K R Williams

A glial endogenous cannabinoid system is upregulated in the brains of macaques with simian immunodeficiency virus-induced encephalitis.

Recent evidence supports the notion that the endocannabinoid system may play a crucial role in neuroinflammation. We explored the changes that some elements of this system exhibit in a macaque model of encephalitis induced by simian immunodeficiency virus. Our results show that profound alterations in the distribution of specific components of the endocannabinoid system occur as a consequence of the viral infection of the brain. Specifically, expression of cannabinoid receptors of the CB2 subtype was induced in the brains of infected animals, mainly in perivascular macrophages, microglial nodules, and T-lymphocytes, most likely of the CD8 subtype. In addition, the endogenous cannabinoid-degrading enzyme fatty acid amide hydrolase was overexpressed in perivascular astrocytes as well as in astrocytic processes reaching cellular infiltrates. Finally, the pattern of CB1 receptor expression was not modified in the brains of infected animals compared with that in control animals. These results resemble previous data obtained in Alzheimers disease human tissue samples and suggest that the endocannabinoid system may participate in the development of human immunodeficiency virus-induced encephalitis, because activation of CB2 receptors expressed by immune cells is likely to reduce their antiviral response and thus could favor the CNS entry of infected monocytes.

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single‐stranded DNA binding protein (ssDBP) UP I were shown to be cross‐reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti‐ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re‐evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic peptide mapping and amino acid analysis in the sub-nanomole range

By ensuring adequate gradient mixing and eliminating all major artifact peaks we have been able to obtain reproducible high-performance liquid chromatographic tryptic peptide maps on less than 50 pmol of protein. Likewise, a 10-20 fold improvement in the sensitivity of amino acid analysis has been achieved by analyzing the phenylthiocarbamyl derivatives of the free amino acids rather than the free amino acids themselves. This approach enables accurate amino acid compositions to be obtained on less than 50 ng of protein, providing that a simple correction is made for the background level of serine and glycine. We have been able to reduce the background level of these two amino acids to ca. 10 pmol each per sample by incinerating the hydrolysis tubes at 500 degrees C prior to introduction of the sample and by using gas-phase as opposed to liquid-phase hydrolysis. Background corrections are not necessary when over 500 ng of protein are hydrolyzed. With this amount of protein, amino acid compositions, based on phenylthiocarbamyl amino acid analyses were, on average, found to be accurate to within +/- 10%.

A molecular characterization of the choroid plexus and stress-induced gene regulation

The role of the choroid plexus (CP) in brain homeostasis is being increasingly recognized and recent studies suggest that the CP has a more important role in physiological and pathological brain functions than currently appreciated. To obtain additional insight on the CP function, we performed a proteomics and transcriptomics characterization employing a combination of high resolution tandem mass spectrometry and gene expression analyses in normal rodent brain. Using multiple protein fractionation approaches, we identified 1400 CP proteins in adult CP. Microarray-based comparison of CP gene expression with the kidney, cortex and hippocampus showed significant overlap between the CP and the kidney. CP gene profiles were validated by in situ hybridization analysis of several target genes including klotho, CLIC 6, OATP 14 and Ezrin. Immunohistochemical analyses were performed for CP and enpendyma detection of several target proteins including cytokeratin, Rab7, klotho, tissue inhibitor of metalloprotease 1 (TIMP1), MMP9 and glial fibrillary acidic protein (GFAP). The molecular functions associated with various proteins of the CP proteome indicate that it is a blood–cerebrospinal fluid (CSF) barrier that exhibits high levels of metabolic activity. We also analyzed the gene expression changes induced by stress, an exacerbating factor for many illnesses, particularly mood disorders. Chronic stress altered the expression of several genes, downregulating 5HT2C, glucocorticoid receptor and the cilia genes IFT88 and smoothened while upregulating 5HT2A, BDNF, TNFα and IL-1b. The data presented here attach additional significance to the emerging importance of CP function in brain health and CNS disease states.

Zinc metalloproteins involved in replication and transcription

RNA polymerase (RPase) from E. coli contains two tightly incorporated Zn(II) ions, while the monomeric RPase from bacteriophage T7 does not contain zinc and does not require Zn(II) in the assay. One of the two Zn(II) ions can be differentially removed from E. coli RPase with p-hydroxymercuriphenylsulfonate (PMPS) combined with EDTA and thiol. The resultant Znl or ZnA RPase shows no alteration in transcription initiation and elongation rate from sigma-specific promoters. Biosynthesis of a Co2 RPase and formation of CoA RPase by similar treatment shows the tetrahedral-type Co(II) d-d absorption bands to be associated only with the Co(II) at the A site with maxima at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. Sulfur to Co(II) charge transfer bands are present at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. The absorption characteristics strongly suggest that the A site is a tetrathiolate site. While DNA polymerases do not in general appear to contain zinc, gene 32 protein (g32P) from bacteriophage T4, an accessory protein essential for DNA replication and recombination and translational control in the T4 life cycle, is a Zn(II) metalloprotein and contains 1 gram atom of tightly incorporated Zn(II). PMPS displaces the zinc by reacting with three SH groups. Apo-g32P shows markedly altered DNA binding properties. Co(II) substitution gives a protein with intense d-d transitions typical of a tetrahedral Co(II) complex with absorption maxima at 680 (epsilon = 480), 645 (epsilon = 660), 605 (epsilon = 430), 355 (epsilon = 2250), and 320 (epsilon = 3175) nm. The data support a 3 Cys, 1 His coordination site located in the middle of the DNA binding domain of g32P. Data thus far suggest that the Zn(II) binding sites in multisubunit RNA polymerases and in accessory proteins involved in polynucleotide biosynthesis are more likely to play structural or allosteric (regulatory) roles rather than directly participating in catalysis.

The size, operation, and technical capabilities of protein and nucleic acid core facilities.

A survey of 40 protein and nucleic acid chemistry facilities has provided data about the capabilities of core facilities and the cost of the services they provide. Approximately 43% of the

Comparative Peptide Mapping by HPLC: Identification of Single Amino Acid Substitutions in Temperature Sensitive Mutants

158,000 average annual operating budget for a typical university facility is derived from service charges. After correcting for the various degrees of subsidization of the different facilities, it was found that it costs a typical university facility

Crystallization of a tryptic core of the single-stranded DNA binding protein of bacteriophage T4☆

65 to carry out an acid hydrolysis and amino acid analysis on a protein. A 25‐residue peptide can be synthesized and cleaved for

A Rat Liver Helix-Destabilizing Protein: Properties and Homology to LDH-5

2078, whereas sequencing the same peptide costs

Use of HPLC Comparative Peptide Mapping in Structure/Function Studies

874. A 25‐residue oligonucleotide can be synthesized for