibited motoneuron death were less effective in inhibiting dendritic regression in the same neurons. In another set of experiments, abdomens were isolated from the ecdysteroid-secreting glands prior to the prepupal peak, and infused with 20-hydroxyecdysone (20-HE). Single injections of CHX delivered just prior to the start of the 20-HE infusion inhibited motoneuron death and pupal cuticle production, but in the range of doses tested, did not prevent dendritic regression. Our findings suggest that protein synthesis is a required step in the steroid-mediated death of proleg motoneurons, and that dendritic regression is less susceptible to inhibition by CHX than is motoneuron death. © 1993 John Wiley & Sons, Inc.     

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits     

Towards novel biocatalysts via protein design: the case of lipases

Abstract: During the past 3 years, the tertiary structures of several lipases have been solved by X-ray analysis. The structures revealed unique features such as hydrophobic 'patches' on the surface, presumably involved in lipid supersubstrate binding, and a lid structure which covers the active site in the absence of substrate. Only very recently the first X-ray structure of a bacterial lipase has been solved, and further structural features different from lipases of eukaryotic origin became apparent. Many lipase genes have been cloned and sequenced recently, and expression systems for the preparation of recombinant enzymes in good yields are available. As an example, the lipase from Rhizopus oryzae has been successfully expressed by us in Escherichia coli , and the resulting inclusion bodies were renatured in high yields. Consequently, the mechanism of action of lipases is now being studied via site-directed mutagenesis, and the rational design of lipases for the selective transformation of substrates is presently addressed in several laboratories.