bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a phosphatidic acid-preferring phospholipase A1 from bovine brain and testis.
Authors: Higgs H N , Glomset J A
Published: May 2006

Recent experiments in several laboratories have provided evidence that phosphatidic acid functions in cell signaling. However, the mechanisms that regulate cellular phosphatidic acid levels remain obscure. Here we describe a soluble phospholipase A1 from bovine testis that preferentially hydrolyzes phosphatidic acid when assayed in Triton X-100 micelles. Moreover, the enzyme hydrolyzes phosphatidic acid molecular species containing two unsaturated fatty acids in preference to those containing a combination of saturated and unsaturated fatty acyl groups. Under certain conditions, the enzyme also displays lysophospholipase activity toward lysophosphatidic acid. The phospholipase A1 is not likely to be a lysosomal enzyme because its optimum pH is 7.5-8.5. Furthermore, it is probably not a general lipid metabolic enzyme because high levels of activity are found in mature testis and brain but no measurable activity is seen in liver, spleen, or heart. The fact that the activity of the phospholipase A1 in mature bovine testis is > 10-fold higher than that in newborn calf testis raises the possibility that the enzyme may play a regulatory role in spermatogenesis or sperm function.
Human plasma vitamin E kinetics demonstrate rapid recycling of plasma RRR-alpha-tocopherol.
Authors: Traber M G , Ramakrishnan R , Kayden H J
Published: May 2006

A kinetic model of vitamin E transport in humans is described using data from our studies with deuterium-labeled stereoisomers of alpha-tocopherol (RRR- and SRR-). In normal subjects, both alpha-tocopherols are present at similar concentrations in chylomicrons, but by 24 hr, RRR-alpha-tocopherol is at higher plasma concentrations because RRR-alpha-tocopherol is preferentially incorporated into very low density lipoproteins, which are then secreted into plasma. In three nondiscriminator patients with familial isolated vitamin E deficiency, the fractional disappearance rates (mean +/- SD) of deuterium-labeled RRR- and SRR-alpha-tocopherols in plasma were 1.4 +/- 0.6 and 1.3 +/- 0.3 pools per day, respectively (difference, 0.1 +/- 0.3). In these patients, plasma concentrations of both RRR- and SRR-alpha-tocopherols decreased similarly to SRR-alpha-tocopherol in controls. In six controls, fractional disappearance rates of deuterium-labeled RRR-alpha-tocopherol (0.4 +/- 0.1 pool per day) were significantly (P < 0.01) slower than for SRR- (1.2 +/- 0.6). The differences (0.8 +/- 0.6 pool per day) between these two rates in controls estimate the rate at which RRR-alpha-tocopherol, which had left the plasma, was returned to the plasma. Although plasma labeled RRR-alpha-tocopherol concentrations in controls appear to change slowly, these data show that both RRR- and SRR-alpha-tocopherols leave the plasma rapidly, but only RRR-alpha-tocopherol is returned to the plasma, likely in nascent very low density lipoproteins. This recycling of RRR-alpha-tocopherol accounts for nearly 1 pool of alpha-tocopherol per day.
Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer.
Authors: Selvin P R , Hearst J E
Published: May 2006

We extend the technique of fluorescence resonance energy transfer (FRET) by introducing a luminescent terbium chelate as a donor and an organic dye, tetramethylrhodamine, as an acceptor. The results are consistent with a Förster theory of energy transfer, provided the appropriate parameters are used. The use of lanthanide donors, in general, and this pair, in particular, has many advantages over more conventional FRET pairs, which rely solely on organic dyes. The distance at which 50% energy transfer occurs is large, 65 A; the donor lifetime is a single exponential and long (millisecond), making lifetime measurements facile and accurate. Uncertainty in the orientation factor, which creates uncertainty in measured distances, is minimized by the donor's multiple electronic transitions and long lifetime. The sensitized emission of the acceptor can be measured with little or no interfering background, yielding a > 25-fold improvements in the signal-to-background ratio over standard donor-acceptor pairs. These improvements are expected to make distances > 100 A measurable via FRET. We also report measurement of the sensitized emission lifetime, a measurement that is completely insensitive to total concentration and incomplete labeling.
Constitutive expression of B-myb can bypass p53-induced Waf1/Cip1-mediated G1 arrest.
Authors: Lin D , Fiscella M , O'Connor P M , Jackman J , Chen M , Luo L L , Sala A , Travali S , Appella E , Mercer W E
Published: May 2006

Overexpression of wild-type p53 protein has been shown to induce arrest in the G1 stage of the cell cycle and to transactivate expression of the gene that encodes the 21-kDa Waf1/Cip1 protein, a potent inhibitor of cyclin-dependent kinase activity. p53-dependent G1 arrest is accompanied by decreased expression of the B-myb gene, a relative of the c-myb cellular oncogene. In this study we show that B-myb expression is required for cells to progress from G1 into S phase and that high levels of ectopic B-myb expression uncoupled from cell cycle regulation rescues cells from p53-induced G1 arrest even in the presence of Waf1/Cip1 transactivation and inhibition of cyclin E/Cdk2 kinase activity. Cotransfection experiments with p53 expression plasmids and expression plasmids encoding in-frame deletion mutations in B-myb coding sequences indicate that the DNA-binding domain of the B-Myb protein is required for this activity. These results provide evidence of a bypass of p53-induced Waf1/Cip1-mediated cell cycle regulatory pathways by a member of the myb oncogene family.
Complete cloning and sequencing of rat gp330/"megalin," a distinctive member of the low density lipoprotein receptor gene family.
Authors: Saito A , Pietromonaco S , Loo A K , Farquhar M G
Published: May 2006

We completed the cDNA cloning and sequencing of gp330, the major kidney glomerular antigen for rat Heymann nephritis. The deduced 4660-aa sequence, expected to constitute a mature protein of M(r) 516,715, consists of a probable N-terminal signal peptide sequence (25 aa), an extracellular region (4400 aa), a single transmembrane domain (22 aa), and a C-terminal cytoplasmic tail (213 aa). The extracellular region contains three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) gene family--36 LDLR ligand-binding repeats forming four clusters of putative ligand-binding domains, 16 growth factor repeats separated by 8 YWTD spacer regions, and 1 C-terminal epidermal growth factor repeat. The cytoplasmic tail contains two copies of the (FX)NPXY motif, which represents a signal for coated pitmediated internalization and an additional similar motif. The overall structure of gp330 is similar to that of the LDLR-related protein (LRP)/alpha 2-macroglobulin receptor and shows even greater similarity to the Caenorhabditis elegans protein, reported as a homologue of LRP. However, gp330 differs from these proteins in (i) the cysteine-rich repeat arrangements found in the extreme extracellular N- and C-terminal regions, (ii) the distribution pattern of cysteine residues in the YWTD spacer regions, (iii) the location of the RX(K/R)R consensus recognition sequence of furin, a precursor processing endoprotease, and (iv) the length and structure of the cytoplasmic tail. We suggest the name megalin (from Greek mega) for gp330, the largest plasma membrane protein identified so far in vertebrates. The cloned cDNA will be useful for studies on the physiological functions of gp330/megalin and for determining its role in Heymann nephritis.
Toxicity of myristic acid analogs toward African trypanosomes.
Authors: Doering T L , Lu T , Werbovetz K A , Gokel G W , Hart G W , Gordon J I , Englund P T
Published: May 2006

New drugs are needed for treatment of diseases caused by African trypanosomes. One possible target for chemotherapy is the biosynthesis of the glycosyl phosphatidyl-inositol (GPI) of this parasite's variant surface glycoprotein (VSG). Unlike mammalian GPIs, the diacylglycerol moiety of the VSG anchor contains only myristate (tetradecanoate), added in unique remodeling reactions. We previously found that 11-oxatetradecanoic acid [i.e., 10-(propoxy)decanoic acid] is selectively toxic to trypanosomes. We have now assayed 244 different fatty acid analogs, most with chain lengths comparable to that of myristate, for trypanocidal effects. In these assays we surveyed the effects on toxicity of systematic alterations in the analogs' steric, conformational, and hydrophobic properties. We also used three 3H-labeled oxatetradecanoic acids to explore the mechanism of analog action. Their incorporation into VSG correlated roughly with toxicity, although they also were incorporated into phospholipids and other proteins. Myristate analogs are useful for studying the mechanism of GPI myristolyation, and they are candidates for antitrypanosomal chemotherapy.
A remarkable cranium of Plesiopithecus teras (Primates, Prosimii) from the Eocene of Egypt.
Authors: Simons E L , Rasmussen D T
Published: May 2006

Between 1991 and 1993 specimens of a highly distinctive primate, named Plesiopithecus teras [Simons, E.L. (1992) Proc. Natl. Acad. Sci. USA 89, 10743-10747], were found at site L-41 in late Eocene deposits of the Fayum Depression, Egypt. The most important of these specimens consists of a nearly complete skull, which facilitates the evaluation of affinities of this primate. Characteristics of the known material now demonstrate that Plesiopithecus is a prosimian, although mandibular molar morphology, in particular, bears similarity to that in molars of archaic members of Anthropoidea. Plesiopithecus has a postorbital bar but lacks postorbital closure, it has upper molars without hypocones, and it may retain four lower premolars. Its familial rank was considered incertae sedis by Simons [Simons, E.L. (1992) Proc. Natl. Acad. Sci. USA 89, 10743-10747]; it can now be demonstrated that Plesiopithecus justifies establishment of a new family and superfamily. The new superfamily apparently lies closer to the toothcomb prosimians (strepsirhines) than to any other known primate group. Under this interpretation the enlarged, procumbent tooth in the jaw of Plesiopithecus is homologous to either the lateral incisor or the canine of the prosimian toothcomb.
A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm.
Authors: Maéno M , Ong R C , Suzuki A , Ueno N , Kung H F
Published: May 2006

The biological effects of endogenous bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta family, on embryonic development of Xenopus laevis were investigated by using a functionally defective mutant of the BMP-4 receptor (delta mTFR11), which blocks the BMP signaling pathway. Injection of delta mTFR11 RNA into either the animal pole area or ventral marginal cells at the two-cell stage induced a dorsal phenotype in the explant of ventral mesoderm with animal pole tissue from stage 10+ embryo, even though the normal fate of this explant is a "mesenchymal ball" containing blood cells. These explants with the dorsal phenotype contained muscle, neural tissue, eye capsule, and cement gland. Northern blot analysis showed an increase of cardiac alpha-actin mRNA and a decrease of T alpha-globin mRNA expression, providing further evidence of a conversion from ventral to dorsal phenotype. Although injection of delta mTFR11 RNA did not induce mesoderm in an animal cap culture, the same tissue injected with delta mTFR11 RNA can alter the differentiation fate of uninjected ventral mesodermal explant from ventral to dorsal type, suggesting specific interaction of animal pole tissue and prospective ventral mesoderm in vivo.
Bruton tyrosine kinase is tyrosine phosphorylated and activated in pre-B lymphocytes and receptor-ligated B cells.
Authors: Aoki Y , Isselbacher K J , Pillai S
Published: May 2006

The gene encoding Bruton tyrosine kinase (Btk) is known to be mutated in human X chromosome-linked agammaglobulinemia and in the Xid mouse. This kinase was examined in B lymphocytes before and after antigen receptor ligation and also in pre-B cells. Btk was found to be catalytically activated and tyrosine phosphorylated in response to anti-IgM stimulation in B cells. This kinase is also constitutively phosphorylated on tyrosine residues in pre-B cells. These findings point to a functional role for Btk in pre-antigen and antigen receptor signaling during B-cell development and provide a biochemical explanation for the X-linked genetic syndromes already linked to this kinase.
Facilitation of GABAergic signaling in the retina by receptors stimulating adenylate cyclase.
Authors: Feigenspan A , Bormann J
Published: May 2006

The gamma-aminobutyric acid type A (GABAA) receptor is the predominant Cl(-)-channel protein mediating inhibition in the retina and elsewhere in the mammalian brain. We have observed a time-dependent increase of GABA-induced whole-cell currents when dopamine was applied externally to rat retinal amacrine cells. After 20 min, the peak current was increased to 208% +/- 10% of its initial value. A comparable effect was observed with the dopamine D1 receptor agonist (+)-1-phenyl-2,3,4,5-tetrahydro(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393) but not with the D2 agonist bromocryptine. The action of dopamine involved phosphorylation of GABAA receptors by protein kinase A, as evident from intracellular application of protein kinase A, cAMP, and forskolin. Both guanosine 5'-[gamma-thio]triphosphate and cholera toxin augmented the GABA response, indicating a role for the guanosine 5'-triphosphate-binding protein Gs in the transduction cascade. Phosphorylation of GABAA receptors shifted the half-maximally effective GABA concentration from 71 microM to 47 microM without affecting the maximal response amplitude. The elevated binding affinity for GABA was caused by an increase of the open probability of the channels from 0.09 to 0.33 (2 microM GABA); conductance and mean open time did not change. Several other receptor agonists such as adenosine, histamine, somatostatin, enkephalin, and vasoactive intestinal peptide were found to couple to the same intracellular phosphorylation pathway. Since some of these cotransmitters colocalize with GABA in amacrine cells, they may fine-tune GABAergic inhibition in the retina.
The aged monkey basal forebrain: rescue and sprouting of axotomized basal forebrain neurons after grafts of encapsulated cells secreting human nerve growth factor.
Authors: Kordower J H , Winn S R , Liu Y T , Mufson E J , Sladek J R , Hammang J P , Baetge E E , Emerich D F
Published: May 2006

Six Rhesus monkeys between 24 and 29 years of age received unilateral transections of the fornix. Three monkeys then received intraventricular transplants of polymer-encapsulated baby hamster kidney (BHK) fibroblasts that had been genetically modified to secrete human nerve growth factor (hNGF). The remaining three monkeys received identical grafts except the cells were not modified to secrete hNGF. Monkeys receiving the fornix transection and control grafts displayed extensive reductions in the number of choline acetyltransferase- (57-75%) and p75 NGF receptor- (53%) immunoreactive medial septal neurons ipsilateral to the lesion/implant. In contrast, monkeys receiving transplants of encapsulated hNGF-secreting cells display only a modest loss of choline acetyltransferase- (0-36%) and p75 NGF receptor-(7-22.4%) immunoreactive septal neurons. Additionally, all monkeys receiving the hNGF-secreting implants, but none receiving control implants, displayed robust sprouting of cholinergic fibers within the septum ipsilateral to the transplant. Just prior to sacrifice, the capsules were retrieved and found to contain viable BHK cells releasing biologically relevant levels of hNGF. These data demonstrate that hNGF can provide trophic and tropic influences to aged primate basal forebrain neurons undergoing lesion-induced degeneration, supporting the contention that hNGF may prevent the degeneration of basal forebrain neurons in Alzheimer disease.
Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis.
Authors: Qin X Q , Livingston D M , Kaelin W G , Adams P D
Published: May 2006

E2F-1 is a transcription factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53. In contrast, coexpression of a naturally occurring loss-of-function RB mutant or wild-type p53 did not suppress the induction of apoptosis under these conditions. Thus, deregulated E2F-1 activity gives rise to proliferative and apoptotic signals. p53 appears to participate in the execution of the latter.
Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development.
Authors: Küppers R , Rajewsky K , Zhao M , Simons G , Laumann R , Fischer R , Hansmann M L
Published: May 2006

Hodgkin disease (HD) is characterized by a small number of putative malignant cells [Hodgkin and Reed-Sternberg (HRS) cells] among a background of lymphocytes and histiocytes. The lineage of HRS cells is still elusive and a clonal origin of these rare cells has not formally been demonstrated. We isolated HRS cells by micromanipulation from histological sections of three cases of Hodgkin lymphoma (each representing a distinct subtype of the disease) and analyzed individual cells for immunoglobulin variable (V) gene rearrangements by PCR. In each of the three cases a single heavy-chain V (VH) (and in one case, in addition, a kappa light-chain) gene rearrangement was amplified from the HRS cells, identifying these cells as members of a single clone. A potentially functional VH rearrangement was obtained from a case of nodular sclerosis HD. Somatic mutations and intraclonal diversity in the VH genes indicate a germinal center B-cell origin of the HRS cells in a case of lymphocyte-predominant HD, whereas in a case of mixed-cellularity HD the sequence analysis revealed only nonfunctional V gene rearrangements, suggesting a pre-B-cell origin. This indicates that HRS cells can originate from B-lineage cells at various stages of development.
Spatially controlled adhesion, spreading, and differentiation of endothelial cells on self-assembled molecular monolayers.
Authors: Spargo B J , Testoff M A , Nielsen T B , Stenger D A , Hickman J J , Rudolph A S
Published: May 2006

Chemically modified glass substrates were used to demonstrate differential adhesion, growth, and differentiation of endothelial cells. Endothelial cells were examined for adhesion and growth on glass, glass treated with N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA), or EDA with a subsequent treatment with physically adsorbed extracellular matrix components human fibronectin and heparin sulfate. EDA and EDA/human fibronectin showed similar abilities to support adhesion, spreading, and proliferation of endothelial cells. In contrast, heparin sulfate inhibited endothelial cell adhesion to EDA. Differentiation of endothelial cells resulting in precapillary cord formation was triggered by addition of basic fibroblast growth factor (bFGF). On EDA and EDA/human fibronectin bFGF causes confluent endothelial cell monolayers to differentiate and form cords, which resulted in a large-scale spatial redistribution of cells on the surface. Formation of organized neovascular assemblies was demonstrated on coplanar molecular patterns of EDA and a nonadhesive perfluorinated alkylsilane (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchloros ilane (13F). Endothelial cells preferentially adhered to the EDA lines and after 24-48 hr, microfilaments aligned with the long axes of the patterned EDA region. Finally, endothelial cells that became confluent within the confines of the EDA region (bound by the nonadhesive, 13F domains) were observed to differentiate into neovascular cords in long-term culture (7-10 days) with bFGF.
Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.
Authors: Hofer F , Fields S , Schneider C , Martin G S
Published: May 2006

The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The interaction of the RalGDS C-terminal segment with Ras is specific, dependent on activation of Ras by GTP, and blocked by a mutation that affects Ras effector function. These characteristics are similar to those previously demonstrated for the interaction between Ras and its putative effector, Raf, suggesting that the RalGDS may also be a Ras effector. Consistent with this idea, the RalGDS was found to inhibit the binding of Raf to Ras.
Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions.
Authors: Choo Y , Klug A
Published: May 2006

In the preceding paper [Choo, Y. & Klug, A. (1994) Proc. Natl. Acad. Sci. USA 91, 11163-11167], we showed how selections from a library of zinc fingers displayed on phage yielded fingers able to bind to a number of DNA triplets. Here, we describe a technique to deal efficiently with the converse problem--namely, the selection of a DNA binding site for a given zinc finger. This is done by screening against libraries of DNA triplet binding sites randomized in two positions but having one base fixed in the third position. The technique is applied here to determine the specificity of fingers previously selected by phage display. We find that some of these fingers are able to specify a unique base in each position of the cognate triplet. This is further illustrated by examples of fingers which can discriminate between closely related triplets as measured by their respective equilibrium dissociation constants. Comparing the amino acid sequences of fingers which specify a particular base in a triplet, we infer that in most instances, sequence-specific binding of zinc fingers to DNA can be achieved by using a small set of amino acid-nucleotide base contacts amenable to a code.
Gene conversion is involved in the transfer of genetic information between naturally occurring inversions of Drosophila.
Authors: Rozas J , Aguadé M
Published: May 2006

The DNA sequences of the ribosomal protein 49 (rp49) region were determined for 34 isochromosomal strains of Drosophila subobscura representing two chromosomal arrangements, the OST and the O3 + 4 gene arrangements, which differ by two overlapping inversions. The data reveal that gene conversion is a mechanism responsible for the transfer of genetic information between naturally occurring inversions of Drosophila. The estimated rate of gene transfer by gene conversion at this region, which is close to an inversion breakpoint, is lower than previous estimates obtained experimentally at the rosy (ry) gene in Drosophila melanogaster. Our data indicate that gene arrangements OST and O3 + 4 are monophyletic and rather old (0.58 and 0.73 million years old, respectively).
The avian vitamin D receptors: primary structures and their origins.
Authors: Elaroussi M A , Prahl J M , DeLuca H F
Published: May 2006

cDNA clones encoding Japanese quail chorioallantoic membrane and chicken kidney 1,25-dihydroxyvitamin D3 receptors were isolated and the total 448-amino acid (aa) sequence was deduced. The sequences of the chicken and quail receptors are identical. The nucleotide and deduced amino acid sequences of the avian receptors are similar but not identical to the reported rat or human receptor sequences. There is a 78% similarity in the nucleotide sequences and 98.5% and 87.5% similarities in the amino acid sequences of the DNA-binding and ligand-binding domains, respectively. Two avian receptor proteins (58 and 60 kDa) arise from a single mRNA transcript by alternate initiation of translation. The avian 1,25-dihydroxyvitamin D3 receptors were produced using a bacterial expression system. Form A receptor was expressed from a cloned cDNA that contains the first translation signal (ATG) and corresponds with the 60-kDa avian receptor protein, and form B receptor was initiated from the third ATG on the same mRNA transcript to give rise to the 58-kDa protein. The cysteine-rich DNA-binding domain is almost conserved among human, rat, and avian receptors. The position of the nine cysteines was conserved in all three sequences. The avian receptor differs in the second zinc finger domain, where a methionine replaces a leucine, a serine replaces an asparagine, and a lysine replaces an arginine at aa 77, 83, and 87, respectively, of the avian sequence. The increased length of the avian receptor results from a 20-aa extension of the N-terminal region. RNA hybridization indicates there is a single mRNA species of approximately 2700 bp for both the chicken and quail receptors compared to 4400 bp for the rat transcript. Surprisingly, the translated avian sequence is larger (448 aa) than the 423-aa rat receptor protein. Therefore, our results confirm that despite the difference in molecular mass between different receptor proteins, there is a similarity in gene organization such that the DNA-binding and hormone-binding domains are positionally conserved from the C terminus.
Crystal structure of intercalated four-stranded d(C3T) at 1.4 A resolution.
Authors: Kang C H , Berger I , Lockshin C , Ratliff R , Moyzis R , Rich A
Published: May 2006

The crystal structure of d(C3T), solved at 1.4 A resolution, reveals that the molecule forms a four-stranded intercalated complex. It consists of two parallel-stranded duplexes, each of which is held together by cytosine-protonated cytosine base pairs. The two duplexes are intercalated with each other and have opposite strand orientation. The molecule has a flat, lath-like appearance, and the covalently bonded cytosines have a slow right-handed twist of 17.1 degrees. However, there is considerable asymmetry. On one of the flat sides, the phosphate groups are rotated away from the center of the molecule. They are held in this orientation by bridging water molecules that bind the NH of cytosine and a phosphate group of an opposite chain. There is also considerable microheterogeneity in the structure. The cytosine hemiprotonation occurs even at pH 7 where stable crystals form.
Protein footprinting by the combined use of reversible and irreversible lysine modifications.
Authors: Hanai R , Wang J C
Published: May 2006

A two-step lysine-modification procedure has been devised to chemically footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a complex, is first treated lightly with a reversible lysine-modifying reagent. The protein is then unfolded and treated extensively with an irreversible lysine reagent to block those lysines that did not react previously; next, the first lysine modification is reversed, and a lysine-specific endoproteinase is used to cleave the tagged polypeptide at the deblocked lysines. Separation of the proteolytic products by size and identification of the tagged fragments map the positions of these lysines. In this procedure, the reversible lysine reagent serves as the chemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macromolecular contacts are identified from differences in proteolytic patterns of the tagged protein when the first lysine modification is done with the protein in the free form and in a complex. Application of the method to vaccinia virus topoisomerase identifies a number of lysines that are involved in its binding to DNA.
Coatomer-rich endoplasmic reticulum.
Authors: Orci L , Perrelet A , Ravazzola M , Amherdt M , Rothman J E , Schekman R
Published: May 2006

We identify in normal cells the existence of two distinct sites of the transitional endoplasmic reticulum (ER), one housing the Sec23p protein complex (the classical transitional element), the other the coatomer protein complex (the coatomer-rich ER). Experimental conditions that reduce transport from the ER to the Golgi complex lead to the overexpression of this newly defined coatomer-rich ER.
Prostaglandin D2-sensitive, sleep-promoting zone defined in the ventral surface of the rostral basal forebrain.
Authors: Matsumura H , Nakajima T , Osaka T , Satoh S , Kawase K , Kubo E , Kantha S S , Kasahara K , Hayaishi O
Published: May 2006

The site of action for the sleep-promoting effect of prostaglandin (PG) D2 was extensively examined in the brain of adult male rats (n = 231). PGD2 was administered at 100 pmol/0.2 microliter per min for 6 hr (2300-0500 hr) through chronically implanted microdialysis probes or infusion cannulae. Among the administrations of PDG2 by dialysis probes (n = 176), only those (n = 8) to a ventro-rostral part of the basal forebrain by the probes implanted on the midline consistently increased slow-wave sleep (SWS), by 51 +/- 6 min (mean +/- SEM) above the baseline value (111 +/- 11 min). Since this area is separated by a cleft into right and left regions, the results were interpreted to mean that, through this cleft, PGD2 diffused in the subarachnoid space over the adjacent ventral surface, where it had the effect of promoting sleep. When PGD2 was directly infused into the subarachnoid space (n = 55), extraordinary increases exceeding 90 min were consistently attained for the SWS at sites located between 0.5 and 2 mm rostral to the bregma and between 0 and 1.2 mm lateral to the midline defined according to the stereotaxic coordinates adopted from the brain atlas of Paxinos and Watson [Paxinos, G. & Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates (Academic, San Diego)]. Thus, we demarcated a "PGD2-sensitive, sleep-promoting zone" within this region in the ventral surface of the rostral basal forebrain. During the bilateral infusion of PGD2 into the subarachnoid space of this zone, the hourly mean SWS level of the nocturnal animals (n = 6) in the night reached the maximum at the second hour of the infusion period; this maximum hourly SWS level, corresponding to the daytime level of the same animals, lasted until the end of PGD2 infusion.
Participation of the ascending serotonergic system in the stimulation of atrial natriuretic peptide release.
Authors: Reis L C , Ramalho M J , Favaretto A L , Gutkowska J , McCann S M , Antunes-Rodrigues J
Published: May 2006

Results obtained in our laboratories have provided evidence for the participation of the hypothalamic atrial natriuretic peptide (ANP) neuronal system in the regulation of water and electrolyte homeostasis. The anterior ventral third ventricular (AV3V) region, a site of the perikarya of the ANP neurons, receives important afferent input from ascending serotoninergic axons. We hypothesized that the ascending serotoninergic tract might be involved in control of the liberation of ANP. Therefore, electrolytic lesions were produced in the mesencephalic dorsal raphé nucleus (DRN), the site of perikarya of serotonin (5-HT) neurons whose axons project to the AV3V region. Rats with sham lesions constituted the control group. In a second group of animals, the serotoninergic system was depleted of 5-HT by lateral ventricular administration of p-chlorophenylalanine (PCPA), an amino acid that causes depletion of 5-HT from the serotoninergic neurons. Control animals were injected with an equal amount of isotonic saline. The DRN lesions induced an increase of water intake and urine output beginning on the first day that lasted for 1 week after lesions were produced. There was a concomitant sodium retention that lasted for the same period of time. When water-loaded, DRN-lesioned and PCPA-injected animals showed diminished excretion of sodium, accompanied by a decrease in basal plasma ANP concentrations, and blockade of the increase in plasma ANP, which followed blood volume expansion by intraatrial injection of hypertonic saline. The results are interpreted to mean that ascending stimulatory serotoninergic input into the ANP neuronal system in the AV3V region produces a tonic stimulation of ANP release, which augments sodium excretion and inhibits water intake. Therefore, in the absence of this serotoninergic input following destruction of the serotoninergic neurons by DRN lesions or intraventricular injection of PCPA, an antinatriuretic effect is obtained that is associated with increased drinking, either because of sodium retention per se or removal of ANP-induced inhibition of release of the dipsogenic peptide, angiotensin II. The serotoninergic afferents also play an essential, stimulatory role in volume expansion-induced release of ANP and the ensuing natriuresis.
African origin of human-specific polymorphic Alu insertions.
Authors: Batzer M A , Stoneking M , Alegria-Hartman M , Bazan H , Kass D H , Shaikh T H , Novick G E , Ioannou P A , Scheer W D , Herrera R J
Published: May 2006

Alu elements are a family of interspersed repeats that have mobilized throughout primate genomes by retroposition from a few "master" genes. Among the 500,000 Alu elements in the human genome are members of the human-specific subfamily that are not fixed in the human species; that is, not all chromosomes carry an Alu element at a particular locus. Four such polymorphic human-specific Alu insertions were analyzed by a rapid, PCR-based assay that uses primers that flank the insertion point to determine genotypes based on the presence or absence of the Alu element. These four polymorphic Alu insertions were shown to be absent from the genomes of a number of nonhuman primates, consistent with their arising as human genetic polymorphisms sometime after the human/African ape divergence. Analysis of 664 unrelated individuals from 16 population groups from around the world revealed substantial levels of variation within population groups and significant genetic differentiation among groups. No significant associations were found among the four loci, consistent with their location on different chromosomes. A maximum-likelihood tree of population relationships showed four major groupings consisting of Africa, Europe, Asia/Americas, and Australia/New Guinea, which is concordant with similar trees based on other loci. A particularly useful feature of the polymorphic Alu insertions is that the ancestral state is known to be the absence of the Alu element, and the presence of the Alu element at a particular chromosomal site reflects a single, unique event in human evolution. A hypothetical ancestral group can then be included in the tree analysis, with the frequency of each insertion set to zero. The ancestral group connected to the maximum-likelihood tree within the African branch, which suggests an African origin of these polymorphic Alu insertions. These data are concordant with other diverse data sets, which lends further support to the recent African origin hypothesis for modern humans. Polymorphic Alu insertions represent a source of genetic variation for studying human population structure and evolution.
"Metabolic switch" for desert survival.
Authors: Merkt J R , Taylor C R
Published: May 2006

Food, like water, is in short supply in the desert. We report a specialized mechanism used by a desert mouse for surviving prolonged food shortages. The key element of this adaptation is a large reduction in resting metabolism. After about 2 weeks of restricted food intake (50% of normal), the desert mouse "switched down" its resting metabolism and was able to survive and maintain its weight indefinitely on these limited rations. When food was again freely available, resting metabolism "switched up," returning to normal levels in a single day. The reduced metabolism occurred without a decrease in body temperature or in levels of activity. In marked contrast, metabolism of the laboratory white mouse increased during food restriction, and the experiments had to be terminated to avoid starvation. We think this "metabolic switch" is common among desert mammals. It may be an amplification of a general metabolic response for coping with food scarcity common to all mammals, including humans.
Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein.
Authors: Frattini M G , Laimins L A
Published: May 2006

The papillomavirus E1 and E2 proteins form heteromeric complexes and individually bind specific sequences within the viral origin of replication. The mechanism by which these proteins are recruited to the origin and the role of the E1/E2 complex in replication remain undefined. To examine the interplay of these replication proteins, we have analyzed the binding of human papillomavirus (HPV) type 31b E1 and E2 proteins to the origin of replication. Binding of E1 to the origin was increased by E2 proteins and required the presence of E2 binding sites. This increase was due to the formation of E1/E2 complexes which preferentially bound E2-responsive sequences, and the magnitude was determined by the relative affinity of the E2 binding sites. While the E1 protein alone bound an A/T-rich sequence at the HPV-31b origin with low affinity, complexes of E1 and E2 bound instead to E2 binding sites with high affinity. The E1/E2 complex bound a similar sequence as E2 homodimers, but only E2 homodimer binding induced a significant increase in hypersensitivity as indicated by DNase I footprinting. In the presence of excess E1, E1 proteins bound both the A/T-rich sequence and E2 binding sites through complex formation with E2. In E2 excess, E1/E2 complexes preferentially formed, and binding was specific for E2 sites. Therefore, changes in the relative amounts of E1 and E2 proteins can dramatically alter the pattern of binding of viral replication factors to the origin. These observations suggest a model whereby modulation of the relative levels of E1 and E2 during the viral life cycle may alter the pattern of origin binding and possibly episomal copy number.
Ras activation of genes: Mob-1 as a model.
Authors: Liang P , Averboukh L , Zhu W , Pardee A B
Published: May 2006

The ras oncogenes function by indirectly controlling expression of a subset of yet-undefined genes that are crucial for cell growth and differentiation. In a differential display strategy, numerous genes were identified on the basis of their differential expression in rat embryo fibroblasts transformed by the cooperation of mutant Ha-ras and p53 genes. We demonstrate here that one such gene, designated mob-1, is a downstream target of the Ras signaling pathway. The 417-bp mob-1 promoter, which contains dual NF-kappa B and AP-1 binding sites, confers the Ras inducibility. Oncogenic Ras as well as serum growth factors that activate endogenous Ras can induce mob-1 expression, but with a fundamental difference in that the oncogenic induction is constitutive whereas the serum induction is transient. mob-1 encodes a small secretory protein with a high degree of homology to IP-10, a member of a proinflammatory cytokine family. These findings link chronic inflammatory response to constitutive ras activation and tumorigenesis. Mob-1 may serve as a secreted marker for oncogenic Ha-ras mutations.
Identification of the TCL1 gene involved in T-cell malignancies.
Authors: Virgilio L , Narducci M G , Isobe M , Billips L G , Cooper M D , Croce C M , Russo G
Published: May 2006

The TCL1 locus on chromosome 14q32.1 is frequently involved in chromosomal translocations and inversions with one of the T-cell receptor loci in human T-cell leukemias and lymphomas. The chromosome 14 region translocated or rearranged involves approximately 350 kb of DNA at chromosome band 14q32.1. Within this region we have identified a gene coding for a 1.3-kb transcript, expressed only in restricted subsets of cells within the lymphoid lineage and expressed at high levels in leukemic cells carrying a t(14;14)(q11;q32) chromosome translocation or a inv(14)(q11;q32) chromosome inversion. The cognate cDNA sequence reveals an open reading frame of 342 nt encoding a protein of 14 kDa. The TCL1 gene sequence, which, to our knowledge, shows no sequence homology with other human genes, is preferentially expressed early in T- and B-lymphocyte differentiation.
Polylysine domain of K-ras 4B protein is crucial for malignant transformation.
Authors: Jackson J H , Li J W , Buss J E , Der C J , Cochrane C G
Published: May 2006

Previous studies have shown that posttranslational modifications are required for both oncogenic K-ras 4B protein membrane binding and transforming activity. In addition, Hancock et al. [Hancock, J. F., Patterson, H. & Marshall, C. J. (1990) Cell 63, 133-139] found that a polylysine domain contained at the C terminus of K-ras 4B was also absolutely essential for K-ras 4B membrane binding but, surprisingly, neither the polylysine domain nor membrane binding was required for transforming activity. We have performed similar studies, but our results are distinctly different. Our studies indicate that the polylysine domain is crucial for K-ras 4B transforming activity. Moreover, we demonstrate that although the polylysine domain increases K-ras 4B membrane binding, significant amounts of membrane binding can occur in the absence of this domain. Finally, while our studies are consistent with the notion that membrane binding is required for K-ras 4B transforming activity, we show that membrane binding, in and of itself, is not sufficient for efficient K-ras 4B transforming activity.
The cAMP-regulated transcription factor CREB interactswith a component of the TFIID complex.
Authors: Ferreri K , Gill G , Montminy M
Published: May 2006

cAMP regulates the expression of a number of genes
through the protein kinase A-mediated phosphorylation of CREB at Ser-133. The
effects of Ser-133 phosphorylation appear to be primarily transmitted through a
modulatory kinase-inducible domain in CREB that functions cooperatively with a
120-amino acid glutamine-rich region (Q2) to stimulate transcription. Indeed,
the kinase-inducible domain activity alone is not sufficient to sustain a
transcriptional response as illustrated by the CREM family of repressors, which
contain the kinase-inducible domain but lack the Q2 region. Here we demonstrate
that Q2 functions as a potent constitutive activator in vitro. The transcription
factor TFIID fraction supports transcriptional activation by Q2, although the
"TATA" binding protein alone does not, suggesting that other
components of the TFIID complex mediate the response to CREB Q2. In fact, Q2
associates with the TATA binding protein-associated factor dTAFII110. As the
transcriptionally inactive CREM alpha and beta proteins lack sequences in Q2
that are necessary for binding dTAFII110, our results suggest that these
proteins may repress transcription because they are unable to interact with the
basal transcription complex.
A and B forms of the androgen receptor are present inhuman genital skin fibroblasts.
Authors: Wilson C M , McPhaul M J
Published: May 2006

Two forms of the androgen receptor (AR) protein
(apparent molecular masses, approximately 110 kDa and approximately 87 kDa) are
present in human genital skin fibroblasts. The 87-kDa isoform (AR-A) contains an
intact C terminus but lacks the normal N terminus found in the 110-kDa isoform
(AR-B). AR-A is the same size as the mutant form of AR produced in fibroblasts
from an androgen-resistant individual (R776) by initiation of AR synthesis at
the internal Met-188 residue of AR-B. The ratio of AR-B to AR-A in fibroblasts
derived from normal individuals is approximately 10:1. The AR isoforms detected
in these experiments resemble the A and B forms of the human progesterone
receptor, which also are encoded by a single gene and differ by the absence or
presence of an N-terminal segment. The A and B forms of the human progesterone
receptor differ in their ability to activate target genes and are regulated
differently in various types of cells. The identification of similar forms of
human AR raises the possibility that AR-A and AR-B also subserve different
functions.
Molecular genetic basis of allelic polymorphism inmalate dehydrogenase (mdh) in natural populations of Escherichia coli andSalmonella enterica.
Authors: Boyd E F , Nelson K , Wang F S , Whittam T S , Selander R K
Published: May 2006

Nucleotide sequences of the mdh gene encoding the
metabolic enzyme malate dehydrogenase (MDH) were determined for 44 strains
representing the major lineages of Escherichia coli and the eight subspecies of
Salmonella enterica. Sequence diversity was four times greater in S. enterica
than in E. coli, and in both species the rate of amino acid substitution was
lower in the NAD(+)-binding domain than in the catalytic domain. Divergence of
the mdh genes of the two species apparently has not involved excess
nonsynonymous substitutions resulting from the fixation of adaptive amino acid
mutations. Allozyme analysis detected 57% of the distinctive amino acid
sequences. Statistical tests of the distribution of polymorphic synonymous
nucleotide sites identified four possible intragenic recombination events, one
involving a single allele of E. coli and three involving alleles of the three
subspecies of S. enterica. But recombination at mdh has not occurred with
sufficient frequency to obscure the phylogenetic relationships among strains
indicated by multilocus enzyme electrophoresis, total DNA hybridization, and
sequence analysis of the gapA and putP genes. These findings provide further
evidence that the effective (realized) rates of horizontal transfer and
recombination for metabolic enzyme and other housekeeping genes are generally
low in these species, in contrast to those for loci encoding or mediating the
structure of cell-surface and other macromolecules for which recombinants may be
subject to strong balancing, directional, or diversifying
selection.
Combinatorially selected guanosine-quartet structureis a potent inhibitor of human immunodeficiency virus envelope-mediated cellfusion.
Authors: Wyatt J R , Vickers T A , Roberson J L , Buckheit R W , Klimkait T , DeBaets E , Davis P W , Rayner B , Imbach J L , Ecker D J
Published: May 2006

The phosphorothioate oligonucleotide T2G4T2 was
identified as an inhibitor of HIV infection in vitro by combinatorial screening
of a library of phosphorothioate oligonucleotides that contained all possible
octanucleotide sequences. The oligonucleotide forms a parallel-stranded
tetrameric guanosine-quartet structure. Tetramer formation and the
phosphorothioate backbone are essential for antiviral activity. The tetramer
binds to the human immunodeficiency virus envelope protein gp120 at the V3 loop
and inhibits both cell-to-cell and virus-to-cell infection.
Molecular cloning and structure of a pre-B-cell growth-stimulating factor.
Authors: Nagasawa T , Kikutani H , Kishimoto T
Published: May 2006

Generation and proliferation of early B-cell progenitors have been known to require stromal cell-derived molecules. A stromal cell line, PA6, was found to produce a soluble mediator, which was distinct from interleukin 7 (IL-7) and stem cell factor and supported the proliferation of a stromal cell-dependent pre-B-cell clone, DW34. A cDNA clone encoding this DW34 growth-stimulating factor was isolated by expression cloning. The nucleotide sequence contained a single substantial open reading frame of 267 nucleotides encoding an 89-amino acid polypeptide. The amino acid sequence of this cytokine, designated pre-B-cell growth-stimulating factor (PBSF), revealed that it is a member of intercrine alpha subfamily. Recombinant PBSF stimulated the proliferation of DW34 cells for itself and, furthermore, synergistically augmented the growth of DW34 as well as bone marrow B-cell progenitors in the presence of IL-7.