bluuebunny/crossref_metadata_2025 huggingface dataset:
Solution conformation of gramicidin S: An intramolecular nuclear Overhauser effect study
Authors: Huang Dee-Hua , Walter Roderich , Glickson Jerry D. , Krishna N. Rama
Published: May 2006

The solution conformation of gramicidin S in deuterated dimethyl sulfoxide was investigated by using the intramolecular nuclear Overhauser effect experiment. Experimental Overhauser enhancements were compared with predicted values for each of the nine most-stable conformations (M1-M9) calculated by Dygert
et al
. on the basis of energy-minimization procedures [Dygert, M., Gō, N. & Scheraga, H. A. (1975)
Macromolecules
8, 750-761]. By using statistical hypothesis testing, the three lowest-energy conformations (M1, M2, and M3) were shown to give the best fit with the experimental data. All other conformations (M4-M9) were found to be inconsistent with the experimental data.
Simian virus 40 tandem repeated sequences as an element of the early promoter.
Authors: Gruss P , Dhar R , Khoury G
Published: May 2006

On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).
Active transport: conditions for linearity and symmetry far from equilibrium.
Authors: Essig A , Caplan S R
Published: May 2006

The impressive linearity of force-flow relationships in epithelial active-transport systems suggests the utility of a linear, nonequilibrium-thermodynamic analysis. We here present a plausibility argument for the appropriateness of such a treatment. Conventional phenomenological equations of nonequilibrium thermodynamics constitute an incomplete description of the processes under study, because a given thermodynamic force may be induced in an infinite number of ways. In general, therefore, flows are nonlinear functions of the forces, and the Onsager reciprocal relations are obeyed only very near equilibrium. If, however, the forces of two coupled processes can be constrained to "proper" pathways, each flow is a linear function of each force, and the phenomenological cross-coefficients are equal far from equilibrium. The nature of such proper pathways is investigated in terms of a simple model of a sodium-active transport system. Where the treatment is appropriate (i.e., for sufficiently small perturbations about a steady state far from equilibrium) it permits a complete thermodynamic characterization of a system, even when only one of the two forces can be controlled experimentally while the other remains constant.
Liponomic control of Ca2+ transport: relationship to mechanism of action of 1,25-dihydroxyvitamin D3.
Authors: Fontaine O , Matsumoto T , Goodman D B , Rasmussen H
Published: May 2006

Isolated vesicles prepared from the brush border membranes of chicken duodenal mucosal cells (enterocytes) take up calcium by a passive but saturable process. The rate of uptake (Vmax) is increased 2.5- to 3-fold, with no change in Km, in vesicles prepared from 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-treated chickens compared to vesicles from vitamin D-deficient controls. Preincubation of vesicles with either cis- or trans-vaccinic acid (cVA or tVA, respectively) or their methyl esters in vitro also alters the rates of calcium transport. Methyl cVA causes an increase in rate of calcium uptake into vesicles from vitamin D-deficient chickens but not in those from 1,25(OH)2D3-treated chickens. This increase is 80-90% of that seen after 1,25(OH)2D3 treatment. Higher concentrations of methyl cVA produce no further increases. Conversely, methyl tVA causes a decrease in rate of calcium uptake in vesicles from 1,25(OH)2D3-treated chickens but no change in vesicles from vitamin D-deficient controls. This decrease reduces the rate of calcium uptake to nearly the same value as seen in vesicles from vitamin D-deficient controls. Higher concentration of methyl tVA produce no further suppression of uptake rate. The changes seen were in the Vmax and not in the Km of the transport process. The fatty acids did not alter the process of Na+-dependent glucose uptake in the same membrane. These data demonstrate that a small alteration in the lipid structure of this membrane can specifically shift the activity of the calcium transport process.
Locations of adenovirus genes required for the replication of adenovirus-associated virus.
Authors: Janik J E , Huston M M , Rose J A
Published: May 2006

We have used DNA transfection to identify several regions of the adenovirus genome needed to induce replication of the defective parvovirus, adenovirus-associated virus (AAV). Previous studies have indicated that only early adenovirus functions are needed to aid the replication of AAV. In this report, we demonstrate that three restriction endonuclease fragments of adenovirus DNA are necessary for production of infectious AAV in 293-31 cells (an adenovirus type 5-transformed human embryonic kidney cell line). These fragments map from 28.5 to 29.4, 59.5 to 75.9, and 89.7 to 100 map units on the adenovirus type 2 genome and correspond to the locations of the VAI RNA gene, early region 2, and early region 4, respectively. The 293-31 cell line, which has been found to express early region 1A and 1B proteins, alone is incapable of supporting AAV replication or even AAV DNA synthesis. Additional experiments with adenovirus type 5 host range mutants (group I, hr1 and group II, hr7) indicate, however, that early region 1A provides an essential function(s) for AAV replication, whereas early region 1B probably does not.
recA protein promotes homologous-pairing and strand-exchange reactions between duplex DNA molecules.
Authors: West S C , Cassuto E , Howard-Flanders P
Published: May 2006

Purified recA protein, product of the recA+ gene, promotes homologous pairing between intact covalent circular duplex DNA and circular single-stranded DNA carrying a short hybridized fragment [West, S. C., Cassuto, E. & Howard-Flanders, P. (1981) Nature (London) 290, 29-33.]. In this paper we investigate the interaction of duplex fragments with circular single-stranded DNA carrying the hybridized fragment and find that recA protein promotes an efficient strand-exchange reaction between interacting DNA molecules. The exchange is dependent upon linear duplex DNA fragments that are homologous to, but extend beyond, the short fragment present on the hybridized DNA substrate. The reactions require stoichiometric amounts of recA protein and the presence of ATP.
Site of attachment of retinal in bacteriorhodopsin.
Authors: Bayley H , Huang K S , Radhakrishnan R , Ross A H , Takagaki Y , Khorana H G
Published: May 2006

The identity of the lysine residue in bacteriorhodopsin to which the chromophore, retinal, is attached as a Schiff's base has been reinvestigated. Retinal is now shown to be linked to lysine-216 and not lysine-41, as had been concluded previously. The retinal in purple membrane was replaced by [15-3H]retinal, the Schiff's base linkage was reduced with NaBH4 while the sample was illuminated, and the resulting [retinyl-3H]bacterio-opsin was cleaved with CNBr. The radioactivity was present exclusively in the COOH-terminal peptide (amino acids 210--248). Sequence analysis showed that the [3H]retinal was attached to lysine-216. The same site was labeled when purple membrane was reduced with NaBH4 in the light at pH greater than 10 or at pH 8 and when membranes modified with ether or solubilized with hexadecyltrimethylammonium bromide were reduced in the dark. Our finding places of Schiff's base linkage close to the midpoint of the putative membrane-spanning alpha-helix that is directly connected to the COOH terminus of bacteriorhodopsin.
Mechanisms for excited state relaxation and dissociation of oxymyoglobin and carboxymyoglobin.
Authors: Reynolds A H , Rand S D , Rentzepis P M
Published: May 2006

The dissociation of carboxymyoglobin (MbCO) and oxymyoglobin (MbO2) induced by 530-nm picosecond excitation in the beta band or the 355-nm delta band has been measured by monitoring the absorbance changes at 420 and 440 nm corresponding to ligand-bound and ligand-detached species, respectively. We find that MbO2 and MbCO dissociate with very similar rates, which do not reflect the 30-fold difference between the quantum yields of the two reactions. Kinetic data suggest that a short-lived intermediate is formed that is responsible for the low quantum efficiency of the MbO2 dissociation.
Evolutionary tree for apes and humans based on cleavage maps of mitochondrial DNA.
Authors: Ferris S D , Wilson A C , Brown W M
Published: May 2006

The high rate of evolution of mitochondrial DNA makes this molecule suitable for genealogical research on such closely related species as humans and apes. Because previous approaches failed to establish the branching order of the lineages leading to humans, gorillas, and chimpanzees, we compared human mitochondrial DNA to mitochondrial DNA from five species of ape (common chimpanzee, pygmy chimpanzee, gorilla, orangutan, and gibbon). About 50 restriction endonuclease cleavage sites were mapped in each mitochondrial DNA, and the six maps were aligned with respect to 11 invariant positions. Differences among the maps were evident at 121 positions. Both conserved and variable sites are widely dispersed in the mitochondrial genome. Besides site differences, ascribed to point mutations, there is evidence for one rearrangement: the gorilla map is shorter than the other owing to the deletion of 95 base pairs near the origin of replication. The parsimony method of deriving all six maps from a common ancestor produced a genealogical tree in which the common and pygmy chimpanzee maps are the most closely related pair; the closest relative of this pair is the gorilla map; most closely related to this trio is the human map. This tree is only slightly more parsimonious than some alternative trees. Although this study has given a magnified view of the genetic differences among humans and apes, the possibility of a three-way split among the lineages leading to humans, gorillas, and chimpanzees still deserves serious consideration.
Culture supernatants of a stimulated T-cell line have helper activity that acts synergistically with interleukin 2 in the response of B cells to antigen.
Authors: Swain S L , Dennert G , Warner J F , Dutton R W
Published: May 2006

Culture supernatants of an antigen-stimulated long-term alloreactive T cell line, C.C3.11.75, contain a T-cell-replacing factor (TRF) activity for the B-cell response to antigen. These same supernatants show little activity in the T-cell growth assay or the costimulator assay. TRF activity was assayed by using spleen cells that were rigorously depleted of both T cells and macrophages. In this assay preparations containing interleukin 2 and supernatants from stimulated C.C3.11.75 cells are relatively inactive if added alone but show marked synergy when added together. We conclude that the C.C3.11.75 TRF activity is not due to interleukin 1 or to interleukin 2 but to a third factor provisionally designated as (DL)TRF. This activity may be equivalent to the (late-acting) TRF described by Schimpl and Wecker. Evidence is presented suggesting that the helper activity (DL)TRF is a product of the T-cell line.
Membranes and phospholipids of liver mitochondria from chronic alcoholic rats are resistant to membrane disordering by alcohol.
Authors: Waring A J , Rottenberg H , Ohnishi T , Rubin E
Published: May 2006

Using the spin probe 5-doxylstearic acid, we studied the structural perturbations of rat liver mitochondrial membranes produced by exposure to ethanol in vitro and by chronic ethanol feeding. The addition of ethanol in vitro to mitochondria from control animals appears to "fluidize" the membranes, as evidenced by a pronounced decrease in the order parameter. By contrast, in membranes from rats fed ethanol chronically, there was no effect on the order parameter. This resistance of the mitochondrial membranes from chronically intoxicated animals to the fluidizing effect of ethanol probably results from a change in the composition of the phospholipids, because the same differential response to ethanol was observed upon using vesicles of mitochondrial phospholipids extracted from control and chronically treated rats. In the presence of 0.025--0.1 M ethanol, a range that prevails in the blood of chronic alcoholics, the order parameter of mitochondrial membranes from rats fed ethanol was comparable to that of control membranes without ethanol in vitro. Analysis of extracted mitochondrial phospholipids showed that the cardiolipin from ethanol-fed animals had fatty acyl residues that are more saturated than those of controls. These findings point to the underlying molecular mechanism of our previous observation that mitochondria from chronic alcoholic rats are more resistant to uncoupling by ethanol at physiological temperature [Rottenberg, H., Robertson, D. E. & Rubin, E. (1980) Lab. Invest. 42, 318--326]. We suggest that an adaptive change in the phospholipid composition leads to structural alterations, which result in increased resistance to disruption of mitochondrial membranes by ethanol. These changes in lipid composition and structure may explain many, if not all, of the mitochondrial abnormalities that have been previously reported to result from chronic ethanol intoxication.
[3H]Bradykinin receptor binding in mammalian tissue membranes.
Authors: Innis R B , Manning D C , Stewart J M , Snyder S H
Published: May 2006

[3H]Bradykinin binds to membranes from a variety of mammalian tissues in a saturable fashion with a dissociation constant of about 5 nM. Highest levels of binding are detected in guinea pig ileum, colon, and duodenum and in the oestrus rat uterus. The relative potencies of bradykinin derivatives in competing for these binding sites in guinea pig ileum membranes correlate with their contractile potencies in the ileum better than with contractile effects in the uterus. Thus, receptor recognition sites may differ in ileum and uterus. Monovalent and divalent cations at physiological concentrations reduce [3H]bradykinin binding. Of the monovalent cations, medium lowers binding 50% at about 80 mM. Among the divalent cations, calcium lowers binding about 50% at 5 mM, suggesting a link to the calcium conductance channel.
Phase transition versus disorder: A criterion derived from a two-dimensional dynamic ferromagnetic model
Authors: Montroll Elliott W. , Reiss Howard
Published: May 2006

The dynamical equations for spin correlation functions for the two-dimensional Ising model are discussed for rapid-quench conditions. The temperature of the bath in which the Ising spins are immersed is postulated to be driven by the hyperbolic cooling program
T
-1
=

T
0

-1
+
bt
(

T
0

being the initial temperature,
t
the time, and
b
a constant). It is shown that for sufficiently large
b
the term in the dynamical equations that would normally drive the system into an ordered state would not have time to become effective so that the system freezes into a disordered state. The correlation length for the disordered state is characterized by the distance a random walker on a two-dimensional lattice would have been able to diffuse in the time required for the quench to reduce the temperature to some preassigned value.
Differential inhibition of multiple forms of DNA polymerase alpha from IMR-32 human neuroblastoma cells.
Authors: Bhattacharya P , Simet I , Basu S
Published: May 2006

Three forms of DNA polymerase (pol) alpha from human neuroblastoma IMR-32 were separated by DEAE column chromatography. All sedimented at approximately 7 S in 5-20% continuous sucrose density gradients. All were heat labile, with pol alpha 2 the most (90% inactivated) and pol alpha 3 the least (50% inactivated) sensitive to heating for 5 min at 50 degrees C. pol alpha 1 and alpha 2 efficiently utilized activated calf thymus DNA as template. The most active form, pol alpha 2, used both poly(dA).(dT)12-18 and poly(dT).(dA)12-18 as template at equal rates. Differential inhibition of DNA polymerase alpha activities was examined in the presence of ricin, hemin, and a nonhistone chromatin protein. All three polymerases were inhibited by both ricin (nonreduced) and hemin, with pol alpha 2 the most (80-90%) and pol alpha 3 the least (60%) sensitive in each case. In contrast, only pol alpha 2 and alpha 3 activities were inhibited (80-85%) by rat liver nonhistone chromatin protein.
Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition.
Authors: Sternglanz R , DiNardo S , Voelkel K A , Nishimura Y , Hirota Y , Becherer K , Zumstein L , Wang J C
Published: May 2006

Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and beta-galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency of Tn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in Salmonella typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.
Complete amino acid sequence of alpha-tubulin from porcine brain.
Authors: Ponstingl H , Krauhs E , Little M , Kempf T
Published: May 2006

The amino acid sequence of alpha-tubulin from porcine brain was determined by automated and manual Edman degradation of eight sets of overlapping peptides. It comprises 450 residues plus a COOH-terminal tyrosine that is present only in 15% of the material. A region of 40 residues at the COOH-terminus is highly acidic, mainly due to 16 glutamyl residues. This high concentration of negative charge suggests a region for binding cations. At least six positions, most of them around position 270, are occupied by two amino acid residues each. Several of these exchange sites were assigned to specific peptides by analysis of the purified corresponding fragments. These data indicate four alpha-tubulins in porcine brain. Although alpha-tubulin on the whole is unrelated to other proteins, there are regions that can be correlated to sequences of the myosin head, to actin, to tropomyosin, and to troponins C and T.
In vivo and in vitro detection of the leader RNA of the histidine operon of Escherichia coli K-12.
Authors: Frunzio R , Bruni C B , Blasi F
Published: May 2006

The DNA of the attenuator region of the histidine operon of Escherichia coli has been transcribed in a purified in vitro system and found to synthesize two major RNA transcripts. The first one, 180 nucleotides long, has been identified as the histidine-specific leader RNA. It contains the coding sequence for the leader peptide [Di Nocera, P. P., Blasi, F., Di Lauro, R., Frunzio, R. & Bruni, C. B. (1978) Proc. Natl. Acad. Sci. USA 75, 4276-4280] and is terminated at the attenuator site. Termination of transcription at this site is extremely efficient in the in vitro system. The leader RNA also has been detected in vivo in a minicell producer strain transformed with plasmids harboring the regulatory region of the histidine operon of E. coli. A second RNA molecule is synthesized in the in vitro system. It has a divergent direction of transcription with respect to the histidine leader RNA, but its role, if any, in the regulation of the histidine operon remains to be ascertained. The existence of the histidine leader RNA lends support to the regulatory mechanism which postulates that regulation of the histidine operon is dependent on the alternative secondary structures that the leader RNA may assume, depending on whether or not the histidine-rich leader peptide is translated.
Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin.
Authors: Trowbridge I S , Omary M B
Published: May 2006

A cell surface glycoprotein antigen with an apparent molecular weight of about 100,000 that is selectively expressed on proliferating cells was purified from deoxycholate-solubilized membranes of a cultured human leukemic thymus-derived (T) cell line by affinity chromatography on a monoclonal antibody-Sepharose column. A conventional xenoantiserum prepared by immunization with the affinity-purified glycoprotein was found to contain antibodies against a serum component that bound tightly to cultured cells. This molecule was shown to be specifically associated with the cell surface glycoprotein purified by immunoprecipitation from lysates of cells. We have identified the serum component as transferrin and conclude that the membrane glycoprotein is the cell surface transferrin receptor.
Selective growth in serum-free hormone-supplemented medium of tumor cells obtained by biopsy from patients with small cell carcinoma of the lung.
Authors: Carney D N , Bunn P A , Gazdar A F , Pagan J A , Minna J D
Published: May 2006

Fifteen tumor-containing specimens were obtained directly from patients with small cell carcinoma of the lung and tested for their ability to grow in serum-supplemented medium and in serum-free medium supplemented with hydrocortisone, insulin, transferrin, estrogen, and selenium (HITES). The tumor cells replicated in 14 of 15 cases (93%) in the HITES medium and in 10 of 15 cases (67%) in the serum-supplemented medium. The neoplastic origin of the cells growing in the HITES medium was confirmed by standard cytologic criteria, by DNA content analysis using flow cytometry, and by their ability to form colonies in agarose and tumors in athymic nude mice. While the tumor cells had very similar morphologies in both media, the serum-free medium did not support the growth of nonmalignant stromal cells, and essentially pure cultures of replicating tumor cells were obtained 7-10 days after plating. The selectivity of the HITES medium was demonstrated by the failure of cells to grow in 20 specimens cytologically negative for small cell carcinoma and in 9 of 10 specimens containing other tumor types (including other types of lung cancer). The results demonstrate that a chemically defined medium, determined by work on tissue culture-adapted human tumor lines, can support the selective growth of tumor specimens obtained directly from patients. Such selective formulas are probably specific for different tumor types and thus could be used for diagnosis, drug sensitivity testing in vitro, and identification of factors regulating tumor growth. All of these have direct application to patient treatment.
Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing.
Authors: Semler B L , Anderson C W , Kitamura N , Rothberg P G , Wishart W L , Wimmer E
Published: May 2006

A partial amino-terminal amino acid sequence of each of the major proteins encoded by the replicase region (P3) of the poliovirus genome has been determined. A comparison of this sequence information with the amino acid sequence predicted from the RNA sequence that has been determined for the 3' region of the poliovirus genome has allowed us to locate precisely the proteolytic cleavage sites at which the initial polyprotein is processed to create the poliovirus products P3-1b (NCVP1b), P3-2 (NCVP2), P3-4b (NCVP4b), and P3-7c (NCVP7c). For each of these products, as well as for the small genome-linked protein VPg, proteolytic cleavage occurs between a glutamine and a glycine residue to create the amino terminus of each protein. This result suggests that a single proteinase may be responsible for all of these cleavages. The sequence data also allow the precise positioning of the genome-linked protein VPg within the precursor P3-1b just proximal to the amino terminus of polypeptide P3-2.
Cloning and sequence of several alpha 2u-globulin cDNAs.
Authors: Unterman R D , Lynch K R , Nakhasi H L , Dolan K P , Hamilton J W , Cohn D V , Feigelson P
Published: May 2006

We describe a simple cloning procedure for alpha 2u-globulin that requires neither enrichment of mRNA for cloning nor purification of a specific probe for screening recombinant colonies. Total adult male liver poly(A)+RNA was used as template for cloning, and the subsequent recombinant colonies were screened by comparing hybridization to radioactive cDNA probes prepared from hepatic male and female mRNA, respectively. Almost all of the selected "male-specific" clones were later shown to contain alpha 2u-globulin sequences. This cloned alpha 2u-globulin cDNA has been shown to specifically hybridize to male rat liver RNA, which, when isolated and translated in vitro, codes for a 21,000-dalton protein (pro-alpha 2u-globulin) immunologically identical to alpha 2u-globulin. When translation occurs in the presence of pancreatic microsomes this in vitro synthesized pro-alpha 2u-globulin is processed to the 19,000-dalton mature form of alpha 2u-globulin. The nucleotide sequence of the alpha 2u-globulin cDNA has been determined, thus elucidating the complete amino acid sequence of alpha 2u-globulin and most of the hydrophobic "leader" sequence of pro-alpha 2u-globulin. The amino acid sequence deduced from the cDNA is in agreement with the partial sequence that we previously determined by sequential Edman degradation of the purified protein. alpha 2u-Globulin cDNA clones contain within the 3'-untranslated region one or both of the two putative polyadenylylation/transcription termination sites (A-A-T-A-A-A and A-A-T-T-A-A-A). Either of these can be used, generating alpha 2u-globulin mRNA species of two lengths. A codon usage analysis of the cDNA showed that, although all six leucine codons are used for the 14 leucine residues in mature alpha 2u-globulin, the seven leucines in the partial leader sequence reported are all encoded by the same codon, CTG. The primary amino acid sequence contains a unique Asn-Gly-Ser sequence, likely to be in beta-turn conformation, as the probable site of glycosylation for this glycoprotein.
Fluctuation x-ray scattering from biological particles in frozen solution by using synchrotron radiation.
Authors: Kam Z , Koch M H , Bordas J
Published: May 2006

Determination of the structure of biological particles, randomly oriented in solution, from spatial correlation analysis of fluctuations in x-ray scattering has recently been proposed. The feasibility of scattering fluctuation measurements was evaluated by using an x-ray synchrotron radiation camera to obtain the spatial correlation for a solution of tobacco mosaic virus along a line. The experimental system, analysis of data, and requirements for the determination of structures in solution are discussed using this example.
Identification of a population of bipotent stem cells in the HL60 human promyelocytic leukemia cell line
Authors: Fontana J. A. , Colbert D. A. , Deisseroth A. B.
Published: May 2006

HL60, the human promyelocytic leukemia cell line, consists of cells that resemble promyelocytes but are transformed into more mature myeloid forms (polymorphonuclear leukocytes) that express most of the functional characteristics of terminally differentiated myeloid forms when incubated in the presence of dimethyl formamide (HCONMe
2
). When HL60 cells are exposed to the phorbol diester 12-
O
-tetradecanoylphorbol 13-acetate [phorbol 12-myristate 13-acetate (PMA)], they shift from suspension to adherent on the surface of the tissue culture vessel, assume the appearance of macrophages, and acquire macrophage-associated surface markers, the enzyme-nonspecific esterase, the isozyme acid phosphatase, and the myeloid-specific esterase. Although data from other laboratories suggest that HL60 consists of stem cells that are bipotent with respect to myeloid or macrophage differentiation, unequivocal proof of bipotency or evidence that ruled out the presence of two different types of stem cells, each committed to different lines of hematopoietic differentiation, has been lacking. To resolve these two alternatives, we have developed a cloned population of HL60 cells and studied the properties of these cells when exposed to inducers of myeloid (HCONMe
2
) or macrophage (PMA) differentiation. After 120 hr of incubation with HCONMe
2
, 95% of the cells acquire myeloid markers and lack specific macrophage markers, whereas the reverse is true in the presence of PMA, clearly establishing that HL60 is able to commit itself to the development of two different programs of hematopoietic differentiation. Moreover, the commitment to macrophage differentiation is irreversible after 6 hr of incubation in PMA whereas development of the myeloid program requires continuous exposure of the cells to HCONMe
2
. Commitment of HL60 to macrophage differentiation by PMA is not affected by subsequent addition of HCONMe
2
, whereas HCONMe
2
-induced myeloid differentiation is overridden by exposure of the cells to PMA. These data suggest that the HL60 cell may provide a model system for studying the process that generates several classes of precursor cells, each committed to different lines of hematopoietic differentiation, as well as the process that mediates the maturation of these committed precursor cells to their terminally differentiated state.
Mechanism for transcriptional action of cyclic AMP in Escherichia coli: entry into DNA to disrupt DNA secondary structure.
Authors: Ebright R H , Wong J R
Published: May 2006

Binding analysis with purified bacterial receptor distinguishes two structural domains in cyclic AMP (cAMP). The first, the cyclic phosphate and furanose, constitutes a binding domain. This region is bound tightly to the receptor. The rest of cAMP is not bound; the adenine moiety of cAMP is exposed. Unlike binding, activity of cAMP requires the adenine moiety. To be active, cAMP must have in domain II the base adenine--specifically, its Watson--Crick atoms N-1 and N-6. Analysis of indoleacetic acid, a compound able to replace cAMP at the L-arabinose operon, indicates a similar distinction between binding and active domains. To be active, the indole must have substitution (carboxyl or amide) electronically comparable to the cAMP N-1 and N-6. On this basis, we propose a detailed mechanism for action of cAMP (or indoleacetic acid) in Escherichia coli. We propose that the exposed adenine of cAMP enters into the DNA. The adenine's N-1 and N-6 form hydrogen bonds to a thymine in DNA. This interaction destabilizes the DNA. It enhances transcription. Marked similarities indicate an identical mechanism for the steroid hormones in eukaryotes.
A nucleotide change at a splice junction in the human beta-globin gene is associated with beta 0-thalassemia.
Authors: Baird M , Driscoll C , Schreiner H , Sciarratta G V , Sansone G , Niazi G , Ramirez F , Bank A
Published: May 2006

beta 0-Thalassemia is a heterogeneous group of disorders associated with absence of beta-globin. In a survey of DNAs from patients with beta 0-thalassemia of diverse ethnic origins, a change at the splice junction at the 5' end of the large intervening sequence (IVS 2) of the human beta-globin gene has been found in one patient of Italian and another two of Iranian ethnic origins. The enzyme Hph I recognizes a change at this site and generates a large-than-normal fragment of DNA, which hybridizes specifically to a beta-globin IVS 2 probe. No other changes in beta-globin gene DNA structure or organization are detectable by extensive restriction endonuclease analysis. The enzyme HinfI which recognizes a sequence beginning three nucleotides from the 5' end of the IVS 2 splice junction, produces normal fragments and localizes the defect to a G-G-T sequence at the 5'-end IVS 2 splice junction. This sequence is known to be remarkably conserved in all globin genes from many species and in most other genes examined to date. Thus, in at least some beta 0-thalassemia patients, the beta 0-thalassemia defect is associated with a nucleotide change at a splice junction. These patients provide unique examples of naturally occurring defects in splice junctions of eukaryotic genes associated with absence of specific gene function.
Production and characterization of monoclonal antibodies to soluble rat lung guanylate cyclase.
Authors: Brandwein H , Lewicki J , Murad F
Published: May 2006

Four monoclonal antibodies to rat lung soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2] have been produced by fusing spleen cells from immunized BALB/c mice with SP-2/0 myeloma cells. The antibodies were detected by their ability to bind immobilized guanylate cyclase and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibody. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid containing 2-5 mg of antibody per ml. The four antibodies obtained had titers of between 1:1580 and 1:3160 but were detectable at dilutions greater than 1:20,000. Soluble guanylate cyclase from several rat tissues were crossreactive with the four monoclonal antibodies, suggesting that the soluble enzyme from different rat tissues is antigenically similar. The antibodies also recognized soluble lung enzyme from rat, beef, and pig, while enzyme from rabbit was not crossreactive and mouse enzyme was recognized by only one of the antibodies. Particulate guanylate cyclase from a number of tissues had only minimal crossreactivity with the antibodies. Immunoprecipitated guanylate cyclase retained catalytic activity, could be activated with sodium nitroprusside, and was inhibited by cystamine. None of the antibodies were inhibitory under the conditions examined. These antibodies will be useful probes for the study of guanylate cyclase regulation and function under a variety of physiological conditions.
C
4
-dicarboxylate transport mutants of
Rhizobium trifolii
form ineffective nodules on
Trifolium repens
Authors: Ronson Clive W. , Lyttleton Pamela , Robertson John G.
Published: May 2006

Mutants of
Rhizobium trifolii
strain 7012 defective in C
4
-dicarboxylate transport were isolated by using a selective procedure based on pH indicator media. The mutant strains CR7098 and CR7099 failed to grow on or transport succinate, fumarate, or malate, but grew at wild-type rates on several other carbon sources. The C
4
-dicarboxylate transport system was inducible in strain 7012, but was expressed constitutively in four out of five succinate-positive revertants of strain CR7098. In the fifth CR7098 revertant (strain CR8008) the system was inducible. However, in contrast to strain 7012, strain CR8008 failed to use the C
4
-dicarboxylates in the presence of a second carbon source. Revertants of strain CR7099 were similar to strain 7012. Both strains CR7098 and CR7099 nodulated white and red clover at a rate similar to that of strain 7012, but nodules formed by the mutant strains were white and ineffective. Microscopic examination showed that the pattern of development of white clover nodules formed by strain CR7098 was similar to that observed with nodules formed by strain 7012, except that large amounts of starch accumulated in bacteroid-filled cells and senescence occurred earlier. Revertant strains were effective, except for strain CR8008, which formed ineffective nodules. The results show that a supply of C
4
-dicarboxylates to bacteroids is essential for nitrogen fixation in clover nodules. However, rhizobia within plant cells must also utilize other carbon sources to support growth and division.
Multiple differences between the nucleic acid sequences of the IgG2aa and IgG2ab alleles of the mouse.
Authors: Schreier P H , Bothwell A L , Mueller-Hill B , Baltimore D
Published: May 2006

To compare the structure of IgG2a alleles we have determined the complete DNA sequence of the constant region, coding sequence, and 3' untranslated region of a cDNA clone, pAB gamma 2a-1, which was derived from the C57BL/6 mouse strain (b allotype). This sequence was compared with the corresponding IgG2a DNA sequence of BALB/c origin (a allotype). The DNA sequences showed 10% differences, and the deduced protein sequences differed by about 15%. These differences were not evenly distributed: most differences were in the hinge region, the CH3 domain and the 3' untranslated region. It is evident that many alterations in the IgG2a alleles have occurred since the a and b haplotypes were separated--some of these changes were point mutations but some appear to have resulted from gene conversion of the IgG2ab allele by the IgG2bb allele.
T cells may express multiple activities: specific allohelp, cytolysis, and delayed-type hypersensitivity are expressed by a cloned T-cell line.
Authors: Dennert G , Weiss S , Warner J F
Published: May 2006

A T-cell line that recognizes private specificities encoded in the H-2 IA subregion in the proliferative response, cytotoxic activity, delayed-type hypersensitivity, and allohelp reaction with B cells has been cloned. The individual clones were assayed for ability to express the activities of the parent line. Results show that the cloned sublines tested are able to perform all the activities of the parent line. It is suggested that T cells may express multiple activities and that the apparent monofunctionality of T cells is due to regulation of these activities.
Thiocyanate and hydroxyl ions inactivate the scrapie agent.
Authors: Prusiner S B , Groth D F , McKinley M P , Cochran S P , Bowman K A , Kasper K C
Published: May 2006

To probe the macromolecular structure of the scrapie agent and explore conditions for monomerization, the stability of the agent in low concentrations of inorganic ions was determined. A reduction by a factor of 10(5) in scrapie titer was found on exposure of the agent to 1 M KSCN or 0.3 M NaOH. In addition to the inactivation by thiocyanate ions, other chaotropic ions such as guanidinium and trichloroacetate inactivate the scrapie agent. Removal of thiocyanate ions by dialysis or glass permeation chromatography prevented the reduction in scrapie agent infectivity. Addition of equimolar amounts of (NH4)2SO4, a nonchaotrope, to preparations containing 1 M KSCN also prevented the loss of scrapie infectivity. In contrast, neutralization of the alkali-treated fractions with HCl did not restore infectivity. Acidification of partially purified fractions did not cause inactivation of the agent but did result in precipitation of the infectious agent. Inactivation by relatively low concentrations of chaotropic ions is consistent with many observations, all of which suggest that the scrapie agent contains a protein component that is essential for the maintenance of infectivity. Thus, it is unlikely that the agent is composed only of a "naked" nucleic acid. Certainly, if the agent were a naked nucleic acid, its lability in alkali virtually eliminates the possibility that it is composed of a single-stranded molecule of DNA.
Translational regulation: identification of the site on bacteriophage T4 rIIB mRNA recognized by the regA gene function.
Authors: Karam J , Gold L , Singer B S , Dawson M
Published: May 2006

The bacteriophage T4 gene regA encodes a protein that diminishes the expression of many unlinked early T4 genes. Previous work demonstrated that regA-mediated repression occurs after transcription. We report here on the identification of the target site on one regA-sensitive mRNA, the message encoding the phage T4 rIIB protein. The target for regA-mediated action overlaps the translational initiation domain of the rIIB messenger. The regA protein may be a repressor that operates translationally on a significant and interesting set of early phage T4 mRNAs.
Xenon NMR: chemical shifts of a general anesthetic in common solvents, proteins, and membranes.
Authors: Miller K W , Reo N V , Schoot Uiterkamp A J , Stengle D P , Stengle T R , Williamson K L
Published: May 2006

The rare gas xenon contains two NMR-sensitive isotopes in high natural abundance. The nuclide 129Xe has a spin of 1/2: 131Xe is quadrupolar with a spin of 3/2. The complementary NMR characteristics of these nuclei provide a unique opportunity for probing their environment. The method is widely applicable because xenon interacts with a useful range of condensed phases including pure liquids, protein solutions, and suspensions of lipid and biological membranes. Although xenon is chemically inert, it does interact with living systems; it is an effective general anesthetic. We have found that the range of chemical shifts of 129Xe dissolved in common solvents is ca. 200 ppm, which is 30 times larger than that found for 13C in methane dissolved in various solvents. Resonances were also observed for 131Xe in some systems; they were broader and exhibited much greater relaxation rates than did 129Xe. The use of 129Xe NMR as a probe of biological systems was investigated. Spectra were obtained from solutions of myoglobin, from suspensions of various lipid bilayers, and from suspensions of the membranes of erythrocytes and of the acetylcholine receptor-rich membranes of Torpedo californica. These systems exhibited a smaller range of chemical shifts. In most cases there was evidence of a fast exchange of xenon between the aqueous and organic environments, but the exchange was slow in suspensions of dimyristoyl lecithin vesicles.
Differentiation of mouse myeloid leukemia cells induced by 1 alpha,25-dihydroxyvitamin D3.
Authors: Abe E , Miyaura C , Sakagami H , Takeda M , Konno K , Yamazaki T , Yoshiki S , Suda T
Published: May 2006

Mouse myeloid leukemia cells can be induced to differentiate into macrophages in vitro by 1 alpha,25-dihydroxyvitamin D3, the active form of vitamin D3. The minimal concentration of 1 alpha,25-dihydroxyvitamin D3 to induce the cell differentiation was 0.12 nM. The degree of cell differentiation in various markers induced by 12 nM 1 alpha,25-dihydroxyvitamin D3 was nearly equivalent to that induced by 1 microM dexamethasone, the most potent known stimulator. Among several markers of the differentiation by 1 alpha,25-dihydroxyvitamin D3, phagocytic activity was induced within 24 hr, and this was followed by induction of lysozyme and locomotive activities. Similar changes were also induced by 0.01-1 microM 1 alpha-hydroxyvitamin D3. 25-Hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 showed only weak inducing activity. These results suggest the possibility that, in addition to its wellknown biological activities in enhancing intestinal calcium transport and bone mineral mobilization, 1 alpha, 25-dihydroxyvitamin D3 is involved in the differentiation of bone marrow cells.
Transformation of frog embryos with a rabbit beta-globin gene.
Authors: Rusconi S , Schaffner W
Published: May 2006

In order to study the fate and possible expression of foreign DNA during embryogenesis of the frog Xenopus laevis, we have injected a rabbit beta-globin gene into fertilized Xenopus eggs. Frog embryo DNA was extracted at various stages of development, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and hybridized to labeled beta-globin recombinant plasmid DNA. It was found that the injected DNA replicated extrachromosomally, reaching, at gastrula stage, a level equivalent to a 10- to 200-fold amplification of input DNA. At later stages, a majority of the foreign DNA was degraded, but a small fraction was maintained. Six-week-old tadpoles as well as six-month-old frogs contained an average of 3-10 copies of the rabbit globin gene per cell. Most of these persisting globin genes were present as long tandem repeats and comigrated in agarose gel electrophoresis with high molecular weight Xenopus DNA. Analysis of globin gene expression by S1 nuclease mapping showed that the rabbit beta-globin promoter was recognized in the frog embryo and that the transcripts were correctly spliced.
Three mouse models of human thalassemia.
Authors: Martinell J , Whitney J B , Popp R A , Russell L B , Anderson W F
Published: May 2006

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes.
Prolongation of murine islet allograft survival by pretreatment of islets with antibody directed to Ia determinants.
Authors: Faustman D , Hauptfeld V , Lacy P , Davie J
Published: May 2006

Islets of Langerhans treated with donor-specific anti-Ia serum and complement were transplanted across a major histocompatibility barrier into nonimmunosuppressed diabetic mice. The allografts survived in all recipients for at least 200 days after transplantation. Rejection of an established allograft could be induced by intravenous injection of donor splenocytes. This demonstrates that allografts can serve as targets for immune rejection and supports the possible role of Ia-positive passenger lymphoid cells in initiation of immune rejection. The results show that immunosuppression of the recipient is not a prerequisite for successful transplantation.