bluuebunny/crossref_metadata_2025 huggingface dataset:
Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase.
Authors: Satoh H , Martin B M , Schulick A H , Christ D D , Kenna J G , Pohl L R
Published: May 2006

Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes of untreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC 3.1.1.1). A similar strategy may be used to purify and characterized neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.
Is there a relationship between DNA sequences encoding peptide ligands and their receptors?
Authors: Goldstein A , Brutlag D L
Published: May 2006

It has been suggested that the coding for a ligand and its receptor may have originated in inverse complementary strands of the same DNA. This would imply a deficiency of stop codons in the complementary strand of the ligand message sequence. We have sought evidence of such deficiencies by an analysis of the usage of selected codons in 23 human neuropeptide and hormone mRNA sequences. We have also searched directly for similarities between substance K or substance P and the substance K receptor. Although bovine proopiomelanocortin has an open reading frame for the full extent of the inverse complement of the coding region, this seems to be a unique case. The data as a whole do not support the hypothesis.
Compact negatively curved manifolds (of dim [unk] 3,4) are topologically rigid
Authors: Farrell F. T. , Jones L. E.
Published: May 2006

Let
M
be a complete (connected) Riemannian manifold having finite volume and whose sectional curvatures lie in the interval [
c
1
,
c
2
] with -∞ <
c
1
[unk]
c
2
< 0. Then any proper homotopy equivalence
h:N
→
M
from a topological manifold
N
is properly homotopic to a homeomorphism, provided the dimension of
M
is >5. In particular, if
M
and
N
are both compact (connected) negatively curved Riemannian manifolds with isomorphic fundamental groups, then
M
and
N
are homeomorphic provided dim
M
[unk] 3 and 4. {If both are locally symmetric, this is a consequence of Mostow's rigidity theorem [Mostow, G. D. (1967)
Publ. Inst. Haut. Etud. Sci.
34, 53-104].} When
M
has infinite volume we can still calculate the surgery
L
-groups of π
1
M
, even when dim
M
= 3, 4, or 5, provided
M
is locally symmetric. An identification of the weak homotopy type of the homeomorphism group of (finite volume)
M
is also made through a stable range. We have previously announced these results for the special case that
c
1
=
c
2
= -1.
Structural significance of an internal water molecule studied by site-directed mutagenesis of tyrosine-67 in rat cytochrome c.
Authors: Luntz T L , Schejter A , Garber E A , Margoliash E
Published: May 2006

The tyrosine-67 to phenylalanine mutated rat cytochrome c is similar to the unmutated protein in its spectral, reduction potential, and enzymic electron-transfer properties. However, the loss of the 695-nm band, characteristic of the ferric form of the normal low-spin physiologically active configuration, occurs 1.2 pH units higher on the alkaline side and 0.7 pH unit lower on the acid side. Similarly, the heme iron-methionine-80 sulfur bond is more stable to temperature, with the midpoint of the transition being 30 degrees C higher, corresponding to an increase in delta H of 5 kcal/mol (1 cal = 4.184 J), partially mitigated by an increase of 11 entropy units in delta S. Urea has only slightly different effects on the two proteins. These phenomena are best explained by considering that the loss of one of the three hydrogen-bonding side chains, tyrosine-67, asparagine-52, and threonine-78, which hold an internal water molecule on the "left, lower front" side of the protein [Takano, T. & Dickerson, R. E. (1981) J. Mol. Biol. 153, 95-115], is sufficient to prevent its inclusion in the mutant protein, leading to a more stable structure, and, as indicated by preliminary proton NMR two-dimensional phase-sensitive nuclear Overhauser effect spectroscopy analyses, a reorganization of this area. This hypothesis predicts that elimination of the hydrogen-bonding ability of residue 52 or 78 would also result in cytochromes c having similar properties. It is not obvious why the space-filling structure involving the internalized water molecule that leads to a destabilization energy of about 3 kcal/mol should be subject to extreme evolutionary conservation, when a more stable and apparently fully functional structure is readily available.
Deletion analysis of the maize mitochondrial superoxide dismutase transit peptide.
Authors: White J A , Scandalios J G
Published: May 2006

The maize mitochondrial superoxide dismutase (SOD; EC 1.15.1.1), a nuclear gene product, has been previously shown to be imported into maize mitochondria. The cDNA for maize mitochondrial SOD was subcloned into a vector containing the T7 promoter. Deletions were made in the transit peptide coding region of the cDNA. The undeleted and deleted proteins were synthetically produced by transcription and translation in vitro. Undeleted preSOD-3 is translocated into isolated maize mitochondria with an efficiency of approximately 30%. Mature SOD-3 subunits are recovered from the matrix but not the membranes of subfractionated mitochondria. These subunits are assembled into the tetrameric holoenzyme. The modified SOD-3 precursors are imported into mitochondria at lower efficiencies than undeleted preSOD-3. The relative import efficiency appears to be dependent upon the deletion size. To our knowledge such analysis of a plant mitochondrial precursor protein has not been reported previously.
A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.
Authors: Lee J M , Greenleaf A L
Published: May 2006

The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts.
Calcium channel activity of purified human synexin and structure of the human synexin gene.
Authors: Burns A L , Magendzo K , Shirvan A , Srivastava M , Rojas E , Alijani M R , Pollard H B
Published: May 2006

Synexin is a calcium-dependent membrane binding protein that not only fuses membranes but also acts as a voltage-dependent calcium channel. We have isolated and sequenced a set of overlapping cDNA clones for human synexin. The derived amino acid sequence of synexin reveals strong homology in the C-terminal domain with a previously identified class of calcium-dependent membrane binding proteins. These include endonexin II, lipocortin I, calpactin I heavy chain (p36), protein II, and calelectrin 67K. The Mr 51,000 synexin molecule can be divided into a unique, highly hydrophobic N-terminal domain of 167 amino acids and a conserved C-terminal region of 299 amino acids. The latter domain is composed of alternating hydrophobic and hydrophilic segments. Analysis of the entire structure reveals possible insights into such diverse properties as voltage-sensitive calcium channel activity, ion selectivity, affinity for phospholipids, and membrane fusion.
Defective virus is associated with induction of murine retrovirus-induced immunodeficiency syndrome.
Authors: Chattopadhyay S K , Morse H C , Makino M , Ruscetti S K , Hartley J W
Published: May 2006

C57BL/6 mice infected with a mixture of murine leukemia viruses (MuLV) develop a syndrome characterized by lymphoproliferation and profound immunodeficiency. Analyses of this viral mixture (LP-BM5 MuLV) showed that it includes replication-competent ecotropic and mink cell focus-inducing MuLV and defective viruses with genome sizes of 3.8-6.5 kilobases. The ecotropic and mink cell focus-inducing MuLV biologically cloned from the mixture did not induce disease, whereas viral preparations containing the ecotropic MuLV and 4.8-kilobase defective virus were active. Cells producing the 4.8-kilobase defective virus expressed an unusual gag-encoded polyprotein of Mr 60,000.
A generalization of the Kostant—Macdonald identity
Authors: Akyildiz E. , Carrell J. B.
Published: May 2006

Let
X
be a smooth projective variety admitting an algebraic vector field
V
with exactly one zero and a holomorphic C
*
-action λ so that the condition
d
λ(
t
)·
V
=

t
p
V

holds for all
t
∈ C
*
. The purpose of this note is to report on a product formula for the Poincaré polynomial of
X
which specializes to the classical identity [Formula: see text] when
X
is the flag variety of a semisimple complex Lie group. A surprising corollary is that the second Betti number of such an
X
is the multiplicity of largest weight of the linear C
*
-action on the tangent space of
X
at the sink of λ. We discuss several examples, including a construction of the rational Fano 3-folds
A
′
22
and
B
5
which is due to Konarski [Konarski, J. (1989) in
C.M.S. Conference Proceedings
, ed. Russell, P. (Am. Math. Soc., Providence, RI), in press].
The 6
1
S
0
-6
3
P
0
transition in thallium isotope ion
204
Tl
+
: A superior atomic clock
Authors: Dehmelt Hans , Yu Nan , Nagourney Warren
Published: May 2006

Experimental and theoretical studies of the forbidden
J
= 0 →
J
′ = 0 optical transition 6
1
S
0
-6
3
P
0
in the odd Hg isotopes are in the literature. From this work natural width and quadratic Zeeman effect have been estimated for the same line in the isoelectronic, even thallium ion
204
Tl
+
, which has a very small nuclear magnetic moment. For the
m
F
= 0 → 0 component a linewidth of <0.01 Hz and a quadratic Zeeman shift <10
-18
at 0.1 Gauss are found, compared to 1 Hz and ≈10
-8
for the hydrogen maser. Electronic quadrupole moments vanish for both
J
and
J
′ states and with them shifts due to electric field gradients. All shifts are orders of magnitude smaller than for Hg
+
and Ba
+
, which have been studied as atomic clock elements.
Barley alpha-amylase genes and the thiol protease gene Aleurain: use of a single poly(A) addition signal associated with a conserved pentanucleotide at the cleavage site.
Authors: Khursheed B , Rogers J C
Published: May 2006

Plant genes usually have multiple potential poly(A) addition signals, and different sites are used for 3' processing of transcripts from a single gene. In contrast, we show here that four barley genes that also have multiple poly(A) addition signals conforming to the plant consensus use only one signal. In each of these genes, the region of cleavage for poly(A) addition is centered on a conserved pentanucleotide. This AGGCA is followed by a conserved sequence homologous to sequences involved in self-cleavage of plant viroid RNA precursors; immediately following, in turn, are four or five nucleotides complementary to the nucleotides immediately preceding AGGCA in each gene. The presence of these conserved sequences and their association with a single region for poly(A) addition in three different gene types (high-pI and low-pI alpha-amylase genes and a thiol protease) that otherwise are not homologous in their 3' untranslated/flanking sequences suggest that they might participate in some common regulatory mechanism shared by these genes.
Identification of a protein associated with p21ras by chemical crosslinking.
Authors: de Gunzburg J , Riehl R , Weinberg R A
Published: May 2006

The products of the ras oncogenes (p21ras) are ubiquitous membrane-associated proteins that bind guanine nucleotides and possess an intrinsic GTPase activity. Because of their functional homologies with regulatory guanine nucleotide-binding proteins, they are thought to be involved in the control of cellular proliferation as transducers of incoming growth signals. In an effort to identify proteins interacting with p21ras, we have used in vivo crosslinking techniques on Rat-1 fibroblasts and derived cell lines overexpressing p21ras and immunoprecipitation with polyclonal anti-p21ras antibodies. Under those conditions, using the homobifunctional crosslinker dithiobis(succinimidyl propionate), a protein of Mr 60,000 (p60) is found to be associated with p21ras, and this association is enhanced by the treatment of quiescent cells with serum. Upon sedimentation of detergent extracts from crosslinked cells on sucrose gradients, a p21-p60 complex could be demonstrated with a Mr of 200,000-300,000, p60 does not appear to be related to pp60src nor to the cytosolic GTPase activating protein that interacts with p21ras to enhance its GTPase activity. The amount of p60 seems to be limiting relative to p21ras in fibroblasts, since similar levels of p60 are immunoprecipitated from Rat-1 cells and transfectants overexpressing Ha-, Ki-, and N-ras p21s; the same protein is also found to associate with p21ras in numerous mammalian cell lines. The relevance of this component to the role of ras proteins in signal transduction is discussed.
Site-directed mutagenesis of the phosphocarrier protein. IIIGlc, a major signal-transducing protein in Escherichia coli.
Authors: Presper K A , Wong C Y , Liu L , Meadow N D , Roseman S
Published: May 2006

The glucose-specific phosphocarrier protein (IIIGlc) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is a major signal transducer that mediates the intricate interplay among extracellular signals (PTS and non-PTS sugars), cytoplasmic and membrane proteins (PTS and non-PTS transporters), and adenylate cyclase. To further define the central role of IIIGlc in these multiplex signaling mechanisms, we have used site-directed mutagenesis to construct three mutant IIIGlc proteins containing single amino acid changes; Phe-3 was replaced with tryptophan [( Trp3]IIIGlc), and His-75 and the active-site His-90 were replaced with glutamine [( Gln75]IIIGlc and [Gln90]IIIGlc, respectively). [Trp3]IIIGlc resembles the wild-type protein in most properties and should be valuable for spectrophotometric experiments. In contrast, clear differences between mutant and wild-type proteins were observed with both [Gln75]IIIGlc and [Gln90]IIIGlc in in vitro sugar phosphorylation assays. As predicted, [Gln90]IIIGlc with a modified active site cannot be phosphorylated. Unexpectedly, [Gln75]IIIGlc accepts but cannot transfer phosphoryl groups, suggesting His-75 may also be a critical amino acid for IIIGlc-mediated signaling mechanisms. The physiological effects of these mutations are briefly described.
Restoration of growth potential in paraclones of human keratinocytes by a viral oncogene.
Authors: Barrandon Y , Morgan J R , Mulligan R C , Green H
Published: May 2006

Human diploid keratinocytes may be divided into three clonal types with differing capacities for proliferation. The paraclone, which has the shortest life span, is limited to 15 divisions, after which all the cells undergo programmed terminal differentiation. By means of a retroviral vector, paraclones which have not completed their life span and which consist of not more than a few hundred cells can be transduced at a high frequency with DNA complementary to the 12S transcript of the adenovirus early region 1A gene. Transformation can be detected within a single cultivation by the formation of progressively growing colonies. The transformants appear to have an unlimited growth potential, and they form a disorganized epidermis when they are grafted as an epithelium onto athymic mice. These experiments clearly show that, in order to be transformed by a viral oncogene, the target cell need not be a stem cell.
Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons.
Authors: Okabe S , Hirokawa N
Published: May 2006

We studied the mechanism of compartmentation of microtubule-associated protein 2 (MAP2) in the dendrites and cell bodies by using microinjection of biotin-labeled MAP2 into mature spinal cord neurons in culture. MAP2 molecules microinjected into the nerve cell body were distributed not only throughout the cytoplasm of the cell body and dendrites, but also in the axon as far as a few millimeters from the cell body within 24 hr after injection. However, when injected cells were incubated for more than 3 days, the amount of biotin-labeled MAP2 in the axon decreased remarkably compared with that in the dendrites. This indicates that there is no sorting mechanism in the cell body for the transport of MAP2 selectively into the dendrites but that the turnover rate of MAP2 in the axons differs from that in the dendrites. To further characterize the mechanism of MAP2 compartmentation, we performed immunoelectron microscopy of injected cells and detergent extraction of microinjected cells prior to immunocytochemistry with anti-biotin. The results strongly suggest that a large part of axonal MAP2 is not associated with cytoskeleton and that this weak association of MAP2 favors selective loss of MAP2 from the axon.
Identification of a point mutation in growth factor repeat C of the low density lipoprotein-receptor gene in a patient with homozygous familial hypercholesterolemia that affects ligand binding and intracellular movement of receptors.
Authors: Soutar A K , Knight B L , Patel D D
Published: May 2006

The coding region of the low density lipoprotein (LDL)-receptor gene from a patient (MM) with homozygous familial hypercholesterolemia (FH) has been sequenced from six overlapping 500-base-pair amplified fragments of the cDNA from cultured skin fibroblasts. Two separate single nucleotide base changes from the normal sequence were detected. The first involved substitution of guanine for adenine in the third position of the codon for amino acid residue Cys-27 and did not affect the protein sequence. The second mutation was substitution of thymine for cytosine in the DNA for the codon for amino acid residue 664, changing the codon from CCG (proline) to CTG (leucine) and introducing a new site for the restriction enzyme PstI. MM is a true homozygote with two identical genes, and the mutation cosegregated with clinically diagnosed FH in his family in which first cousin marriages occurred frequently. The amino acid change occurs in the center of growth factor repeat C in the epidermal growth factor precursor-homology domain of the protein, a region of highly conserved sequence between bovine and human LDL receptors, and results in slowed, but complete, maturation of the precursor to the mature form of the receptor and, despite its remoteness from the ligand-binding domain, in impaired binding of LDL. LDL receptors in MM's skin fibroblasts bind less LDL than normal and with reduced affinity. Thus this naturally occurring single point mutation affects both intracellular transport of the protein and ligand binding and occurs in growth factor-like repeat C, a region that has not previously been found to influence LDL binding.
Cytotoxic activity of a recombinant fusion protein between interleukin 4 and Pseudomonas exotoxin.
Authors: Ogata M , Chaudhary V K , FitzGerald D J , Pastan I
Published: May 2006

A recombinant chimeric toxin in which the cell binding domain of Pseudomonas exotoxin (PE) was replaced by murine interleukin 4 (IL-4) was produced in Escherichia coli. This chimeric protein, IL-4-PE40, was cytotoxic to murine IL-4 receptor-bearing cell lines but had little effect on human cell lines lacking receptors capable of binding murine IL-4. A mutant form of IL-4-PE40 (termed IL-4-PE40 asp553) with very low ADP-ribosylating activity displayed mitogenic activity similar to that of IL-4 rather than cytotoxic activity. Because the cytotoxic effects of IL-4-PE40 were blocked by excess IL-4 or by neutralizing antibody to IL-4 (11B11), we conclude that the cytotoxic effect of IL-4-PE40 is specifically mediated through IL-4 receptors. IL-4-PE40 could be a useful reagent for specific elimination of cells bearing IL-4 receptors.
Import of cytochrome c into mitochondria: reduction of heme, mediated by NADH and flavin nucleotides, is obligatory for its covalent linkage to apocytochrome c.
Authors: Nicholson D W , Neupert W
Published: May 2006

The covalent attachment of heme to apocytochrome c, and therefore the import of cytochrome c into mitochondria, is dependent on both NADH plus a cytosolic cofactor that has been identified to be FMN or FAD. NADH in concert with flavin nucleotides mediates the reduction of heme. Heme in the reduced state is a prerequisite for its covalent attachment to apocytochrome c by the enzyme cytochrome c heme lyase and thus for subsequent translocation of cytochrome c across the outer mitochondrial membrane during import.
Conserved terminal hairpin sequences of histone mRNA precursors are not involved in duplex formation with the U7 RNA but act as a target site for a distinct processing factor.
Authors: Vasserot A P , Schaufele F J , Birnstiel M L
Published: May 2006

The hairpin loop structure and the downstream spacer element of histone mRNA precursors are both needed for efficient 3' end formation in vivo and in vitro. Though generally considered as a single processing signal, these two motifs are involved in different types of interaction with the processing machinery. Whereas RNA duplex formation between the downstream spacer element and the U7 small nuclear RNA is essential for processing, we show here that base pairing between the histone stem-loop structure and the U7 RNA is not relevant. Our experiments demonstrate that a processing factor other than the U7 RNA makes contact with the highly conserved hairpin structure of the histone precursor. The recognition of the target site by the processing factor is structure and sequence specific. Prevention of this interaction results in an 80% decrease of 3' cleavage efficiency in vitro. The hairpin binding factor is Sm-precipitable and can be partially separated from the U7 small nuclear ribonucleoprotein particle on a Mono Q column.
Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression.
Authors: Cai X Y , Maxon M E , Redfield B , Glass R , Brot N , Weissbach H
Published: May 2006

Studies by Urbanowski et al. [Urbanowski, M. L., Stauffer, L. T., Plamann, L. S. & Stauffer, G. V. (1987) J. Bacteriol. 169, 1391-1397] have identified a regulatory locus, called metR, required for the expression of the metE and metH genes. We recently purified the MetR protein from Escherichia coli and showed that it could stimulate the in vitro expression of the metE gene and autoregulate its own synthesis. In the present study, the purified MetR protein has been shown to stimulate the in vitro expression of the metH gene. Also, the in vitro synthesized MetE, MetH, and MetR proteins were shown to be biologically active. The transcription start sites for the metE and metR genes have been determined, and DNA footprinting experiments have identified regions in the metE-metR intergenic sequence that are protected by either the MetR or MetJ proteins.
DNA polymerase III gene of Bacillus subtilis.
Authors: Sanjanwala B , Ganesan A T
Published: May 2006

The Bacillus subtilis dnaF (polC) gene that codes for the alpha subunit of the DNA polymerase III holoenzyme has been sequenced. It consists of 4005 base pairs coding for 1335 amino acids (from the start to the stop codon), giving a molecular weight of 151,273. A mutation (azp-12) that confers resistance to the antimicrobial drug 6-(p-hydroxyphenylazo)-uracil is due to a single base change at nucleotide 3523, from TCA to GCA, resulting in a change of the 1175th amino acid, serine, to alanine. It is in the active site and located at the C-terminal part of the enzyme. The amino acid composition in an N-terminal domain has 26% homology to the epsilon subunit coded by the dnaQ gene of Escherichia coli, which is a 3'----5' proofreading exonuclease, supporting an earlier observation that this function is an integral part of the polymerase molecule in B. subtilis.
Colchicine analogues that bind reversibly to tubulin define microtubular requirements for newly synthesized protein secretion in rat lacrimal gland.
Authors: Herman G , Busson S , Gorbunoff M J , Mauduit P , Timasheff S N , Rossignol B
Published: May 2006

The role of microtubules in 3H-labeled protein secretion in rat lacrimal glands was probed by the use of colchicine and two of its analogues that reversibly bind to tubulin. These analogues were 2-methoxy-5-(2,3,4,4'-trimethoxyphenyl)-2,4,6-cycloheptatriene-1-o ne and 2,3,4,4'-tetramethoxy-1,1'-biphenyl, the latter having been synthesized for these studies. Immunofluorescence revealed that untreated exocrine acinar cells contained an intact microtubule network, which was totally abolished by drug addition. Subsequent drug removal restored the network for the two reversibly binding drugs--more rapidly so for the biphenyl, but this was not the case with colchicine. The protein-secretory process was examined by adding the three drugs at various stages--prepulse incubation, pulse, maturation, apical storage of granules, and discharge under cholinergic stimulation. Comparison with the kinetics of microtubular network restoration, which differed for the two reversibly binding drugs, led to the conclusion that the microtubular system is critical to the maturation phase of secretion.
Single amino acid exchanges in the finger domain impair the function of the Drosophila gene Krüppel (Kr).
Authors: Gaul U , Redemann N , Jäckle H
Published: May 2006

Mutations in the Drosophila gene Krüppel (Kr) cause deletions of segments in the embryo and also affect Malpighian tubule development. In the hypomorphic Kr alleles that were analyzed, the defects in the segment pattern and the Malpighian tubules are parallel in strength. We have sequenced the DNA of four Kr alleles that show normal spatial and temporal patterns of Kr-encoded protein expression. Three of the four alleles have single base pair mutations that result in a single amino acid change. The exchanges occur in the putative DNA-binding domain of the Kr protein, which is characterized by four repeats of the zinc finger motif. Sequence comparison of the finger motifs helped to define the structural requirements for the folding of the finger domain to some extent. Our data on the Kr mutants support the view that has emerged from the evolutionary analysis.
A monoclonal anti-double-stranded DNA autoantibody binds to a 94-kDa cell-surface protein on various cell types via nucleosomes or a DNA-histone complex.
Authors: Jacob L , Viard J P , Allenet B , Anin M F , Slama F B , Vandekerckhove J , Primo J , Markovits J , Jacob F , Bach J F
Published: May 2006

A crude supernatant of hybridoma secreting a monoclonal anti-double-stranded (ds)DNA antibody (PME77 mAb), used to stain fibroblasts (CVI cells) in immunofluorescence, gives a punctuated staining of variable intensity. We had suggested that anti-DNA antibodies bind to cell-surface protein(s) of several cells. When the mAb of this crude supernatant was purified on a dsDNA-cellulose column and a histone-Trisacryl column, the mAb no longer bound to the cell surface. Only when dsDNA plus purified histones was added to the purified antibody did the immune complex strongly and uniformly stain again the cell surface of CVI cells. No significant staining was observed if either DNA or histones were omitted. A signal 94-kDa protein from membrane fractions of CVI, Raji, and RINm cell lines was visualized in immunoblots when mAb-DNA-histone complexes were applied to the nitrocellulose strips. No polypeptide was seen if one component was omitted. This 94-kDa protein behaved like a plasma membrane protein since it required the use of detergent to be solubilized and was quantitatively recovered in the Triton X-114 detergent-rich phase. Moreover, a brief treatment of living cells with trypsin cleared off this protein. Purified nucleosomes could be substituted to DNA-histone complexes, giving rise to identical results. Finally, purified polyclonal anti-DNA antibodies from sera of systemic lupus erythematosus patients labeled a 94-kDa protein provided that DNA-histone complexes were added. Anti-DNA autoantibodies could be pathogenic when they are bound to nucleosomes.
Plant retrotransposon from Lilium henryi is related to Ty3 of yeast and the gypsy group of Drosophila.
Authors: Smyth D R , Kalitsis P , Joseph J L , Sentry J W
Published: May 2006

The lily retrotransposon del 1-46 is 9345 base pairs (bp) long. It has long terminal repeats (LTRs) of 2406 bp (left) and 2415 bp (right), which differ in sequence by 1.4%. Sequences similar to those involved in priming DNA synthesis in retroviruses occur in the internal region. Near the left LTR is a sequence complementary to 18 residues at the 3' end of methionine initiator tRNA of three plant species, and a run of 12 purines occurs close to the right LTR. One internal reading frame of del 1-46 has relatively few stop codons. The 1462-codon product from this frame has motifs, in N to C terminus order, corresponding to those identified with RNA binding, protease, reverse transcriptase, RNase H, and integrase functions in retroviruses and certain other retrotransposons. Amino acid sequence comparisons of three conserved pol regions show del to be closely related to the Ty3 retrotransposon of yeast (37-40% identity). del is also related to the gypsy group of Drosophila (17.6, 297, gypsy/mdg4, and 412), showing closer identity with their reverse transcriptase (32-38%) and RNase H (36-45%) domains than with their integrase domain (21-26%). It is proposed that a gypsy group ancestor exchanged the integrase region with a more distantly related element since its divergence from a del/Ty3 common ancestor. The occurrence of related retrotransposons in three different kingdoms (plants, animals, and fungi) strongly implies their horizontal transmission in recent evolutionary time.
Regulation of tumor necrosis factor gene expression in colorectal adenocarcinoma: in vivo analysis by in situ hybridization.
Authors: Beissert S , Bergholz M , Waase I , Lepsien G , Schauer A , Pfizenmaier K , Krönke M
Published: May 2006

Tumor necrosis factor (TNF) produced by macrophages is thought to contribute to the host defense against development of cancer. However, since tumor cells themselves are able to produce TNF, it is conceivable that TNF may also play an adverse pathological role in carcinogenesis. To better understand the functional significance of TNF in neoplastic disease, we have determined the cellular source of TNF activity produced in 10 patients with colorectal cancer. Northern blot analysis of RNAs extracted from fresh biopsy specimens revealed detectable TNF mRNA levels in all instances. By using in situ hybridization of frozen sections, scattered cells expressing TNF mRNA could be discerned. Based on morphological criteria, these TNF-positive cells most likely belong to the macrophage lineage. Macrophages in normal tissue surrounding the tumor did not express TNF mRNA, suggesting that macrophage activation occurs locally at the site of neoplastic transformation. Immunohistochemistry using anti-TNF monoclonal antibodies revealed that less than 1% of tumor-infiltrating macrophages synthesize TNF protein. Thus we present evidence that in colorectal cancer only a small proportion of tumor-infiltrating macrophages produces TNF, indicating that the microenvironment of the tumor provides adequate, yet suboptimal, conditions for macrophage activation.
Monoclonal antibodies specific to native murine T-cell receptor gamma delta: analysis of gamma delta T cells during thymic ontogeny and in peripheral lymphoid organs.
Authors: Itohara S , Nakanishi N , Kanagawa O , Kubo R , Tonegawa S
Published: May 2006

Three hamster monoclonal antibodies (mAbs), all recognizing different epitopes present on the native form of the murine T-cell antigen receptor (TCR) gamma delta subunits, have been generated. mAb 3A10 is specific to a pan-murine TCR gamma delta, recognizing a C delta constant region determinant. mAb 8D6 is specific to a subset of T cells expressing V gamma 4- and V delta 5-encoded gamma delta TCR, and mAb 5C10 is clonotypic. Using these and other mAbs directed against a variety of T-cell surface markers, we quantitated and characterized gamma delta T cells present in developing thymuses as well as in the conventional lymphatic organs by flow cytometry. These studies revealed that (i) many gamma delta thymocytes and peripheral T cells bear CD4 and/or CD8 molecules, (ii) T cells bearing both alpha beta and gamma delta TCRs are scarce, and (iii) thymocyte subsets bearing TCR gamma delta encoded by different combinations of V gamma and V delta gene segments appear in waves during ontogeny.
Susceptibility to rheumatoid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR beta-1 chain and the Epstein-Barr virus glycoprotein gp110.
Authors: Roudier J , Petersen J , Rhodes G H , Luka J , Carson D A
Published: May 2006

Rheumatoid arthritis is associated with the HLA antigen HLA-DR4. Disease susceptibility maps to the amino acid sequence QKRAA located in the third hypervariable region of the DR beta-1 chain. This region is thought to be a site of recognition for the T-cell antigen receptor. We searched for an antigen in the human environment that could induce T-cell recognition of this sequence. An analysis of protein and DNA databases revealed that the Epstein-Barr virus glycoprotein gp110, which is encoded by the BALF4 open reading frame, contains the sequence QKRAAQRAA, which is highly homologous to the rheumatoid arthritis susceptibility determinant. Experiments using antibodies to synthetic peptides showed that the QKRAA determinant is expressed on the gp110 protein. Humans with serologic evidence of Epstein-Barr virus infection had serum antibodies to gp110 and peripheral blood T cells that recognized peptides from gp110 and HLA-Dw4 encompassing the QKRAA determinant.
Rate of internalization of an immunotoxin correlates with cytotoxic activity against human tumor cells.
Authors: Wargalla U C , Reisfeld R A
Published: May 2006

The relationship between the cellular internalization of an anti-ganglioside GD2 monoclonal antibody (14.G2a) and the toxic effect of its ricin A-chain immunotoxin (14.G2a-RA) was examined on GD2-bearing M21 human melanoma and T293 small cell lung carcinoma cell lines. The capacity for ligand uptake was determined by examining the parameters that contribute to this constant, including the number of cell-surface binding sites and the internalization rate constant (ke). The maximum uptake of 14.G2a is 11-fold greater for M21 than for T293 cells, due to a 2.7-fold difference in binding sites and a 4-fold difference in the rate of antibody internalization. The capacity for ligand uptake correlates with the cytotoxic activity of the 14.G2a-RA immunotoxin against these two cell lines. Furthermore, we were able to demonstrate that the consequence of internalization of 14.G2a-RA is the intracellular release of undegraded ricin A-chain from the antibody. These studies indicate that the rate of internalization is a quantitative parameter that plays a key role in predicting the cytotoxic potency of this immunotoxin.
Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts.
Authors: Soos M A , O'Brien R M , Brindle N P , Stigter J M , Okamoto A K , Whittaker J , Siddle K
Published: May 2006

The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-[3H]glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of [3H]thymidine was stimulated approximately 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor beta-subunit in cells prelabeled with [32P]phosphate was increased 10- to 20-fold within 5 min of adding insulin, with a half-maximal effect at approximately 3 nM insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only approximately 30% that of insulin. Two antibodies (25-49 and 83-14) showed a similar concentration dependence to insulin in their metabolic effects and in the inhibition of 125I-labeled insulin binding to cells. The other two antibodies (83-7 and 18-44) were somewhat less potent and did not inhibit insulin binding. None of the antibodies significantly increased insulin receptor phosphorylation at concentrations up to 100 nM, which at least in the case of 25-49 and 83-14 was sufficient for full receptor occupancy. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway.
Multiple enhancer-like sequences in the HLA-B7 gene.
Authors: Ganguly S , Vasavada H A , Weissman S M
Published: May 2006

Enhancer-like sequences have previously been identified in the promoter region of the mouse major histocompatibility complex (MHC) class I genes. We have screened for such sequences in and around a human MHC class I gene, HLA-B7. Various restriction fragments of the B7 gene were assayed for their ability to enhance transcription of a bacterial chloramphenicol acetyltransferase gene from a simian virus 40 promoter in transiently transfected mouse LTA cells. Our results demonstrate that enhancer activity is located in introns 3 and 5 as well as 5' to the transcription initiation site. RNase protection experiments corroborate the results. Preliminary experiments indicate that B7 enhancers are active in various cell types. The role of these enhancers in B7 gene expression is not known at present. We speculate that the position of the enhancer elements may be related to the occurrence of Hpa II tiny fragment islands.
Identification of a transcriptional silencer in the 5'-flanking region of the human epsilon-globin gene.
Authors: Cao S X , Gutman P D , Dave H P , Schechter A N
Published: May 2006

We have studied the 5'-flanking sequences required for the transcriptional regulation of human epsilon-globin gene expression. A series of deletion mutants of the human epsilon-globin gene 5'-flanking sequences were constructed and linked to the bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in HeLa cells and the human erythroleukemia K-562 cells. By measuring chloramphenicol acetyltransferase activities and mRNA levels we found that the sequence between -177 and -392 base pairs (bp) relative to the mRNA initiation site exerts a negative effect on epsilon-globin promoter activity. This effect is more pronounced in HeLa cells compared with K-562 cells. To further characterize the negative control region we cloned the DNA sequence between -177 and -392 bp either 5' or 3' of the epsilon-globin promoter and in either orientation. Our data indicate that this negative control region inhibits the epsilon-globin promoter activity in a position- and orientation-independent manner, thus suggesting that it is a silencer. In addition, the silencer also inhibits the expression from the Herpesvirus thymidine kinase promoter. Sequence comparison reveals that there are three short regions within the silencer that share extensive homology with those found in other negative control DNA elements. Our results therefore indicate that an upstream silencer element is present in the epsilon-globin gene and that it may play an important role in the control of epsilon-globin gene expression during development.
Characterization of the termini and transposition products of Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis.
Authors: Hecht D W , Thompson J S , Malamy M H
Published: May 2006

We have isolated a 9.6-kilobase conjugal transposon, Tn4399 from Bacteroides fragilis, that is capable of mobilizing nonconjugal plasmids in cis. Here we characterize the ends of the transposon, its target-site requirements, and the products of transposition into the B. fragilis chromosome and two sets of B. fragilis-Escherichia coli shuttle vectors. With the exception of an additional cytosine residue in the left end, there are perfect 13-base-pair (bp) inverted repeats at the ends of Tn4399. Insertion of Tn4399 resulted in a 3-bp target-site repeat in 8 out of 12 independent transpositions and showed a high insertion-site specificity. A remarkable feature of Tn4399 insertions is the presence of an additional 5 bp located between the right inverted repeat and the target-site repeat. Four sequence variations of the 5 bp were found, with absolute conservation at positions 1, 2, and 5. Only two of the variations were present in junction fragments of all three copies of Tn4399 contained in the chromosome of the original donor strain, B. fragilis TM4.2321. Tn4399 appears to represent a new type of conjugal transposon. In contrast to Tn916 and Tn1545, described in streptococci, Tn4399 creates a target-site repeat and contains an additional 5 bp at the right end only, between the transposon and the target sequence. In addition, Tn4399 can mobilize plasmids in cis.
Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor.
Authors: Deuschle U , Pepperkok R , Wang F B , Giordano T J , McAllister W T , Ansorge W , Bujard H
Published: May 2006

Systems that stringently regulate the expression of individual genes within a complex genetic background have contributed greatly to the analysis of gene function. In this report the development of a highly regulated expression system in mammalian cells is described in which transcription of a foreign gene is mediated by the bacteriophage T3 RNA polymerase under the control of the Escherichia coli lac repressor. Rabbit kidney cell lines have been established that constitutively express the phage RNA polymerase and lac repressor. The two bacterial proteins regulate the transcription of the coding sequence of the firefly luciferase, which has been placed under the control of a T3 promoter/lac operator fusion. In the presence of the inducer isopropyl beta-D-thiogalactoside, efficient T3 polymerase-dependent transcription is observed, which is tightly repressed in the absence of inducer. Translation of the T3 transcripts can be mediated by vaccinia virus functions. The demonstration that a specific transcription activity can be regulated over a range of several orders of magnitude in higher eukaryotic cells by using a highly specific and nontoxic inducer has broad implications for a variety of studies.
Shuttle vectors for the archaebacterium Halobacterium volcanii.
Authors: Lam W L , Doolittle W F
Published: May 2006

Progress in archaebacterial molecular biology requires tools for genetic analysis. We describe vectors that can be selected and maintained in either Halobacterium volcanii or Escherichia coli. A genetic determinant for resistance to the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor mevinolin was isolated by "shotgun cloning" into a derivative of the endogenous H. volcanii plasmid pHV2, to form pWL2, which transforms sensitive H. volcanii to mevinolin resistance at high frequency. The resistance determinant, portions of pHV2, and an ampicillin- and tetracycline-resistance-conferring pBR322 derivative, pAT153, were ligated together to form the shuttle vectors pWL101 and pWL102. We describe conditions for the use of these vectors and provide preliminary definition of regions essential for drug resistance and for plasmid replication and maintenance.
Identification and localization of a tau peptide to paired helical filaments of Alzheimer disease.
Authors: Iqbal K , Grundke-Iqbal I , Smith A J , George L , Tung Y C , Zaidi T
Published: May 2006

Amino acid sequencing of a CNBr digest of the tau protein isolated from bovine brain revealed an amino acid sequence of 17 residues, Pro-Gly-Leu-Lys-Glu-Ser-Pro-Leu-Gln-Ile-Gly-Ala-Ala-Pro-Gly-Leu-Lys, which we call peptide I, with heterogeneity at position 11 of glycine (peptide Ia) and proline (peptide Ib); peptide I showed no homology with the previously reported cDNA-derived mouse and human tau sequences. Antisera raised to synthetic peptides corresponding to peptides Ia and Ib labeled all the bovine tau polypeptides recognized by other monoclonal and polyclonal antibodies to bovine tau. Antisera to peptide Ib did not label any mouse tau polypeptides; however, an anti-Ia antiserum labeled two of the four mouse tau polypeptides. Antisera to both peptides labeled paired helical filaments (PHF) as neurofibrillary tangles, plaque neurites, and neuropil threads in Alzheimer disease brain and PHF polypeptides on immunoblots. Immunostaining with anti-Ia antisera of PHF in tissue sections and PHF polypeptides, but not bovine tau, on immunoblots was markedly increased when pretreated with alkaline phosphatase. These studies suggest that (i) the amino acid sequences of some isoforms of tau peptide might be different from that predicted from cDNAs, (ii) a tau peptide that is absent in the predicted sequences is present in PHF in Alzheimer disease, and (iii) tau in PHF is abnormally phosphorylated.