bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat liver endocytic coated vesicles do not exhibit ATP-dependent acidification in vitro.
Authors: Fuchs R , Ellinger A , Pavelka M , Mellman I , Klapper H
Published: May 2006

Coated vesicle fractions from a variety of tissues have been found to contain a vacuolar proton ATPase. Since these fractions contain both plasma membrane- and Golgi-derived coated vesicles, we sought to determine specifically whether endocytic coated vesicles from rat liver contain an active vacuolar proton ATPase. Endocytic vesicles (coated vesicles and endosomes) were selectively labeled with pH-sensitive endocytic tracers (fluorescein isothiocyanate-dextran or -asialoorsomucoid). Coated vesicles were then separated from endosomes by sucrose density gradient centrifugation. Although the endosomal fractions were found to exhibit significant ATP-dependent acidification activity, highly purified coated vesicles containing pH-sensitive endocytic tracers were unable to generate a pH gradient in response to ATP addition. The coated vesicles could be passively acidified, however, by creating potassium diffusion potentials, indicating that they were in fact capable of maintaining proton gradients. Moreover, significant ATP-dependent acidification activity was observed when the coated vesicle fractions were assayed using the nonselective externally added pH probe acridine orange. Thus, it appears that rat liver endocytic coated vesicles do not contain a functional proton pump. The active vacuolar proton ATPase found in these fractions instead reflected the presence of Golgi-derived coated vesicles or contaminating membranes.
The calcitonin gene is expressed in salmon gills.
Authors: Martial K , Maubras L , Taboulet J , Jullienne A , Berry M , Milhaud G , Benson A A , Moukhtar M S , Cressent M
Published: May 2006

Calcitonin is an important physiological regulator of salmon gills. Although the calcitonin receptor was found in salmon gills, the critical question concerning the source of the hormone remained unanswered. In this communication, evidence is presented for expression of calcitonin mRNA and its encoded peptide in gills of the pink salmon, Oncorhynchus gorbuscha. The expression of calcitonin gene transcripts was demonstrated by reverse transcription-polymerase chain reaction, Southern hybridization, and sequencing. The sequencing identified a sequence corresponding to that of exon 4 of the salmon calcitonin gene. Expression of the encoded calcitonin gene in gills was detected by radioimmunoassay in gill extracts. This synthesis of calcitonin in gills, which also possess specific receptors to the peptide, suggests function of an autocrine or paracrine process producing calcitonin in this tissue. These observations confirm and extend previous reports on the physiological role of calcitonin in fish gills.
Specific association between the human DNA repair proteins XPA and ERCC1.
Authors: Li L , Elledge S J , Peterson C A , Bales E S , Legerski R J
Published: May 2006

Processing of DNA damage by the nucleotide-excision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specifically interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that mediate this interaction. These results suggest that XPA may play a role in the localization or loading of an incision complex, composed of ERCC1 and possibly other repair factors, onto a damaged site.
Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.
Authors: Fu C , Javedan S , Moshiri F , Maier R J
Published: May 2006

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product.
Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.
Authors: Savard L , Li P , Strauss S H , Chase M W , Michaud M , Bousquet J
Published: May 2006

We have estimated the time for the last common ancestor of extant seed plants by using molecular clocks constructed from the sequences of the chloroplastic gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the nuclear gene coding for the small subunit of rRNA (Rrn18). Phylogenetic analyses of nucleotide sequences indicated that the earliest divergence of extant seed plants is likely represented by a split between conifer-cycad and angiosperm lineages. Relative-rate tests were used to assess homogeneity of substitution rates among lineages, and annual angiosperms were found to evolve at a faster rate than other taxa for rbcL and, thus, these sequences were excluded from construction of molecular clocks. Five distinct molecular clocks were calibrated using substitution rates for the two genes and four divergence times based on fossil and published molecular clock estimates. The five estimated times for the last common ancestor of extant seed plants were in agreement with one another, with an average of 285 million years and a range of 275-290 million years. This implies a substantially more recent ancestor of all extant seed plants than suggested by some theories of plant evolution.
Expression of cytokeratin confers multiple drug resistance.
Authors: Bauman P A , Dalton W S , Anderson J M , Cress A E
Published: May 2006

The cytokeratin network is an extensive filamentous structure in the cytoplasm whose biological function(s) is unknown. Based upon previous data showing the modification of cytokeratin by mitoxantrone, we investigated the ability of cytokeratin networks to influence the survival response of cells to chemotherapeutic agents. We have compared the survival of mouse L fibroblasts lacking cytokeratins with that of L cells transfected with cytokeratins 8 and 18 in the presence of chemotherapeutic drugs. The expression of cytokeratins 8 and 18 conferred a multiple drug resistance phenotype on cells exposed to mitoxantrone, doxorubicin, methotrexate, melphalan, Colcemid, and vincristine. The degree of drug resistance was 5-454 times that of parental cells, depending upon the agent used. Drug resistance could not be attributed to altered growth characteristics, altered drug accumulation, or an altered drug efflux in the transfected cells. Cytokeratin does not confer resistance to ionizing radiation, which damages DNA independently of intracellular transport mechanisms. These data suggest a role for cytokeratin networks in conferring a drug resistance phenotype.
p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein.
Authors: Slebos R J , Lee M H , Plunkett B S , Kessis T D , Williams B O , Jacks T , Hedrick L , Kastan M B , Cho K R
Published: May 2006

The cell cycle regulatory tumor suppressor proteins p53 and pRB are targeted for inactivation by several tumor viruses, including the high-risk types of human papillomaviruses (HPVs) via interactions of the HPV E6 and E7 oncoproteins with p53 and pRB, respectively. p53 plays a central role in a signal transduction pathway that mediates G1 arrest after DNA damage, though the mechanism by which G1 arrest occurs has not been elucidated. The cyclin-associated protein p21waf1/cip1 has recently been shown to be induced by p53 and to inhibit cyclin complex-mediated phosphorylation of pRB in vitro. Thus, we investigated a possible role for pRB in the p53-mediated DNA damage response. After gamma-irradiation, cells expressing wild-type p53 arrested in G1, contained increased levels of WAF1/CIP1 mRNA, and demonstrated accumulation of hypophosphorylated pRB. In contrast, cell lines with abnormal p53 genes or with p53 functionally inactivated by the E6 oncoprotein of HPV16 (a high-risk HPV) failed to arrest in G1, did not elevate WAF1/CIP1 mRNA, and did not accumulate hypophosphorylated pRB. Despite apparently normal elevation of p53 protein and WAF1/CIP1 mRNA after irradiation, cells expressing HPV16 E7 also failed to arrest in G1 and did not accumulate hypophosphorylated pRB. Disruption of RB genes alone did not totally abrogate this G1 arrest. Our results suggest that p53 indirectly regulates phosphorylation of pRB and that pRB and/or other pRB-like molecules that bind to HPV16 E7 participate in the DNA damage-mediated G1 arrest signal. In the process of HPV infection, the HPV E6 and E7 oncoproteins may undermine this cell cycle checkpoint, contributing to the accumulation of genetic alterations during tumorigenesis.
Mapping of herpes simplex virus 1 genes with mutations which overcome host restrictions to infection.
Authors: Brandimarti R , Huang T , Roizman B , Campadelli-Fiume G
Published: May 2006

Earlier studies have shown that the thymidine kinase-negative baby hamster kidney (BHKTK-) cell lines expressing constitutively the herpes simplex virus 1 (HSV-1) glycoprotein D (gD), designated BJ, restrict infection by HSV-1 at the level of virus entry. U10, a HSV-1 mutant not restricted by the BJ cells, carried the substitution of proline for Leu25 in the gD gene, suggesting that gD encodes a specialized domain which precludes virus entry into cells expressing gD. Analyses of a new series of 36 unrestricted viral mutants showed the following. (i) Only two mutants contained mutations at a site which did not overlap with the previously reported mutation. A representative of a previously mapped mutant and one of the two new mutants were examined in detail. Thus, in the gD of mutant U30 Ala185 was replaced by threonine, whereas in gD of U21, Ala185 and Leu25 were replaced with threonine and proline, respectively. U30 and U21 multiplied better than the wild-type parent virus in the parental BHKTK- cells. (ii) Transfer of the gD gene from U21 or U30 to wild-type parent virus or to the gD- virus FgD beta yielded recombinants which, while capable of infecting BJ cells, were considerably less efficient than the parent unrestricted mutants, suggesting that the latter contained additional mutations which were responsible in part for the unrestricted phenotype. Conversely, marker rescue of mutant viruses with wild-type gD reduced but did not abrogate entirely the unrestricted phenotype. (iii) Mutations in gD which conferred the unrestricted phenotype were not random. (iv) gD plays a role in the restriction, inasmuch as preincubation of cells expressing gD with antibodies to gD abolished restriction. (v) In mutant R5000, the gD substitution Ser140 to Asn was capable of overcoming a restriction of a BHKTK- clonal line which does not express gD but conferred very low ability to replicate on BJ cells. We conclude that (a) uncloned stocks of BHKTK- cells exhibit a low level restriction to infection with wild-type virus, (b) clonal lines of BHKTK- cells which vary with respect to the stringency of restriction express either allelic genes differing in the properties of their products or products of different genes, and (c) both the restricted and unrestricted phenotypes reflect the interactions of gD with these cellular products. The implications of these conclusions with respect to the restriction imposed on BHK cells by the expression of gD are discussed.
Proton conduction within the reaction centers of Rhodobacter capsulatus: the electrostatic role of the protein.
Authors: Maróti P , Hanson D K , Baciou L , Schiffer M , Sebban P
Published: May 2006

Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor QB. In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the QB binding site, all of which lack the protonatable amino acids Glu-L212 and Asp-L213. These strains are the photosynthetically incompetent site-specific mutant Glu-L212/Asp-L213-->Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from QB (class 3 strain, Ala-Ala + Arg-M231-->Leu; class 4 strain, Ala-Ala + Asn-M43-->Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to QB compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain > 2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (> or = 60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone QA to QB. The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of QB and to increase an apparent "functional" pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long-range electrostatic effects.
Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase: identification of the biotin carboxylase and biotin-carrier domains.
Authors: Song J , Wurtele E S , Nikolau B J
Published: May 2006

Soybean genomic clones were isolated based on hybridization to probes that code for the conserved biotinylation domain of biotin-containing enzymes. The corresponding cDNA was isolated and expressed in Escherichia coli through fusion to the bacterial trpE gene. The resulting chimeric protein was biotinylated in E. coli. Antibodies raised against the chimeric protein reacted specifically with an 85-kDa biotin-containing polypeptide from soybean and inhibited 3-methylcrotonoyl-CoA carboxylase (EC 6.4.1.4) activity in cell-free extracts of soybean leaves. Thus, the isolated soybean gene and corresponding cDNA code for the 85-kDa biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase. The nucleotide sequence of the cDNA and portions of the genomic clones was determined. Comparison of the deduced amino acid sequence of the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase with sequences of other biotin enzymes suggests that this subunit contains the functional domains for the first half-reaction catalyzed by all biotin-dependent carboxylases--namely, the carboxylation of biotin. These domains are arranged serially on the polypeptide, with the biotin carboxylase domain at the amino terminus and the biotin-carboxyl carrier domain at the carboxyl terminus.
Pure chromosome-specific PCR libraries from single sorted chromosomes.
Authors: VanDevanter D R , Choongkittaworn N M , Dyer K A , Aten J , Otto P , Behler C , Bryant E M , Rabinovitch P S
Published: May 2006

Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases.
Functionally and spatially distinct Ca2+ stores are revealed in cultured vascular smooth muscle cells.
Authors: Tribe R M , Borin M L , Blaustein M P
Published: May 2006

Sarcoplasmic reticulum Ca2+ in vascular smooth muscle may be separated into at least two types of Ca2+ stores, one releasable by inositol 1,4,5-trisphosphate and the other releasable by caffeine and ryanodine. We employed digital imaging with fura-2 to study the effects of thapsigargin (which blocks Ca2+ sequestration in the inositol trisphosphate-sensitive store) and caffeine on the cytosolic free Ca2+ concentration ([Ca2+]cyt) in cultured arterial myocytes. These agents increased [Ca2+]cyt by depleting different Ca2+ stores in the absence of extracellular Ca2+. Moreover, Ca2+ could be transferred between the two stores, as prior application of caffeine, which alone evoked little or no rise in [Ca2+]cyt, significantly augmented the response to thapsigargin. Conversely, a substantial caffeine-induced rise in [Ca2+]cyt was observed only after the ability of the thapsigargin-sensitive Ca2+ store to sequester Ca2+ was inhibited. This suggests that the caffeine-sensitive store may have a thapsigargin-insensitive Ca(2+)-sequestration mechanism. To complement these fura-2 experiments, chlortetracycline was used to visualize the Ca2+ stores directly. The latter studies revealed spatial differences in the location of the thapsigargin-sensitive and caffeine-sensitive Ca2+ stores (measured as thapsigargin-sensitive and caffeine-sensitive chlortetracycline fluorescence). Thus, these two types of stores appear to be both functionally and spatially distinct.
Chromosome 18 DNA markers and manic-depressive illness: evidence for a susceptibility gene.
Authors: Berrettini W H , Ferraro T N , Goldin L R , Weeks D E , Detera-Wadleigh S , Nurnberger J I , Gershon E S
Published: May 2006

In the course of a systematic genomic survey, 22 manic-depressive (bipolar) families were examined for linkage to 11 chromosome 18 pericentromeric marker loci, under dominant and recessive models. Overall logarithm of odds score analysis for the pedigree series was not significant under either model, but several families yielded logarithm of odds scores consistent with linkage under dominant or recessive models. Affected sibling pair analysis of these data yielded evidence for linkage (P < 0.001) at D18S21. Affected pedigree member analysis also suggests linkage, with multilocus results for five loci giving P < 0.0001 and P = 0.0007 for weighting functions f(p) = 1 and 1/square root p, respectively, where p is the allele frequency. These results imply a susceptibility gene in the pericentromeric region of chromosome 18, with a complex mode of inheritance. Two plausible candidate genes, a corticotropin receptor and the alpha subunit of a GTP binding protein, have been localized to this region.
Protection of mice from endotoxic death by 2-methylthio-ATP.
Authors: Proctor R A , Denlinger L C , Leventhal P S , Daugherty S K , van de Loo J W , Tanke T , Firestein G S , Bertics P J
Published: May 2006

The lethal effects of endotoxin, a bacterial product shed into the blood during bacteremia, are thought to be due to macrophage release of mediators such as tumor necrosis factor alpha and interleukin 1. Although much is known about the pathophysiology of endotoxemia, relatively little is known about the cellular signaling mechanisms that are involved. The data in this study suggest that extracellular adenine nucleotides can influence the development of endotoxin shock. An adenine nucleotide analog, 2-methylthio-ATP, inhibited the endotoxin-stimulated release of toxic mediators (i.e., tumor necrosis factor alpha and interleukin 1), and it protected mice from endotoxin-induced death. These studies suggest a fundamental and unusual role for adenine nucleotides on endotoxin action, and they provide a potentially new therapeutic approach for the control of the pathophysiology of Gram-negative septicemia.
Human and rat TR4 orphan receptors specify a subclass of the steroid receptor superfamily.
Authors: Chang C , Da Silva S L , Ideta R , Lee Y , Yeh S , Burbach J P
Published: May 2006

We have identified a member of the steroid receptor superfamily and cloned it from human and rat hypothalamus, prostate, and testis cDNA libraries. The open reading frame between first ATG and terminator TGA can encode 615 (human) and 596 (rat) amino acids with calculated molecular mass of 67.3 (human) and 65.4 (rat) kDa. The amino acid sequence of this protein, called TR4 orphan receptor, is closely related to the previously identified TR2 orphan receptor. The high homology between TR2 and TR4 orphan receptor suggests that these two orphan receptors constitute a unique subfamily within the steroid receptor superfamily. These two orphan receptors are differentially expressed in rat tissues. Unlike TR2 orphan receptors, the TR4 orphan receptor appears to be predominantly located in granule cells of the hippocampus and the cerebellum, suggesting that it may play some role(s) in transcriptional regulation in these neurons.
N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes.
Authors: Pierotti A R , Prat A , Chesneau V , Gaudoux F , Leseney A M , Foulon T , Cohen P
Published: May 2006

N-Arg dibasic convertase is a metalloendopeptidase from rat brain cortex and testis that cleaves peptide substrates on the N terminus of Arg residues in dibasic stretches. By using both an oligonucleotide and antibodies to screen a rat testis cDNA library, a full-length cDNA was isolated. The sequence contains an open reading frame of 1161 codons corresponding to a protein of 133 kDa that exhibits 35% and 48% similarity with Escherichia coli protease III (pitrilysin, EC 3.4.99.44) and rat or human insulinase (EC 3.4.99.45), respectively. Moreover, the presence of the HXXEH amino acid signature (XX = FL) clearly classifies N-Arg dibasic convertase as a member of the pitrilysin family of zinc-metalloendopeptidases. In addition, a Cys residue that may be responsible for the thiol sensitivity of the insulinase and N-Arg dibasic convertase was proposed. The protein sequence contains a distinctive additional feature consisting of a stretch of 71 acidic amino acids. We hypothesize that this metalloendopeptidase may be a member of a distinct class of processing enzymes.
Two cellular proteins that bind to wild-type but not mutant p53.
Authors: Iwabuchi K , Bartel P L , Li B , Marraccino R , Fields S
Published: May 2006

p53 is a tumor-suppressor protein that can activate and repress transcription. Using the yeast two-hybrid system, we identified two previously uncharacterized human proteins, designated 53BP1 and 53BP2, that bind to p53. 53BP1 shows no significant homology to proteins in available databases, whereas 53BP2 contains two adjacent ankyrin repeats and a Src homology 3 domain. In vitro binding analyses indicate that both of these proteins bind to the central domain of p53 (residues 80-320) required for site-specific DNA binding. Consistent with this finding, p53 cannot bind simultaneously to 53BP1 or 53BP2 and to a DNA fragment containing a consensus p53 binding site. Unlike other cellular proteins whose binding to p53 has been characterized, both 53BP1 and 53BP2 bind to the wild-type but not to two mutant p53 proteins identified in human tumors, suggesting that binding is dependent on p53 conformation. The characteristics of these interactions argue that 53BP1 and 53BP2 are involved in some aspect of p53-mediated tumor suppression.
"RFLP subtraction": a method for making libraries of polymorphic markers.
Authors: Rosenberg M , Przybylska M , Straus D
Published: May 2006

We developed a method, "RFLP subtraction," that isolates large numbers of unique sequence restriction fragment length polymorphisms (RFLPs) in a single experiment. The technique purifies small restriction fragments from one genome containing sequences that reside on large fragments in a related genome. We first isolate samples containing the small restriction fragments from two polymorphic strains. Subtractive hybridization then removes the fragments that are present in both samples. The remaining sequences are RFLPs: they occur on small fragments in one strain but not in the other. Here we use RFLP subtraction to make a library of hundreds of unique sequence RFLPs from two inbred mouse strains. We analyze and map a subset of the RFLPs and show that the genetic linkage of these markers can be rapidly determined by an efficient dot blot mapping technique. Several other potential applications of RFLP subtraction, including isolating region specific markers, are discussed.
Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.
Authors: Ebisawa T , Karne S , Lerner M R , Reppert S M
Published: May 2006

Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family.
Primary structure and characterization of a small-conductance inwardly rectifying potassium channel from human hippocampus.
Authors: Périer F , Radeke C M , Vandenberg C A
Published: May 2006

We have isolated a human hippocampus cDNA that encodes an inwardly rectifying potassium channel, termed HIR (hippocampal inward rectifier), with strong rectification characteristics. Single-channel recordings indicate that the HIR channel has an unusually small conductance (13 pS), distinguishing HIR from other cloned inward rectifiers. RNA blot analyses show that HIR transcripts are present in heart, skeletal muscle, and several different brain regions, including the hippocampus.
Three distinct human thymopoietins are derived from alternatively spliced mRNAs.
Authors: Harris C A , Andryuk P J , Cline S , Chan H K , Natarajan A , Siekierka J J , Goldstein G
Published: May 2006

Thymopoietin (TP) was originally isolated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular transmission and was subsequently observed to affect T-cell differentiation and function. We now report the isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, which we have named TP alpha (75 kDa), TP beta (51 kDa), and TP gamma (39 kDa), contain identical N-terminal regions, including sequences nearly identical to that of the originally isolated 49-aa protein, but divergent C-terminal regions. TP mRNAs are expressed in many tissues, most abundantly in adult thymus and fetal liver of the tissues so far examined. Distinct structural domains and functional motifs in TPs alpha, beta, and gamma suggest that the proteins have unique functions and may be directed to distinct subcellular compartments.
Functional redundancy: the respective roles of the two sloppy paired genes in Drosophila segmentation.
Authors: Cadigan K M , Grossniklaus U , Gehring W J
Published: May 2006

The sloppy paired (slp) locus consists of two genes, slp1 and slp2, both of which encode proteins containing a forkhead domain (a DNA-binding motif). Previous work has shown that a severe segmentation phenotype is obtained only when both slp genes are deleted. Here we examine the functional redundancy of the locus in more detail. The phenotypes of embryos containing various combinations of functional slp genes suggest that for early slp function, until gastrulation, only slp1 is required. At later times, there is still a greater requirement for slp1, but in many respects the two slp genes are completely redundant. Both slp genes produce similar phenotypes when ubiquitously expressed via a heat shock promoter. We propose that the slp proteins are biochemically equivalent and that the greater requirement for slp1 in some functions can be explained in large part by its earlier expression.
The plasma flux and oxidation rate of ornithine adaptively decline with restricted arginine intake.
Authors: Castillo L , Sánchez M , Chapman T E , Ajami A , Burke J F , Young V R
Published: May 2006

We hypothesized recently that arginine homeostasis is achieved in humans largely by modulating the rate of arginine degradation. We have tested this hypothesis further by measuring in vivo the whole body rate of conversion of arginine to ornithine and ornithine oxidation in six healthy young adults. Subjects received for 6 days an L-amino acid-based diet supplying an arginine-rich or arginine-free intake and on day 7, following an overnight fast, an 8-h tracer protocol (first 3 h, fast state; next 5 h, fed state) was conducted; L-[guanidino-15N2; 5,5-2H]arginine and L-[5-13C]ornithine were given as primed, constant intravenous tracers; measurements of the abundances of various isotopologs of arginine, ornithine, and citrulline in plasma were made, and from these the various kinetic parameters of the metabolism of these amino acids were derived. Arginine and ornithine fluxes were significantly (P < 0.001) reduced in the fed state with arginine-free feeding. The rates of conversion (mumol.kg-1.h-1; mean +/- SD) of plasma arginine to ornithine for arginine-rich were 12.9 +/- 2.6 and 24.7 +/- 4.8 for fast and fed states. These values were 11.1 +/- 3.5 and 9.6 +/- 1.2 (P > 0.05 and P < 0.001), respectively, with an arginine-free diet. [13C]Ornithine oxidation was reduced (P < 0.001) by 46% during the fed state when the arginine-free diet was given. The findings strengthen our hypothesis that homeostasis of arginine metabolism in the human host depends importantly upon a regulation in the rate of arginine degradation with, perhaps, little involvement in the de novo net rate of arginine synthesis.
Nitric oxide mediates sexual behavior in female rats.
Authors: Mani S K , Allen J M , Rettori V , McCann S M , O'Malley B W , Clark J H
Published: May 2006

Nitric oxide (NO), an active free radical formed during the conversion of arginine to citrulline by the enzyme NO synthase (NOS), mediates vasorelaxation, cytotoxicity, and neurotransmission. Neurons containing NOS (NOergic) are located in the hypothalamus. These NOergic neurons control the release of several hypothalamic peptides. Release of NO from these NOergic neurons stimulates pulsatile release of luteinizing hormone-releasing hormone (LHRH) in vivo and LHRH release in vitro. LHRH not only induces LH release, which induces ovulation, but also facilitates female sexual behavior. Sexual behavior can be induced reliably in estrogen-primed ovariectomized female rats by progesterone (P). This behavior consists of proceptive behavior to attract the male and the assumption of a clear characteristic posture, lordosis, when mounted by the male. To ascertain the role of NO in the control of sexual behavior in female rats, an inhibitor of NOS, NG-monomethyl-L-arginine was microinjected into the third cerebral ventricle (3V) of conscious, ovariectomized, estrogen-primed rats with indwelling cannulae. NG-Monomethyl-L-arginine (10-1000 micrograms) prevented P-facilitated lordosis when administered intracerebroventricularly into the 3V, 20 min prior to the 3V injection of P. NG-Monomethyl-D-arginine, which does not inhibit NOS, did not inhibit lordosis under the same experimental conditions. Microinjection into the 3V of sodium nitroprusside (SNP), which spontaneously releases NO, facilitated lordosis in estrogen-primed rats in the absence of P. The facilitation of lordosis induced by either P or SNP was prevented by intracerebroventricular injection of hemoglobin, which binds NO. Lordosis facilitated by P or SNP was blocked by injection of LHRH antiserum into the 3V. The results are interpreted to mean that the P-facilitated lordosis response is mediated by LHRH release. Furthermore, since NO release from SNP also facilitates lordosis in the absence of P and this response could be blocked by LHRH antiserum, we conclude that P brings about the release of NO, which stimulates LHRH release that facilitates lordosis. Thus, the results indicate that NO induces LHRH release and that LHRH then plays a crucial role in mediation of sexual behavior in the female rats.
Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides.
Authors: Ma P W , Knipple D C , Roelofs W L
Published: May 2006

Sex pheromone biosynthesis in a number of moth species is induced by a conserved 33-amino acid amidated neuropeptide PBAN (pheromone biosynthesis-activating neuropeptide). We have isolated and characterized the Helicoverpa zea PBAN cDNA corresponding to a 766-nucleotide mRNA that is expressed in the subesophageal ganglion of adult moths. This mRNA is encoded on a transcription unit comprising 6 exons. The longest open reading frame of the cDNA encodes a 194-amino acid precursor protein that contains the PBAN peptide sequence. Proteolytic processing of this protein, which has structural features consistent with its being a preprohormone, is predicted to generate Hez-PBAN and four additional neuropeptides having a common C-terminal pentapeptide motif, Phe-Xaa-Pro-(Arg or Lys)-Leu (Xaa = Gly, Ser, or Thr), which is also found in insect pyrokinin and myotropin peptide families.
Creation and phenotypic analysis of alpha-lactalbumin-deficient mice.
Authors: Stinnakre M G , Vilotte J L , Soulier S , Mercier J C
Published: May 2006

alpha-Lactalbumin is an abundant milk-specific calcium metalloprotein which has an evolutionary relationship to lysozyme. It modifies the substrate specificity of a Golgi galactosyltransferase by forming the lactose synthetase binary complex. Lactose, together with other sugars and diffusible ions, is responsible for the osmotic pressure of milk. To assess the involvement of alpha-lactalbumin in lactogenesis, alpha-lactalbumin-deficient mice were created by disrupting the gene by homologous recombination in embryonic stem cells. Homozygous mutant mice are viable and fertile but females cannot feed their offspring. They produce a highly viscous milk that pups appear to be unable to remove from the mammary gland. This milk is rich in fat and protein and is devoid of alpha-lactalbumin and lactose. The phenotype of heterozygous mice was found to be intermediate, with a 40% decrease in alpha-lactalbumin but only a 10-20% decrease in the lactose content of their milk compared with wild-type animals. These results emphasize the key function of alpha-lactalbumin in lactogenesis and open new opportunities to manipulate milk composition.
Reversal of cell determination in yeast meiosis: postcommitment arrest allows return to mitotic growth.
Authors: Honigberg S M , Esposito R E
Published: May 2006

When yeast from the early stages of meiosis are transferred from sporulation to growth medium, they can reenter the mitotic cell cycle directly. In contrast, cells from later stages of meiosis (after the initiation of the first nuclear division) will complete meiosis and sporulation despite the shift to growth medium, a phenomenon known as "commitment to meiosis." This study reports the surprising finding that when the normal progression of meiosis is arrested, cells from later stages of meiosis can return to growth. Cells were arrested after the first or second meiotic division by three independent means: the spo14 mutation, the spo3-1 mutation, and a high-temperature arrest of wild-type cells. In every case, the arrested cells were able to form buds after transfer to growth medium. These cells, however, experienced a delay upon return to growth relative to uncommitted cells. We propose that the commitment phenomenon results from a transient delay of mitotic growth, which occurs specifically during meiosis, and that commitment does not involve an irreversible inhibition of mitosis as previously thought.
A yeast system for expression of human cystathionine beta-synthase: structural and functional conservation of the human and yeast genes.
Authors: Kruger W D , Cox D R
Published: May 2006

Human cystathionine beta-synthase (CBS; EC 4.2.1.22) deficiency results in a recessive genetic disorder whose clinical and biochemical manifestations vary greatly among affected individuals. In an effort to identify and analyze mutations in the human CBS gene, we have developed a yeast expression system for human CBS. We have cloned and sequenced a human cDNA that codes for CBS and have expressed the human CBS protein in yeast cells lacking endogenous CBS. The human enzyme produced in yeast is functional both in vitro and in vivo. We have also cloned and sequenced the yeast gene, CYS4, that codes for CBS. The predicted human and yeast CBS proteins are 38% identical and 72% similar to each other, as well as sharing significant similarity with bacterial cysteine synthase. These results demonstrate the evolutionary conservation of CBS and establish the utility of a yeast expression system for studying human CBS.
The low density lipoprotein receptor-related protein mediates the cellular degradation of tissue factor pathway inhibitor.
Authors: Warshawsky I , Broze G J , Schwartz A L
Published: May 2006

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) is a cell-surface glycoprotein of 4525 amino acids that functions as a hepatic endocytosis receptor for several plasma proteins. These include alpha 2-macroglobulin-protease complexes, free plasminogen activators as well as plasminogen activators complexed with their inhibitors, and beta-migrating very low density lipoproteins complexed with either apolipoprotein E or lipoprotein lipase. In the current study we used human and rat hepatoma cell lines to demonstrate that LRP can mediate the degradation of tissue factor pathway inhibitor (TFPI), a Kunitz-type plasma serine protease inhibitor that regulates tissue factor-induced blood coagulation. The cellular degradation of 125I-labeled TFPI (125I-TFPI) was inhibited more than 80% both by antibodies directed against LRP and by the LRP-associated 39-kDa protein, a protein that inhibits the binding and/or cell-mediated degradation of all ligands by LRP. Using rat hepatoma cells, we report that at 4 degrees C, 125I-TFPI binds to approximately 2 x 10(6) sites per cell with an equilibrium dissociation constant of approximately 30 nM. 125I-TFPI binding to the cell surface is not inhibited by the 39-kDa protein. Taken together, our results suggest that TFPI binds to an as-yet-unidentified cell surface molecule. After binding, LRP mediates the cellular degradation of TFPI.
Molecular cloning of the human nucleotide-excision-repair gene ERCC4.
Authors: Thompson L H , Brookman K W , Weber C A , Salazar E P , Reardon J T , Sancar A , Deng Z , Siciliano M J
Published: May 2006

ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.
Fine mapping of a replication origin of human DNA.
Authors: Giacca M , Zentilin L , Norio P , Diviacco S , Dimitrova D , Contreas G , Biamonti G , Perini G , Weighardt F , Riva S
Published: May 2006

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.
Overexpression of ERBB2 in human mammary epithelial cells signals inhibition of transcription of the E-cadherin gene.
Authors: D'souza B , Taylor-Papadimitriou J
Published: May 2006

Overexpression of the ERBB2 receptor in transfectants of a human mammary epithelial cell line (MTSV1-7) is associated with a reduced ability to undergo morphogenesis in vitro and with a decreased level of expression of the E-cadherin and alpha 2 integrin genes. The inhibition of expression of the adhesion molecules has been shown to be at the level of transcription by using nuclear run-on assays and by following transcription of a reporter gene fused to 5' sequences of the E-cadherin gene. To relate the effects on gene transcription to a functional ERBB2 protein, signaling from the receptor was inhibited by the antibody 4D5, which blocks phosphorylation of ERBB2 on tyrosine residues and association of the protein with the GRB2/Sem5 protein. After treatment with the antibody 4D5, the ERBB2 transfectants regain the ability to form three-dimensional structures in collagen gels and the rates of transcription of the genes encoding the E-cadherin and the alpha 2 integrin subunit are restored to the levels seen in MTSV1-7neo cells. These results demonstrate that the inhibition of morphogenesis and transcription of specific adhesion molecules in human mammary epithelial cells can be affected by signals generated by the ERBB2 receptor and suggest a role for ERBB2 overexpression in tumor progression and metastasis.
The gene for a recessively inherited human childhood progressive epilepsy with mental retardation maps to the distal short arm of chromosome 8.
Authors: Tahvanainen E , Ranta S , Hirvasniemi A , Karila E , Leisti J , Sistonen P , Weissenbach J , Lehesjoki A E , de la Chapelle A
Published: May 2006

A recently delineated childhood epilepsy has hitherto been observed only in a small geographic region in northern Finland, where, with the exception of one, both parents of all of the 11 sibships with affected individuals descend from one or two founding couples. The disease is characterized by generalized tonic-clonic seizures with onset at 5-10 years and progressive, severe mental retardation with onset 2-5 years after the first seizures. In this study the gene locus is assigned to the telomeric region of chromosome 8p by linkage. Analyses of recombinations place the locus in the 7-centimorgan interval between AFM185xb2 and D8S262 in which three markers, D8S504, D8S264, and AFM077yg5, show no recombinations with the phenotype. Haplotypes comprising alleles at the above five loci support the hypothesis of a single founding mutation for all affected chromosomes except the one belonging to the unrelated parent, who has a very different haplotype, suggesting another mutation or a very old ancestry of a single mutation. This study raises to three the number of heritable epilepsies whose gene loci have been mapped and provides a starting point for the cloning of the gene. It also suggests the possibility that the disease might not be limited to the northern Finnish population.