Analysis of sequential stages in serum bactericidal reactions

Michael, J. Gabriel (House of the Good Samaritan, Children's Hospital Medical Center, Boston, Mass.), and Werner Braun. Analysis of sequential stages in serum bactericidal reaction. J. Bacteriol. 87:1067-1072. 1964.-The bactericidal reaction of "normal" human serum against Escherichia coli was found to be separable into two distinctive stages. The early (first) stage of the reaction lasts for a relatively short period of time, and involves factors that are present in sufficient amounts only in slightly diluted serum. The later (second) stage needs more time and requires factors present in highly diluted serum. The first stage depends on the presence of Ca(++) and Mg(++) and on the activity of all components of complement; the second stage does not require divalent cations and C'1, C'2, and C'4, but requires factors that can be removed by zymosan. Under our conditions, removal of lysozyme did not influence either stage of the reaction. Bacteria exposed to concentrated serum for a short time, during the first stage, are essentially unaffected as far as their potential for subsequent multiplication is concerned; the actual damage to cellular integrity occurs only during the second stage of the reaction. In the absence of cell division, the "sensitization" produced during the first stage can be preserved for prolonged periods, and the bactericidal reaction can be completed later by exposure to antibody-free, highly diluted serum (second stage). Cell multiplication abolishes the sensitizing effects of the first stage.     

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.     

Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli.

The fec region of the Escherichia coli chromosome determines a citrate-dependent iron(III) transport system. The nucleotide sequence of fec revealed five genes, fecABCDE, which are transcribed from fecA to fecE. The fecA gene encodes a previously described outer membrane receptor protein. The fecB gene product is formed as a precursor protein with a signal peptide of 21 amino acids; the mature form, with a molecular weight of 30,815, was previously found in the periplasm. The fecB genes of E. coli B and E. coli K-12 differed in 3 nucleotides, of which 2 gave rise to conservative amino acid exchanges. The fecC and fecD genes were found to encode very hydrophobic polypeptides with molecular weights of 35,367 and 34,148, respectively, both of which are localized in the cytoplasmic membrane. The fecE product was a rather hydrophilic but cytoplasmic membrane-bound protein of Mr 28,189 and contained regions of extensive homology to ATP-binding proteins. The number, structural characteristics, and locations of the FecBCDE proteins were typical for a periplasmic-binding-protein-dependent transport system. It is proposed that after FecA- and TonB-dependent transport of iron(III) dicitrate across the outer membrane, uptake through the cytoplasmic membrane follows the binding-protein-dependent transport mechanism. FecC and FecD exhibited homologies to each other, to the N- and C-terminal halves of FhuB of the iron(III) hydroxamate transport system, and to BtuC of the vitamin B12 transport system. FecB showed some homology to FhuD, suggesting that the latter may function in the same manner as a binding protein in iron(III) hydroxamate transport. The close homology between the proteins of the two iron transport systems and of the vitamin B12 transport system indicates a common evolution for all three systems.     

Tissue distribution of cocaine in the pregnant rat

Cocaine hydrochloride was administered by single intraperitoneal (IP) doses to pregnant rats at day 18 or 19 of gestation. Plasma and tissue cocaine and norcocaine concentrations were measured by high-pressure liquid chromatography. Pharmacokinetic analysis of concentration versus time data showed rapid distribution of cocaine and its metabolite to maternal and fetal tissues. The area under the cocaine concentration versus time curve (AUC) in fetus compared to maternal plasma was 3.33. The half-life of cocaine in the maternal plasma and fetus was 46 and 55 minutes, respectively, similar to values reported for cocaine elimination half-life in human plasma. The order of cocaine concentrations was placenta greater than fetal liver greater than maternal heart greater than whole fetus greater than fetal brain greater than maternal brain = maternal plasma. Norcocaine concentrations were usually less than 20% of cocaine concentrations in plasma and tissues. These results support extensive fetal exposure to cocaine following administration to pregnant rodents. Pharmacodynamic studies of cocaine in pregnancy should consider the effects of the drug on the developing fetus.     

The xylem sap of maple (Acer platanoides) trees--sap obtained by a novel method shows changes with season and height

Xylem sap of log pieces of maple trees was collected by a novelpressure/decompression method developed recently for the mechanicaldrying of timber. Seasonal changes and spatial distributionsof the osmotic potential, the pH and the concentrations of majorsolutes and of the plant stress-hormone abscisic acid (ABA)were investigated. Sucrose and quebrachitol were the main components of the xylemsap. The sucrose concentration varied between 10 mM and 25 mMduring the winter months and declined to a minimum during theperiod of bud burst. Quebrachitol was found in concentrationsof up to 7 mM with a high variability throughout the year. Highconcentrations of ABA were measured during the summer seasonand in mid-winter. Rainfall caused an increase of ABA in somesamples. The osmotic potential of the xylem sap increased with the heightof the collection site. The pH of the sap decreased by approximatelyone unit between the base of the trunk and the crown. The increaseof the osmotic potential was mainly due to sucrose, quebrachitoland potassium. Malate contributed to the decrease of the pH.ABA of the xylem sap increased with decreasing moisture contentof the wood. Key words: Pressure/decompression method, xylem sap, abscisic acid, sucrose, quebrachitol, Acer platanoides     

Sperm viability is influenced in vitro by the bovine seminal protein aSFP: effects on motility, mitochondrial activity and lipid peroxidation

The 13 kDa acidic seminal fluid protein (aSFP) is a major component of bovine semen exerting growth factor-like activity. The influence of the pure protein on sperm viability was observed by evaluating sperm motility using computer-assisted semen analysis. Furthermore, mitochondrial dehydrogenase activity as a parameter of sperm metabolism and the integrity of sperm membranes using a metal catalyzed lipid peroxidation assay were measured. Over a wide physiological range (0.003 to 4 g/l) aSFP did not influence motility and average-path velocity of sperm, but at the highest concentration (6 g/l) a significant reduction in motility could be observed. Mitochondrial activity was significantly stimulated at medium concentrations (0.125 to 2 g/l), whereas a 40% suppression was observed at maximum levels (4 g/l). A dose-dependent inhibition of lipid peroxidation could be demonstrated for medium and high concentrations of aSFP (0.125 to 4 g/l). Compared with other reducing agents, aSFP showed the highest potency in preventing oxidative stress. Such effects might be explained by the remarkable redox behavior of the protein. We suggest that in the bull aSFP may play a role in the regulation of sperm metabolism and the protection of sperm membranes from oxidative damage.     

Molecular evaluation of an Alu repeat including a polymorphic variable poly(dA) (AluVpA) in the vitamin D binding protein (DBP) gene

We investigated an Alu element at the end of intron 8 of the human vitamin D-binding protein (hDBP, group-specific component, GC) gene that shows a polymorphic poly(A) tail due to a variable number of tandem repeats (AluVpA) forming the 3 end of this member of the most abundant class of short interspersed repeated DNA element (SINES). The Alu element sequence in intron 8 of the GC gene was identical in all three common GC alleles (GC*1F, GC*1S, and GC*2) and could be classified as an Alu-Sa or Alu class-II sequence. The polymerase chain reaction was used to amplify selectively a fragment of about 200 bp containing the identified (TAAA)n repeat from genomic DNA of 188 unrelated human subjects. The size of the amplified products was determined by polyacrylamide gel electrophoresis. Four alleles (named GC-18*6, GC-I8*8, GC-I8*10, and GC-18*11) were found that differed in size by multiples of four nucleotides. The allele frequencies ranged from 0.0053 to 0.8511 and the observed heterozygosity was 26%. The stable inheritance of this polymorphic patterned poly(A) sequence was confirmed by a segregation study of a highly informative family with 19 members. Statistically significant linkage disequilibrium between the AluVpA and the GC isoelectric focusing (IEF) phenotypes was found in a sample of 188 unrelated individuals and delta values were calculated from the observed haplotype distribution.