Manganese (II) electron spin resonance and cadmium-113 nuclear magnetic resonance evidence for the nature of the calcium binding site in alpha-lactalbumins

Bovine and goat alpha-lactalbumins were substituted with 113Cd(II) or Mn(II) at the strong calcium site [Murakami, K., Andree, P.J., & Berliner, L.J. (1982) Biochemistry 21, 5488-5494] and studied by 113 Cd NMR and electron spin resonance. The 113Cd chemical shifts were in the -80 to -85 ppm range vs. Cd(ClO4)2, which was almost identical with that found for several nearly octahedral (oxygen-coordinated) calcium binding proteins such as calmodulin, parvalbumin, and troponin C. The electron spin resonance spectra of bound Mn(II)-alpha-lactalbumin complexes at 9 or 35 GHz were also confirmatory of a highly symmetric (cubic) environment around the Mn(II) with only slight distortions. The near identity of this site in alpha-lactalbumin to those of calcium binding proteins containing an "EF hand domain" was remarkable despite the absence of such a domain sequence in the alpha-lactalbumin structure.     

Metabolism of prostaglandin D2 in the monkey.

[3H7]Prostaglandin D2 was biosynthesized and infused into an unanesthetized monkey. The urinary metabolites were isolated and subsequently identified by gas chromatography-mass spectrometry. Two pathways of prostaglandin D2 metabolism were identified and resulted in metabolites with prostaglandin D (3-hydroxycyclopentanone) and prostaglandin F (cyclopentane-1,3-diol) ring structures. The major prostaglandin D ring metabolite was identified as 9,20-dihydroxy-11,15-dioxo-2,3-dinorprost-5-en-1-oic acid. Nine other prostaglandin D ring metabolites were identified reflecting various combinations of metabolism by beta and omega oxidation, 15 dehydrogenation, and 13-14 reduction. In greater abundance were those prostaglandin D2 metabolites which had the prostaglandin F ring structure. The major prostaglandin D2 metabolite which had the prostaglandin F ring structure was identified as 9,11,15-trihydroxy-2,3-dinorprosta-5,13-dien-1-oic acid (dinor prostaglandin F2 alpha). Nine other metabolites with the prostaglandin F ring structure were identified, including prostaglandin F2 alpha itself. These, for the most part, were the structural counterparts of the metabolites with the prostaglandin D ring. Since many prostaglandin D2 metabolites were found to be identical with the metabolites of prostaglandin F2 alpha, quantitative determinations of prostaglandin F ring metabolites may not be a specific indicator of prostaglandin F2 alpha biosynthesis. Likewise, data involving the measurement of a biological effect of prostaglandin D2 must be re-examined to account for the possible contribution of prostaglandin F2 alpha, a metabolite of prostaglandin D2, to the biological response.     

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.     

Interactions between parallel pathways during biosynthesis of rosmarinic acid in cell suspension cultures of Coleus blumei

Phenylalanine ammonia-lyase from an over-producer strain of Coleus blumei Benth. cell cultures accumulating high levels of rosmarinic acid (RA) has been shown to possess no special feed-back sensitivity to RA or its precursors. No tyrosine-3-hydroxylase activity could be detected in culture extracts and no specific inhibitors of tyrosine incorporation into RA were found. L--aminooxy--phenyl propionic acid, however, was effective in specifically blocking phenylalanine incorporation. This block also led to an accumulation of label from tyrosine in 4-hydroxyphenyllactic acid rather than in 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenyllactic acid. These observations require a re-evaluation of the possible role of DOPA as a major biogenic precursor to RA.Abbreviations AOPP -aminooxy--phenylpropionic acid - DOPA 3,4-dihydroxyphenylalanine - RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Methods For Removing Uranyl Acetate Precipitate From Ultrathin Sections

Precipitate resulting from en bloc staining with uranyl acetate was removed by treating sections with 15% oxalic acid in 50% methanol for 30 minutes at 40 C. Precipitate resulting from poststaining sections with hot uranyl acetate was removed by rinsing sections in 0.25-0.50% aqueous oxalic acid for 10-15 seconds at room temperature. Rinsing sections for longer than 30 seconds removed uranyl precipitate and also destained the sections. These procedures did not damage the embedding medium or cellular detail.     

Partial reduction of aquomethemoglobin on a sephadex G-25 column as detected by magnetic circular dichroism spectroscopy and revised extinction coefficients for aquomethemoglobin

In a typical preparation of aquomethemoglobin, oxyhemoglobin is oxidized with potassium ferricyanide, and the resultant mixture of methemoglobin and potassium ferro- and ferricyanides is separated on a Sephadex G-25 column. We find that about 1% of the heme is reduced on the column and is eluted with the methemoglobin. Magnetic circular dichroism spectra show that the reduced species is oxyhemoglobin. Magnetic circular dichroism is more sensitive than is absorption spectroscopy to small amounts of oxyhemoglobin in such solutions; we can detect its presence at the 0.1% level. A redetermination of the extinction coefficients for methemoglobin gives a value of 0.80 for the absorbance ratio A⁵⁷⁰/A⁶³⁰ at pH 6. This value lies clearly outside the currently accepted range of 0.83 to 0.87.     

Malaria in Birmingham and a London teaching hospital.

During the past five years the incidence of imported malaria increased among patients seen in East Birmingham Hospital and in St Thomas''s Hospital, London. Plasmodium vivax was the predominant species in Birmingham, and was almost always acquired by Asian immigrants visiting the Indian subcontinent. In St Thomas''s P falciparum was most commonly imported, usually by African immigrants visiting Nigeria and Ghana. Two patients (one Irish, one Japanese) died of falciparum malaria after visiting tropical Africa. In both hospitals the immigrant patients had seldom taken prophylactic drugs, and the few who had, ceased to do so on arrival in the UK and sometimes before leaving the malarious country. Apparently immigrants who visit their homeland do not consult their general practitioners before travelling, are given inappropriate advice, or do not take appropriate advice when given. Since the incidence of imported falciparum malaria in the UK is rising, the following points should be considered: the infection may be lethal, particularly in patients lacking immunity; it can mimic other diseases, which may lead to delayed diagnosis; severe disease may be associated with few parasites on a blood film, and even if the result is negative further tests should be performed; clinicians and hospital pharmacists should be aware of the need to keep permanent stocks of parenteral chloroquine and quinine preparations.