Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli.

The gene for xylanase A of alkalophilic Bacillus sp. strain C-125 was cloned in Escherichia coli with pBR322. The plasmid pCX311 contained 2.6- and 2.0-kilobase-pair HindIII fragments. The characteristics of the purified pCX311-encoded xylanase were the same as those of purified xylanase A from alkalophilic Bacillus sp. strain C-125.     

Stoichiometric translocation of adipocyte insulin receptors from the cell-surface to the cell-interior. Studies using a novel method to rapidly remove detergent and concentrate soluble receptors

A rapid one-step method was developed for harvesting and concentrating insulin receptors from solubilized adipocytes, which entails precipitating soluble receptors with polyethylene glycol and resuspending the receptor-containing pellet in a reduced volume of binding buffer. With this procedure 90-100% of receptors were recovered, while 80% of cellular protein was removed, thus resulting in a marked reduction of both ligand and receptor proteases and about a 5-fold purification of the receptor. More importantly, greater than 98% of the Triton X-100 detergent was removed during this procedure so that the reduced receptor affinity observed in solubilized extracts (due to detergent) was restored to normal. Reconstituted receptors exhibited normal binding characteristics similar to those observed for plasma membrane receptors. The general utility of our receptor precipitation-reconstitution method is highlighted by studies on insulin-induced translocation of receptors from the cell-surface to the cell-interior of adipocytes and studies on the assessment of the binding affinity of nascent intracellular receptors. The results of these studies are consistent with the following. 1) Insulin initiates endocytotic uptake of insulin receptors, which then recycle back to the cell-surface. 2) Chloroquine impairs the recycling of internalized receptors while preventing receptor degradation, resulting in the progressive trapping and accumulation of receptors within cells during insulin treatment. 3) Receptor translocation during acute insulin-induced down-regulation is stoichiometric in that receptors lost from the cell-surface can be quantitatively recovered within the cell-interior. 4) In the absence of ligand, these receptors within adipocytes are mainly newly synthesized receptors enroute to the cell-surface, and they possess an affinity similar, if not identical, to mature receptors on the plasma membrane.     

Structural relationships of the three stimulatory factors of RNA polymerase II from Ehrlich ascites tumor cells

The structural relationships of S-II, S-II', and S-I(b) stimulatory proteins of RNA polymerase II purified from Ehrlich ascites tumor cells were investigated. From analysis of the amino acid compositions and tryptic peptide maps of these proteins labeled with radioiodinated Bolton-Hunter reagent, it was concluded that S-I(b) is a part of S-II located at either the amino- or carboxyl-terminal and that only this region mainly contains radioiodinatable amino acid residues when labeled using 125I. On chymotryptic digestion, S-II was cleaved to 21- and 18-kDa fragments in the presence of DNA. The 21-kDa fragment was found to be sufficient for stimulation of RNA polymerase II. It was suggested that S-II' is formed by phosphorylation of S-II in the domain containing the 18-kDa fragment.     

Ontogenesis of gastrin cells in the pyloric antrum and duodenum of the mouse

Summary Ontogenesis of gastrin cells was studied in the pyloroduodenal mucosa of the mouse using anti-human G17 serum, R-1301, and anti-human G34(1–15) serum, R-2703. R-1301-immunostained cells first appeared in the pyloric mucosa of 14-day-old fetuses. Cells stained with both R-1301 and R-2703 appeared immediately after birth, and gradually increased in number to the adult level. Most R-1301-reactive cells were also reactive to R-2703, whereas some cells that reacted with R-1301 exhibited very weak or no reaction with R-2703. The discrepancy between these two immunoreactivities is discussed.In the duodenum, a considerable number of R-1301-reactive cells were present from the perinatal stage and through out adult development. A few R-2703-reactive cells were seen in the duodenum of young mice but not of the adult.     

Molecular cloning and expression of cellulase genes of alkalophilic Bacillus sp. strain N-4 in Escherichia coli.

The genes for cellulases of alkalophilic Bacillus sp. strain N-4 were cloned in Escherichia coli with pBR322. Plasmids pNK1 and pNK2 were isolated from the transformants producing carboxymethyl cellulase, and the carboxymethyl cellulase genes cloned were in 2.0- and 2.8-kilobase-pair HindIII fragments, respectively. On the DNA level, the pNK1 fragment had a different restriction map from that of the pNK2 fragment, but the genomic hybridization experiments showed partial homology among these fragments. A total of 74 and 34% of the enzyme activities were observed in the periplasmic space of E. coli carrying the plasmids pNK1 and pNK2 , respectively. The carboxymethyl cellulase thus produced had broad pH activity curves (pH of 5 to 10.9) and was stable up to 75 degrees C.     

Culture of intramural cardiac ganglia of the newborn guinea-pig

Summary The present study describes the ultrastructure of non-neuronal cells and their interrelationships with intracardiac neurones present in cultures dissociated atria and interatrial septum from newborn guinea-pig. When compared with the in situ preparation, most of these features in culture were similar to those observed in situ, but some differences were also apparent. Both mature and immature Schwann cells were observed in culture, and as in situ, the latter were closely associated with intracardiac neurones, whilst the former were more widely separated. The ultrastructure of satellite cells was more variable in culture than in situ: three general types were distinguished on the basis of their 10-nm filament content. This variation could be due to conditions of culture. Interstitial cells were present in culture and closely resembled those described in situ, although there was less space between cultured interstitial cells and their associated cells. Many fibroblasts, some myoblasts and a few mast cells were also found in the culture preparations.     

Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane.

Two plasmids containing the penicillinase gene of alkalophilic Bacillus sp. strain 170, pEAP1 and pEAP2, were constructed. Most of the penicillinase produced by Escherichia coli, which carried these plasmids, was found in the culture medium. This excretion is caused by the cloned DNA fragment which contains some component that changes the outer membrane of E. coli.