bluuebunny/crossref_metadata_2025 huggingface dataset:
Major histocompatibility complex haplotypes and class II genes in non-Jewish patients with pemphigus vulgaris.
Authors: Ahmed A R , Wagner R , Khatri K , Notani G , Awdeh Z , Alper C A , Yunis E J
Published: May 2006

Previous studies demonstrated that HLA-DR4 was markedly increased among Ashkenazi Jewish patients with pemphigus vulgaris (PV), almost entirely as the common Jewish extended haplotype [HLA-B38, SC21, DR4, DQw8] or as the haplotype HLA-B35, SC31, DR4, DQw8, and that HLA-DR4, DQw8 was distributed among patients in a manner consistent with dominant expression of a class II (D-region or D-region-linked) susceptibility gene. In the present study of major histocompatibility complex (MHC) haplotypes in 25 non-Jewish PV patients, DR4, DQw8 was found in 12 of the patients and DRw6, DQw5 was found in 15. Only 3 patients had neither. Only 1 of the DR4, DQw8 haplotypes was [HLA-B38, SC21, DR4, DQw8] and 2 were HLA-B35, SC31, DR4, DQw8; most were the presumed fragments (SC31, DR4, DQw8) or (SC21, DR4, DQw8) or DR4, DQw8 with some other complotype. Of the patients with DRw6, DQw5, all were DRw14, DQw5, and 6 had a rare Caucasian haplotype, HLA-Bw55, SB45, DRw14, DQw5. Four of 6 of these were found in patients of Italian extraction, as was the 1 normal example. The non-Jewish patients were of more Southern European extraction than our controls. This suggests that there are two major MHC susceptibility alleles in American patients with PV. The more ancient apparently arose on a haplotype in the Jews, HLA-B38(35), SC21(SC31), DR4, DQw8, and spread to other populations largely as D-region segments. The other arose in or near Italy on the haplotype HLA-Bw55, SB45, DRw14, DQw5 and has also partially fragmented so that many patients carry only DRw14, DQw5. The available data do not permit the specific localization of either the DR4, DQw8- or the DRw14, DQw5-linked susceptibility genes.
Clusters of clusters: self-organization and self-similarity in the intermediate stages of cluster growth of Au-Ag supraclusters.
Authors: Teo B K , Zhang H
Published: May 2006

A systematic structural investigation of a new series of high-nuclearity Au-Ag clusters containing 25, 37, 38, and 46 metal atoms led to the description of these clusters as "clusters of clusters" based on vertex-sharing icosahedra as building blocks. Based on the observed structures, a growth sequence is proposed here for the formation of these secondary clusters (clusters of clusters) from a single 13-atom icosahedron to a 127-atom icosahedron of icosahedra via successive additions of vertex-sharing icosahedral units. This cluster-of-clusters growth mechanism parallels the atom-by-atom growth pathway for the primary clusters from a single atom to a 13-atom icosahedron. It is hypothesized that the formation of these clusters of clusters is a manifestation of the spontaneous self-organization and self-similarity processes often observed in nature. It is conceivable that the concept of cluster of clusters may be important in the intermediate stages of some cluster growth as exemplified by the polyicosahedral growth of Au-Ag supraclusters.
Periodic and chaotic host-parasite interactions in human malaria.
Authors: Kwiatkowski D , Nowak M
Published: May 2006

It has been recognized since ancient times that malaria fever is highly periodic but the mechanism has been poorly understood. Malaria fever is related to the parasite growth cycle in erythrocytes. After a fixed period of replication, a mature parasite (schizont) causes the infected erythrocyte to rupture, releasing progeny that quickly invade other erythrocytes. Simultaneous rupture of a large number of schizonts stimulates a host fever response. Febrile temperatures are damaging to Plasmodium falciparum, particularly in the second half of its 48-hr replicative cycle. Using a mathematical model, we show that these interactions naturally tend to generate periodic fever. The model predicts chaotic parasite population dynamics at high multiplication rates, consistent with the classical observation that P. falciparum causes less regular fever than other species of parasite.
Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes.
Authors: Thomas L , Leduc R , Thorne B A , Smeekens S P , Steiner D F , Thomas G
Published: May 2006

Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrine-precursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursor-processing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processing-deficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg- sites flanking corticotropin. The -Lys-Arg- within beta-lipotropin was less efficiently cleaved to release beta-endorphin. Expression of PC2 together with PC3 resulted in efficient conversion of beta-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin in the regulated secretory pathway. Finally, the processing selectivities of PC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffin cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.
Possible role for calmodulin and the Ca2+/calmodulin-dependent protein kinase II in postsynaptic neurotransmission.
Authors: Siekevitz P
Published: May 2006

The theory presented here is based on results from in vitro experiments and deals with three proteins in the postsynaptic density/membrane-namely, calmodulin, the Ca2+/calmodulin-dependent protein kinase, and the voltage-dependent Ca2+ channel. It is visualized that, in vivo in the polarized state of the membrane, calmodulin is bound to the kinase; upon depolarization of the membrane and the intrusion of Ca2+, Ca2(+)-bound calmodulin activates the autophosphorylation of the kinase. Calmodulin is visualized as having less affinity for the phosphorylated form of the kinase and is translocated to the voltage-dependent Ca2+ channel. There, with its bound Ca2+, it acts as a Ca2+ sensor, to close off the Ca2+ channel of the depolarized membrane. At the same time, it is thought that the configuration of the kinase is altered by its phosphorylated states; by interacting with Na+ and K+ channels, it alters the electrical properties of the membrane to regain the polarized state. Calmodulin is moved to the unphosphorylated kinase to complete the cycle, allowing the voltage-dependent Ca2+ channel to be receptive to Ca2+ flux upon the next cycle of depolarization. Thus, the theory tries to explain (i) why calmodulin and the kinase reside at the postsynaptic density/membrane site, and (ii) what function autophosphorylation of the kinase may play.
Adsorption/desorption of human serum albumin on hydroxyapatite: a critical analysis of the Langmuir model.
Authors: Mura-Galelli M J , Voegel J C , Behr S , Bres E F , Schaaf P
Published: May 2006

We studied the adsorption of human albumin onto synthetic hydroxyapatite, using a radiotracer technique and a special flow cell. Adsorption was studied under various conditions corresponding to different thermodynamic paths. It appears that (i) as is the usual case, the isotherms obtained within a short time range (a few hours) do not correspond to a true equilibrium situation; (ii) when the adsorption process is followed for longer times, which is necessary at low bulk concentrations, one always reaches the plateau surface adsorption; (iii) this plateau value is independent of the "history" of the adsorption process and corresponds well to the jamming limit predicted by the random sequential adsorption model; and (iv) surface denaturation, leading to enhanced surface binding and thus decreasing desorption constants, is the important phenomenon that can partly and qualitatively explain our observations. Its time dependence, however, remains to be clarified.
DNA looping and Sp1 multimer links: a mechanism for transcriptional synergism and enhancement.
Authors: Mastrangelo I A , Courey A J , Wall J S , Jackson S P , Hough P V
Published: May 2006

Using conventional and scanning transmission electron microscopy, we have examined the physical basis of long-range enhancer effects between distal and proximal elements in a eukaryotic promoter. Specifically, we have studied binding of human transcription factor Sp1 to 10-base-pair G+C-rich elements ("GC boxes") located at -100 and +1700 relative to the RNA start site. It was previously observed that the distantly located site functions in synergism with the promoter-proximal site to strongly activate transcription in vivo. Here we demonstrate that this synergism is likely to be a direct consequence of interactions between remote and local Sp1, the remote Sp1 translocated to the promoter by a DNA loop. Scanning transmission electron microscopy shows that Sp1 initially forms a tetramer and subsequently assembles multiple tetramers stacked in register at the DNA loop juncture. This unexpected finding not only provides the physical basis for loop formation but also defines a biological process leading to strongly increased concentration of activator protein at the promoter. The mechanism may unify the problem of transcriptional activation by removing enhancer action as a separate class of regulatory activity.
Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica).
Authors: Miura T , Yamauchi K , Takahashi H , Nagahama Y
Published: May 2006

The importance of gonadotropins and androgens for spermatogenesis is generally accepted in vertebrates, but the role played by specific hormones has not been clarified. Under cultivation conditions, male Japanese eels (Anguilla japonica) have immature testes containing only premitotic spermatogonia, type A and early-type B spermatogonia. In the present study, a recently developed organ-culture system for eel testes was used to determine in vitro effects of various steroid hormones on spermatogenesis. After 9 days of culture in serum-free, chemically defined medium containing 11-ketotestosterone (10 ng/ml), a major androgen in male eels, type A and early-type B spermatogonia began mitosis, producing late-type B spermatogonia. After 18 days, zygotene spermatocytes with synaptonemal complexes appeared, indicating that meiosis had already started by this time. In testis fragments cultured for 21 days, round spermatids and spermatozoa were observed with spermatogenic cells at all stages of development. Addition of 11-ketotestosterone to the culture medium also caused a marked cytological activation of Sertoli cells. No other steroid hormones tested had such stimulatory effects. These results, together with our earlier observations, suggest the following sequence for the hormonal induction of spermatogenesis in eel testes; gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone, which, in turn, activates the Sertoli cells leading to the completion of spermatogenesis. This is, thus, an example of an animal system in which all stages of spermatogenesis have been induced by hormonal manipulation in vitro.
Heterozygosity at individual amino acid sites: extremely high levels for HLA-A and -B genes.
Authors: Hedrick P W , Whittam T S , Parham P
Published: May 2006

The amino acid heterozygosities per site for HLA-A and -B loci are determined to be extremely high by combining population serotypic frequencies with amino acid sequences. For the 54 amino acid sites thought to have functional importance, the average heterozygosity per site is 0.301. Sixteen positions have heterozygosities greater than 0.5 at one or both loci and the frequencies of amino acids at a given position are very even, resulting in nearly the maximum heterozygosity possible. Furthermore, the high heterozygosity is concentrated in the peptide-interacting sites, whereas the sites that interact with the T-cell receptor have lower heterozygosity. Overall, these results indicate the importance of some form of balancing selection operating at HLA loci, maybe even at the individual amino acid level.
Substitution of leucine for isoleucine in a sequence highly conserved among retroviral envelope surface glycoproteins attenuates the lytic effect of the Friend murine leukemia virus.
Authors: Sitbon M , d'Auriol L , Ellerbrok H , André C , Nishio J , Perryman S , Pozo F , Hayes S F , Wehrly K , Tambourin P
Published: May 2006

Friend murine leukemia virus is a replication-competent retrovirus that contains no oncogene and that exerts lytic and leukemogenic properties. Thus, newborn mice inoculated with Friend murine leukemia virus develop severe early hemolytic anemia before appearance of erythroleukemia. To identify the retroviral determinants regulating these effects, we used chimeric infectious constructions and site-directed point mutations between a virulent Friend murine leukemia virus strain and a naturally occurring variant attenuated in lytic and leukemogenic effects. We found that severe hemolytic anemia was always associated with higher numbers of blood reticulocytes with budding retroviral particles. Furthermore, a remarkably conservative leucine to isoleucine change in the extracellular SU component of the retroviral envelope was sufficient to attenuate this lytic effect. Also, this leucine at position 348 of the envelope precursor protein was located within the only stretch of five amino acids that is conserved in the extracellular SU component of all murine, feline, and primate type C and type D retroviral envelopes. This observation suggested an important structural function for this yet undescribed conserved sequence of the envelope. Lastly, we observed that lytic and leukemogenic effects were attenuated by a deletion of a second repeat in the transcriptional enhancer region of the viral long terminal repeats of the variant strain.
Molecular mimicry by Trypanosoma cruzi: the F1-160 epitope that mimics mammalian nerve can be mapped to a 12-amino acid peptide.
Authors: Van Voorhis W C , Schlekewy L , Trong H L
Published: May 2006

Antigenic mimicry by Trypanosoma cruzi antigens that share epitopes with mammalian tissues may drive autoreactive B- or T-cell clones to expand and cause autoimmune pathogenesis. We have been studying one of these antigens, F1-160, a 160-kDa protein on the surface of T. cruzi that antigenically mimics a 48-kDa protein found in mammalian axonal and myenteric plexus cells. The F1-160 antigen has been characterized by cloning and expression of T. cruzi DNA encoding F1-160 in Escherichia coli. Recombinant peptides from various regions of the F1-160 gene were expressed and used to compete with affinity-purified polyclonal anti-F1-160 antibodies binding to nerve. Recombinant 48-amino acid peptide (48X) derived from expression of base pairs 611-761 of the DNA sequence completely inhibited anti-F1-160 binding to nerve. Recombinant peptides expressed from DNA lacking this region did not inhibit anti-F1-160 binding to nerve. Three peptides were synthesized to encompass the 48X peptide, a 12-amino acid peptide and two 18-amino acid peptides. The 12-amino acid peptide TPQRKTTEDRPQ (12X), corresponding to bases 615-651, completely inhibited the binding of anti-F1-160 antibodies to nerve at a concentration of 80 ng/ml (30 microM). The two 18-residue peptides did not inhibit, even at 10 micrograms/ml. Thus, the epitope of F1-160 crossreactive with nervous tissue can be mapped to a 12-amino acid peptide. Some humans with T. cruzi infection make antibodies to F1-160 and to the 48X and 12X peptides. Control sera from uninfected persons did not react with these antigens. Anti-48X antibodies, immunoselected from human serum with 48X peptide, bind to human nerve axons. This demonstrates that some individuals infected with T. cruzi make antibodies to the F1-160 epitope crossreactive with nervous tissues.
Fibroblast cell interactions with human melanoma cells affect tumor cell growth as a function of tumor progression.
Authors: Cornil I , Theodorescu D , Man S , Herlyn M , Jambrosic J , Kerbel R S
Published: May 2006

It is known from a variety of experimental systems that the ability of tumor cells to grow locally and metastasize can be affected by the presence of adjacent normal tissues and cells, particularly mesenchymally derived stromal cells such as fibroblasts. However, the comparative influence of such normal cell-tumor cell interactions on tumor behavior has not been thoroughly investigated from the perspective of different stages of tumor progression. To address this question we assessed the influence of normal dermal fibroblasts on the growth of human melanoma cells obtained from different stages of tumor progression. We found that the in vitro growth of most (4 out of 5) melanoma cell lines derived from early-stage radial growth phase or vertical growth phase metastatically incompetent primary lesions is repressed by coculture with normal dermal fibroblasts, suggesting that negative homeostatic growth controls are still operative on melanoma cells from early stages of disease. On the other hand, 9 out of 11 melanoma cell lines derived from advanced metastatically competent vertical growth phase primary lesions, or from distant metastases, were found to be consistently stimulated to grow in the presence of dermal fibroblasts. Evidence was obtained to show that this discriminatory fibroblastic influence is mediated by soluble inhibitory and stimulatory growth factor(s). Taken together, these results indicate that fibroblast-derived signals can have antithetical growth effects on metastatic versus metastatically incompetent tumor subpopulations. This resultant conversion in responsiveness to host tissue environmental factors may confer upon small numbers of metastatically competent cells a growth advantage, allowing them to escape local growth constraints both in the primary tumor site and at distant ectopic tissue sites.
The number of positively charged amino acids in the basic domain of Tat is critical for trans-activation and complex formation with TAR RNA.
Authors: Delling U , Roy S , Sumner-Smith M , Barnett R , Reid L , Rosen C A , Sonenberg N
Published: May 2006

The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines.
Molecular cloning of a membrane-associated human FK506- and rapamycin-binding protein, FKBP-13.
Authors: Jin Y J , Albers M W , Lane W S , Bierer B E , Schreiber S L , Burakoff S J
Published: May 2006

The 12-kDa FK506-binding protein (FKBP-12) is a cytosolic receptor for the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and subcellular localization of a 13-kDa FKBP (FKBP-13), which has a 21-amino acid signal peptide and appears to be membrane-associated. Although no internal hydrophobic region, and thus no transmembrane domain, is apparent within the 120 amino acids of mature FKBP-13, a potential endoplasmic reticulum retention sequence (Arg-Thr-Glu-Leu) is found at its C terminus. FKBP-13 has 51% nucleotide sequence identity and 43% amino acid sequence identity to FKBP-12; the N-terminal sequences are divergent, but the 92-amino acid C-terminal sequence of FKBP-13 has 46 identical and 20 related residues when compared with FKBP-12. The conserved residues that comprise the drug binding site and rotamase active site of FKBP-12 are completely conserved in FKBP-13. Therefore, the three-dimensional structures of FKBP-12 and the FKBP-12/FK506 complex are likely to be excellent models of the corresponding FKBP-13 structure.
Neuronal activity up-regulates astroglial gene expression.
Authors: Steward O , Torre E R , Tomasulo R , Lothman E
Published: May 2006

Neuronal gene expression is known to be modulated by functional activity. This modulation is thought to play a key role in determining the differentiation of developing neurons and regulating the operation of mature neurons. Here we describe a regulation of astroglial gene expression by neuronal activity. We report that intense neuronal activity (electrically induced seizures) in rat hippocampus leads to rapid and dramatic increases in mRNA for glial fibrillary acidic protein (GFAP), an astroglia-specific intermediate filament protein. GFAP mRNA levels increased at sites of stimulation as well as in areas that were synaptically activated by the resultant seizures. When seizures were induced repetitively for many days, levels of GFAP mRNA remained chronically elevated. However, GFAP mRNA returned to control levels within a few days after the cessation of stimulation. The coupling between astroglial gene expression and neuronal activity may be a mechanism through which neuronal activity modulates the function of supporting cells that are responsible for regulating the extracellular microenvironment of the brain.
Human type VII collagen: cDNA cloning and chromosomal mapping of the gene.
Authors: Parente M G , Chung L C , Ryynänen J , Woodley D T , Wynn K C , Bauer E A , Mattei M G , Chu M L , Uitto J
Published: May 2006

A human keratinocyte cDNA expression library in bacteriophage lambda gt11 was screened with the purified IgG fraction of serum from a patient with epidermolysis bullosa acquisita, which had a high titer of anti-type VII collagen antibodies. Screening of approximately 3 x 10(5) plaques identified 8 positive clones, the largest one (K-131) being approximately 1.9 kilobases in size. Dideoxynucleotide sequencing of K-131 indicated that it consisted of 1875 base pairs and contained an open reading frame coding for a putative N-terminal noncollagenous domain of 439 amino acids and a collagenous C-terminal segment of 186 amino acids. The collagenous domain was characterized by repeating Gly-Xaa-Yaa sequences that were interrupted in several positions by insertions or deletions of 1-3 amino acids. The deduced amino acid sequence also revealed a peptide segment that had a high degree of identity with a published type VII collagen protein sequence. Northern hybridization of the K-131 cDNA with human epidermal keratinocyte and skin fibroblast RNA revealed an mRNA of approximately 8.5 kilobases. The fusion protein produced by the K-131 cDNA, when incubated with epidermolysis bullosa acquisita serum, bound to antibodies that reacted in Western blots with type VII collagen. The genomic location of the type VII collagen gene (COL7A1) was determined by chromosomal in situ hybridization with the K-131 cDNA. The results mapped the COL7A1 to the locus 3p21. The cDNA clones characterized in this study will be valuable for understanding the protein structure and gene expression of type VII collagen present in anchoring fibrils and its aberrations in the dystrophic forms of heritable epidermolysis bullosa.
Large-scale functional reorganization in adult monkey cortex after peripheral nerve injury.
Authors: Garraghty P E , Kaas J H
Published: May 2006

In adult monkeys, peripheral nerve injuries induce dramatic examples of neural plasticity in somatosensory cortex. It has been suggested that a cortical distance limit exists and that the amount of plasticity that is possible after injury is constrained by this limit. We have investigated this possibility by depriving a relatively large expanse of cortex by transecting and ligating both the median and the ulnar nerves to the hand. Electrophysiological recording in cortical areas 3b and 1 in three adult squirrel monkeys no less than 2 months after nerve transection has revealed that cutaneous responsiveness is regained throughout the deprived cortex and that a roughly normal topographic order is reestablished for the reorganized cortex.
Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles.
Authors: Mortensen R M , Zubiaur M , Neer E J , Seidman J G
Published: May 2006

The alpha i2 subunit of the inhibitory heterotrimeric guanine nucleotide-binding proteins is highly conserved in mammals and is expressed in all cell types, but its exact function is not yet defined. We have investigated the role of this protein by producing embryonic stem (ES) cells lacking a functional alpha i2 gene. These alpha i2-null cell lines regulate adenylyl cyclase and grow and differentiate in vitro the same as wild-type ES cells. Homologous recombination was used to sequentially inactivate both copies of the alpha i2 gene. The first allele was inactivated by insertion of a neomycin-resistance gene. We modified the hygromycin B-resistance gene for improved expression in ES cells and used this gene to inactivate the remaining normal allele. The techniques used should be generally applicable to other genes whether or not they are expressed in ES cells.
Inhibition of calcium influx and calcium current by gamma-aminobutyric acid in single synaptic terminals.
Authors: Heidelberger R , Matthews G
Published: May 2006

Inhibition of Ca influx and Ca current by gamma-aminobutyric acid (GABA) was studied in single synaptic terminals of isolated retinal bipolar neurons. Measurements of intracellular Ca concentration [( Ca]i) using the fluorescent Ca indicator fura-2 showed that GABA potently inhibited Ca influx into the terminal elicited by high extracellular K concentration ([K]o). This inhibition was attributed to GABA type A (GABAA) receptor-activated chloride ion conductance that prevented bipolar neurons from depolarizing sufficiently to activate the Ca current, even in response to increased [K]o. Patch-clamp recordings of the Ca current revealed a second effect of GABA: GTP-dependent inhibition of the Ca current. This inhibition was not mediated by GABAA receptors, but baclofen, which binds to the GABA type B (GABAB) receptor and is known to inhibit the Ca current in other systems, was not able to mimic the action of GABA. This suggests the involvement of a different type of GABAB-like receptor in the inhibition of Ca current by GABA. GABA did not cause an overall suppression of the Ca current; rather, the voltage-dependence of Ca-channel activation was shifted to more depolarized potentials. Thus, maximal inhibition of the Ca current by GABA occurred in the physiological range of potential.
Human liver glucokinase gene: cloning and sequence determination of two alternatively spliced cDNAs.
Authors: Tanizawa Y , Koranyi L I , Welling C M , Permutt M A
Published: May 2006

A human liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) cDNA was isolated from a liver cDNA library. This cDNA (hLGLK1) appeared to be full length [2548 base pairs (bp) plus additional poly(A) residues], as its size was consistent with a single 2.8-kilobase (kb) glucokinase mRNA on Northern blot analysis of liver poly(A)+ RNA. The cDNA contained an open reading frame of 1392 bp that predicted a protein of 464 amino acids and a molecular mass of 52 kDa; this protein has 97% identity to rat liver glucokinase. Fourteen residues on the amino terminus of the predicted human liver glucokinase, however, differed completely from those of the predicted rat liver enzyme and could be explained by alternative splicing of a 124-bp cassette exon in human cDNA. A second glucokinase cDNA (hLGLK2), missing the 124-bp cassette exon, was isolated by PCR amplification of human liver cDNA. The hLGLK2 cDNA contained an open reading frame of 1398 bp from an ATG codon at position 164, encoding a predicted protein of 466 residues, 98% identical to the rat enzyme, but different from the predicted protein of hLGLK1 cDNA by 16 amino-terminal residues. In contrast, hLGLK1 cDNA contains multiple initiator codons upstream of the predicted initiator codon at position 294 within the cassette exon. Translation of the two mRNAs in vitro by a reticulocyte lysate system resulted in proteins of the expected size (52 kDa) for both mRNAs; yet hLGLK2 mRNA was translated four to six times more efficiently. These results suggested that the alternative splicing of a cassette exon in hLGLK1 resulted in an mRNA with an upstream initiator codon and reduced function. The relative biological activity of the two isoforms of human glucokinase and their possible developmental and/or metabolic regulation remain to be determined.
Modeling the cell division cycle: cdc2 and cyclin interactions.
Authors: Tyson J J
Published: May 2006

The proteins cdc2 and cyclin form a heterodimer (maturation promoting factor) that controls the major events of the cell cycle. A mathematical model for the interactions of cdc2 and cyclin is constructed. Simulation and analysis of the model show that the control system can operate in three modes: as a steady state with high maturation promoting factor activity, as a spontaneous oscillator, or as an excitable switch. We associate the steady state with metaphase arrest in unfertilized eggs, the spontaneous oscillations with rapid division cycles in early embryos, and the excitable switch with growth-controlled division cycles typical of nonembryonic cells.
Differential expression of epidermal growth factor-related proteins in human colorectal tumors.
Authors: Ciardiello F , Kim N , Saeki T , Dono R , Persico M G , Plowman G D , Garrigues J , Radke S , Todaro G J , Salomon D S
Published: May 2006

Amphiregulin (AR) and cripto are proteins that are structurally related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is also functionally related to this family of growth regulatory molecules and is able to bind and activate the 170-kDa EGF receptor (EGFR). Human EGFR-3 (HER3)/ERBB3 is a recently identified protein related to the EGFR that is widely expressed in breast carcinomas and is a candidate receptor for EGF-like growth factors. Differential expression of these putative ligands and receptors in transformed cells suggests that they may function in an autocrine manner to regulate tumor cell growth. Specific mRNA transcripts for TGF-alpha [4.8 kilobases (kb)], AR (1.4 kb), cripto (2.2 kb), and HER3 (6.2 kb) were expressed in a majority of human colon cancer cell lines. HER3 mRNA was detected in 55% of primary or metastatic human colorectal carcinomas but in only 22% of normal colon mucosa and 32% of normal liver samples. In contrast, cripto and AR mRNA were expressed in 60-70% of primary or metastatic human colorectal cancers but in only 2-7% of normal human colonic mucosa. Immunostaining also detected AR protein in primary and metastatic colorectal tumors but not in normal colon or uninvolved liver. These findings suggest that cripto and AR may be useful markers to discriminate between normal and malignant colonic epithelium and may provide a selective growth advantage for colorectal carcinomas.
Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues.
Authors: Dawson T M , Bredt D S , Fotuhi M , Hwang P M , Snyder S H
Published: May 2006

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.
Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy.
Authors: Huber B E , Richards C A , Krenitsky T A
Published: May 2006

An approach involving retroviral-mediated gene therapy for the treatment of neoplastic disease is described. This therapeutic approach is called "virus-directed enzyme/prodrug therapy" (VDEPT). The VDEPT approach exploits the transcriptional differences between normal and neoplastic cells to achieve selective killing of neoplastic cells. We now describe development of the VDEPT approach for the treatment of hepatocellular carcinoma. Replication-defective, amphotrophic retroviruses were constructed containing a chimeric varicella-zoster virus thymidine kinase (VZV TK) gene that is transcriptionally regulated by either the hepatoma-associated alpha-fetoprotein or liver-associated albumin transcriptional regulatory sequences. Subsequent to retroviral infection, expression of VZV TK was limited to either alpha-fetoprotein- or albumin-positive cells, respectively. VZV TK metabolically activated the nontoxic prodrug 6-methoxypurine arabinonucleoside (araM), ultimately leading to the formation of the cytotoxic anabolite adenine arabinonucleoside triphosphate (araATP). Cells that selectively expressed VZV TK became selectively sensitive to araM due to the VZV TK-dependent anabolism of araM to araATP. Hence, these retroviral-delivered chimeric genes generated tissue-specific expression of VZV TK, tissue-specific anabolism of araM to araATP, and tissue-specific cytotoxicity due to araM exposure. By utilizing such retroviral vectors, araM was anabolized to araATP in hepatoma cells, producing a selective cytotoxic effect.
Measurement of ligand-receptor interactions.
Authors: Helm C A , Knoll W , Israelachvili J N
Published: May 2006

One distinguishing feature of "life" is that the physical forces between biological molecules and membrane surfaces are often highly specific, in contrast to nonspecific interactions such as van der Waals, hydrophobic, and electrostatic (Coulombic) forces. We have used the surface-forces-apparatus technique to study the specific "lock and key" or "ligand-receptor" interaction between two model biomembrane surfaces in aqueous solution. The membranes were lipid bilayers supported on mica surfaces; one carrying streptavidin receptors, the other exposing biotin ligand groups. We found that, although no unusual or specific interaction occurs between two avidin or two biotin surfaces, an avidin and a biotin surface exhibit a very strong, very short-range (less than 1 nm) attraction and that the binding mechanism involves equally specific molecular rearrangements. The results also show that highly specific biological interactions such as are involved in immunological recognition and cell-cell contacts may be studied at the molecular level and in real time by the surface-forces-apparatus technique.
Mechanism of synergistic transcriptional transactivation by the human glucocorticoid receptor.
Authors: Wright A P , Gustafsson J A
Published: May 2006

Induction of transcription from a promoter with two upstream glucocorticoid response elements is 10- to 20-fold greater than that from a similar promoter with only one response element. We have shown that interactions involving the major transactivation domain of the glucocorticoid receptor (tau 1) are the sole determinant of such synergistic transactivation by the receptor. The other transactivation domain of the receptor (tau 2) did not mediate synergistic transactivation, and therefore the ability to synergize is operationally distinct from the transactivation function per se. The level of synergistic transactivation observed in vivo can be accounted for by the level of cooperative DNA binding seen in vitro for a glucocorticoid receptor derivative containing only the tau 1 and DNA-binding domains. Cooperative DNA binding was also observed using a tau 1-DNA-binding domain protein, which was expressed in Escherichia coli and extensively purified. Therefore, it is likely that direct protein-protein interactions between tau 1 domains mediate the cooperative DNA binding. The role of cooperative DNA binding for synergistic transactivation in vivo is discussed in relation to other possible mechanisms.
Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins.
Authors: Kornberg L J , Earp H S , Turner C E , Prockop C , Juliano R L
Published: May 2006

The integrin family of cell adhesion receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound influences on cell behavior, it seems likely that integrins transduce biochemical signals across the cell membrane. The nature of these putative signals has, thus far, remained elusive. Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-beta 1 integrin monoclonal antibody for 30 min on ice followed by incubation at 37 degrees C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115- to 130-kDa complex of proteins termed pp130. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, pp130 showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal pp130 phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. pp130 phosphorylation depended on the amount of anti-integrin antibody present. Additionally, the tyrosine phosphorylation of pp130 showed specificity since it was stimulated by antibodies to the integrin alpha 3 and beta 1 subunits but not by antibodies to other integrin alpha subunits or to nonintegrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that pp130 is not itself a beta 1 integrin. It is postulated, therefore, that the integrin-stimulated tyrosine phosphorylation of pp130 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior.
Sp1-dependent activation of a synthetic promoter by human immunodeficiency virus type 1 Tat protein.
Authors: Kamine J , Subramanian T , Chinnadurai G
Published: May 2006

The Tat protein coded by human immunodeficiency virus (HIV) is a strong activator of viral gene expression from the long terminal repeat (LTR). It appears that Tat-mediated trans-activation of the HIV LTR is predominantly a transcriptional event. It has been reported that Tat acts at the level of both transcriptional initiation and elongation through interaction with a nascent RNA target sequence termed TAR (for trans-activation response element). However, the precise mechanism(s) by which Tat mediates TAR-dependent transcriptional activity is not known. To determine whether Tat functions similarly to other eukaryotic transcriptional activators through any of the conventional promoter elements, we tested Tat activity on synthetic promoters containing consensus sequences required for binding transcription factor Sp1 and a TATA box. Here, we report that a chimeric Tat protein targeted to the promoter region by the DNA-binding domain of yeast transcription factor GAL4 activates the synthetic promoter. Because this trans-activation depends on Sp1-binding sites, Tat can apparently mediate transcriptional activation through its interaction with Sp1. Mutational analysis of the gal4-tat chimeric gene reveals that the N-terminal 48-amino acid region of Tat constitutes the activation region for Sp1-dependent trans-activation. This region of Tat exhibits substantially more activity than the N-terminal 58 amino acids of Tat, which includes the arginine-rich basic region. Effects of specific mutations in the 48-amino acid Tat region of GAL4-Tat on trans-activation of the synthetic promoter mimic the effects of these specific mutations on Tat-mediated trans-activation of the HIV-1 LTR, suggesting that trans-activation of both the synthetic promoter and the intact LTR occurs by a common mechanism.
Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.
Authors: Orci L , Ravazzola M , Meda P , Holcomb C , Moore H P , Hicke L , Schekman R
Published: May 2006

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.
X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution.
Authors: Kuriyan J , Kong X P , Krishna T S , Sweet R M , Murgolo N J , Field H , Cerami A , Henderson G B
Published: May 2006

Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidine conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. We report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from -2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4 degrees in the domains that form the site, with relative shifts of as much as 2-3 A in residue positions. These results provide a detailed view of the residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.
Solvent reorganizational red-edge effect in intramolecular electron transfer.
Authors: Demchenko A P , Sytnik A I
Published: May 2006

Polar solvents are characterized by statistical distributions of solute-solvent interaction energies that result in inhomogeneous broadening of the solute electronic spectra. This allows photoselection of the high interaction energy part of the distribution by excitation at the red (long-wavelength) edge of the absorption bands. We observe that intramolecular electron transfer in the bianthryl molecule from the locally excited (LE) to the charge-transfer (CT) state, which requires solvent relaxation and does not occur in vitrified polar solutions, is dramatically facilitated in low-temperature propylene glycol glass by the red-edge excitation. This allows one to obtain spectroscopically the pure CT form and observe its dependence upon the relaxational properties of the solvent. A qualitative potential model of this effect is presented.
Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa.
Authors: Dryja T P , Hahn L B , Cowley G S , McGee T L , Berson E L
Published: May 2006

We searched for point mutations in every exon of the rhodopsin gene in 150 patients from separate families with autosomal dominant retinitis pigmentosa. Including the 4 mutations we reported previously, we found a total of 17 different mutations that correlate with the disease. Each of these mutations is a single-base substitution corresponding to a single amino acid substitution. Based on current models for the structure of rhodopsin, 3 of the 17 mutant amino acids are normally located on the cytoplasmic side of the protein, 6 in transmembrane domains, and 8 on the intradiscal side. Forty-three of the 150 patients (29%) carry 1 of these mutations, and no patient has more than 1 mutation. In every family with a mutation so far analyzed, the mutation cosegregates with the disease. We found one instance of a mutation in an affected patient that was absent in both unaffected parents (i.e., a new germ-line mutation), indicating that some "isolate" cases of retinitis pigmentosa carry a mutation of the rhodopsin gene.