bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of pTiC58 plasmid-encoded proteins for virulence in Agrobacterium tumefaciens.
Authors: Hagiya M , Close T J , Tait R C , Kado C I
Published: May 2006

Analyses were made of the host-dependent-variation (hdv) locus of the virulence (vir) region of the pTiC58 plasmid of Agrobacterium tumefaciens. The hdv locus is comprised of at least four genes that encode polypeptides of 13, 15, 29, and 28 kDa. Insertion of transposon Tn5 in the first gene abolishes the expression of all four genes in vitro and in vivo. Nucleotide sequence analysis of the hdv locus revealed four open reading frames tandemly arranged with spacer sequences having no promoter-like sequences and lacking the ability to bind A. tumefaciens RNA polymerase. These studies suggest that the hdv locus is comprised of at least four genes arranged in an operon in the vir region. The protein products of these genes are likely to function in some aspect of the host-range determination of A. tumefaciens.
Isolation of a cDNA clone for the liver cell adhesion molecule (L-CAM).
Authors: Gallin W J , Prediger E A , Edelman G M , Cunningham B A
Published: May 2006

Liver cell adhesion molecule (L-CAM) is a calcium-dependent cell adhesion molecule found in very early vertebrate embryos and on liver and other epithelial cells in adults. To describe the genes coding for the molecule and study its synthesis, we have cloned cDNA from poly(A)+ RNA of 10-day embryonic chicken liver using the delta gt11 expression vector. One clone, lambda L301, has been characterized and used in analyses of L-CAM mRNA and genomic DNA. Clone lambda L301 produced a fusion protein that reacted strongly with polyclonal antibodies that recognize L-CAM (Mr 124,000) and its Mr 81,000 NH2-terminal fragment, Ft1, released from liver membranes by trypsin. This result indicates that lambda L301 contains a cDNA insert complementary to protein coding sequence within the two-thirds of the mRNA coding region beginning at the 5' end. The 220-base-pair cDNA insert was isolated and used as a probe in hybridization experiments. RNA transfer blot analysis of poly(A)+ RNA showed a single 4-kilobase mRNA; Southern blot analysis showed multiple components consistent with the presence of one to three L-CAM genes. To test whether different tissues express different forms of L-CAM message, poly(A)+ RNA from eight embryonic organs was analyzed. Only organs that expressed L-CAM protein contained poly(A)+ RNA that hybridized to the lambda L301 probe; in all cases a single band, with the same mobility as that in liver, was observed. The L-CAM mRNA in each tissue was present in proportions similar to those detected previously for the L-CAM protein in these tissues. The combined results suggest that any possible heterogeneity in the L-CAM genes is not reflected in the size of either the mRNA or protein.
The telomeric region of the human X chromosome long arm: presence of a highly polymorphic DNA marker and analysis of recombination frequency.
Authors: Oberlé I , Drayna D , Camerino G , White R , Mandel J L
Published: May 2006

A DNA fragment (named St14) derived from the human X chromosome reveals a small family of related sequences that have been mapped to the Xq26-Xq28 region by using a panel of rodent-human somatic cell hybrids. The probe detects in human DNA digested by Taq I a polymorphic system defined by a series of at least eight allelic fragments with a calculated heterozygosity in females of 80%. With Msp I, we found three additional restriction fragment length polymorphisms, each of them being defined by two alleles. These polymorphisms are also common in Caucasian populations. The genetic locus defined by probe St14 has been localized more precisely to the distal end of the X chromosome (in band q28) by linkage analysis to other polymorphic DNA markers. The results obtained suggest that the frequency of recombination is distributed very unevenly in the q27-qter region of the X chromosome, with a cluster of seven tightly linked loci in q28 showing about 30% recombination with the gene for coagulation factor IX located in the neighboring q27 band. Probe St14 reveals one of the most polymorphic loci known to date in the human genome, and 17 different genotypes have already been observed. It constitutes the best marker on the X chromosome and should be of great use for the genetic study of three important diseases: hemophilia A, mental retardation with a fragile X chromosome, and adrenoleukodystrophy.
Plasmid-induced "hit-and-run" tumorigenesis in Chinese hamster embryo fibroblast (CHEF) cells.
Authors: Lau C C , Gadi I K , Kalvonjian S , Anisowicz A , Sager R
Published: May 2006

The Chinese hamster embryo fibroblast cell line CHEF/18 is readily transfected by plasmid DNA. In the present transfection studies with CHEF/18 cells, focus formation induced by plasmids containing the mutant human c-Ha-ras gene EJ was compared with that of control plasmids without the EJ insert. The focus-forming activity of the transfected plasmid J132, a recombinant of the Harvey murine sarcoma virus LTR and the normal human c-Ha-ras1 in pBR322, also was assessed. Foci were recovered after transfection with either pSV2gpt or pSV2neo at about 10% the frequency obtained with the EJ-containing plasmids, and J132 gave a similar frequency, all well above background obtained with salmon sperm DNA. Whereas foci from transfection with EJ-containing plasmids contained the EJ DNA, no plasmid DNA was detected in either tumorigenic or tumor-derived cells from foci transfected with pSVgpt, pSVneo, or J132. Evidence that genomic changes were induced by plasmid transfection is based on finding chromosomal aberrations in all expanded foci and tumor-derived cells examined. The results suggest the occurrence of "hit-and-run" tumorigenesis induced by transient plasmid transfection.
A molecular basis for the Ia.2 and Ia.19 antigenic determinants.
Authors: Landais D , Matthes H , Benoist C , Mathis D
Published: May 2006

The murine Ia antigens, heterodimeric glycoproteins on the surface of immunocompetent cells, restrict immune recognition by their influence on cell-cell interactions. Many serological specificities have been mapped to these molecules, and monoclonal antibodies directed against some of these determinants block antigen presentation to T lymphocytes. As a step toward a better understanding of Ia function, we sought to define the molecular basis of Ia.2 and Ia.19, specificities found on the A alpha chain of only the k or of both k and r haplotypes, respectively. We report nucleotide sequences for the A alpha chain cDNAs of the k, r, and s haplotypes, which, when compared to previously published A alpha sequences, demonstrate the existence of one k-specific amino acid residue and of another present only in the k and r haplotypes. These residues must thus play an important role in the generation of Ia.2 and Ia.19 specificities.
Molecular analysis of an EL4 cell line that expresses H-2Db but not H-2Kb or beta 2-microglobulin.
Authors: Potter T A , Zeff R A , Schmitt-Verhulst A M , Rajan T V
Published: May 2006

EL4/Mar is a variant cell line that expresses H-2Db but neither H-2Kb nor beta 2-microglobulin (beta 2m). Southern and RNA blot analysis and immunoprecipitation of metabolically labeled proteins established that the B2m gene(s), beta 2m mRNA, and beta 2m protein are normal in this cell line. Somatic cell hybridization showed that the defect in this cell line was in the synthesis of H-2Kb, and RNA blot analysis with an H-2Kb specific oligonucleotide established that the H-2Kb gene(s) in this cell line was not transcribed into a stable mRNA species. The apparent absence of beta 2m on the surface of this cell line suggests that there may be some feature of the H-2Db molecule that allows it to be expressed in the absence of detectable beta 2m.
A mouse tumor-specific transplantation antigen is a heat shock-related protein.
Authors: Ullrich S J , Robinson E A , Law L W , Willingham M , Appella E
Published: May 2006

A tumor-specific transplantation antigen has been purified to homogeneity from the cytosol of a methylcholanthrene-induced tumor, Meth A. The purified antigen is highly immunogenic and specific against challenge with Meth A, providing greater than 95% inhibition of tumor growth in immunized syngeneic mice. Immunofluorescence analysis of Meth A showed that the antigen is a highly abundant cytosolic protein but that it is also present at the cell surface and, therefore, accessible to the host's immune system. The antigen consists of two polypeptide isoforms present in equimolar amounts, having similar masses (84 and 86 kDa), pI values (4.95 and 4.90), and amino acid compositions. Both are phosphoproteins, and neither is glycosylated. The NH2-terminal sequences of the two isoforms are identical except that each chain contains a portion of unique sequence. Comparison of the NH2-terminal and CNBr-fragment sequence data to the sequences of the yeast and Drosophila heat shock proteins (Hsp90 and Hsp83, respectively) reveals that 73 of 91 residues compared are identical. In addition, an anti-Meth A tumor antigen serum that defects the isoforms from a variety of tumors also immunoprecipitates proteins of identical mass and pI from both normal and heat-shocked mouse embryo cells.
Overproduction-induced mislocalization of a yeast vacuolar protein allows isolation of its structural gene.
Authors: Rothman J H , Hunter C P , Valls L A , Stevens T H
Published: May 2006

Using an immunological screening procedure that allows the detection of yeast cells aberrantly secreting vacuolar proteins, we have isolated a cloned DNA fragment containing the structural gene for the vacuolar enzyme proteinase A (PrA; EC 3.4.23.6). A large portion of PrA is misdirected to the cell surface in cells harboring the PrA structural gene on a multicopy plasmid. This mislocalized PrA traverses the late stages of the secretory pathway and differs slightly in apparent molecular weight from the vacuolar form. A deletion in the genomic copy of the PrA structural gene eliminates immunoreactive PrA as well as the enzymatic activities of at least three other vacuolar hydrolases. In the case of the vacuolar enzyme carboxypeptidase Y (EC 3.4.16.1), the lack of activity is due to the absence of proteolytic activation of the zymogen. Thus, PrA may be required for in vivo processing of a number of yeast vacuolar hydrolases.
Generalized random fields related to self-intersections of the Brownian motion
Authors: Dynkin E. B.
Published: May 2006

Let

T
k

ε
(λ;
t
1
,...,

t
k

) = ρ(

X
t

1
)
q
ε
(

X
t

2
-

X
t

1
)...
q
ε
(

X

t
k

-

X

t
k

-1), where

X
t

is a Brownian motion in R
2
, λ(
dx
) = ρ(
x
)
dx
, and
q
ε
converges to Dirac's δ function as ε ↓ 0. The self-intersection local times of order
k
are described by a generalized random field

T
k

(λ;
t
1
,...,

t
k

) = lim
ε↓0

T
k

ε
(λ;
t
1
,...,

t
k

) for 0 <
t
1
<... <

t
k

. The field “blows up” as

t
i

-

t
j

→ 0 for some
i
≠
j
. I show that with a proper choice of the coefficients

B
k
l

(ε), a generalized random field [unk]

k

(λ;
t
1
,...,

t
k

) = lim
ε↓0
[

T
k

ε
(λ;
t
1
,...,

t
k

) + Σ

l
=1

k
-1

[

B
k
l

(ε)

T
l

ε
](λ;
t
1
,...,

t
k

)] is well defined for all 0 ≤
t
1
≤... ≤

t
k

and it coincides with

T
k

(λ;
t
1
,...,

t
k

) for
t
1
<... <

t
k

.
Predicting DNA duplex stability from the base sequence.
Authors: Breslauer K J , Frank R , Blöcker H , Marky L A
Published: May 2006

We report the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions. We obtained the relevant thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. We show how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence. We illustrate our method of calculation by using the nearest-neighbor data to predict transition enthalpies and free energies for a series of DNA oligomers. These predicted values are in excellent agreement with the corresponding values determined experimentally. This agreement demonstrates that a DNA duplex structure thermodynamically can be considered to be the sum of its nearest-neighbor interactions. Armed with this knowledge and the nearest-neighbor thermodynamic data reported here, scientists now will be able to predict the stability (delta G degree) and the melting behavior (delta H degree) of any DNA duplex structure from inspection of its primary sequence. This capability should prove valuable in numerous applications, such as predicting the stability of a probe-gene complex; selecting optimal conditions for a hybridization experiment; deciding on the minimum length of a probe; predicting the influence of a specific transversion or transition on the stability of an affected DNA region; and predicting the relative stabilities of local domains within a DNA duplex.
Keratin-like proteins that coisolate with intermediate filaments of BHK-21 cells are nuclear lamins.
Authors: Goldman A E , Maul G , Steinert P M , Yang H Y , Goldman R D
Published: May 2006

Four proteins of Mr approximately equal to 60,000, 65,000, 67,000, and 70,000 coisolate with the major intermediate filament (IF) structural proteins of BHK-21 cells. These proteins are keratin-like, they form distinctive paracrystals in vitro, and they are concentrated at the nuclear surface. Since these properties indicate similarities with the nuclear lamins, we have prepared conventional fractions of BHK-21 nuclei from which the same type of paracrystal is obtained. Furthermore, biochemical and immunological data demonstrate that the lamins are identical to the Mr 60,000-70,000 proteins found in IF preparations, and both sets of proteins are similar to keratin. These results suggest that an IF-like protein network is present in the nuclear lamina. We speculate that in some unknown way this network connects to the cytoplasmic IF network that courses from the juxtanuclear region to the cell surface. These proposed interconnecting networks may form part of the infrastructure of cytoskeletal-nuclear matrix-connecting links involved in signal transmission between the nuclear and cytoplasmic compartments of eukaryotic cells.
Evidence for selection as a mechanism in the concerted evolution of Lycopersicon esculentum (tomato) genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.
Authors: Pichersky E , Bernatzky R , Tanksley S D , Cashmore A R
Published: May 2006

The nuclear gene sequences encoding RBCS, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from several plants show extensive interspecific divergence but little intraspecific divergence, suggesting that these genes are evolving in concert within a genome. In this study, the nucleotide sequences of two tomato (Lycopersicon esculentum) RBCS genes and a cDNA clone containing the entire coding region of a third tomato RBCS gene were determined. The three genes, designated Rbcs-1, Rbcs-2A, and Rbcs-3A, each belong to a different one of the three RBCS loci in the tomato genome. The nucleotide sequence of Rbcs-1 differs from that of Rbcs-2A and Rbcs-3A by 13.9% and 13.1%, respectively. Rbcs-2A and Rbcs-3A differ from each other by 10.7%. A recently published RBCS gene sequence from tobacco (Nicotiana tabacum) [Mazur, B. J. & Chui, C.-F. (1985) Nucleic Acids Res. 13, 2373-2386] differs by 10.6% and 11.3% from Rbcs-2A and Rbcs-3A, respectively, and by 15.0% from Rbcs-1. Thus the tobacco gene seems to be phylogenetically as closely related to the tomato genes Rbcs-2A and Rbcs-3A as the latter two are to each other, and more closely related to them than Rbcs-1 is. However, the mature part of the polypeptide encoded by the tobacco RBCS gene differs by five and six amino acids from the corresponding region in the polypeptides encoded by Rbcs-2A and Rbcs-3A, respectively, while these two tomato RBCS polypeptides differ from each other in the mature part by a single amino acid. Rbcs-1, whose nucleotide sequence shows higher divergence from both the tobacco RBCS gene and Rbcs-2A and Rbcs-3A, encodes a polypeptide whose mature part differs by eight amino acids from the corresponding region in the tobacco polypeptide but only by three and four amino acids from the corresponding regions of Rbcs-2A- and Rbcs-3A-encoded polypeptides, respectively. Thus, it appears that in the tomato selection has maintained near uniformity of the coding information in the portion of the RBCS genes encoding the mature polypeptides.
Conversion of low-affinity interleukin 2 receptors to a high-affinity state following fusion of cell membranes.
Authors: Robb R J
Published: May 2006

The cellular receptors for interleukin 2 (IL-2) exist in at least two forms, one with a particularly high affinity and a second, more numerous class, with a much lower affinity for IL-2. Indirect evidence suggests that both classes of receptors use the same p55 glycoprotein as their ligand-binding component. L cells transfected with cDNA encoding this protein, however, displayed only low-affinity IL-2 binding. To determine if such receptors could be converted to a high-affinity state, L-cell membranes containing the murine p55 protein were fused with membranes from human T cells displaying high-affinity receptors. The anti-Tac antibody was used to block ligand binding to human p55 on the fusion product. The results showed that a fraction of the murine p55 chains were converted to a dramatically higher affinity following fusion. Fusion of the L-cell membranes with themselves or with membrane preparations from human T-cell lines lacking the IL-2 receptor resulted in little or no affinity modulation. One explanation of the results is that cofactors present in receptor-positive T-cell lines crossed species lines and combined with the murine p55 chain to create "high-affinity" binding sites. Thus, depending upon its environment, the same p55 molecule can apparently form either a low- or high-affinity IL-2 receptor.
[3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine: an antagonist radioligand for adenosine receptors.
Authors: Jacobson K A , Ukena D , Kirk K L , Daly J W
Published: May 2006

An amine-functionalized derivative of 1,3-dipropyl-8-phenylxanthine has been prepared in tritiated form as a xanthine amine congener ([3H]XAC) for use as an antagonist radioligand for adenosine receptors. [3H]XAC has higher receptor affinity, higher specific activity, lower nonspecific membrane binding, and more favorable hydrophilicity than 1,3-diethyl-8-[3H]phenylxanthine, the xanthine commonly used for adenosine receptor binding. In rat cerebral cortical membranes, [3H]XAC exhibits saturable, specific binding with a Kd of 1.23 nM and a Bmax of 580 fmol/mg of protein at 37 degrees C. N6-(R-Phenylisopropyl)adenosine is a more potent inhibitor of [3H]XAC binding than is 5'-N-ethylcarboxamidoadenosine, indicating that binding is to an A1-adenosine receptor. In the absence of GTP, the inhibition curves for adenosine agonists versus [3H]XAC binding are biphasic, indicating that [3H]XAC is binding to low- and high-affinity agonist states of the A1 receptor. In the presence of GTP, adenosine analogs exhibit monophasic, low-affinity inhibition of binding of [3H]XAC. Inhibition of [3H]XAC binding by theophylline or by various 8-phenylxanthines is monophasic, and the potencies are commensurate with the potencies of these xanthines as adenosine receptor antagonists. The receptor sites in calf brain membranes exhibit a higher affinity (Kd = 0.17 nM) for [3H]XAC, whereas sites in guinea pig exhibit a slightly lower affinity (Kd = 3.0 nM). Densities of [3H]XAC binding sites are similar in brain membranes from all species.
Characterization of human prorenin expressed in mammalian cells from cloned cDNA.
Authors: Fritz L C , Arfsten A E , Dzau V J , Atlas S A , Baxter J D , Fiddes J C , Shine J , Cofer C L , Kushner P , Ponte P A
Published: May 2006

Human preprorenin was synthesized in Chinese hamster ovary (CHO) cells transfected with an expression vector containing renin cDNA sequences. These cells secrete an inactive form of renin (EC 3.4.23.15) that can be activated by trypsin. This inactive renin is precipitable by antibody generated against purified human renal renin and also by antisera generated to a synthetic peptide derived from the amino acid sequence of the pro segment of preprorenin (anti-propeptide), indicating that the secreted inactive enzyme is a form of prorenin. Analysis of [35S]methionine-labeled proteins immunoprecipitated from CHO cell conditioned culture medium indicates that prorenin is expressed in CHO cells as two distinct forms that differ in their degree of glycosylation. In vitro trypsin activation of prorenin cleaves approximately 4.5 kDa from the protein, rendering it unreactive with the antipropeptide antiserum but still recognizable by anti-renal renin antibody. These results show directly that the prorenin expressed by CHO cells is an inactive enzyme that is activated by trypsin cleavage of the pro segment. The ability to express human renin in this form will allow for the purification of both active and inactive forms of the enzyme in quantities sufficient for detailed physiological and structural studies.
Light quality regulates expression of chloroplast genes and assembly of photosynthetic membrane complexes
Authors: Glick Richard E. , McCauley Steven W. , Gruissem Wilhelm , Melis Anastasios
Published: May 2006

The concentrations of photosystem I (PSI) and photosystem II (PSII) reaction centers and the level of chloroplast reaction center gene transcripts were determined in pea plants grown under different light-quality regimes. In plants grown in light primarily absorbed by PSI (“red” light), the PSII/PSI reaction center ratio was 2-fold greater than that in plants grown in PSII-sensitizing (“yellow”) light. In addition, the ratio of a PSII gene (
psbB
) transcript to a PSI gene (
psaA
) transcript was 2.6 times greater in red-grown plants relative to yellow-grown plants. Thus, a differential reaction-center concentration in the thylakoid membrane was accompanied by a differential expression of reaction center genes, suggesting that the synthesis of chloroplast membrane complexes and the assembly of photosystems are regulated by light quality at the transcriptional and/or post-transcriptional level.
Rapid isolation of monoclonal antibodies specific for cell surface differentiation antigens.
Authors: Barclay S L , Smith A M
Published: May 2006

Two immunization procedures were compared for their ability to yield monoclonal antibodies that react with plasma membrane-bound differentiation antigens of Dictyostelium. In the first method, hybridomas prepared from BALB/c mice immunized with aggregating amoebae produced monoclonal antibodies that recognized antigens present on both growing and aggregating Dictyostelium amoebae. None of the monoclonal antibodies reacted with only the injected aggregation-stage cell type. In contrast, monoclonal antibodies that reacted with differentiation antigens were easily obtained by primary immunization of BALB/c mice with living aggregation-stage cells, followed by secondary immunization with a preparation of plasma membrane from aggregating cells or intact aggregating cells mixed with polyclonal BALB/c antiserum raised against undifferentiated cells. By this method, approximately 20% of all anti-Dictyostelium monoclonal antibodies obtained in a fusion are specific for differentiation antigens. The properties and developmental regulation of several of these antigens are described. The possible uses of this immunological method to detect unique determinants on other kinds of cells and the likely immune mechanisms that make it successful are discussed.
The gene encoding the T-cell surface protein T4 is located on human chromosome 12.
Authors: Isobe M , Huebner K , Maddon P J , Littman D R , Axel R , Croce C M
Published: May 2006

The surface glycoproteins T4 and T8 define functionally distinct populations of T lymphocytes. We have obtained cDNA and genomic clones encoding the T4 molecule and used these as probes to determine the chromosomal location of this gene. Genomic blotting experiments, along with in situ hybridization analyses, indicate that the T4 gene resides on the short arm of human chromosome 12, at region p12-pter. Thus, the T4 gene is not linked to any known member of the immunoglobulin gene family, including its counterpart gene, T8, which resides on human chromosome 2 immediately distal to the immunoglobulin kappa locus.
Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains.
Authors: Wobbe C R , Dean F B , Murakami Y , Weissbach L , Hurwitz J
Published: May 2006

We have evidence for the formation of a stable preelongation complex during the replication of simian virus 40 (SV40) origin containing DNA (ori+ DNA) in vitro. Preincubation of ori+ DNA with HeLa cytosolic extracts and SV40-encoded large tumor antigen (T antigen) in the absence of deoxynucleoside triphosphates eliminates a lag that normally precedes replication. This effect requires ATP and is inhibited by RNase A; subsequent elongation is inhibited by aphidicolin but not by RNase A. A T antigen and SV40 origin-dependent complex can be isolated by gel-filtration chromatography of preincubation reaction mixtures. In both cases, the products formed by replication after complex formation resemble those formed during in vitro replication reactions described previously. HeLa cytosolic extract was separated into two ammonium sulfate fractions: a 0-40% fraction (AS 40) that shows low levels of DNA synthesis and a 40-65% fraction (AS 65) that is inactive by itself but stimulates synthesis when added to the AS 40 fraction. DNA synthesis by these combined fractions has the same requirements as crude extract, occurs in two stages as described above, and is sensitive to RNase A. Pretreatment of both fractions with micrococcal nuclease eliminated replication activity, whereas the combination of a pretreated fraction (either AS 40 or 65) with an untreated fraction was active. A heat-inactivated (55 degrees C, 5 min) AS 65 fraction restored replication activity to the combination of micrococcal nuclease-treated AS 40 and AS 65 fractions.
Autonomous functions of structural domains on human tissue-type plasminogen activator.
Authors: van Zonneveld A J , Veerman H , Pannekoek H
Published: May 2006

Transfected mouse Ltk- cells were employed for transient expression of recombinant human tissue-type plasminogen activator (t-PA; EC 3.4.21.31) or of recombinant-t-PA deletion proteins, encoded by SV40-pBR322-derived t-PA cDNA plasmids. The t-PA cDNA deletion mutants have two features in common, i.e., cDNA programming the signal peptide and the coding region for the light chain. Consequently, recombinant t-PA mutant proteins are efficiently secreted and display plasminogen activator activity. The gene encoding the amino-terminal heavy chain [an array of structural domains homologous to other plasma proteins (finger, epidermal growth factor, and kringle domains)] was mutated using restriction endonucleases to delete one or more structural domains. The stimulatory effect of fibrinogen fragments on the plasminogen activator activity of t-PA was demonstrated to be mediated by the kringle K2 domain and to a lesser extent by the finger/epidermal growth factor region but not by the kringle K1 domain. These data correlate well with the fibrin-binding properties of the recombinant t-PA deletion proteins, indicating that the stimulation of the activity by fibrinogen fragments is based on aligning the substrate plasminogen and t-PA on the fibrin matrix. Our results support the evolutionary concept of exon shuffling, arranging structural domains that constitute autonomous functions of the protein.
Multiple cis-acting elements modulate the translational efficiency of GCN4 mRNA in yeast.
Authors: Tzamarias D , Alexandraki D , Thireos G
Published: May 2006

The expression of the GCN4 gene in yeast is regulated at the translational level: growth of yeast cells under amino acid starvation conditions results in an increase in the translational efficiency of GCN4 mRNA. A sequence within the 5' untranslated region of this mRNA, which contains four small open reading frames, acts in cis to suppress translation when growth occurs in rich media. In this report, we have analyzed the effects on translation of a series of deletion, insertion, and substitution mutations in the 5' untranslated region of GCN4 mRNA. This analysis showed that at least two distinct cis elements located within the region of the small upstream open reading frames are required, in conjunction with trans positive elements, for the translational activation of GCN4 mRNA. We propose that the translational efficiency of GCN4 mRNA is modulated by the rate of translation initiation at the upstream AUG codons.
Characterization of somatomedins from human fetal brain: identification of a variant form of insulin-like growth factor I.
Authors: Sara V R , Carlsson-Skwirut C , Andersson C , Hall E , Sjögren B , Holmgren A , Jörnvall H
Published: May 2006

A fetal form of somatomedin (insulin-like growth factor) that crossreacts in the fetal brain radioreceptor assay has been proposed to exist in humans. Using this assay to monitor activity during purification, we have isolated a variant form of insulin-like growth factor I (IGF-I) from human fetal brain tissue. The variant IGF-I showed potent crossreaction in the fetal brain radioreceptor assay and stimulated DNA synthesis in fetal brain cells in vitro. Structural analysis revealed the variant IGF-I to have a truncated NH2-terminal region compared to IGF-I isolated from serum. An additional peptide, which displayed less potent crossreaction in the fetal brain radioreceptor assay, was also isolated from the human fetal brain. Partial amino acid sequence analysis revealed identity to insulin-like growth factor II.
Olfactory GTP-binding protein: signal-transducing polypeptide of vertebrate chemosensory neurons.
Authors: Pace U , Lancet D
Published: May 2006

The sense of smell involves the stimulation of sensory neurons by odorants to produce depolarization and action potentials. We show that olfactory responses may be mediated by a GTP-binding protein (G protein), a homolog of the visual, hormonal, and brain signal transducing polypeptides. The olfactory G protein is identified in isolated dendritic membranes (olfactory cilia preparations) of chemosensory neurons from three vertebrate species and is shown to mediate the stimulation by odorants of the highly active adenylate cyclase in these membranes. The G protein of olfactory neurons is most similar to Gs, the hormonal stimulatory GTP-binding protein. Its alpha subunit has a molecular weight of about 42,000, and it undergoes ADP-ribosylation catalyzed by cholera toxin that leads to adenylate cyclase activation. The slight difference in molecular weights of the frog olfactory and the liver Gs alpha subunits and the higher sensitivity of olfactory adenylate cyclase to nonhydrolyzable GTP analogs are consistent with the possible existence of different Gs variants. Signal amplification due to the olfactory G protein may be responsible for the unusual acuity of the sense of smell.
Identification of three forms of human myelin basic protein by cDNA cloning.
Authors: Kamholz J , de Ferra F , Puckett C , Lazzarini R
Published: May 2006

We have isolated cDNA clones encoding three separate forms of human myelin basic protein (MBP), 21.5, 18.5, and 17.2 kDa, and have determined the nucleotide sequence of each. The three forms share a common sequence but differ by the inclusion of a 26-residue amino acid sequence near the N terminus of the 21.5-kDa protein or by the absence of an 11-residue amino acid sequence near the C terminus of the 17.2-kDa protein. The sequences either added to or deleted from the major 18.5-kDa MBP correspond exactly to exons 2 and 5 of the mouse MBP gene, suggesting that the human and mouse genes have similar exon structures. We have also identified the 21.5-kDa human MBP on immunoblots using antisera raised to a peptide encoded by the mouse exon 2 sequence. Southern blotting studies of human genomic DNA reveal a simple pattern consistent with a single human MBP gene. Thus, the three MBP mRNAs are likely to arise from alternative splicing of a primary human MBP transcript. Conservation of the 26 amino acid mouse exon 2 sequence in human MBP suggests an important role for this sequence in myelination.
Truncation of the c-myb gene by a retroviral integration in an interleukin 3-dependent myeloid leukemia cell line.
Authors: Weinstein Y , Ihle J N , Lavu S , Reddy E P
Published: May 2006

Among a series of myeloid leukemia cell lines, one (NFS-60) was found to have a rearrangement of the c-myb locus. The rearrangement involved the integration of a retrovirus into the region of the gene corresponding to the sixth exon of the avian c-myb locus. The insertion is associated with the production of a truncated RNA and the introduction of a terminator codon at the juncture of the long terminal repeat and the c-myb locus. The properties of the NSF-60 cells were compared with those of other myeloid cell lines, and the known sequence of differentiation induced by interleukin 3. Similar to other myeloid cell lines, the NFS-60 cells do not terminally differentiate in response to interleukin 3, granulocyte/macrophage, or granulocyte colony-stimulating factor suggesting that the cells are transformed with regard to their ability to differentiate. The NFS-60 cells are totally dependent on interleukin 3 for growth and maintenance of viability in vitro but also proliferate in response to granulocyte colony-stimulating factor. The properties of the cells support the concept that the c-myb protooncogene is involved in the control of normal differentiation of hematopoietic cells.
Expression of proenkephalin messenger RNA by mouse spermatogenic cells.
Authors: Kilpatrick D L , Millette C F
Published: May 2006

The presence of proenkephalin mRNA in germ cells purified from adult mouse testis were examined using RNA gel- and dot-blot analyses. Both pachytene spermatocytes and round spermatids were shown to contain concentrations of this transcript that are 2- to 3-fold greater than that found for whole mouse testis, on a per microgram of polyadenylylated RNA basis. The detection of proenkephalin mRNA in purified spermatocytes and spermatids could not be accounted for by contamination by either Leydig or Sertoli cells. No proenkephalin mRNA was detectable in extracts of mature sperm. These data suggest that developing germ cells may be a major site of proenkephalin synthesis in the adult testis and that proenkephalin-derived peptides may function as germ cell-associated hormones or autocrine/paracrine factors.
tRNA anticodon replacement experiments show that ribosomal frameshifting can be caused by doublet decoding.
Authors: Bruce A G , Atkins J F , Gesteland R F
Published: May 2006

The expression of certain normal genes requires a specific ribosomal frameshift event because the mRNA has the coding information for one protein in two different reading frames. One of several possible mechanisms for this involves recognition of a nontriplet codon by a noncognate tRNA. The AGUC-decoding Escherichia coli tRNASer3 reads a GCA alanine codon to cause a -1 frameshift. Replacement of the anticodon of tRNAPhe with the anticodon of tRNASer3 allows the constructed tRNA to cause this frameshifting. By altering the anticodon loop nucleotides at positions 33-36 in the constructed tRNAPhe molecules, the tRNA was found to recognize a 2-base codon. Instead of the usual anticodon, positions 34-36, the nucleotides in positions 34 and 35 form essential base pairs with the first two positions of the alanine codon. The uridine in position 36 is also required but not for base pairing.
Early replication and expression of oocyte-type 5S RNA genes in a Xenopus somatic cell line carrying a translocation.
Authors: Guinta D R , Tso J Y , Narayanswami S , Hamkalo B A , Korn L J
Published: May 2006

In Xenopus somatic cells, the somatic-type 5S RNA genes replicate early in S phase, bind the transcription factor TFIIIA, and are expressed; in contrast, the late replicating oocyte-type genes do not bind TFIIIA and are transcriptionally inactive. These facts support a model in which the order of replication of the somatic-type versus the oocyte-type 5S genes causes their differential expression in somatic cells due to sequestration of TFIIIA by the early-replicating somatic genes. Here we provide further evidence for the model by showing that in one Xenopus cell line in which some oocyte-type 5S genes are translocated, some oocyte-type 5S genes replicate early and are expressed.
Production of a monoclonal antibody specific for seminomas and dysgerminomas.
Authors: Bailey D , Baumal R , Law J , Sheldon K , Kannampuzha P , Stratis M , Kahn H , Marks A
Published: May 2006

A monoclonal antibody (M2A, IgG2a) was produced against a cultured human ovarian epithelial adenocarcinoma cell line, HEY. Monoclonal antibody M2A reacted with a glycoprotein of molecular weight 40,000 on the surface of HEY cells. The affinity constant of the monoclonal antibody M2A for HEY cells was 10(9) M-1, and the number of binding sites on HEY cells was 2 X 10(4) per cell. The monoclonal antibody produced positive immunoperoxidase staining of fetal (but not adult) testis and of seminomas and dysgerminomas but did not stain various normal adult tissues or other gonadal or extragonadal tumors. Monoclonal antibody M2A may be useful for confirming a histological diagnosis of seminoma and dysgerminoma.
Urinary kallikrein: a physiological regulator of epithelial Na+ absorption.
Authors: Lewis S A , Alles W P
Published: May 2006

The apical membrane of the mammalian urinary bladder contains two populations of ionic conductances--one Na+ selective and amiloride blockable, the other cation selective and amiloride insensitive (a leak channel). Addition of kallikrein (an enzyme of unknown function normally found in urine) to the mucosal solution of the mammalian urinary bladder epithelium resulted in the loss (over a 2-hr period) of amiloride-sensitive Na+ current and an increase in the leak current that is amiloride insensitive. The rate of hydrolysis of Na+ channels is a first-order process that is concentration (activity) dependent and described by simple Michaelis-Menten kinetics with a maximum rate of 9.5 X 10(-3) min-1. At the activities measured in human urine, the corresponding rate constant will decrease Na+ channel density by 99.5% in 24 hr. Amiloride protects the amiloride-sensitive Na+ channels from degradation but not the leak pathway. The rate of hydrolysis of the leak pathway as well as the kinetics of hydrolysis are the same as that described for the Na+ channel. Of interest is that the leak pathway is hydrolyzed into a form that seems to partition between the apical membrane and mucosal solution (an unstable leak pathway). These results and previous findings suggest a regulatory role for kallikrein in salt and water homeostasis.
Cloning and expression of cDNA for anti-müllerian hormone.
Authors: Picard J Y , Benarous R , Guerrier D , Josso N , Kahn A
Published: May 2006

Messenger RNA, prepared from fetal bovine testicular tissue, was used to construct a cDNA library in lambda gt11 phage. The library was screened with an antibody probe directed against bovine anti-Müllerian hormone and three positive clones were isolated. Cross-hybridizing cDNA inserts carried by clones 4 and 5 (1.2 and 0.08 kilobases long, respectively) code for a fragment of authentic anti-Müllerian hormone, as shown by the ability of the anti-epitope antibodies eluted from fusion protein 4 to bind strongly to anti-Müllerian hormone on immunoblots and by the capacity of anti-epitope antibodies 4 and 5 to precipitate radioiodinated bovine anti-Müllerian hormone. A probe prepared from insert 4 hybridizes with an mRNA present only in tissues that are known producers of anti-Müllerian hormone, such as the fetal testis and adult ovarian follicles. The amount of specific mRNA in tissues of males and females is related to the rate of their anti-Müllerian hormone production. The 2.1-kilobase size of this mRNA species is large enough to code for the Mr 62,000 anti-Müllerian hormone polypeptide chain. Insert 4 also hybridizes with an mRNA of similar size in human and rat fetal testicular tissue. The third isolated clone, clone 8, which does not cross-hybridize with the others, carries a cDNA insert coding for a ubiquitous protein, smaller than anti-Müllerian hormone, with which it apparently shares an epitope.
Isolated liver gap junctions: gating of transjunctional currents is similar to that in intact pairs of rat hepatocytes.
Authors: Spray D C , Saez J C , Brosius D , Bennett M V , Hertzberg E L
Published: May 2006

We have shown previously that conductance of rat liver gap junctions is blocked by an affinity-purified polyclonal antibody generated against rat liver junctional membranes, is not affected by moderate transjunctional or transmembrane potentials, and is reversibly decreased by cytoplasmic acidification and perfusion with octanol. We have now recorded currents from isolated liver gap junctions using patch electrodes dipped through a layer of mixed lipids whose concentrations match those of isolated liver appositional membranes. These currents are blocked by the same polyclonal antibody, are insensitive to moderate voltages imposed across the pipette tip, and are reversibly blocked by similar concentrations of H ions and octanol as are junctions in situ. The currents are likely to be gap junctional in origin; their block by low pH and other agents indicates that the gating mechanisms are intrinsic to the gap junctions themselves and presumably result from conformational change in the channel-forming protein.
Epidermal growth factor receptor is increased in multidrug-resistant Chinese hamster and mouse tumor cells.
Authors: Meyers M B , Merluzzi V J , Spengler B A , Biedler J L
Published: May 2006

Multidrug-resistant sublines of Chinese hamster lung and mouse tumor cells selected for high levels of resistance to vincristine or actinomycin D have increased numbers of epidermal growth factor (EGF) receptors compared to control cells. Evidence for this increase was found in six of six resistant cell lines with the use of receptor binding or immunoprecipitation techniques. Levels of 125I-labeled EGF binding to intact actinomycin D-resistant cells derived from DC-3F or CLM-7 Chinese hamster lines are increased 3- to 10-fold compared to controls. Scatchard analysis of these data suggests that increased binding is a result of increased receptor number rather than altered affinity of receptor for ligand. Affinity-labeling and immunoprecipitation studies confirmed the finding of increased receptor amount in resistant hamster and mouse cells. Multidrug-resistant variants of DC-3F cells overproduce a plasma membrane glycoprotein (gp150-180) with several physicochemical properties that resemble those of EGF receptor. However, electrophoretic transfer blots with a polyclonal antibody to gp150-180 show that EGF receptor and gp150-180 are probably different molecules. Resistant cells described in this report manifest a normalized phenotype compared to transformed, tumorigenic, drug-sensitive parental cells. EGF receptor increase in resistant variants may be associated with this reverse transformation.
Characterization of a female-specific cDNA derived from a developmentally regulated mRNA in the human blood fluke Schistosoma mansoni.
Authors: Bobek L , Rekosh D M , van Keulen H , LoVerde P T
Published: May 2006

We have isolated and characterized a cDNA clone that is derived from a developmentally regulated mRNA found only in mature female schistosomes. The mRNA is approximately 950 nucleotides in length and is not detectable in immature female schistosomes isolated from single-sex infections, in male worms, or in eggs. During normal bisexual infections, the mRNA species is first detected 28 days after infection (the time of worm pairing) and increases to a high level 35 days after infection, coinciding with the start of egg production. The nucleotide sequence of the cDNA shows two large open reading frames in the coding strand. Several features of the clone, including the deduced sequence of the polypeptide encoded by one of the reading frames, suggest a relationship to the silk moth chorion (egg shell) gene family. The isolation of this clone provides us with a probe for further studies of female schistosome development and is a first step toward a detailed understanding of this process at the molecular level.
Natural killer susceptibility of human cells may be regulated by genes in the HLA region on chromosome 6.
Authors: Harel-Bellan A , Quillet A , Marchiol C , DeMars R , Tursz T , Fradelizi D
Published: May 2006

Natural killer (NK) cells exist in each individual in the absence of any intentional immunization. They are able to kill a wide range of targets from tumoral as well as from normal origin. However, their exact physiologic role is not clearly understood. In this study we report results about a human Epstein-Barr virus-transformed B-cell line from which variants perturbed in the expression of HLA molecules have been derived. Our results indicate that in these cell lines an inverse relationship exists between expression of HLA antigens and susceptibility to NK lysis. The original cell line is highly resistant to NK lysis. On the contrary, the variant perturbed in class I antigen expression is highly susceptible. Variant perturbed in class II antigen expression is intermediate in susceptibility. Interferon, which induces HLA class I expression and NK resistance in the unrelated classical K-562 target cells, does not change either HLA expression or NK susceptibility in the variant cell lines. The difference between the original cell line and the variants does not reside in the ability to be bound by NK effectors. Our results suggest a different role for HLA molecules. By some unknown mechanism discussed here, the presence of HLA molecules at the surface of a cell would prevent this cell from being killed by NK cells. The loss of this "good health" signal would lead to the elimination of the cell through NK lysis.
Characterization of the fertilization antigen 1 for the development of a contraceptive vaccine.
Authors: Naz R K , Phillips T M , Rosenblum B B
Published: May 2006

A fertilization antigen, FA-1, was purified from either deoxycholate- or lithium diiodosalicylate-solubilized murine testes by immunoaffinity chromatography using a monoclonal antibody, MA-24, which inhibited fertilization in vitro. The FA-1 was recovered at high (11.4) or low (2.8) pH using stepwise elution procedures of the deoxycholate or lithium diiodosalicylate extracts, respectively. Both of these fractions showed a single band of 47 kDa when analyzed by NaDodSO4/PAGE and silver staining. Following removal of the detergent and extensive dialysis at pH 5.8 or treatment with 0.15 M NaCl, even in the presence of detergent, a monomer of 23 kDa was detected. Two-dimensional PAGE of FA-1 showed, four or five polypeptides in the 47-kDa or 23-kDa range. The dialyzed FA-1 contained a major 23-kDa and a minor 48-kDa band when separated on both sucrose and cesium chloride gradients. High performance size-exclusion chromatography showed a major peak at 23 kDa and a minor peak at 50 kDa. Further analysis of the 23-kDa peak by reverse-phase chromatography resolved the antigen into three peaks, which gave similar two-dimensional gel patterns as the native FA-1. Lectin affinity chromatography on a lens culinaris column demonstrated that a part of the antigen was bound to the lectin while the rest was not. The FA-1 revealed a positive reaction with periodic-Schiff reagent and contained glucose and mannose, which together constituted 18.8% of the total antigen mass. Amino acid analysis showed a high percentage of aspartic acid, glutamic acid, serine, and glycine. As a single injection of MA-24 significantly reduced fertilization rates in vivo, the purified FA-1 is an attractive candidate for the development of contraceptive vaccine.