bluuebunny/crossref_metadata_2025 huggingface dataset:
Production by activated human T cells of interleukin 4 but not interferon-gamma is associated with elevated levels of serum antibodies to activating malaria antigens.
Authors: Troye-Blomberg M , Riley E M , Kabilan L , Holmberg M , Perlmann H , Andersson U , Heusser C H , Perlmann P
Published: May 2006

T cells play a crucial role in antibody-mediated and antibody-independent immunity against Plasmodium falciparum malaria. Therefore, a vaccine immunogen should include parasite-derived B- and T-cell epitopes capable of giving rise to protective responses in both systems. The P. falciparum antigen Pf155/ring-infected erythrocyte surface antigen (RESA), a vaccine candidate, contains immunodominant T- and B-cell epitopes located in the central (5') and C-terminal (3') invariant repeat regions of the molecule. To relate Pf155/RESA-peptide-specific responses of T cells to function, T cells from P. falciparum immune donors were activated with peptides corresponding to these immunodominant regions. Activation was measured as induction of interferon-gamma secretion, T-cell proliferation (DNA synthesis), or transcription and translation of interleukin 4 (IL-4) mRNA. Peptides from both regions were shown to induce interferon-gamma, IL-4, proliferation, or any combination. In individual donors, there was no correlation between these different activities. Rather, they were negatively correlated, demonstrating the importance of examining multiple parameters of T-cell activation when estimating the proportion of individuals responding to a given epitope. However, IL-4 mRNA and intracellular IL-4 could be induced in T cells of donors who had elevated concentrations of serum antibodies to the same peptide that was used for T-cell activation. These results suggest that a causal relationship exists between the activation of IL-4-producing T-cell subsets and production of the anti-Pf155/RESA-specific antibodies in individuals in which immunity has been induced by natural infection. This finding has implications that should be considered for the selection of immunogens to be included in a future P. falciparum subunit vaccine and for vaccine development in general.
Enhanced immunogenicity of a sequence derived from hepatitis B virus surface antigen in a composite peptide that includes the immunostimulatory region from human interleukin 1.
Authors: Rao K V , Nayak A R
Published: May 2006

The effect on immunogenicity of coupling the immunostimulatory nonapeptide sequence (residues 163-171) from human interleukin 1 beta (IL-1 beta) to a small immunogen was examined. A 21-amino acid sequence spanning positions 12-32 on the large protein of hepatitis B surface antigen was chosen as a model. Three peptides were synthesized corresponding to the IL-1 beta-derived sequence [peptide IL-(163-171)], the hepatitis B surface antigen-derived sequence [peptide S1-(12-32)] and a composite peptide that included both these sequences separated by a spacer of two glycine residues [peptide S1-(12-32)-IL-(163-171)]. In an in vitro thymocyte proliferation assay, both peptides S1-(12-32)-IL-(163-171) and IL-(163-171) showed comparable activity, whereas peptide S1-(12-32) was inactive. Groups of five to seven mice each from C3H/CH, BALB/c, SJL/J, and C57BL/6 strains were immunized with equimolar amounts of either peptide S1-(12-32), peptide S1-(12-32)-IL-(163-171), or a mixture of peptides S1-(12-32) and IL-(163-171), and sera were screened for anti-S1-(12-32) antibodies. In all strains, peptide S1-(12-32)-IL-(163-171) elicited an increased primary and secondary anti-S1-(12-32) antibody response compared to the other two groups. Further, peptide S1-(12-32)-IL-(163-171) also induced an increased number of responders to primary immunization, though the number of responders was quantitative in all groups following secondary immunization. At least part of the enhanced immunogenicity of the S1-(12-32) sequence in peptide S1-(12-32)-IL-(163-171) appears to be due to augmented T-helper cell activity. These results suggest that coupling of the immunostimulatory IL-1 beta-derived sequence in tandem with an immunogen may confer inbuilt adjuvanticity.
Identification of calcium-activated neutral protease as a processing enzyme of human interleukin 1 alpha.
Authors: Kobayashi Y , Yamamoto K , Saido T , Kawasaki H , Oppenheim J J , Matsushima K
Published: May 2006

We describe here the involvement of calcium-activated neutral protease (CANP or calpain, EC 3.4.22.17) in calcium-dependent proteolytic processing of the precursor of human interleukin 1 alpha (IL-1 alpha) into mature IL-1 alpha. Calcium ionophore ionomycin enhanced proteolytic processing of pre-IL-1 alpha and the release of mature IL-1 alpha either from lipopolysaccharide (LPS)-activated human adherent mononuclear cells or from a human bladder carcinoma cell line (HTB9 5637) that constitutively produces human IL-1 alpha and -beta. The proteolytic processing of pre-IL-1 alpha was completely inhibited by EGTA. Similar calcium-dependent proteolytic processing of pre-IL-1 alpha was also observed with lysates of either LPS-activated human adherent mononuclear cells or HTB9 5637 cells. Since the optimal pH for processing was between 7 and 8, and E-64 (a cysteine protease inhibitor) and leupeptin (a serine and cysteine protease inhibitor) both inhibited this processing by cell lysates, we hypothesized that a calcium-activated neutral protease, CANP, might be responsible for this processing. This hypothesis was supported by data showing that the specific CANP inhibitor peptide inhibited this proteolysis in cell lysates in a dose-dependent fashion (IC50 = 0.05 microM) and that treatment of pre-IL-1 alpha with purified CANP yielded the 17-kDa mature form of IL-1 alpha, which has an amino terminus identical with that reported for mature human IL-1 alpha. Taken together, these findings indicate that calcium-dependent proteolytic processing of pre-IL-1 alpha is selectively mediated by CANP.
The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family.
Authors: Van Wart H E , Birkedal-Hansen H
Published: May 2006

The general applicability of the "cysteine-switch" activation mechanism to the members of the matrix metalloproteinase (MMP) gene family is examined here. All currently known members of the MMP gene family share the characteristic that they are synthesized in a latent, inactive, form. Recent evidence suggests that this latency in human fibroblast collagenase (HFC) is the result of formation of an intramolecular complex between the single cysteine residue in its propeptide domain and the essential zinc atom in the catalytic domain, a complex that blocks the active site. Latent HFC can be activated by multiple means, all of which effect the dissociation of the cysteine residue from the complex. This is referred to as the "cysteine-switch" mechanism of activation. The propeptide domain that contains the critical cysteine residue and the catalytic domain that contains the zinc-binding site are the only two domains common to all of the MMPs. The amino acid sequences surrounding both the critical cysteine residue and a region of the protein chains containing two of the putative histidine zinc-binding ligands are highly conserved in all of the MMPs. A survey of the literature shows that many of the individual MMPs can be activated by the multiple means observed for latent HFC. These observations support the view that the cysteine-switch mechanism is applicable to all members of this gene family. This mechanism is unprecedented in enzymology as far as we know and offers the opportunity for multiple modes of physiological activation of these important enzymes. Since conditions in different cells and tissues may match those necessary to effect one of these activation modes for a given MMP, this may offer metabolic flexibility in the control of MMP activation.
Where metal ions bind in proteins.
Authors: Yamashita M M , Wesson L , Eisenman G , Eisenberg D
Published: May 2006

The environments of metal ions (Li+, Na+, K+, Ag+, Cs+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+) in proteins and other metal-host molecules have been examined. Regardless of the metal and its precise pattern of ligation to the protein, there is a common qualitative feature to the binding site: the metal is ligated by a shell of hydrophilic atomic groups (containing oxygen, nitrogen, or sulfur atoms) and this hydrophilic shell is embedded within a larger shell of hydrophobic atomic groups (containing carbon atoms). That is, metals bind at centers of high hydrophobicity contrast. This qualitative observation can be described analytically by the hydrophobicity contrast function, C, evaluated from the structure. This function is large and positive for a sphere of hydrophilic atomic groups (characterized by atomic solvation parameters, delta sigma, having values less than 0) at the center of a larger sphere of hydrophobic atomic groups (characterized by delta sigma greater than 0). In the 23 metal-binding molecules we have examined, the maximum values of the contrast function lie near to observed metal binding sites. This suggests that the hydrophobicity contrast function may be useful for locating, characterizing, and designing metal binding sites in proteins.
Complete sequence of human vinculin and assignment of the gene to chromosome 10.
Authors: Weller P A , Ogryzko E P , Corben E B , Zhidkova N I , Patel B , Price G J , Spurr N K , Koteliansky V E , Critchley D R
Published: May 2006

We have determined the complete sequence of human vinculin, a cytoskeletal protein associated with cell-cell and cell-matrix junctions. Comparison of human and chicken embryo vinculin sequences shows that both proteins contain 1066 amino acids and exhibit a high level of sequence identity (greater than 95%). The region of greatest divergence falls within three 112-amino acid repeats spanning residues 259-589. Interestingly, nematode vinculin lacks one of these central repeats. The regions of human vinculin that are N- and C-terminal to the repeats show 54% and 61% sequence identity, respectively, to nematode vinculin. Southern blots of human genomic DNA hybridized with short vinculin cDNA fragments indicate that there is a single vinculin gene. By using a panel of human-rodent somatic cell hybrids, the human vinculin gene was mapped to chromosome 10q11.2-qter.
Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies.
Authors: Akamizu T , Ikuyama S , Saji M , Kosugi S , Kozak C , McBride O W , Kohn L D
Published: May 2006

A rat thyrotropin (thyroid-stimulating hormone, TSH) receptor cDNA was isolated that encoded a protein of 764 amino acids, Mr 86,528. Transfection of the cDNA caused COS-7 cells to develop a TSH-sensitive adenylate cyclase response and the ability to bind 125I-labeled TSH; both activities were similar to those of rat FRTL-5 thyroid cells and not duplicated by lutropin. The gene represented by the cDNA was assigned to mouse chromosome 12 and human chromosome 14. Northern analyses identified two species of mRNA, 5.6 and 3.3 kilobases, in FRTL-5 thyroid cells; the transcripts appeared to differ only in the extent of their 3' noncoding sequences. There were minimal amounts of the two mRNAs in rat ovary, and neither was detected in RNA preparations from rat testis, liver, lung, brain, spleen, and FRT thyroid cells, which do not have a functional TSH receptor. TSH decreased both mRNA species 3- to 4-fold within 8 hr in FRTL-5 thyroid cells; down-regulation was dependent on TSH concentration and duplicated by forskolin, cholera toxin, or 8-bromo-cAMP but not by a phorbol ester. Down-regulation was also duplicated by thyroid-stimulating autoantibodies, which increased cAMP levels, but not by thyrotropin binding-inhibiting auto-antibodies, which actually increased TSH receptor mRNA levels.
Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2.
Authors: Mueller B M , Reisfeld R A , Gillies S D
Published: May 2006

Recombinant techniques allow one to engineer an antibody molecule and, in this way, manipulate its properties and functions. We engineered a chimeric human/mouse antibody to the tumor-associated antigen ganglioside GD2, with the aim of decreasing its serum half-life, maintaining its full antigen-binding capacity, and deleting its effector functions, thus making it a potentially useful reagent for the radioimaging of tumors. To this end, the constant region of the human gamma 1 chain was mutated by deleting the second domain (CH2). Here we show that the CH2-deleted antibody (ch14.18-delta CH2) was cleared from the blood of athymic (nu/nu) mice bearing human melanoma tumors with the same kinetics as human IgG F(ab')2. At a beta t1/2 of 12 hr, 0.9% of the injected dose of 125I-labeled ch14.18-delta CH2 was found per milliliter of blood 24 hr after i.v. injection. In biodistribution experiments, 125I-labeled ch14.18-delta CH2 targeted specifically to melanoma xenografts, achieving optimal tumor-to-tissue ratios 12-16 hr after i.v. injection. ch14.18-delta CH2 was localized to the melanoma tumors more rapidly and with better localization ratios than the intact chimeric antibody ch14.18. Sixteen hours after i.v. injection, the tumor-to-blood and tumor-to-liver ratios of ch14.18-delta CH2 were 5 and 12, respectively, while optimal localization ratios obtained for ch14.18 were 1 and 5, respectively, but 96 hr after injection. A reagent such as ch14.18-delta CH2 should be useful for radioimmunodetection of human tumors because of reduced immunogenicity, increased targeting specificity, and rapid clearance from circulation.
G protein gamma subunits contain a 20-carbon isoprenoid.
Authors: Mumby S M , Casey P J , Gilman A G , Gutowski S , Sternweis P C
Published: May 2006

A small subset of cellular proteins are covalently modified by the addition of isoprenoid groups. These include p21ras, fungal mating factors, and nuclear lamins, which are isoprenylated at carboxyl-terminal cysteine residues with a 15-carbon farnesyl group. The similarity of the carboxyl-terminal sequences of these proteins with the alpha and gamma subunits of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins) prompted examination of isoprenylation of G protein subunits. PC-12 cells were incubated with the isoprenoid precursor [3H]mevalonolactone. The beta and gamma subunits were isolated by specific association with an affinity column of immobilized alpha subunits. The gamma subunits were radiolabeled, and the tritiated lipid released from them by treatment with methyl iodide comigrated chromatographically with the 20-carbon isoprenoid geranylgeraniol. Label was not detected in G protein alpha or beta subunits. Isoprenylation of gamma subunits by the geranylgeranyl group is presumed to contribute to the association of G proteins with membranes.
Mutation in NS2, a nonstructural protein of influenza A virus, extragenically causes aberrant replication and expression of the PA gene and leads to generation of defective interfering particles.
Authors: Odagiri T , Tobita K
Published: May 2006

Several consecutive undiluted passages of infectious virus are usually required to obtain defective interfering particles of influenza virus. In contrast, a reassortant (Wa-182) of influenza A/WSN, which we isolated, whose NS gene was replaced with the NS gene of A/Aichi, was readily converted to defective interfering form after only a single high-multiplicity infection. The defective interfering particles of Wa-182 were devoid of the PA gene (RNA segment 3) but possessed several species of subgenomic RNAs of the PA gene origin. Such aberrant replication of the PA gene was shown to be caused by an extragenic effect of the NS gene of Wa-182, because, when the NS gene of Wa-182 was singly transferred to the wild-type A/Ann Arbor/6/60 virus, the recipient showed exactly the same features. Analysis of nucleotide sequence demonstrated that the NS gene of Wa-182 contained three point mutations relative to the wild-type NS gene that resulted in two amino acid substitutions in the nonstructural protein NS2, suggesting that the mutation in NS2 protein affected the normal replication of the PA gene of Wa-182. The results also suggest that the NS2 protein plays an important role in the synthesis of intact genome RNAs.
Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells.
Authors: Satoh T , Endo M , Nakafuku M , Nakamura S , Kaziro Y
Published: May 2006

The ras gene product (p21) is a GTP-binding protein and is thought to play an important role in signal transduction of growth and differentiation in many types of mammalian cells. The p21.GTP complex is an active conformation, as described previously for polypeptide chain elongation factors (EF-Tu and EF-G) and heterotrimeric GTP-binding proteins (G proteins). In the study reported here, we measured the amounts of p21-bound guanine nucleotides under various conditions in the G54 cell line, a derivative of Swiss 3T3 cells that overexpresses normal c-Ha-ras. More p21.GTP complexes were present in growing cells than in quiescent cells. When quiescent cells were stimulated with fetal bovine serum to promote DNA synthesis, p21.GTP increased approximately 2-fold. Among a number of purified growth factors, platelet-derived growth factor enhanced the formation of p21.GTP, whereas the combination of bombesin and insulin, which also induces DNA synthesis, did not. These results strongly suggest that p21 is a transducer of the growth signal from the platelet-derived growth factor receptor in Swiss 3T3 cells and that the signal is transmitted through a p21.GTP complex.
Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations.
Authors: Pathak V K , Temin H M
Published: May 2006

We determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus (SNV). A method was developed to clone integrated proviruses of retroviral shuttle vectors by exploiting the tight binding of the lac operator to the lac repressor protein. The vectors contained the lacZ alpha gene as a reporter of mutations. Thirty-seven of the 16,867 proviruses recovered contained five classes of mutations, including substitutions and frameshifts. Runs of 9 and 10 identical base pairs and a direct repeat of 110 base pairs were mutational hotspots. In addition, two copies of a provirus contained 15 G-to-A substitutions. Such proviruses, which we name hypermutants, may arise through the action of an error-prone polymerase and could significantly contribute to the genetic variation in retroviral populations.
Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals.
Authors: Frostig R D , Lieke E E , Ts'o D Y , Grinvald A
Published: May 2006

We have shown previously the existence of small, activity-dependent changes in intrinsic optical properties of cortex that are useful for optical imaging of cortical functional architecture. In this study we introduce a higher resolution optical imaging system that offers spatial and temporal resolution exceeding that achieved by most alternative imaging techniques for imaging cortical functional architecture or for monitoring local changes in cerebral blood volume or oxygen saturation. In addition, we investigated the mechanisms responsible for the activity-dependent intrinsic signals evoked by sensory stimuli, and studied their origins and wavelength dependence. These studies enabled high-resolution visualization of cortical functional architecture at wavelengths ranging from 480 to 940 nm. With the use of near-infrared illumination it was possible to image cortical functional architecture through the intact dura or even through a thinned skull. In addition, the same imaging technique proved useful for imaging and discriminating sensory-evoked, activity-dependent changes in local blood volume and oxygen saturation (oxygen delivery). Illumination at 570 nm allowed imaging of activity-dependent blood volume increases, whereas at 600-630 nm, the predominant signal probably originated from activity-dependent oxygen delivery from capillaries. The onset of oxygen delivery started prior to the blood volume increase. Thus, optical imaging based on intrinsic signals is a minimally invasive procedure for monitoring short- and long-term changes in cerebral activity.
Preferential utilization of conserved immunoglobulin heavy chain variable gene segments during human fetal life.
Authors: Schroeder H W , Wang J Y
Published: May 2006

The ability to respond to specific antigens develops in a programmed fashion. Although the antibody repertoire in adults is presumably generated by stochastic combinatorial joining of rearranged heavy variable, diversity, and joining (VH-DH-JH) and light (VL-JL) chains, experimental evidence in the mouse has shown nonrandom utilization of variable gene segments during ontogeny and in response to specific antigens. In this study, we have performed sequence analysis of 104-day human fetal liver-derived, randomly isolated constant region C+ mu transcripts and demonstrate a consistent preference during fetal life for a small subset of three highly conserved VH3 family gene segments. In addition, the data show that this preferential gene segment utilization extends to the DHQ52 and the JH3 and JH4 loci. Sequence analysis of two "sterile" DH-JH transcripts suggests that transcriptional activation of the JH-proximal DHQ52 element may precede initiation of DH-JH rearrangement and influence fetal DH utilization. Sequence comparisons reveal striking nucleotide polymorphism in allelic gene segments which is poorly reflected in the peptide sequence, implying considerable evolutionary selection pressure. Although vertebrate species utilize a variety of strategies to generate their antibody repertoire, preferential utilization of VH3 elements is consistently found during early development. These data support the hypothesis that VH3 gene segments play an essential role in the development of the immune response.
Physicochemical characterization of a polypeptide present in uremic serum that inhibits the biological activity of polymorphonuclear cells.
Authors: Hörl W H , Haag-Weber M , Georgopoulos A , Block L H
Published: May 2006

A granulocyte inhibitory protein was isolated and characterized from uremic serum by using ion-exchange column chromatography, high-performance size-exclusion chromatography, and immunochemical procedures. The purification process concentrated the protein 240-fold and to a purity of greater than 95%. An overall recovery of 45% was achieved; the purified protein had a specific activity of 104 units per mg of protein. The polypeptide had a molecular weight of approximately 28,000 and an isoelectric point of 4.0-4.5. Amino acid sequencing of the NH2 terminus revealed a single sequence (Asp-Ile-Val-Met-Thr-Gln-Ser-Pro-Gly-Thr-Leu-Ser-Val-Ser-Pro-Gly-Glu-Arg-Ala- Thr) that proved to be nonhomologous with other serum proteins that appear during an inflammatory state. The polypeptide inhibited the uptake of deoxyglucose, chemotaxis, oxidative metabolism, and intracellular bacterial killing by polymorphonuclear leukocytes. A specific rabbit polyclonal antibody raised against the protein nullified these inhibitory changes. We contend that the protein is responsible for the leukocyte dysfunction that is commonly seen in patients with uremia.
Early events in hepatitis C virus infection of chimpanzees.
Authors: Shimizu Y K , Weiner A J , Rosenblatt J , Wong D C , Shapiro M , Popkin T , Houghton M , Alter H J , Purcell R H
Published: May 2006

The cytoplasmic antigen and ultrastructural changes we described previously for chimpanzees (Pan troglodytes) infected with hepatitis C virus (HCV) or with hepatitis D virus have recently been shown to be indirect measures of viral replication and appear to represent a host response to the expression or action of interferon. The time of appearance of these changes in hepatocytes during HCV infection, when compared with similar changes in hepatitis D virus infection, suggests a very early replicative phase for HCV. To investigate the early events in HCV infection, we infected two chimpanzees with HCV and obtained blood and liver biopsy samples from them daily during the first 10 days of infection. The early stage of infection with regard to HCV replication, antigen expression, and ultrastructural changes was similar in both chimpanzees. When tested by cDNA/polymerase chain reaction, HCV sequences became detectable in the serum as early as 3 days after inoculation and remained positive through the peak of aminotransferase elevations. In one chimpanzee the peak of virus production appeared to be 7 weeks after inoculation, which was coincident with rising enzyme values. The cytoplasmic antigen, detected by immunofluorescence, and ultrastructural changes, detected by electron microscopy, became positive in hepatocytes 3 and 6 days, respectively, after HCV sequences were first detected in serum. Circulating anti-HCV appeared 13 weeks and 32 weeks after inoculation, respectively, in the chimpanzees. These data indicate a very early replicative phase for HCV and a potentially long period of infectivity before the appearance of anti-HCV.
1 alpha,25-dihydroxyvitamin D3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells.
Authors: Billecocq A , Emanuel J R , Levenson R , Baron R
Published: May 2006

1 alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of the steroid hormone vitamin D, is a potent regulator of macrophage and osteoclast differentiation. The mature osteoclast, unlike the circulating monocyte or the tissue macrophage, expresses high levels of carbonic anhydrase II (CAII). This enzyme generates protons and bicarbonate from water and carbon dioxide and is involved in bone resorption and acid-base regulation. To test whether 1,25(OH)2D3 could induce the differentiation of myelomonocytic precursors toward osteoclasts rather than macrophages, we analyzed its effects on the expression of CAII in bone marrow cultures containing precursors common to both cell types. The expression of CAII was markedly increased by 1,25(OH)2D3 in a dose- and time-dependent manner. In bone marrow, this increase occurred at the mRNA and protein levels and was detectable as early as 24 hr after stimulation. 1,25(OH)2D3 was also found to induce CAII expression in a transformed myelomonocytic avian cell line. These results suggest that 1,25(OH)2D3 regulates the level at which myelomonocytic precursors express CAII, an enzyme that is involved in the function of the mature osteoclast.
Single base mutation in the type II procollagen gene (COL2A1) as a cause of primary osteoarthritis associated with a mild chondrodysplasia.
Authors: Ala-Kokko L , Baldwin C T , Moskowitz R W , Prockop D J
Published: May 2006

A cosmid clone was isolated that contained an allele for the type II procollagen gene previously shown to be coinherited with primary generalized osteoarthritis in a large family. Affected members of the family had evidence of a mild chondrodysplasia, but they developed progressive osteoarthritic changes in many joints that had no epiphyseal deformities. The clone contained 52 of the 54 exons of the gene. Nucleotide sequencing of greater than 20,000 base pairs from the clone demonstrated that all the coding sequences and all the intron-exon boundaries were normal except for a single base mutation that converted the codon for arginine at position 519 of the alpha 1(II) chain to a codon for cysteine, an amino acid not found in type II collagen from humans or a variety of other species. The mutation was found in all affected members of the family but not in unaffected members or in 57 unrelated individuals.
High concentrations of recombinant soluble CD4 are required to neutralize primary human immunodeficiency virus type 1 isolates.
Authors: Daar E S , Li X L , Moudgil T , Ho D D
Published: May 2006

There is substantial evidence supporting the CD4 molecule as the principal cellular receptor for the human immunodeficiency virus type 1 (HIV-1). A number of truncated recombinant soluble CD4 (sCD4) molecules have been produced and shown to easily neutralize infection of laboratory strains of HIV-1 in vitro, and clinical trials using these sCD4 preparations have begun in patients with AIDS. Infectious HIV-1 titers in the plasma and peripheral blood mononuclear cells of five patients receiving sCD4 at 30 mg/day were sequentially monitored. No significant decrease in viral titers was found during therapy. Furthermore, plasma samples from eight patients with AIDS were titrated for HIV-1 with and without the addition of sCD4 ex vivo. Despite the addition of sCD4 at up to 1 mg/ml, there was little change in plasma viral titers. Subsequently, 10 primary HIV-1 isolates were tested for their susceptibility to neutralization in vitro by one preparation of sCD4. Neutralization of these clinical isolates required 200-2700 times more sCD4 than was needed to inhibit laboratory strains of HIV-1. Similar results were observed using one other monomeric sCD4 preparation and two multimeric CD4-immunoglobulin hybrid molecules. We conclude that unlike laboratory strains, primary HIV-1 isolates require high concentrations of sCD4 for neutralization. This phenomenon may pose a formidable problem for sCD4-based therapeutics in the treatment of HIV-1 infection.
Purification of two transcription factors required for initiation by mammalian RNA polymerase II.
Authors: Kawaguchi T , Kitajima S , Niho Y , Oda T , Germino J , Weissman S M , Yasukochi Y
Published: May 2006

We have purified two general transcription factors (FA and FE) necessary for specific transcription by mammalian RNA polymerase II to near homogeneity. Both activities are associated with peptides of approximately 33 kDa. FA and FE do not replace one another and show different kinetics of action in a sarkosyl block assay. In particular, FE participated in a rapid reaction after the formation of an initial complex with the other transcription factors. Furthermore, FE can associate with purified calf thymus RNA polymerase II.
Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.
Authors: Elroy-Stein O , Moss B
Published: May 2006

A mouse cell line that constitutively synthesizes the bacteriophage T7 RNA polymerase was constructed. Fluorescence microscopy indicated that the T7 RNA polymerase was present in the cytoplasmic compartment. The system provided, therefore, a unique opportunity to study structural elements of mRNA that affect stability and translation. The in vivo activity of the bacteriophage polymerase was demonstrated by transfection of a plasmid containing the chloramphenicol acetyltransferase (CAT) gene flanked by T7 promoter and termination signals. Synthesis of CAT was dependent on the presence of a cDNA copy of the untranslated region of encephalomyocarditis virus (ECMV) RNA downstream of the T7 promoter, consistent with the absence of RNA-capping activity in the cytoplasm. CAT expression from a plasmid, pT7EMCAT, containing the T7 and EMCV regulatory elements was detected within 4 hr after transfection and increased during the next 20 hr, exceeding that obtained by transfection of a plasmid with the CAT gene attached to a retrovirus promoter and enhancer. Nevertheless, the presumably cap-independent transient expression of CAT from pT7EMCAT was increased more than 500-fold when the transfected cells also were infected with wild-type vaccinia virus. A protocol for high-level expression involved the infection of the T7 RNA polymerase cell line with a single recombinant vaccinia virus containing the target gene regulated by a T7 promoter and EMCV untranslated region.
Pathway of proton transfer in bacterial reaction centers: replacement of serine-L223 by alanine inhibits electron and proton transfers associated with reduction of quinone to dihydroquinone.
Authors: Paddock M L , McPherson P H , Feher G , Okamura M Y
Published: May 2006

The pathway of proton transfer in the reaction center (RC) from Rhodobacter sphaeroides was investigated by site-directed mutagenesis. Ser-L223, a putative proton donor that forms a hydrogen bond with the secondary quinone acceptor QB, was replaced with Ala and Thr. RCs with Ala-L223 displayed reduced electron transfer and proton uptake rates in the reaction QA-QB- + 2H+----QAQBH2. The rate constant for this reaction, k(2)AB, was found to be reduced approximately 350-fold to 4.0 +/- 0.2 s-1. Proton uptake measurements using a pH indicator dye showed a rapid uptake of 1 H+ per RC followed by a slower uptake of 1 H+ per RC at a rate of 4.1 +/- 0.1 s-1; native RCs showed a rapid uptake of 2H+ per RC. Evidence is provided that these changes were not due to gross structural changes in the binding site of QB. RCs with Thr-L223 showed little reduction in the rates of electron and proton transfer. These results indicate that proton transfer from the hydroxyl group of Ser-L223 or Thr-L223 is required for fast electron and proton transfer associated with the formation of the dihydroquinone QH2. In contrast, previous work showed that replacing Glu-L212, another putative proton donor to QB, with Gln slowed proton uptake from solution without significantly altering electron transfer. We propose a model that involves two distinct proton transfer steps. The first step occurs prior to transfer of the second electron to QB and involves proton transfer from Ser-L223. The second step occurs after this electron transfer through a pathway involving Glu-L212.
Use of brefeldin A to define sites of glycosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to the brefeldin A block.
Authors: Young W W , Lutz M S , Mills S E , Lechler-Osborn S
Published: May 2006

Brefeldin A (BFA) induces the rapid redistribution of the Golgi complex into the endoplasmic reticulum (ER), causing the glycoproteins that are retained in the ER to be processed by Golgi enzymes. We have examined the effects of BFA on the synthesis of glycosphingolipids (GSL) to map the intracellular sites of GSL synthesis. In several cultured cell types, BFA inhibited the synthesis of the neutral GSL gangliotriaosylceramide (GA2) and monosialoganglioside GM2 and disialoganglioside GD2, where GD2 is GalNAc(beta 1----4)- [NeuAc(alpha 2----8)NeuAc(alpha 2----3)]Gal(beta 1----4)GlcCer, GM2 lacks the NeuAc(alpha 2----8) unit, and GA2 lacks both NeuAc(alpha 2----8) and NeuAc(alpha 2----3) units. The observed decrease in labeling of GA2, GM2, and GD2 in the presence of BFA was not due either to enhanced degradation of these glycolipids or to shedding of these glycolipids from the cells. In rat liver all three of these glycolipids have been shown by others to be synthesized by the same enzyme, GA2/GM2/GD2 synthase, which catalyzes the addition of N-acetylgalactosamine to lactosylceramide (Lac-Cer), GM3 [NeuAc(alpha 2----3)Gal(beta 1----4)GlcCer], and GD3 [NeuAc(alpha 2----8)NeuAc-(alpha 2----3)Gal(beta 1----4)GlcCer], respectively. Studies with a fluorescent glycolipid analog indicated that BFA redistributed the trans-Golgi stacks into a reticular pattern characteristic of the ER. These studies localize GA2/GM2/GD2 synthase, a key enzyme involved in the synthesis of complex gangliosides, to a compartment late in the intracellular trafficking pathway, which remains functionally distinct from the ER in the presence of BFA.
Rapid, sensitive analysis of protein mixtures by mass spectrometry.
Authors: Beavis R C , Chait B T
Published: May 2006

We have developed a method for determining the molecular masses of proteins in complex mixtures by mass spectrometry. The method has the capacity to examine the components of mixtures without using any chromatographic separation steps and will tolerate relatively large amounts of buffers and inorganic contaminants. It allows the simultaneous determination of protein molecular masses from 1 to 40 kDa with an accuracy of +/- 0.01% and above 40 kDa with reduced accuracy. The lower limit for practical detection of a protein is a concentration of approximately 0.1 microM, and less than 1 microliter of such a solution is consumed. The analysis is very fast: less than 15 min is necessary to perform the complete analysis, including sample preparation, introduction into the mass spectrometer, mass spectrum collection, and data reduction. The mass spectrum that is obtained does not require elaborate interpretation because there is no fragmentation of the ionized protein (or protein subunit) molecule. Therefore, there is a one-to-one correspondence between the peaks in the mass spectrum and the proteins present in the original mixture. The spectra assume the appearance of chromatograms, with the abscissa being mass-to-charge ratio rather than chromatographic retention time.
Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.
Authors: Key N S , Vercellotti G M , Winkelmann J C , Moldow C F , Goodman J L , Esmon N L , Esmon C T , Jacob H S
Published: May 2006

Latent infection of vascular cells with herpes-viruses may play a pathogenic role in the development of human atherosclerosis. In a previous study, we found that cultured human umbilical vein endothelial cells (HUVECs) infected with herpes simplex virus 1 (HSV-1) became procoagulant, exemplified both by their enhanced assembly of the prothrombinase complex and by their inability to reduce adhesion of platelets. We now report two further procoagulant consequences of endothelial HSV infection: loss of surface thrombomodulin (TM) activity and induction of synthesis of tissue factor. Within 4 hr of infection of HUVECs, TM activity measured by thrombin-dependent protein C activation declined 21 +/- 3% (P less than 0.05) and by 18 hr, 48 +/- 5% (P less than 0.001). Similar significant TM decrements accompanied infection of bovine aortic endothelial cells. Identical TM loss was induced with HSV-2 infection but not with adenovirus infection. Decreased surface expression of TM antigen (measured by the specific binding of a polyclonal antibody to bovine TM) closely paralleled the loss of TM activity. As examined by Northern blotting, these losses apparently reflected rapid onset (within 4 hr of HSV infection) loss of mRNA for TM. In contrast, HSV infection induced a viral-dose-dependent increase in synthesis of tissue factor protein, adding to the procoagulant state. The results indicate that loss of endothelial protein-synthetic capacity is not a universal effect of HSV infection. We suggest that the procoagulant state induced by reduction in TM activity and amplified tissue factor activity accompanying HSV infection of endothelium could contribute to deposition of thrombi on atherosclerotic plaques and to the "coagulant-necrosis" state that characterizes HSV-infected mucocutaneous lesions.
Trans-activation of HLA-DR gene by hepatitis B virus X gene product.
Authors: Hu K Q , Vierling J M , Siddiqui A
Published: May 2006

Hepatocellular injury during hepatitis B virus (HBV) infection has been postulated to result from a human leukocyte antigen (HLA)-restricted T-lymphocyte host immune response against HBV antigens. Although HLA expression is enhanced in the presence of hepatic inflammation, whether HBV itself can induce HLA expression on infected hepatocytes is unknown. In this study, we demonstrate the induction of HLA-DR expression on human hepatoma cell lines transfected with HBV DNA sequences. The HBV X gene alone was capable of inducing HLA-DR expression. This induction correlated with elevated HLA-DR RNA, and this resulted directly from transcriptional trans-activation of the HLA-DR gene by the HBV X protein. These studies suggest that the HBV X protein can regulate the expression of HLA-DR and thus raise the possibility of participation by the X gene in the immunopathogenesis of HBV infection.
High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function.
Authors: Mårtensson J , Lai J C , Meister A
Published: May 2006

Glutathione, an essential cellular antioxidant required for mitochondrial function, is not synthesized by mitochondria but is imported from the cytosol. Rat liver mitochondria have a multicomponent system that underlies the remarkable ability of mitochondria to take up and retain glutathione. At external glutathione levels of less than 1 mM, glutathione is transported into the mitochondrial matrix by a high-affinity component (Km, approximately 60 microM; V max, approximately 0.5 nmol/min per mg of protein), which is saturated at levels of 1-2 mM and stimulated by ATP. Another component has lower affinity (Km, approximately 5.4 mM; Vmax, approximately 5.9 nmol/min per mg of protein) and is stimulated by ATP and ADP. Both components are inhibited by carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), glutamate, and ophthalmic acid. Increase of extramitochondrial glutathione promotes uptake and exchange; the intermembranous space seems to function as a recovery zone that promotes efficient recycling of matrix glutathione. The findings are in accord with in vivo data showing that (i) rapid exchange occurs between mitochondrial and cytosolic glutathione, (ii) lowering of cytosolic glutathione levels (produced by administration of buthionine sulfoximine) decreases export of glutathione from mitochondria to cytosol, and (iii) administration of glutathione esters increases glutathione levels in mitochondria more than those in the cytosol.
Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells.
Authors: Udagawa N , Takahashi N , Akatsu T , Tanaka H , Sasaki T , Nishihara T , Koga T , Martin T J , Suda T
Published: May 2006

We previously reported that osteoclast-like cells were formed in cocultures of a mouse marrow-derived stromal cell line (ST2) with mouse spleen cells in the presence of 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone. In this study, we developed a new coculture system to determine the origin of osteoclasts. When relatively small numbers of mononuclear cells (10(3)-10(5) cells per well) obtained from mouse bone marrow, spleen, thymus, or peripheral blood were cultured for 12 days on the ST2 cell layers, they formed colonies with a linear relationship between the number of colonies formed and the number of hemopoietic cells inoculated. Tartrate-resistant acid phosphatase (TRAPase)-positive mononuclear and multinucleated cells appeared in the colonies (TRAPase-positive colonies) in response to 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone. When hemopoietic cells suspended in a collagen-gel solution were cultured on the ST2 cell layers to prevent their movement, TRAPase-positive colonies were similarly formed, indicating that each colony originated from a single cell. All of the colonies consisted of nonspecific esterase-positive cells. The monocyte-depleted population prepared from peripheral blood failed to form colonies, whereas the monocyte-enriched population produced a large number of TRAPase-positive colonies. In addition, alveolar macrophages formed TRAPase-positive colonies most efficiently on the ST2 cell layers in the presence of the two hormones. Salmon 125I-labeled calcitonin specifically bound to the TRAPase-positive cells. Resorption lacunae were formed on dentine slices on which cocultures were performed. When direct contact between the peripheral blood cells and the ST2 cells was inhibited by a collagen-gel sheet, no TRAPase-positive cells were formed. These results indicate that osteoclasts are also derived from the mature monocytes and macrophages when a suitable microenvironment is provided by bone marrow-derived stromal cells.
Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily.
Authors: Kozma S C , Ferrari S , Bassand P , Siegmann M , Totty N , Thomas G
Published: May 2006

Recently we reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A)+ mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.
An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection.
Authors: Ashorn P , McQuade T J , Thaisrivongs S , Tomasselli A G , Tarpley W G , Moss B
Published: May 2006

The activity of the human immunodeficiency virus (HIV) protease is essential for processing of the gag-pol precursor proteins and maturation of infectious virions. We have prepared a peptidomimetic inhibitor, U-75875, that inhibited HIV-1 gag-pol protein processing in an essentially irreversible manner. Noninfectious virus particles produced in the presence of the drug contained gag precursors and were morphologically immature. In human peripheral blood mononuclear cells and in a continuous cell line, U-75875 completely blocked HIV replication; in the latter case, no spread occurred over a period of 4 weeks. U-75875, on a molar basis, was as potent as 3'-azido-3'-deoxythymidine in blocking HIV-1 replication in human lymphocytes and also inhibited HIV-2 and simian immunodeficiency virus proteases, demonstrating that it has broad activity. These results provide further evidence for the therapeutic potential of protease inhibitors in HIV infection.
cDNA cloning of a quail homeobox gene and its expression in neural crest-derived mesenchyme and lateral plate mesoderm.
Authors: Takahashi Y , Le Douarin N
Published: May 2006

We report here the cloning of a quail cDNA related to the Drosophila gene msh and to the mouse genes Hox-7 and Hox-7.1. For this reason we called this cDNA Quox-7. The amino acid homology of Quox-7 cDNA with the above mentioned genes is high (83%) for the homeobox and its 5'- and 3'-flanking sequences, and the homology is medium (43%) for another stretch of amino acids upstream of the homeobox; elsewhere the sequences of quail and mouse cDNAs have diverged significantly. In quail embryos of day 2-5, Quox-7 transcripts were found essentially in the ventral mesenchyme (neural crest-derived mesectoderm of the face and hypobranchial structures, somatopleure, and limbs) and also in a narrow dorsomedial band of cells of the superficial ectoderm and neural tube. This pattern is fundamentally similar to that reported for Hox-7/7.1, suggesting that the products of these genes play a similar role in the development of the different classes of vertebrates.