bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of a multidrug-resistance gene in human tumors and tissues.
Authors: Fojo A T , Ueda K , Slamon D J , Poplack D G , Gottesman M M , Pastan I
Published: May 2006

The identification and cloning of a segment of a human multidrug resistance gene (mdr1) was reported recently. To examine the molecular basis of one type of multidrug resistance, we have prepared RNA from human tumors and normal tissues and measured their content of mdr1 RNA. We find that the mdr1 gene is expressed at a very high level in the adrenal gland; at a high level in the kidney; at intermediate levels in the lung, liver, lower jejunum, colon, and rectum; and at low levels in many other tissues. The mdr1 gene is also expressed in several human tumors, including many but not all tumors derived from the adrenal gland and the colon. In addition, increased expression was detected in a few tumors at the time of relapse following initial chemotherapy. Although controlled clinical studies will be required, our results suggest that measurement of mdr1 RNA may prove to be a valuable tool in the design of chemotherapy protocols.
Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies.
Authors: Venkateswaran P S , Millman I , Blumberg B S
Published: May 2006

An aqueous extract of the plant Phyllanthus niruri inhibits endogenous DNA polymerase of hepatitis B virus and binds to the surface antigen of hepatitis B virus in vitro. The extract also inhibits woodchuck hepatitis virus (WHV) DNA polymerase and binds to the surface antigen of WHV in vitro. The extract, nontoxic to mice, was tested for antiviral activity in woodchucks (Marmota monax). In a trial using six long-term WHV-carrier woodchucks, five treated animals showed a faster decrease in woodchuck hepatitis virus surface antigen titer compared to one untreated control. In animals recently infected with WHV, the extract was effective when administered i.p. in three out of four animals in reducing and within 3-6 weeks eliminating both the surface antigen titer and DNA polymerase activity in serum. The treatment was discontinued after 10 weeks, and the treated animals have remained free of detectable markers of WHV for more than 45 weeks. In contrast, three untreated controls remained positive for both markers for WHV. One of the controls died after 8 weeks; the other two controls have remained positive for WHV markers for more than 45 weeks. In a third trial with long-term carriers, test animals treated subcutaneously with the extract for 12 weeks did not respond; but on switching the mode of administration to i.p., two out of the five animals showed a significant decrease in woodchuck hepatitis virus surface antigen titer compared to controls.
Involvement of a specific protein in the regulation of a circadian rhythm in Aplysia eye.
Authors: Yeung S J , Eskin A
Published: May 2006

Our previous results indicated that protein synthesis was necessary for serotonin (5-hydroxytryptamine, 5-HT) to regulate the phase of the biological clock in the Aplysia eye. Also, we showed that 5-HT appeared to increase the synthesis of a 34-kDa protein with an isoelectric point of 7.2. Subsequent studies were carried out to quantitate the effect of 5-HT on the 34-kDa protein and to examine whether the 34-kDa protein was involved in the circadian timing system. The regional specificity of the effect of 5-HT on the 34-kDa protein was investigated. The proximal portion of the eye appeared to synthesize much more of the 34-kDa protein than the distal portion. Also, 5-HT had a much larger effect on the synthesis of the 34-kDa protein in the proximal portion than in the distal portion. The proximal location of synthesis and the 5-HT effect on the synthesis of the 34-kDa protein correlate with the proximal location of cells and processes that are necessary for the expression of the circadian rhythm. The relationship between the effect of 5-HT on the circadian rhythm and the effect of 5-HT on the 34-kDa protein was also examined. As 5-HT causes phase shifts in the rhythm by activating adenylate cyclase to increase cAMP, forskolin and 8-benzylthioadenosine 3',5'-cyclic monophosphate mimicked the effect of 5-HT on the 34-kDa protein. We also found that 5-HT significantly increased the synthesis of the 34-kDa protein at phases when 5-HT delays or advances the phase of the rhythm but did not increase the synthesis of the 34-kDa protein at a phase when 5-HT did not phase shift. This phase-dependent effect of 5-HT on the 34-kDa protein qualitatively accounts for the phase dependence of the effect of 5-HT on the circadian rhythm. These results, when considered together with our earlier data, suggest that the synthesis of the 34-kDa protein is directly involved in the phase shift produced by 5-HT. The 34-kDa protein is worthy of future investigation as a candidate for a component of the circadian oscillator.
Acute insulin action requires insulin receptor kinase activity: introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin.
Authors: Morgan D O , Roth R A
Published: May 2006

The role of the insulin receptor tyrosine kinase (protein-tyrosine kinase, EC 2.7.1.112) in various rapid insulin effects was studied by injecting four different cell types (by osmotic lysis of pinocytotic vesicles) with a monoclonal antibody that specifically inhibits the kinase activity of the insulin receptor and the closely related receptor for insulin-like growth factor (IGF)-I. Injection of this inhibitory antibody resulted in a decreased ability of insulin to stimulate the uptake of 2-deoxyglucose in Chinese hamster ovary cells and freshly isolated rat adipocytes, ribosomal protein S6 phosphorylation in CHO cells, and glycogen synthesis in the human hepatoma cell line HepG2. The ability of insulin, IGF-I, and IGF-II to stimulate glucose uptake in TA1 mouse adipocytes was also inhibited. Studies with CHO cells demonstrated that these effects of the inhibitory antibody were specific, since there was no change in phorbol ester-stimulated glucose uptake and injection of a noninhibiting antibody to the kinase had no effect on insulin action. These studies indicate that the tyrosine kinase activity of the insulin receptor is important in mediating several rapid insulin effects in a variety of different cell types.
Accessible surface areas as a measure of the thermodynamic parameters of hydration of peptides.
Authors: Ooi T , Oobatake M , Némethy G , Scheraga H A
Published: May 2006

A method is described for the inclusion of the effects of hydration in empirical conformational energy computations on polypeptides. The free energy of hydration is composed of additive contributions of various functional groups. The hydration of each group is assumed to be proportional to the accessible surface area of the group. The constants of proportionality, representing the free energy of hydration per unit area of accessible surface, have been evaluated for seven classes of groups (occurring in peptides) by least-squares fitting to experimental free energies of solution of small monofunctional aliphatic and aromatic molecules. The same method has also been applied to the modeling of the enthalpy and heat capacity of hydration, each of which is computed from the accessible surface area.
Apparent lack of discrimination in the reading of certain codons in Mycoplasma mycoides.
Authors: Samuelsson T , Guindy Y S , Lustig F , Borén T , Lagerkvist U
Published: May 2006

We report a cluster of four tRNA genes from Mycoplasma mycoides as well as the sequence of the alanine, proline, and valine tRNAs and the serine tRNA reading the UCN codons (where N stands for G, A, C, or U). This brings the total number of tRNA genes that we have so far characterized in this organism to 14, 6 of which code for tRNAs that read the codons of family boxes. In each of these latter cases, we found only one gene per family box, and the gene sequence contains a thymidine in the position corresponding to the wobble nucleotide, with the exception of the arginine tRNA gene that has an adenosine in this position. Furthermore, all of the tRNA structures reported here have an unsubstituted uridine in the wobble position. These findings are similar to those reported for mitochondria, especially yeast mitochondria, that contain an arginine tRNA with the anticodon ACG. However, the resemblance is not complete since we have demonstrated the presence of two isoacceptor tRNAs for threonine having uridine and adenosine, respectively, in the wobble position. It is suggested that in the M. mycoides at least some of the family codon boxes are read by only one tRNA each, using an unconventional method without discrimination between the nucleotides in the third codon position.
Expression of p21ras in normal and malignant human tissues: lack of association with proliferation and malignancy.
Authors: Chesa P G , Rettig W J , Melamed M R , Old L J , Niman H L
Published: May 2006

Proteins encoded by cellular ras oncogenes (p21ras) are expressed in a wide variety of malignant tumors, including carcinomas, lymphomas, and neuroectodermal tumors. The function of p21ras in these tumors and the distribution and role of p21ras in corresponding normal tissues are unclear. This immunohistochemical study examined the relationship between p21ras expression and malignant transformation, cellular differentiation, and proliferative activity in vivo. p21ras was found to be widely expressed in normal tissues, but within those tissues expression was often sharply restricted to cells at specific stages of differentiation; terminally differentiated cells generally showed stronger reactivity with antibodies to p21ras than did rapidly proliferating cells. Fetal and adult tissues had corresponding patterns of p21ras expression, and the distribution of p21ras in neoplasms paralleled the pattern in normal tissue from which they were derived. Thus, p21ras seems to play a role in many fully differentiated cell types, and levels of p21ras expression do not correlate with proliferative activity in normal cell or, in contrast to past reports, with the transformed phenotype.
The very late antigen family of heterodimers is part of a superfamily of molecules involved in adhesion and embryogenesis.
Authors: Takada Y , Strominger J L , Hemler M E
Published: May 2006

The very late antigen (VLA) protein family contains at least five related heterodimers, including a fibronectin receptor structure, and probably other cell substrate adhesion receptors. These cell-surface VLA proteins were immunopurified from human placenta (VLA-1, VLA-3, and VLA-5), platelets (VLA-2), and Molt-4 cells (VLA-4) using a series of monoclonal antibody-Sepharose immunoaffinity columns. After further purification by gel electrophoresis, the N-terminal amino acid sequence for each of the five VLA alpha subunits was determined. In the first 14 positions, the five VLA alpha subunits showed an average of 42% homology to each other, rising to 59% including conservative amino acid substitutions. In addition, the alpha subunits from the LFA-1, Mac-1 (CR-3), and p150,95 family of heterodimers, the vitronectin receptor-platelet GPIIb/IIIa family, and a position-specific (PS) antigen important in Drosophila embryogenesis each showed average homologies of 31-40% to individual VLA alpha sequences and 46-52% homology to VLA alpha subunits including conservative substitutions. Taken together, these results suggest that the VLA proteins, the LFA-1, Mac-1, and p150,95 family, the GPIIb/IIIa, vitronectin receptor family, and the Drosophila PS antigens have evolved as four subgroups in a highly conserved supergene family of receptors involved in fundamentally important functions, such as cell adhesion, migration, and embryogenesis.
Growth factor(s) produced during infection with an adenovirus variant stimulates proliferation of nonestablished epithelial cells.
Authors: Quinlan M P , Sullivan N , Grodzicker T
Published: May 2006

Infection of primary baby rat kidney cells with an adenovirus variant that encodes only the 12S gene of the E1A region, adenovirus type 5 (Ad5) 12S, results in the production of a growth factor that stimulates primary epithelial cells to proliferate. Increased epithelial cell DNA synthesis and proliferation is detectable between 24 and 36 hr after the addition of conditioned medium from Ad5 12S infected cells and not from cells infected with an E1A deletion mutant virus, Ad5 dl312. This mitogenic factor(s) is effective in the absence of serum and can override the inhibitory effect of serum on primary epithelial cells. Furthermore, there is a requirement for the continued presence of the growth factor(s) in the Ad5 12S conditioned medium to maintain epithelial cell proliferation, and the conditioned medium can maintain these cells in a proliferative state for at least 6 wk. The stimulatory activity in Ad5 12S conditioned medium is associated with large molecular weight complexes, from which it can be released by 4 M NaCl. Several characteristics of the growth factor(s) indicate that it is a unique mitogen for epithelial cells.
Structure and expression of human dihydropteridine reductase.
Authors: Lockyer J , Cook R G , Milstien S , Kaufman S , Woo S L , Ledley F D
Published: May 2006

Dihydropteridine reductase (DHPR; EC 1.6.99.7) catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pterin-dependent aromatic amino acid hydroxylating systems. A cDNA for human DHPR was isolated from a human liver cDNA library in the vector lambda gt11 using a monospecific antibody against sheep DHPR. The nucleic acid sequence and amino acid sequence of human DHPR were determined from a full-length clone. A 112 amino acid sequence of sheep DHPR was obtained by sequencing purified sheep DHPR. This sequence is highly homologous to the predicted amino acid sequence of the human protein. Gene transfer of the recombinant human DHPR into COS cells leads to expression of DHPR enzymatic activity. These results indicate that the cDNA clone identified by antibody screening is an authentic and full-length cDNA for human DHPR.
Neurofilament gene expression: a major determinant of axonal caliber.
Authors: Hoffman P N , Cleveland D W , Griffin J W , Landes P W , Cowan N J , Price D L
Published: May 2006

Within the wide spectrum of axonal diameters occurring in mammalian nerve fibers, each class of neurons has a relatively restricted range of axonal calibers. The control of caliber has functional significance because diameter is the principal determinant of conduction velocity in myelinated nerve fibers. Previous observations support the hypothesis that neurofilaments (NF) are major intrinsic determinants of axonal caliber in large myelinated nerve fibers. Following interruption of axons (axotomy) by crushing or cutting a peripheral nerve, caliber is reduced in the proximal axonal stumps, which extend from the cell bodies to the site of axotomy. (The distal axonal stumps, which are disconnected from the cell bodies, degenerate and are replaced by the outgrowth of regenerating axonal sprouts arising from the proximal stump). This reduction in axonal caliber in the proximal stumps is associated with a selective diminution in the amount of NF protein undergoing slow axonal transport in these axons, with a decrease in axonal NF content, and with reduced conduction velocity. The present report demonstrates that changes in axonal caliber after axotomy correlate with a selective alteration in NF gene expression. Hybridization with specific cDNAs was used to measure levels of mRNA encoding the 68-kDa neurofilament protein (NF68), beta-tubulin, and actin in lumbar sensory neurons of rat at various times after crushing the sciatic nerve. Between 4 and 42 days after axotomy by nerve crush, the levels of NF68 mRNA were reduced 2- to 3-fold. At the same times, the levels of tubulin and actin mRNAs were increased several-fold. These findings support the hypothesis that the expression of a single set of neuron-specific genes (encoding NF) directly determines axonal caliber, a feature of neuronal morphology with important consequences for physiology and behavior.
RETRACTED: Calcium-dependent effect of the thymic polypeptide thymopoietin on the desensitization of the nicotinic acetylcholine receptor.
Authors: Revah F , Mulle C , Pinset C , Audhya T , Goldstein G , Changeux J P
Published: May 2006

The effects of the thymic polypeptide thymopoietin (Tpo) on the properties of the nicotinic acetylcholine receptor (AcChoR) were investigated by patch clamp techniques on mouse C2 myotubes and by biochemical assays on AcChoR-rich membrane fragments purified from the Torpedo marmorata electric organ. At high concentrations (greater than 100 nM), Tpo inhibits the binding of cholinergic agonists to the AcChoR in a Ca2+-insensitive manner. At lower concentrations (2 nM), Tpo applied on C2 myotubes simultaneously with nondesensitizing concentrations of acetylcholine results in the appearance of long closed times separating groups of openings. This effect depends on the presence of Ca2+ in the external medium. Outside-out recordings, performed with various concentrations of EGTA in the intracellular medium, suggest that Ca2+ acts on the cytoplasmic face of the membrane after entry through acetylcholine-activated channels. Parallel studies with T. marmorata AcChoR-rich membranes show that in the presence of Ca2+ Tpo causes a decrease in the apparent equilibrium dissociation constant of the noncompetitive blocker [3H]phencyclidine, enhances, at low concentrations, the binding of [3H]acetylcholine, and also alters the binding kinetics of the fluorescent agonist 6-(5-dimethylamino-1-naphthalenesulfonamido)-n-hexanoic acid beta-(N-trimethylammonium bromide) ethyl ester to the AcChoR. It was concluded that, in the presence of Ca2+, Tpo displaces the conformational equilibrium of the AcChoR towards a high-affinity desensitized state and increases the transition rate towards the same state.
Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase.
Authors: Leblanc G G , Trimmer B A , Landis S C
Published: May 2006

Neuropeptide Y (NPY) is widely distributed in the sympathetic nervous system, where it is colocalized with norepinephrine. We report here that NPY-immunoreactive neurons are also abundant in three cranial parasympathetic ganglia, the otic, sphenopalatine, and ciliary, in the rat. High-performance liquid chromatographic analysis of the immunoreactive material present in the otic ganglion indicates that this material is very similar to porcine NPY and indistinguishable from the NPY-like immunoreactivity present in rat sympathetic neurons. These findings raise the possibility that NPY acts as a neuromodulator in the parasympathetic as well as the sympathetic nervous system. In contrast to what has been observed for sympathetic neurons, NPY-immunoreactive neurons in cranial parasympathetic ganglia do not contain detectable catecholamines or tyrosine hydroxylase (EC 1.14.16.2) immunoreactivity, and many do contain immunoreactivity for vasoactive intestinal peptide and/or choline acetyltransferase (EC 2.3.1.6). These findings suggest that there is no simple rule governing coexpression of NPY with norepinephrine, acetylcholine, or vasoactive intestinal peptide in autonomic neurons. Further, while functional studies have indicated that NPY exerts actions on the peripheral vasculature which are antagonistic to those of acetylcholine and vasoactive intestinal peptide, the present results raise the possibility that these three substances may have complementary effects on other target tissues.
Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues.
Authors: Prody C A , Zevin-Sonkin D , Gnatt A , Goldberg O , Soreq H
Published: May 2006

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ "true" acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from lambda gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.
Rapid enhancement of beta 2-interferon/B-cell differentiation factor BSF-2 gene expression in human fibroblasts by diacylglycerols and the calcium ionophore A23187.
Authors: Sehgal P B , Walther Z , Tamm I
Published: May 2006

The expression in human fibroblasts of the beta 2-interferon (IFN-beta 2) gene, which is now recognized to be identical to the gene encoding B-cell differentiation factor BSF-2, is enhanced by several cytokines that affect cell growth (tumor necrosis factor, interleukin 1, platelet-derived growth factor, and beta 1-interferon). We have examined the possibility that IFN-beta 2 gene expression is regulated through activation, by diacylglycerol, of the protein kinase C pathway. The synthetic diacylglycerols 1,2-dioctanoylglycerol (diC8) and 1-oleoyl-2-acetylglycerol strongly enhanced IFN-beta 2, but not IFN-beta 1, gene expression in human fibroblasts (FS-4 strain). An increase in IFN-beta 2 mRNA level was detected within 15 min after addition of diC8 (290 microM) to FS-4 cells and was maximal approximately 20 hr later. An increase in IFN-beta 2 gene transcription was detected within 5 min of addition of diC8, and the rate of transcription was near-maximal by 15-30 min. The enhancement of IFN-beta 2 gene expression by diC8, interleukin 1, or tumor necrosis factor was not prevented by H8, a preferential inhibitor of cAMP- and cGMP-dependent protein kinases, but was blocked by H7, an inhibitor of protein kinase C as well as of cyclic nucleotide-dependent protein kinases. diC8 was found to protect FS-4 cells from the cytopathic effect of vesicular stomatitis virus; this protection was blocked by polyclonal or monoclonal antibodies that neutralize IFN-beta, suggesting that the antiviral effect was due to the secretion of IFN-beta 2 by the diC8-treated fibroblasts. The calcium ionophore A23187 (1-10 microM) also elicited an increase in the level of IFN-beta 2 mRNA in FS-4 fibroblasts; appropriate combinations of A23187 and diC8 had at least an additive effect in enhancing IFN-beta 2 mRNA levels. These results show that protein kinase C-activating or [Ca2+]-elevating agents rapidly increase the expression of the IFN-beta 2 gene in human fibroblasts.
Spectral and kinetic evidence for the existence of two forms of bathorhodopsin.
Authors: Einterz C M , Lewis J W , Kliger D S
Published: May 2006

Transient-absorption difference spectra from 320 nm to 700 nm were obtained at times ranging from 30 ns to 1200 ns after 532-nm photolysis of rhodopsin at room temperature. Kinetics on this time scale at various wavelengths are also presented. The isosbestic points between spectra acquired at successive times after photolysis shift from 510 nm to 530 nm. This shift is inconsistent with a simple process of one bathorhodopsin (BathoR) intermediate being transformed into one lumirhodopsin (LumiR) intermediate on this time scale. The kinetics at 425 nm, 515 nm, and 575 nm could not be fit well to a single-exponential expression. The data are consistent with the existence of two forms of BathoR (BathoR1 and BathoR2) that exhibit different spectra and decay kinetics. The BathoR1 absorption maximum lies near 565 nm, and the BathoR1/LumiR1 isosbestic point is near 430 nm. The BathoR2 absorption maximum lies near 535 nm, and the BathoR2/LumiR2 isosbestic point is near 480 nm. The kinetics at the isosbestic wavelengths were fit to single-exponential expressions corresponding to BathoR1 and BathoR2 lifetimes of 170 +/- 20 ns and 36 +/- 15 ns, respectively.
Nucleotide sequence and evolution of ETn elements.
Authors: Sonigo P , Wain-Hobson S , Bougueleret L , Tiollais P , Jacob F , Brûlet P
Published: May 2006

The ETn (for "early transposon") family of long repeated sequences in abundantly transcribed in early mouse embryos from retroviral-like long terminal repeats. Nucleotide sequencing of two elements does not reveal any long open reading frame nor significant homology to retroviral proteins. The genetic polymorphism, monitored by Southern blotting within and across mouse species, reflects a concerted mode of evolution for the ETn sequences.
Interleukin 4 is a growth factor for activated thymocytes: possible role in T-cell ontogeny.
Authors: Zlotnik A , Ransom J , Frank G , Fischer M , Howard M
Published: May 2006

We have shown that recombinant or natural interleukin 4 (IL-4) (formerly called B-cell stimulatory factor 1) induces proliferation of activated adult or fetal thymocytes. In the case of adult thymocytes, IL-4 in combination with Con A or phorbol 12-myristate 13-acetate (PMA) stimulated the proliferation of peanut agglutinin (PNA)-negative (-) thymocytes, while PNA-positive (+) thymocytes showed only marginal responses. Further investigation revealed that day 14-17 fetal thymocytes, purified L3T4- LyT2- double-negative adult thymocytes, and single positive L3T4+ LyT2- or L3T4- LyT2+ thymocytes failed to respond to IL-4 or PMA alone but proliferated strongly with both IL-4 and PMA. In contrast, purified double-positive L3T4+ LyT2+ adult thymocytes showed only a marginal proliferative response to these stimuli. Responsiveness of thymic subpopulations to PMA and IL-4 could be inhibited with anti-IL-4 but not with anti-IL-2 monoclonal antibodies, indicating that they were IL-2 independent. Finally, we have observed that supernatants from calcium ionophore and PMA-stimulated adult double-negative L3T4- LyT2- thymocytes induce proliferation of double-negative adult thymocytes. This latter response is inhibited by anti-IL-4 monoclonal antibodies, suggesting that under appropriate stimulation conditions, these immature thymocytes are able to produce IL-4. These observations suggest a role for IL-4 in T-cell ontogeny.
Evidence from x-ray and neutron powder diffraction patterns that the so-called icosahedral and decagonal quasicrystals of MnAl
6
and other alloys are twinned cubic crystals
Authors: Pauling Linus
Published: May 2006

It is shown that the x-ray powder diffraction patterns of rapidly quenched MnAl
6
and Mg
32
(Al,Zn)
49
and the neutron powder diffraction pattern of MnAl
6
are compatible with the proposed 820-atom primitive cubic structure [Pauling, L. (1987)
Phys. Rev. Lett.
58, 365-368]. The values found for the edge of the unit cube are 23.365 Å (x-ray) and 23.416 Å (neutron) for MnAl
6
and 24.313 Å (x-ray) for Mg
32
(Al,Zn)
49
.
Bidirectional assembly of tobacco mosaic virus
in vitro
Authors: Fukuda Megumi , Okada Yoshimi
Published: May 2006

The timing of the bidirectional growth in the assembly reaction of tobacco mosaic virus has been the subject of controversy: Does elongation actually occur simultaneously to 5′ and 3′ ends or sequentially, first to the 5′ end and then to the 3′ end? To determine the timing of elongation toward the 3′ end directly, using the S1 nuclease mapping method on a cloned cDNA with micrococcal nuclease-digested tobacco mosaic virus RNA, we analyzed encapsidation of the RNA region that was located downstream from the assembly origin. The results clearly showed that elongation toward the 3′ end did not occur for at least the first 4 min. Actually it was first observed at 8 min. It is concluded that, in the first 5-7 min, a rapid elongation of the nucleation complex occurs only toward the 5′ end of the RNA and that this gives rise to an intermediate particle 260 nm long. Furthermore, the lengths of the RNA that were protected against S1 nuclease digestion showed a clear banding pattern that had a spacing of ≈100 nucleotides. This supports the hypothesis that the 20S aggregate is kinetically favored as the protein source for elongation to the 3′ end.
Structure of the glycosaminoglycan domain in the type IX collagen-proteoglycan.
Authors: McCormick D , van der Rest M , Goodship J , Lozano G , Ninomiya Y , Olsen B R
Published: May 2006

Type IX collagen represents 5-20% of the total collagen in hyaline cartilage. The molecules of this collagen are composed of three genetically distinct polypeptide subunits. One of these subunits, alpha 2(IX), contains covalently bound glycosaminoglycan (chondroitin sulfate or dermatan sulfate). We report here on the structure of the glycosaminoglycan attachment site of type IX collagen-proteoglycan. We show, by a combination of cDNA and peptide sequencing, that the attachment region contains the sequence Gly-Ser-Ala-Asp, located within the noncollagenous domain NC3 of the alpha 2(IX) chain. By comparing the exons encoding the NC3 domain in the alpha 2(IX) and alpha 1(IX) genes, we find that the exon coding for the glycosaminoglycan attachment site in the alpha 2(IX) gene is 48 base pairs long, whereas the homologous alpha 1(IX) exon is 33 base pairs. The NC3 domain is, therefore, five amino acid residues longer in alpha 2(IX) than in alpha 1(IX). The extra sequence in alpha 2(IX), Val-Glu-Gly-Ser-Ala, provides a simple explanation for the kink observed at the NC3 domain of type IX molecules when examined by electron microscopy. The inserted block of amino acid residues also provides the NC3 domain of alpha 2(IX) chains with a serine residue, not present in alpha 1(IX) that serves as attachment site for a glycosaminoglycan side chain. Our data show that the amino acid sequence that surrounds the glycosylated serine residue in type IX collagen-proteoglycan differs from glycosylated sequences in noncollagenous core proteins. The data also provide strong evidence that glycosylation of type IX collagen is not a chance glycosylation of a serine residue in a noncollagenous domain, but is a specific post-translational modification of this unusual collagen molecule.
Nucleotide sequence of medium-chain acyl-CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue.
Authors: Kelly D P , Kim J J , Billadello J J , Hainline B E , Chu T W , Strauss A W
Published: May 2006

Medium-chain acyl-CoA dehydrogenase (MCAD; acyl-CoA: (acceptor) 2,3-oxidoreductase, EC 1.3.99.3) is one of three similar enzymes that catalyze the initial step of fatty acid beta-oxidation. Definition of the primary structure of MCAD and the tissue distribution of its mRNA is of biochemical and clinical importance because of the recent recognition of inherited MCAD deficiency in humans. The MCAD mRNA nucleotide sequence was determined from two overlapping cDNA clones isolated from human liver and placental cDNA libraries, respectively. The MCAD mRNA includes a 1263-base-pair coding region and a 738-base-pair 3'-nontranslated region. A partial amino acid sequence (137 residues) determined on peptides derived from MCAD purified from porcine liver confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human MCAD cDNA with the partial protein sequence of the porcine MCAD revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis shows that MCAD mRNA is expressed in a variety of rat (2.2 kilobases) and human (2.4 kilobases) tissues. Blot hybridization of RNA prepared from cultured skin fibroblasts from a patient with MCAD deficiency disclosed that mRNA was present and of similar size to MCAD mRNA derived from control fibroblasts. The isolation and characterization of MCAD cDNA is an important step in the definition of the defect underlying MCAD deficiency and in understanding its metabolic consequences.
Human chromosomes 6 and 21 are required for sensitivity to human interferon gamma.
Authors: Jung V , Rashidbaigi A , Jones C , Tischfield J A , Shows T B , Pestka S
Published: May 2006

The human interferon gamma receptor has previously been assigned to chromosome 6. Chromosome 6 also encodes HLA, the human class I major histocompatibility antigens. However, the presence of chromosome 6 in hamster-human hybrids is by itself insufficient to confer sensitivity to human immune interferon as measured by the induction of human HLA. Human chromosome 21 was found to be the second chromosome essential for HLA inducibility. Similar results were found with mouse-human somatic cell hybrids. Thus, at least two steps are involved in the action of human interferon gamma: the binding of interferon gamma to its receptor coded by chromosome 6 and the linkage of this binding event through a factor coded by chromosome 21 to trigger biological action. Both of these steps are species-specific.
Genetic transformation of the fungal pathogen responsible for rice blast disease
Authors: Parsons Kenneth A. , Chumley Forrest G. , Valent Barbara
Published: May 2006

The analysis of complex genetic determinants that control the ability of a fungus to colonize its host has been impaired by the lack of sophisticated genetic tools for characterizing important pathogens. We have developed a system for the genetic transformation of
Magnaporthe grisea
, the causal agent of rice blast disease, to overcome this limitation. A
M. grisea
arginine auxotroph was shown to contain a mutation (
arg3-12
) that abolishes ornithine carbamoyltransferase activity.
M. grisea
strains that contain
arg3-12
were used as recipients in transformation experiments with plasmid pMA2, which carries the
ArgB
+
gene from
Aspergillus nidulans
. Stable prototrophic transformants arose at a frequency of about 35 per microgram of plasmid DNA. Integration of single or multiple plasmid copies occurred at a single site in the genome of each transformant; rearrangements were often created during integration. When
M. grisea
genomic segments were incorporated into pMA2, the presence of any one of five different
M. grisea
segments did not greatly affect the efficiency of transformation. Integration via homologous recombination occurred when the donor plasmid was linearized by cleaving at a unique restriction site within the
M. grisea
segment.
Nonreciprocal recombination between alleles of the chloroplast 23S rRNA gene in interspecific
Chlamydomonas
crosses
Authors: Lemieux Claude , Lee Robert W.
Published: May 2006

The inheritance of six polymorphic loci mapping in the rRNA-encoding (rDNA) region of the inverted repeat sequence of chloroplast DNA (cpDNA) was scored in hybrid subclones derived from reciprocal interspecific crosses between the green algae
Chlamydomonas eugametos
and
Chlamydomonas moewusii
. In order to enhance the detection of cells that had undergone recombination between parental cpDNAs, hybrids were selected that inherited a chloroplast antibiotic-resistance marker contributed by the mating-type-minus(
mt
-
) parent, the parent that normally contributes fewer cpDNA molecules. The major findings of this study can be summarized as follows. (
i
) The majority of the hybrids (14/17) were recombinant for cpDNA markers in the 10-kilobase-pair rDNA region under study. (
ii
) Only one allele of each polymorphic cpDNA locus was ever detected in the hybrids, thus suggesting that newly recombined rDNA sequences in one copy of the inverted repeat are rapidly spread to the other by a copy-correction mechanism. (
iii
) Chloroplast streptomycin-resistance (
sr-2
) and erythromycin-resistance (
er-nM1
) loci, although showing little or no genetic linkage, were mapped to the 16S and 23S rRNA gene regions of the cpDNA, respectively, by virtue of their perfect coinheritance with polymorphic markers within these genes. (
iv
) cpDNA markers associated with a putative intron of the
C. eugametos
23S rRNA gene were inherited by all 17 hybrids. Such a result is similar to that observed for certain alleles of the large rRNA gene of yeast mitochondria in crosses between ω
+
and ω
-
strains.
Hyperinducibility of Ia antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis.
Authors: Massa P T , ter Meulen V , Fontana A
Published: May 2006

In search of a phenotypic marker determining genetically controlled susceptibility to delayed-type hypersensitivity (DTH) reactions in the brain--in particular, experimental autoimmune encephalomyelitis (EAE)--we have compared the gamma-interferon (IFN-gamma) induction of Ia molecules on astrocytes and macrophages from rat and mouse strains that are susceptible or resistant to this disease. We focused on Ia expression because DTH reactions to self or foreign antigens are largely mediated by lymphocytes restricted by class II (Ia) antigens of the major histocompatibility complex (MHC). Our data demonstrate that Lewis (fully susceptible) and Brown Norway (BN) (fully resistant) rats are very different in that Lewis astrocytes express much higher levels of Ia than BN astrocytes. Similar data were obtained from an analysis of EAE-susceptible and -resistant mouse strains (SJL and BALB/c, respectively), which suggests that this phenomenon may be universal and not limited to only one mammalian species. At least one gene responsible for Ia hyperinduction is located outside the rat RT-1 or the mouse MHC locus. Animals congenic at the RT-1 or MHC locus of the resistant strain but with background genes of the susceptible strain exhibit intermediate levels of Ia compared to fully resistant and susceptible rodents, which fits well with the reduced EAE susceptibility of these congenic animals. Furthermore, hyperinduction of Ia is astrocyte specific, since peritoneal macrophages of susceptible and resistant strains exhibit identical profiles of Ia induction. Thus, astrocyte Ia hyperinducibility may be a major strain- and tissue-specific factor that contributes to Ia-restricted DTH reactions in the brain.
Helper T-cell antigenic site identification in the acquired immunodeficiency syndrome virus gp120 envelope protein and induction of immunity in mice to the native protein using a 16-residue synthetic peptide.
Authors: Cease K B , Margalit H , Cornette J L , Putney S D , Robey W G , Ouyang C , Streicher H Z , Fischinger P J , Gallo R C , DeLisi C
Published: May 2006

Much effort has been devoted to the analysis of antibodies to acquired immunodeficiency syndrome virus antigens, but no studies, to our knowledge, have defined antigenic sites of this virus that elicit T-cell immunity, even though such immunity is important in protection against many other viruses. T cells tend to recognize only a limited number of discrete sites on a protein antigen. Analysis of immunodominant helper T-cell sites has suggested that such sites tend to form amphipathic helices. An algorithm based on this model was used to identify two candidate T-cell sites, env T1 and env T2, in the envelope protein of human T-lymphotropic virus type IIIB that were conserved in other human immunodeficiency virus isolates. Corresponding peptides were synthesized and studied in genetically defined inbred and F1 mice for induction of lymph node proliferation. After immunization with a 426-residue recombinant envelope protein fragment, significant responses to native gp 120, as well as to each peptide, were observed in both F1 combinations studied. Conversely, immunization with env T1 peptide induced T-cell immunity to the native gp 120 envelope protein. The genetics of the response to env T1 peptide were further examined and revealed a significant response in three of four independent major histocompatibility haplotypes tested, an indication of high frequency responsiveness in the population. Identification of helper T-cell sites should facilitate development of a highly immunogenic, carrier-free vaccine that induces T-cell and B-cell immunity. The ability to elicit T-cell immunity to the native viral protein by immunization with a 16-residue peptide suggests that such sites represent potentially important components of an effective vaccine for acquired immunodeficiency syndrome.
cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.
Authors: Lübbert H , Hoffman B J , Snutch T P , van Dyke T , Levine A J , Hartig P R , Lester H A , Davidson N
Published: May 2006

We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA.
Characterization of the promoter region of the human c-erbB-2 protooncogene.
Authors: Ishii S , Imamoto F , Yamanashi Y , Toyoshima K , Yamamoto T
Published: May 2006

Three overlapping genomic clones that contain the 5'-terminal portion of the human c-erbB-2 gene (ERBB2) were isolated. The promoter region was identified by nuclease S1 mapping with c-erbB-2 mRNA. Seven transcriptional start sites were identified. DNA sequence analysis showed that the promoter region contains a "TATA box" and a "CAAT box" about 30 and 80 base pairs (bp), respectively, upstream of the most downstream RNA initiation site. Two putative binding sites for transcription factor Sp1 were identified about 50 and 110 bp upstream of the CAAT box, and six GGA repeats were found between the CAAT box and the TATA box. This region had strong promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into monkey CV-1 cells. These data indicate that the promoter of the human c-erbB-2 protooncogene is different from that of the protooncogene c-erbB-1 (epidermal growth factor receptor gene), which does not contain either a TATA box or a CAAT box. Comparison of the promoter sequences and activities of the two protooncogenes should be helpful in analysis of the regulatory mechanism of expression of their gene products, which are growth-factor receptors.
Cell lines and peripheral blood leukocytes derived from individuals with chronic myelogenous leukemia display virtually identical proteins phosphorylated on tyrosine residues.
Authors: Huhn R D , Posner M R , Rayter S I , Foulkes J G , Frackelton A R
Published: May 2006

An aberrant p210BCR-ABL protein that possesses constitutive protein-tyrosine kinase activity is presumed to be involved in the development of the neoplastic phenotype in chronic myelogenous leukemia (CML). Using a highly specific antibody against phosphotyrosine, we have isolated the tyrosine-phosphorylated p210BCR-ABL and several other proteins containing phosphotyrosine from a variety of CML cell lines. p210BCR-ABL isolated by the monoclonal anti-phosphotyrosine antibody possessed protein-tyrosine kinase activity in vitro comparable to that of the p210BCR-ABL isolated by antibody to a specific peptide sequence in the ABL protein-tyrosine kinase. Other prominent proteins containing phosphorylated tyrosine residues were observed at 185, 150, 120, 105, 63, 56, 36, and 32 kDa, and less prominent proteins were observed at 195, 155, 94, 53, 40, and less than 29 kDa. Staphylococcal V8 peptide mapping indicated that proteins of similar molecular weights were highly homologous to each other across cell lines, despite the diverse hematopoietic lineages of these cells and the genetic heterogeneity of the patients from whom the CML cell lines were derived. Phosphopeptide mapping also revealed that these proteins were distinct from each other as well as from p210BCR-ABL. Because virtually identical phosphotyrosine-containing proteins were found in peripheral blood leukocytes taken directly from CML patients, these proteins are not an artifact of long-term tissue culture but appear to be an integral part of the CML phenotype.
Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase alpha subunit.
Authors: Yutani K , Ogasahara K , Tsujita T , Sugino Y
Published: May 2006

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, we have constructed a series of variant alpha subunits of tryptophan synthase from Escherichia coli substituted by each of 20 amino acids at position 49, which is buried in the interior of the protein. The stabilities were quantitatively examined except for the mutant protein substituted by arginine, which was not obtained in enough quantity. The Gibbs energy of unfolding in water and the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride for each protein were compared at pH 7.0 and pH 9.0. The Gibbs energy of unfolding in water at pH 7.0 varied from 0.72 to 1.92 times that of the wild-type protein by the substitutions, but the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride varied only from 0.95 to 1.03 times that of the wild-type protein. Moreover, the stability of the protein substituted at this position, which is buried in the interior of the molecule, tended to increase linearly with increasing hydrophobicity of the substituted residue, unless the volume of the substituted residue was over a certain limit.
Pst I restriction fragment length polymorphism of the human placental alkaline phosphatase gene in normal placentae and tumors.
Authors: Tsavaler L , Penhallow R C , Kam W , Sussman H H
Published: May 2006

The structure of the human placental alkaline phosphatase gene from normal term placentae was studied by restriction enzyme digestion and Southern blot analysis using a cDNA probe to the gene for the placental enzyme. The DNA digests fall into three distinct patterns based on the presence and intensity of an extra 1.1-kilobase Pst I band. The extra 1.1-kilobase band is present in 9 of 27 placenta samples, and in 1 of these samples the extra band is present at double intensity. No polymorphism was revealed by digestion with restriction enzymes EcoRI, Sma I, BamHI, or Sac I. The extra Pst I-digestion site may lie in a noncoding region of the gene because no correlation was observed between the restriction fragment length polymorphism and the common placental alkaline phosphatase alleles identified by starch gel electrophoresis. In addition, because placental alkaline phosphatase is frequently re-expressed in neoplasms, we examined tissue from ovarian, testicular, and endometrial tumors and from BeWo choriocarcinoma cells in culture. The Pst I-DNA digestion patterns from these cells and tissues were identical to those seen in the normal ovary and term placentae. The consistent reproducible digestion patterns seen in DNA from normal and tumor tissue indicate that a major gene rearrangement is not the basis for the ectopic expression of placental alkaline phosphatase in neoplasia.