Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

The Amount of BCL6 in B Cells Shortly after Antigen Engagement Determines Their Representation in Subsequent Germinal Centers

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Nuttallia balearicae sp. n., an Avian Piroplasm from Crowned Cranes (Balearica spp.)

An avian piroplasm, Nuttallia balearicae sp. n., is described from captive Balearica p. pavonina and B. p. gibbericeps. Other avian piroplasms and their validity and taxonomic status are discussed. The possible route of transmission of these parasites is also considered.     

An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem

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TonB protein and energy transduction between membranes

TonB protein couples cytoplasmic membrane electrochemical potential to active transport of iron-siderophore complexes and vitamin B12 through high-affinity outer membrane receptors of Gram-negative bacteria. The mechanism of energy transduction remains to be determined, but important concepts have already begun to emerge. Consistent with its function, TonB is anchored in the cytoplasmic membrane by its uncleaved amino terminus while largely occupying the periplasm. Both the connection to the cytoplasmic membrane and the amino acid sequences of the anchor are essential for activity. TonB directly associates with a number of envelope proteins, among them the outer membrane receptors and cytoplasmic membrane protein ExbB. ExbB and TonB interact through their respective transmembrane domains. ExbB is proposed to recycle TonB to an active conformation following energy transduction to the outer membrane. TonB most likely associates with the outer membrane receptors through its carboxy terminus, which is required for function. In contrast, the novel prolinerich region of TonB can be deleted without affecting function. A model that incorporates this information, as well as tempered speculation, is presented.     

Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus

The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.