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Low yields of extracted cell-free DNA (cfDNA) from plasma limit continued development of liquid biopsy in cancer, especially in early-stage cancer diagnostics and cancer screening applications. We investigate a novel liquid-phase-based DNA isolation method that utilizes aqueous two-phase systems to purify and concentrate circulating cfDNA. The PHASIFY MAX and PHASIFY ENRICH kits were compared to a commonly employed solid-phase extraction method on their ability to extract cfDNA from a set of 91 frozen plasma samples from cancer patients. Droplet digital PCR (ddPCR) was used as the downstream diagnostic to detect mutant copies. Compared to the QIAamp Circulating Nucleic Acid (QCNA) kit, the PHASIFY MAX method demonstrated 60% increase in DNA yield and 171% increase in mutant copy recovery, and the PHASIFY ENRICH kit demonstrated a 35% decrease in DNA yield with a 153% increase in mutant copy recovery. A follow-up study with PHASIFY ENRICH resulted in the positive conversion of 9 out of 47 plasma samples previously determined negative with QCNA extraction (all with known positive tissue genotyping). Our results indicate that this novel extraction technique offers higher cfDNA recovery resulting in better sensitivity for detection of cfDNA mutations compared to a commonly used solid-phase extraction method.

Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies for early-stage cancer diagnostics and cancer screening applications. Conventional nucleic acid extraction methods have limitations in capturing low levels of cfDNA and risk capturing high levels of high molecular weight contaminating genomic DNA. We developed a liquid-phase based DNA extraction method (PHASIFY™ ENRICH; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify, concentrate, and enrich for cfDNA to improve mutant signal detection. Methods: The PHASIFY ATPS consists of unique formulations that are optimized to drive cfDNA into one liquid phase and plasma contaminants into the other, achieving simultaneous target isolation and concentration. In a previous study using the PHASIFY technique, we demonstrated improved cfDNA and mutant copy yield over solid-phase methods. We further developed an additional size-selection feature (PHASIFY ENRICH) to remove high molecular weight genomic DNA (>500 bp) and to reduce signal noise for molecular diagnostics. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY ENRICH cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., low grade serious ovarian, appendiceal, thyroid, and pancreatic cancer), with known tissue mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). In a follow up study, we tested a similar cohort of plasma samples that were previously tested negative for cfDNA mutation status when using QCNA cfDNA extraction, despite known positive mutation status from corresponding tumor tissue genotyping. Results: In the head-to-head study against QCNA, 57 plasma samples from 23 unique patients with diverse advanced cancers with known tissue mutation status were used for cfDNA extraction. The PHASIFY ENRICH method resulted in a 153% increase in median mutant copy recovery compared to QCNA (P = 0.01 Wilcoxon Signed-Rank). In the follow up study, 47 plasma samples from 31 patients with diverse advanced cancers and prior cfDNA negative mutation status with tissue positive mutation status were used for extraction. Of the 47samples extracted by PHASIFY ENRICH, 9 samples resulted in a conversion to positive cfDNA mutation status compared to no mutant signals when previously using QCNA. Conclusion: Our results indicate that PHASIFY ENRICH provides improved cfDNA mutation detection by increasing cfDNA recovery and reducing contaminating genomic DNA background. Citation Format: Filip Janku, Helen Huang, David Y. Pereira, Masae Kobayashi, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA mutation detection with novel liquid-phase extraction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 575.

Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies especially in early-stage cancer diagnostics and cancer screening applications. We developed a novel liquid-liquid based DNA isolation method (PHASIFY™ MAX; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify and concentrate circulating cfDNA, which can result in increase of cfDNA recovery and mutant signal detection. Methods: The PHASIFY™ ATPS consists of unique formulations of polymer and salt components that can intrinsically separate into two distinct phases. The formula can be optimized to drive cfDNA into one phase and contaminants from plasma into the other, achieving simultaneous target isolation and concentration. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY™ MAX cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from 1mL of plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., thyroid, pancreatic), with known tissue BRAF, KRAS, or NRAS mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based (BRAF, KRAS, NRAS) Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). Results: Plasma samples from 89 patients with diverse advanced cancers (colorectal, N=36; ovarian, N=18; lung N=7; thyroid, N=6; pancreatic, N=6; others, N=16) with known BRAF, KRAS or NRAS mutation status were used for cfDNA extraction with standard QCNA and PHASIFY™ MAX kits. On average, PHASIFY™ MAX demonstrated a 288% increase in total cfDNA recovery compared to the QCNA kit (p-value Conclusion: Our early results indicate that the PHASIFY™ MAX extraction technique offers higher cfDNA recovery resulting in higher sensitivity of cfDNA mutation detection compared to the industry standard. Citation Format: Filip Janku, Helen J. Huang, David Y. Pereira, Masae Kobayashi, Chung Hei Chiu, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA and mutant copy recovery from plasma with novel liquid-liquid extraction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1979.

The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle1,2. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population3 into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.

Analysis of the carboxymethylated subunit of human cartilage oligomeric matrix protein (COMP) by matrix-assisted laser desorption time-of-flight mass spectrometry indicated a protonated molecular mass of 86949 ± 149 Da, compared with 83547.0 Da calculated from the sequence. Treatment withN-glycanase caused a reduction in mass of 3571 ± 219 Da, but there was no loss of mass after treatment withO-glycanase or neuraminidase. Peptides containing two putative sites of N-glycosylation were purified and characterized. Analysis of the masses of these afterN-glycanase treatment indicated that one was substituted at Asn-101 with an oligosaccharide of mass 1847.2 ± 6.6 Da, and the other was unsubstituted at Asn-124. The remaining site of attachment, at Asn-721, was, therefore, also substituted with an oligosaccharide of mass 1724 ± 226 Da. Analysis of the total monosaccharide content by chemical methods indicated that there were no additional oligosaccharide substituents. The MALDI-TOF mass spectra of COMP from bovine fetal and adult cartilage were compared, indicating a more heterogeneous pattern of substitution at Asn-101 in the fetal form. Since COMP is distributed throughout the pericellular and territorial environments in developing cartilage but occupies the interterritorial zone in mature cartilage, these changes in glycosylation may allow for different intermolecular interactions.

Characteristic of chronic myelogenous leukemia (CML) is the presence of the chimeric p210(bcr-abl) protein possessing elevated protein tyrosine kinase activity relative to normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and associates with the p120 ras GTPase-activating protein (GAP). We have purified p62(dok) from a hematopoietic cell line expressing p210(bcr-abl). p62(dok) is a novel protein with features of a signaling molecule. Association of p62(dok) with GAP correlates with its tyrosine phosphorylation. p62(dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases.

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.

The transforming growth factor beta (TGF-beta) family of growth modulators play critical roles in tissue development and maintenance. Recent data suggest that individual TGF-beta isoforms (TGF-beta 1, -beta 2 and -beta 3) have overlapping yet distinct biological actions and target cell specificities, both in developing and adult tissues. The TGF-beta 3 isoform was purified to homogeneity from both natural and recombinant sources and characterized by laser desorption mass spectrometry, by protein sequencing, by amino acid analysis and by biological activity. TGF-beta 3 was the major TGF-beta isoform in umbilical cord (230 ng/g), and was physically and biologically indistinguishable from recombinant TGF-beta 3 and from the tumor growth inhibitory (TGI) protein found in umbilical cord. Immunohistochemistry using antipeptide TGF-beta 3 specific antibody showed TGF-beta 3 localization in perivascular smooth muscle.

Protein kinase CKII (formerly casein kinase II) can be isolated as a heterotetramer, containing two catalytic (alpha or alpha') and two regulatory (beta) subunits. We have characterized the forms of CKII in HeLa cells using antibodies specific for the alpha or alpha' subunits. Following metabolic labeling with [35S]methionine, whole cell soluble extracts were analyzed by immunoprecipitation and gel electrophoresis. Both alpha and alpha' coprecipitate with beta and with each other. However, when extracts are depleted of alpha, a pool of CKII containing only alpha' and beta is identified. Similarly, depletion of alpha' revealed a pool exclusively of alpha and beta. Therefore, we propose that there are three distinct isoforms of CKII within HeLa cells with different catalytic subunit stoichiometries (alpha 2 beta 2, alpha alpha' beta 2, and alpha' 2 beta 2). With our immunodepletion procedure we have characterized the isoforms by activity analysis, turnover of pulse-labeled subunits, and by localization in subcellular fractions obtained from labeled cells. We have also analyzed complex formation between the catalytic and regulatory subunits by examining the differences in the rate of signal incorporation into subunits in immunoprecipitates obtained from continuously labeled and pulse-labeled cells. We have found that the alpha 2 beta 2 and alpha alpha' beta 2 isoforms assemble relatively slowly (12-16 h), whereas complex formation of the alpha' 2 beta 2 isoform occurs more rapidly (2-4 h). Analysis of isoform complex formation in subcellular fractions from pulse-labeled cells revealed that the majority of nuclear CKII is assembled in the nucleus from free catalytic and regulatory subunit polypeptides.

A 64-kDa protein was purified from an octyl glucoside/cholate extract of spinach thylakoids. N-Terminal analysis yielded 23 residues of sequence, of which the first 15 were identical to a sequence reported [Gal, A., Herrmann, R. G., Lottspeich, F., & Ohad, I. (1992) FEBS Lett. 298, 33-35] for a protein kinase with specificity toward the photosystem II light-harvesting complex (LHC-II). We report the complete sequence of this 64-kDa protein, deduced from cDNA clones. The transit peptide has a chloroplast import signal at the N-terminus and a C-terminal hydrophobic span bounded by basic amino acids that predicts localization of the protein to the thylakoid lumen. The mature protein sequence is about 50% identical to several polyphenol oxidases (PPOs). Canonical protein kinase motifs are absent, as are sequences characteristic of ATP-binding sites. The mature protein resembles arthropodan hemocyanin (Hc), possessing three major domains. The N-terminal domain is rich in cysteine residues and predicted alpha-helices. The central domain has a conserved motif, N-terminal to a presumptive Cu-A site, that is not found in tyrosinases or Hc and is proposed as the provider of a third imidazole ligand to Cu-A. An unusual 13-residue, glutamine-rich link begins a C-terminal domain containing 7 predicted beta-strands which, by analogy with Hc, may form an antiparallel beta-barrel. We conclude that this 64-kDa polypeptide is a lumenal PPO and the precursor of a 42.5-kDa PPO form described previously [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].(ABSTRACT TRUNCATED AT 250 WORDS)

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

Accumulation of the amyloid A beta peptide, which is derived from a larger precursor protein (APP), and the formation of plaques, are major events believed to be involved in the etiology of Alzheimer's disease. Abnormal regulation of the metabolism of APP may contribute to the deposition of plaques. APP is an integral membrane protein containing several putative phosphorylation sites within its cytoplasmic domain. We report here that APP is phosphorylated at Thr668 by p34cdc2 protein kinase (cdc2 kinase) in vitro, and in a cell cycle-dependent manner in vivo. At the G2/M phase of the cell cycle, when APP phosphorylation is maximal, the levels of mature APP (mAPP) and immature APP (imAPP) do not change significantly. However, imAPP is altered qualitatively. Furthermore, the level of the secreted extracellular N-terminal domain (APPS) is decreased and that of the truncated intracellular C-terminal fragment (APPCOOH) is increased. These findings suggest the possibility that phosphorylation-dependent events occurring during the cell cycle affect the metabolism of APP. Alterations in these events might play a role in the pathogenesis of Alzheimer's disease.

An Epstein-Barr virus-encoded protein, ZEBRA, mediates the switch from latency to the viral lytic life cycle. ZEBRA's domain structure and DNA binding specificity resemble that of cellular transcriptional activators such as c-Fos/c-Jun. We show that ZEBRA, like c-Jun, is phosphorylated by casein kinase II (CKII). The principal site of phosphorylation is serine-173 (S173), five amino acids upstream of the basic DNA recognition domain. CKII phosphorylation abrogated ZEBRA's capacity to bind its target DNA sequences. S173 is a functional component of ZEBRA's DNA binding domain, since mutation of S173 to alanine (S173A) reduced DNA binding in vitro to 10% of wild-type levels. Transcriptional activation of a native viral promoter in vivo by mutant S173A was also reduced markedly. Reversible phosphorylation of S173 is likely to be an important means of regulating ZEBRA's activity in vivo.

We report a method for optimizing the specificity of product formation in the polymerase chain reaction (PCR). This technique is based on the use of dimethyl sulfoxide (DMSO) and takes into account primer Tm. The reduction in Tm by DMSO is directly correlated with renaturation temperature such that a DMSO gradient reflects a temperature gradient. We use this relationship to show that optimum product formation usually occurs at or within several degrees of the midpoint Tm of a given primer pair. We illustrate these correlations using three examples deriving PCR products from a human cDNA library, representing the casein kinase II alpha and beta subunits as well as the 5' untranslated region for the beta subunit. By following product formation as a function of renaturation temperature, we postulate rules for cycle design based on primer Tm. Implications for the use of degenerate primers are discussed.

The activity of p34cdc2 kinase is regulated in the phases of vertebrate cell cycle by mechanisms of phosphorylation and dephosphorylation. In this paper, we demonstrate that casein kinase II (CKII) phosphorylates p34cdc2 in vivo and in vitro at Ser39 during the G1 phase of HeLa cell division cycle. Human p34cdc2 shows a typical phosphorylation sequence motif site for CKII at Ser39 (ES39EEE). In our experiments, either p34cdc2 expressed and purified from bacteria or p34cdc2 immunoprecipitated from HeLa cells enriched in G1 by elutriation were substrates for in vitro phosphorylation by CKII. Phosphoamino acid analysis, N-chlorosuccinimide mapping, and two-dimensional tryptic mapping of p34cdc2 phosphorylated in vitro were performed to determine the phosphorylation site. A synthetic peptide spanning residues 33-50 of human p34cdc2, including the CKII site, was used to map the site. In addition, phosphorylation at Ser39 also occurs in vivo, since p34cdc2 is phosphorylated during G1 on serine, and its two-dimensional tryptic map shows two phosphopeptides that comigrate exactly with the synthetic peptides used as standard.

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

Several small, acid-soluble, basic peptides with anti-microbial properties have been isolated from maize (inbred B73) kernels. One of these peptides (MBP-1) has been purified to homogeneity and characterized. The peptide has a molecular weight of 4127.08 as determined by plasma desorption mass spectroscopy, has no free cysteines, and is predominantly alpha-helical as determined by circular dichroism. The primary sequence of the peptide (33 residues) has been determined by Edman degradation and shows no homology to the thionins, a group of cysteine-rich peptides found in some cereals including wheat, barley, and sorghum, as well as several dicot species. Like the thionins, however, MBP-1 has been found to have antimicrobial properties in vitro. MBP-1 inhibits spore germination or hyphal elongation of several plant pathogenic fungi, including two seed pathogens of maize (Fusarium moniliforme Sheld. and Fusarium graminearum (Gibberella zeae (Schw.) Petsch)), and several bacteria, including a bacterial pathogen of maize (Clavibacter michiganense ssp. nebraskense). A synthetic MBP-1 peptide, air-oxidized and purified by reverse phase chromatography, was equally antifungal as compared with the naturally occurring peptide.

The Schwann cell myelin protein (SMP), previously defined in quail and chick by a monoclonal antibody, is in vivo exclusively expressed by myelinating and nonmyelinating Schwann cells and oligodendrocytes. The isolation of the complete nucleotide sequence of SMP is reported here. The predicted polypeptide chain reveals that SMP is a transmembrane molecule of the immunoglobulin superfamily showing sequence similarities with several surface glycoproteins expressed in the nervous and immune systems. In spite of a 43.5% overall sequence identity between rat myelin-associated glycoprotein (MAG) and quail SMP, SMP does not seem to be the avian homolog of MAG, since their expression, regulation, and functions are significantly different. Unusual sequence arrangements shared by SMP, MAG, and two lymphoid antigens suggest the existence of a particular subgroup in the immunoglobulin superfamily.

Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 microM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexes of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinase and their regulation throughout the cell division cycle.

To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.

Pancreatic thread protein (PTP) forms double helical threads in the neutral pH range after purification, undergoing freely reversible, pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Gross et al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing greater than 99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH2-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca2(+)-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.

Primary cultures of human cells provide an increasingly important alternative to using virally transformed or otherwise immortalized cell lines or to using cloned cell lines derived from human or animal tumors. Advances in primary cell culture techniques, media formulations, and other reagents have enabled routine culture of primary cells derived from human tissues for biomedical research and drug discovery approaches such as high content screening. That primary cells retain the phenotypic characteristics of the original tissue is one main advantage over immortalized cell lines. However, securing reliable supplies of primary cells on a large scale has been problematic. Here, two primary differentiating cell types, preadipocytes and preosteoclasts, are described to illustrate the utility of commercially produced cell systems in discovery research and rapid-throughput applications.

Primary cultures of human cells provide an increasingly important alternative to using virally transformed or otherwise immortalized cell lines or to using cloned cell lines derived from human or animal tumors. Advances in primary cell culture techniques, media formulations, and other reagents have enabled routine culture of primary cells derived from human tissues for biomedical research and drug discovery approaches such as high content screening. That primary cells retain the phenotypic characteristics of the original tissue is one main advantage over immortalized cell lines. However, securing reliable supplies of primary cells on a large scale has been problematic. Here, two primary differentiating cell types, preadipocytes and preosteoclasts, are described to illustrate the utility of commercially produced cell systems in discovery research and rapid-throughput applications.

Three groups of male F344 rats were exposed to a water-soluble metal working fluid (MWF) aerosol at concentrations of 20, 60 or 180 mg/m3 for 6 h/day, five days a week, for 13 weeks in inhalation chambers. The aerosol particles were normally distributed and the mass median aerodynamic diameter was 1.56 mm. Despite the absence of clinical findings or significant changes in body weight during the 13-week exposure period, the numbers of white blood cells and lung weights were significantly higher at the end of the 13-week exposure period. Exposure to 20 mg/m3 of the MWF aerosol was found to have an effect on the respiratory system, including an accumulation of foamy macrophages in the bronchoalveolar lavage (BAL) fluid and thickening of the alveolar walls in the histopathology. The level of histamine and number of polymorphonuclear (PMN) cells were also higher in the BAL fluid from the rats exposed to 60 mg/m3 of the MWF aerosol, while the respiratory inflammation was most pronounced in the rats exposed to 180 mg/m3 of the MWF aerosol, including the accumulation of PMNs and foamy macrophages in the BAL cells, lung weight increase and thickening of the alveolar walls. Immunoglobulin IgG2a level was also lower in the sera from the rats exposed to 180 mg/m3 of the MWF aerosol. Therefore, even though no clinical symptoms were observed in the rats exposed to the high MWF concentration, respiratory inflammation was still induced by a relatively low concentration of the MWF, while the immune system was affected by the high MWF concentration.

Rats were exposed for 6 h per day in inhalation chambers to a 10 mg/m(3) concentration of metalworking fluid (MWF) contaminated with endotoxin at concentrations of 1813 (low dose) and 20,250 eu/m(3) (high dose) 5 days per week for 8 weeks. It was found that 94.7% of the MWF aerosol particles had diameters in the range of 0.42-4.6 microm, with geometric mean diameter of 1.56 microm. The body weight and pulmonary function parameters were measured every week during the 8 weeks of exposure, whereas bronchoalveolar lavage (BAL) fluid was prepared to measure the inflammatory markers and cytokines after the 8 weeks of exposure. There were no changes in body weight and respiratory function (tidal volume and respiratory frequency) during the 8 weeks of exposure to the MWF containing endotoxins, yet lung weight increased significantly (P < 0.05) in the rats exposed to the MWF both with and without endotoxins. The number of polymorphonuclear (PMN) cells in the BAL fluid increased significantly (P < 0.05) in the rats exposed to MWF with endotoxins, and the levels of cytokines such as IL-4, INF-gamma, IL-1beta, and TNF-alpha also were significantly increased (P < 0.05) compared to the control. The NOx production activity of the BAL cells increased significantly (P < 0.05) in the rats exposed to the MWF with and without endotoxins. Increases in lung weight, number of PMN cells, and levels of extracellular cytokines and NOx were all more significant in the rats exposed to the MWF with endotoxins rather than in those exposed to MWF without endotoxins. In spleen cell cultures, T-cell proliferation activity was decreased, yet cytokine levels (INF-gamma, IL-1beta, IL-4, and TNF-alpha) remained unchanged after repeated exposure to MWF with and without endotoxins. Although the levels of total IgG(1), IgG(2a), and IgE antibodies in the serum were not changed, the levels of endotoxin-specific antibodies, including IgG(2a) and IgE, were increased significantly (P < 0.05) in the rats exposed to endotoxins, but there was not a significant increase in endotoxin-specific IgG(1). When taken together, the results indicate that lung inflammatory responses can be induced without changing pulmonary function after repeated exposure to MWFs contaminated with endotoxins. In addition, endotoxin-specific IgG(2a) and IgE may be effective biomarkers for workers exposed to MWFs contaminated with endotoxins in the workplace.

Male Sprague-Dawley rats were exposed to a water-soluble metal working fluid (MWF) (5% v/v) contaminated with endotoxins (10,000 eu/ml or 100,000 eu/ml) at 10 mg/m3 for six hours per day for three days (acute exposure) or two weeks (subacute exposure). The geometric mean diameter of the MWF aerosols was 1.56 μm, and the airborne endotoxin concentrations ranged from 1,231 to 2,173 eu/m3 (10,000 eu/ml in the bulk MWF) for the low dose and 19,263–27,386 eu/m3 (100,000 eu/ml in the bulk MWF) for the high dose. Minimal effects were observed after exposure to 10 mg/m3 of the MWF without endotoxins for three days or two weeks. However, an increase in the number of polymorphonuclear cells (PMNs) and the level of protein was noted in the bronchoalveolar lavage (BAL) fluid from the rats acutely exposed to the MWF with endotoxins. The acute exposure produced a greater increase in the number of PMNs and total cell number in the BAL fluid than the subacute exposure. The number of white blood cells in the peripheral blood and the weight of the lungs both increased after the subacute exposure to the MWF aerosol with endotoxins, indicating increased vascular permeability in response to the endotoxin exposure. The levels of cyotokines such as IL-4, INF-γ, and IL-1β in the BAL fluid from the rats exposed to the MWF with or without endotoxins remained unchanged. Although the level of nitric oxide (NO x ) in the BAL supernatant did not show any change, the induction of NO x from the alveolar macrophages increased in the rats acutely or subacutely exposed to the MWF contaminated with endotoxins. The ConA-induced proliferation response showed no change, yet the LPS-induced proliferation response was significantly increased in the splenocytes from the rats subacutely exposed to the MWF with and without endotoxins. The level of TNF-α in the spleen cell culture obtained from the rats exposed to the MWF with or without endotoxins increased without changing the levels of IL-1β, IL-4, and INF-γ. The level of endotoxin-specific IgE in the serum obtained from the rats exposed to the MWF with endotoxins increased dose-dependently, while the levels of total immunoglobulins (IgG1, IgG2a and IgE) and endotoxin-specific IgG1 and IgG2a remained unchanged. Accordingly, the current results indicate that lung inflammation can be immediately induced by acute or subacute exposure to an MWF contaminated with endotoxins, and macrophages would appear to play a role in the induction of inflammation along with B-cell functions rather than T-cell functions, after subacute exposure to an MWF with endotoxins. In addition, endotoxin-specific IgE is an early marker for endotoxin exposure in the workplace.

We have recently reported that protein kinase CK2 phosphortylates both in vivo and in vitro residue serine-46 of the cell cycle regulating protein Cdc28 of budding yeast Saccharomyces cerevisiae, confirming a previous observation that the same site is phosphorylated in Cdc2/Cdk1, the human homolog of Cdc28. In addition, S. cerevisiae in which serine-46 of Cdc28 has been mutated to alanine show a decrease of 33% in both cell volume and protein content, providing the genetic evidence that CK2 is involved in the regulation of budding yeast cell division cycle, and suggesting that this regulation may be brought about in G1 phase of the mammalian cell cycle. Here, we extended this observation reporting that the mutation of serine-46 of Cdc28 to glutamic acid doubles, at least in vitro, the H1-kinase activity of the Cdc28/cyclin A complex. Since this mutation has only little effects on the cell size of the cells, we hypothesize multiple roles of yeast CK2 in regulating the G1 transition in budding yeast.

To investigate the biological function of CKII, we have identified proteins that interact with the subunits of CKII using the yeast two-hybrid system. Here we report that SAG, an antioxidant protein containing Ring-H2 finger motif, is a cellular partner associating with the beta subunit of CKII. SAG does not interact with the alpha subunit of CKII. Analysis of SAG deletion mutants indicates that the Ring-H2 motif of SAG is necessary and sufficient for its binding to the beta subunit of CKII. Recombinant SAG can be phosphorylated by CKII in vitro, providing evidence that the beta subunit mediates the interaction of CKII enzyme with substrate proteins. Overlay experiment shows that SAG and the beta subunit of CKII associate directly in vitro and that CKII-mediated phosphorylation of SAG does not affect the interaction between SAG and the beta subunit of CKII. Northern blot analysis indicates that both SAG and the beta subunit of CKII were relatively rich in human heart, liver, skeletal muscle, and pancreas, but were detected in only trace amounts in brain, placenta, and lung. Our present results suggest that CKII may play a role in the regulation of SAG function.

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

Protein kinase CKII (CKII) is a heterotetramer composed of two catalytic (alpha or alpha') and two regulatory (beta) subunits. Using the yeast two-hybrid system, we have identified the highly basic, ribosomal protein L41 as a cellular protein capable of interacting with the beta subunit of CKII. We show, furthermore, using purified proteins, that L41 protein and CKIIbeta associate directly in vitro. L41 protein is not a substrate for CKII phosphorylation, and it does not stimulate CKII activity with either beta-casein or synthetic peptide substrate (RRREEETEEE). However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIalpha by CKII by 2.5 times. Additionally, L41 protein enhances the autophosphorylation of CKIIalpha. The data indicate that L41 protein associates with CKII and can modulate its activity toward a specific substrate or substrates. The direct interaction of CKIIbeta with ribosomal proteins also suggests that CKIIbeta itself or CKII holoenzyme may be involved in ribosome assembly or translational control.

Casein kinase II (CKII) usually exists as a heterotetramer with alpha 2 beta 2, alpha alpha'beta 2, or alpha'2 beta 2. The alpha or alpha' subunits catalyze protein phosphorylation, whereas the function of the beta subunit remains unclear. One of the possible functions of the beta subunit may be to mediate the interaction of the catalytic subunit with target proteins. To identify proteins capable of associating with the beta subunit in vivo, we have used a two-hybrid system. One protein identified is human ribosomal protein L5. The protein L5 does not interact with the alpha or alpha' subunits of CKII, supporting the idea that the beta subunit can determine a substrate specificity of CKII. These results furthermore suggest a novel role for CKII in ribosomal L5 phosphorylation, in ribosomal assembly, or ribosomal transport in the intact cells. The protein L5 may act as a regulator of the activity or subcellular localization of CKII.

We are developing methods to identify Gi-phase substrates of cyclin-Cdk: complexes. In earlier years of this project, we optimized two-dimensional gels for visualization of phosphorylation of Cdc28 substrates, and we developed monoclonal antibodies with reactivity to phospho-Ser-Pro or phospho-Thr- Pro. We identified one important in vivo substrate, Sic 1. However, it appears that these methods by themselves are insufficiently powerful to identify many substrates, since most substrates seem to be very rare. We have therefore supplemented these methods with three other approaches: (1) purification of affinity tagged cyclins, to look for co-purifying proteins; (2) complete genome two-hybrid screens with Cdc28 and with Gi cyclins, to look for interacting proteins; and (3) genome-wide analysis of cell cycle regulated transcription, on the basis that some important substrates might be regulated at the transcriptional level as well as at the activity level. All three approaches are going very well, and we have excellent candidates for new substrates, which will be characterized in part using the 2D gel system.

S100 beta, a calcium-binding protein synthesized by CNS astrocytes, has trophic effects in vitro (neurite extension and glial proliferation). In Alzheimer's disease and Down's syndrome, severely afflicted brain regions exhibit up to 20-fold higher levels of S100 beta protein, and astrocytes surrounding neuritic plaques exhibit highly elevated levels of S100 beta immunostaining. A major constituent of plaques, beta-amyloid, has been reported to have neurotoxic and neurotrophic effects in vitro. In our study we examined the responses of CNS glia to beta-amyloid. C6 glioma cells and primary rat astrocyte cultures were treated with beta A(1-40) peptide at doses up to 1 microM. Weak mitogenic activity, measured by [3H]thymidine incorporation, was observed. Northern blot analysis revealed increases of S100 beta mRNA within 24 h in a dose-dependent manner. Nuclear run-off transcription assays showed that beta A(1-40) specifically induced new synthesis of S100 beta mRNA in cells maintained in serum, but under serum-free conditions, there was a general elevation of several mRNA species. Corresponding increases of S100 beta protein synthesis were observed by immunoprecipitation of 35S-labeled cellular proteins. To evaluate whether this effect of beta-amyloid was mediated via neurokinin receptors or by calcium fluxes, various agonists and antagonists were tested and found to be ineffective at stimulating S100 beta synthesis. In sum, these in vitro data suggest that in neuropathological conditions, beta-amyloid itself is an agent which may provoke chronic gliosis and the production of trophic substances by astrocytes.

Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro.

Publisher Summary In the biopharmaceutical industry, C-terminal sequence information can be essential for the early characterization of recombinant proteins, as well as for the routine batch analysis, and several automated chemical degradation schemes, analogous in principle to the Edman method for amino-terminal (N-terminal) sequencing, have been proposed. This chapter explores specific examples, wherein the application of chemistry, especially the Perkin-Elmer method can be used as an additional analytical tool for protein characterization. The chapter discusses the chemistry's robustness in confirming the expected sequence of recombinant proteins, and that this method can identify post-translational modifications and sample heterogeneity. The sequencing method has been demonstrated on proteins obtained from several independent laboratories, and the information obtained has been complimentary and consistent with data obtained from other analytical techniques, such as peptide mapping, amino acid analysis, and mass spectrometry. Although, some amino acid residues interfere with sequencing all protein samples, however, many proteins have been sequenced successfully by this new C-terminal sequencing method, which continues to improve, and thereby provides a useful analytical tool.

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these ‘down-regulated’ cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.

The mechanisms by which human immunodeficiency virus (HIV) infection provokes progressive neurodegeneration and dementia in acquired immunodeficiency syndrome (AIDS) remain obscure. In HIV-infected (HIV+) individuals, we found that the brain cells preferentially infected by HIV, viz. the microglia, were abundant, activated, and intensely immunopositive for interleukin-1 alpha (IL-1 alpha), an immune response-generated cytokine that increases the synthesis and processing of beta-amyloid precursor proteins (beta-APP) and promotes proliferation and activation of astroglia. We also found an increase in the number of activated astroglia expressing elevated levels of S100 beta, a cytokine that increases intraneuronal calcium levels and promotes excessive growth of neuronal processes (neurites). These glial changes were accompanied by increased expression of beta-APP immunoreaction product in neurons and overgrown (dystrophic) neurites. In addition, some neurons contained monoclonal antibody Tau-2 immunopositive, neurofibrillary tangle-like structures. Our findings provide evidence that glial activation with increased expression of IL-1 alpha and S100 beta may be important in the neuropathogenesis of AIDS dementia. We propose that HIV infection promotes excessive microglial IL-1 alpha expression with consequent astrogliosis and increased expression of S100 beta. Overexpression of these two cytokines may then be involved in AIDS neuropathogenesis by inducing gliosis, growth of dystrophic neurites, and calcium-mediated neuronal cell loss in AIDS.

Protein F1/GAP43 is neuron-specific, associated with neurite outgrowth during development and a substrate for PKC. This protein is present in high levels in serotonergic neurons which in culture sprout in response to the glial-derived S100b, the beta-beta homodimer. As an initial step in determining whether S100b acts on F1/GAP43 we studied the regulation by S100b of PKC phosphorylation of F1/GAP43. Either the S100b or a mixture of S100a and S100b, both from a brain glial cell source, inhibited in vitro phosphorylation of purified F1/GAP43 by purified PKC in a dose-dependent manner. Using recombinant PKC subtypes, purified S100b preferentially inhibited the F1/GAP43 phosphorylation by the beta subtype. The IC50 of S100b for beta I and beta II PKC was 8 microM while for alpha and gamma PKC it was 64 microM. S100b inhibition was thus subtype-selective. Histone III-S phosphorylation by the four PKC subtypes was not inhibited by S100b. S100b inhibition was thus substrate-selective. Moreover, the effect of S100b on phosphorylation could not be explained by a direct inhibition of kinase activity. Together with earlier studies implicating a role for S100 in synaptic plasticity and neurite outgrowth, the present results suggest that S100b may regulate such functions through its inhibition of neuron-specific PKC substrate (F1/GAP43) phosphorylation. The regulation of this neuron-specific substrate phosphorylation by glial S100 suggests the potential for a novel neuro-glial interaction. Finally, the location of S100 gene on chromosome 21, trisomic in Down's syndrome, and over-expressed in this disorder, as well as in Alzheimer's disease, suggests a link to cognitive impairments in human.

p53 is a sequence-specific DNA-binding oligomeric protein that can activate transcription from promoters bearing p53-binding sites. Whereas the activation region of p53 has been identified within the amino terminus, the location of the specific DNA-binding domain has not been reported. Thermolysin treatment of p53 protein generates a stable protease-resistant fragment that binds with marked specificity to p53 DNA-binding sites. Amino-terminal sequencing of the fragment located the thermolysin cleavage site to residue 91. Because the fragment does not contain the cdc2 phosphorylation site at Ser-315, we conclude that the the site-specific DNA-binding domain of p53 spans the central region of the protein. The vast majority of the mutations in oncogenically derived p53 proteins are located within this central portion of the molecule. Such mutant p53 proteins exhibit defective sequence-specific DNA-binding. Although thermolysin digestion of mutant p53 proteins generates proteolytic patterns that differ from wild-type protein, one mutant tested, His-273, generates a resistant fragment that migrates with a similar electrophoretic mobility to the wild-type protease-resistant fragment. Interestingly, although intact mutant His-273 protein binds to DNA at 20 degrees C, the thermolysin-resistant mutant fragment does not. In addition, the central protease-resistant, site-specific binding region of wild-type p53 does not demonstrate nonspecific DNA-binding. Thus, although sequences outside of the central region of p53 contribute to both nonspecific DNA-binding and oligomerization, they are not required for sequence-specific DNA-binding.

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.