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Biomedical subjects

B Greenberg

Publications and source records attributed to B Greenberg.

At least 199 records · Page 11Linked to original sources
PubMed1974

Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae.

Two steps in the uptake of DNA by Diplococcus pneumoniae were characterized by analyzing mutants defective in transformation. A strain deficient in the two major deoxyribonucleases of D. pneumoniae takes up DNA normally and converts it to single strands within the cell and oligonucleotide fragments outside the cell. Extracts of this strain contain a residual deoxyribonuclease that produces similar oligonucleotide fragments in vitro. This enzyme is missing in transformation-defective mutants blocked in the second or entry step. Cells of this mutant class bind large amounts of DNA to their surface in a form accessible to external agents. Another class of nontransformable mutants fails to bind DNA at all. Their deoxyribonuclease content is unchanged, and they are apparently blocked in the first or binding step of DNA uptake. The binding step requires a source of energy and prior activation of the cells by competence factor. Entry may be independent of these requirements and may come about by action of the deoxyribonuclease on one strand of DNA with energy for the transport of the intact strand deriving from hydrolysis of the degraded strand. The enzyme may thus act as a DNA translocase.

Binding Sites↗
PubMed1973

Competence for deoxyribonucleic acid uptake and deoxyribonuclease action external to cells in the genetic transformation of Diplococcus pneumoniae.

A mutant of Diplococcus pneumoniae that apparently does not require activator can become competent for uptake of deoxyribonucleic acid (DNA) when grown in dilute cultures or in the presence of trypsin. Development of competence in both mutant and wild strains is temperature dependent, being 10-fold greater at 30 C than at 37 C. Induction of competence on a shift from 37 to 30 C requires protein synthesis and the presence of Mg(2+) and Ca(2+); uptake of DNA does not require protein synthesis. Competence decays exponentially at higher temperatures. As well as taking up DNA, competent cells release oligonucleotide fragments of donor DNA in the medium external to the cells. Normal strains release fragments comparable in amount to the DNA taken up; but, in a mutant selected for inability to degrade DNA in agar, the amount of fragments formed external to the cells is only 40% of DNA uptake. Requirements for external deoxyribonuclease action are identical to those for DNA uptake: prior development of competence and the presence during treatment with DNA of Mg(2+) ions and a source of energy.

Calcium↗
PubMed1970

Factors affecting the transmission of salmonella by flies: natural resistance to colonization and bacterial interference.

Groups of immobilized gnotobiotic houseflies, Musca domestica L., were mono-contaminated with graded doses of Salmonella typhimurium, bicontaminated with S. typhimurium and Proteus mirabilis, and multicontaminated with S. typhimurium and a mouse fecal flora. Of 45 flies fed an average of 22 Salmonella cells alone, 26 showed bacterial multiplication, up to 1.4 x 10(7) in one case. There was a fairly consistent increase in percentage of Salmonella excreters with increasing input, so that all flies given 10(4) and 10(5) cells shed Salmonella some time during an observation period of 8 to 13 days. Multiplication, however, reached higher levels in flies given 10(2) or 10(3) organisms. A high input was not necessary to produce a high proportion of Salmonella excreters. Among 22 flies given an average of 90 Salmonella cells, multiplication occurred in 17 and 18 became excreters. Antagonism between P. mirabilis and S. typhimurium, which was previously described in the blowfly maggot tract, also occurred in the gut of the adult housefly. With an input of 10(3) cells each of Salmonella and Proteus, there was a reduction of Salmonella excreters to 27% on the first day and elimination by the second day. With a normal fecal flora which contained no demonstrable P. mirabilis, 37% of the flies were still excreting Salmonella on the fourth day, but not by day 6; among flies fed Salmonella alone at the same input, the organism was excreted by 87% on the first day and 62% on the second day, and outputs continued for at least 8 days. Salmonella was lodged mainly in the mid- and hindgut 3 days after the infective meal, minimizing transmission through vomit. Comparison studies with the common green bottle fly, Phaenicia sericata Mg., showed it to be more resistant than the housefly to establishment and multiplication of S. typhimurium.

Journal Article↗
PubMed1969

Salmonella suppression by known populations of bacteria in flies.

Survivorship of Salmonella typhimurium, Streptococcus faecalis, Proteus mirabilis, and Pseudomonas aeruginosa was studied in dibiotic and tribiotic interactions in vitro and in various regions of the digestive tract of the blow fly, Calliphora vicina. In dibiotic interactions, Salmonella typhimurium dominated Streptococcus faecalis and was dominated by P. mirabilis, but in neither case was it eliminated from the larval gut. In tribiotic interactions, there was synergic suppression and a definite trend toward elimination of Salmonella typhimurium from the gut. This trend approaches but does not match the total exclusion of S. typhimurium from the gut of maggots with a normal flora. Bacterial survival in the gut of the fly is discussed in relation to doubling time, sweep-out rate of the maggot and prepupal gut, and the midgut bactericide.

Animals↗
PubMedDate not supplied

Comparison of batch and perfusion culture in combination with pilot-scale expanded bed purification for the production of soluble recombinant beta-secretase.

beta-Secretase is one of the prime targets for therapeutic intervention of Alzheimer's disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant beta-secretase is a prerequisite. Therefore various culture modes were investigated using HEK-293 cells stably transfected with soluble recombinant beta-secretase. The coupling of the Fc part of human IgG1 to the ectodomain of beta-secretase (residues 1-460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 x 10(6) cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS-PAGE, and the final product contained both the mature and the pro form of beta-secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.

Amino Acid Sequence↗