bluuebunny/crossref_metadata_2025 huggingface dataset:
Unusual retention of rhodamine 123 by mitochondria in muscle and carcinoma cells.
Authors: Summerhayes I C , Lampidis T J , Bernal S D , Nadakavukaren J J , Nadakavukaren K K , Shepherd E L , Chen L B
Published: May 2006

Mitochondria in cardiac muscle cells and myoblast-fused myotubes display unusually long (3-5 days) retention times of rhodamine 123, a mitochondria-specific fluorescent probe, in living cells. Among 50 keratin-positive carcinoma or transformed epithelial cell lines tested, mitochondria with prolonged rhodamine 123 retention are detected in most of the transitional cell carcinoma, adenocarcinoma, and chemical carcinogen-transformed epithelial cell lines and in some squamous cell carcinoma lines but not in any oat cell carcinoma lines. The presence of mitochondria having unusual dye retention may be useful for diagnosis and exploitable for chemotherapy of certain human carcinomas.
Dependence of locally measured cellular deformability on position on the cell, temperature, and cytochalasin B.
Authors: Petersen N O , McConnaughey W B , Elson E L
Published: May 2006

We describe an approach to exploring cell surface-cytoskeleton interactions through direct measurements of the mechanical resistance of living cells to locally applied forces. These measurements are sensitive to variations in structure across the cell and at various depths below its surface. We find that local cellular deformability depends on the temperature and on the integrity of the cytoskeleton. Cytochalasin B increases the deformability of all regions of the cell except the nucleus.
Mta, a maternally inherited cell surface antigen of the mouse, is transmitted in the egg.
Authors: Lindahl K F , Bürki K
Published: May 2006

Mta is a maternally transmitted cell surface antigen found in most mouse strains. It serves as a target for unrestricted T killer cells. All 49 embryos transferred between positive and negative mothers, even as early as 10-15 hr after fertilization, developed into mice with the Mta type of their original mothers. Therefore the genetic element that determines expression of Mta must be transmitted by the egg. Mta+ and Mta- lymphocytes, coexisting for months in lethally irradiated F1 mice reconstituted with a mixture of parental bone marrow cells, retained their Mta type. Thus Mta does not spread by infection.
Physical mechanism for regulation of proton solute symport in Escherichia coli.
Authors: Konings W N , Robillard G T
Published: May 2006

The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers. A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity. Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition. These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide. Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline. The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate. The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+). These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.
Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence.
Authors: Palva I , Sarvas M , Lehtovaara P , Sibakov M , Kääriäinen L
Published: May 2006

We describe a secretion vector system for introducing foreign genes into Bacillus subtilis. We constructed secretion vectors from the plasmid pUB110 and the promoter and signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens. Foreign structural genes can be inserted into the various vectors after the signal sequence region of the alpha-amylase gene. Demonstrating secretion of a foreign gene product from Bacillus, we here report that the Escherichia coli beta-lactamase gene, devoid of its own signal sequence coding region, can be expressed in B. subtilis by the aid of the secretion vectors so that greater than 95% of the enzyme activity is secreted to the growth medium. Efficient secretion of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is observed if the complete signal sequence coding region of the alpha-amylase gene precedes the beta-lactamase structural gene. However, an incomplete alpha-amylase signal peptide lacking the six carboxy-terminal amino acid residues does not promote secretion of the fused beta-lactamase, which remains unprocessed and cell-associated.
DNA repair in Escherichia coli: identification of the uvrD gene product.
Authors: Maples V F , Kushner S R
Published: May 2006

A 2.9-kilobase (kb) Pvu II DNA fragment that contains the uvrD gene of Escherichia coli K-12 has been cloned in both low-copy and multiple-copy plasmid vehicles. The low-copy uvrD plasmid (pVMK49) complements a variety of uvrD, uvrE, and recL mutations. In contrast, the same strains carrying the 2.9-kb fragment in a multiple-copy plasmid (pVMK45) remain sensitive to ultraviolet light (UV). Additionally, pVMK45 transformants of wild-type E. coli are sensitive to UV and methyl methanesulfonate and appear to be recombination deficient. The cloned uvrD gene does not complement the dominant uvrD3 allele. The 2.9-kb Pvu II insert in these plasmids encodes a single 76,000-dalton protein, which, on the basis of insertional inactivation experiments with the Tn1000 transposon, must be the uvrD gene product. These data confirm earlier genetic analysis which suggested that recL, uvrE, and uvrD were all allelic. The direction of transcription of the uvrD gene has also been determined.
Herpesvirus-dependent amplification and inversion of cell-associated viral thymidine kinase gene flanked by viral a sequences and linked to an origin of viral DNA replication.
Authors: Mocarski E S , Roizman B
Published: May 2006

The genome of herpes simplex virus 1 or 2 consists of two components, L and S, which invert relative to each other during infection. As a result, viral DNA consists of four equimolar populations of molecules differing solely in the relative orientations of the L and S components. Previous studies have shown that the a sequences, located in the same orientation at the genomic termini and in inverted orientation at the L-S junction, play a key role in the inversion of L and S components. In this report we describe a virus-dependent system designed to allow identification of the viral genes capable of acting in trans to invert DNA flanked by inverted copies of a sequences. In this system, cells are converted to the thymidine kinase-positive phenotype with a chimeric plasmid carrying the thymidine kinase gene flanked by inverted copies of the a sequence and linked to an origin of viral DNA replication derived from the S component. The DNA introduced into the cells is retained and propagated in its original sequence arrangement as head-to-tail concatemers. Infection of these cells with herpes simplex virus 1 or 2 results in as much as 100-fold amplification of the plasmid sequences and inversion of the DNA flanked by copies of the a sequence. In infected cells, the amplified resident DNA accumulates in head-to-tail concatemers and no rearrangement other than the inversions could be detected. These results suggest that the a sequence-dependent inversions required trans-acting viral gene products.
Cell surface antigens of human astrocytoma defined by mouse monoclonal antibodies: identification of astrocytoma subsets.
Authors: Cairncross J G , Mattes M J , Beresford H R , Albino A P , Houghton A N , Lloyd K O , Old L J
Published: May 2006

The surface antigens of cultured human malignant astrocytomas were analyzed by using mouse monoclonal antibodies. BALB/c mice were immunized repeatedly with either SK-MG-1 [a glial fibrillary acidic protein (GFA)-negative astrocytoma line] or SK-AO2 (a GFA-positive astrocytoma line). After fusion with NS/1 mouse myeloma cells, 12 antibody-producing clones were selected for detailed study. Serological analysis permitted the identification of nine distinct antigenic systems. Four monoclonal antibodies (Ab AJ225, Ab AO10, Ab AJ8, and Ab AO122) identified cell surface antigens preferentially expressed on tumors of neuroectodermal origin, and these antibodies subdivided the astrocytoma panel into distinguishable subsets. The determinant detected by Ab AO10 and Ab AJ8 showed mutually exclusive expression on the astrocytoma lines. The AO10 and AJ8 phenotypes appeared to reflect the differentiation state of the cultured cells; 4/7 AO10-positive astrocytomas expressed GFA, an intracellular astrocyte differentiation antigen, whereas all AJ8-positive astrocytoma (9/9) were GFA-negative. Five antibodies (Ab AJ10, Ab AJ9, Ab AJ17, Ab AJ425, and Ab AJ2) recognized determinants widely distributed on normal and malignant cells. Four antibodies defined in this study precipitated proteins from reduced preparations of radioisotope-labeled SK-MG-1 and SK-AO2 cells: Ab AJ225 (Mr 145,000); Ab AO122 (Mr265,000); Ab AJ10 (Mrs 195,000 and 165,000); and Ab AJ2 (Mrs 170,000, 140,000, 140,000, and 28,000).
Monoclonal radioimmunoassays for hepatitis B surface antigen: demonstration of hepatitis B virus DNA or related sequences in serum and viral epitopes in immune complexes.
Authors: Shafritz D A , Lieberman H M , Isselbacher K J , Wands J R
Published: May 2006

High-affinity monoclonal IgM and IgG antibodies (anti-HBs) to hepatitis B surface antigen (HBsAg) have recently been developed and used by us in the construction of highly sensitive radioimmunoassays for the detection of HBsAg-associated determinants in serum. We now report that selected serum samples demonstrating monoclonal immunoreactive material also contain nucleic acid sequences complementary to hepatitis B virus (HBV) DNA by molecular hybridization analysis. In addition, monoclonal radioimmunoassays can detect viral determinants in HBsAg-anti-HBs immune complexes formed in anti-HBs excess; such determinants are undetectable by commercial radioimmunoassay. These and our previous studies suggest that there are HBV or "HBV-related" agents present in human serum that are detected by monoclonal antibodies but are not identified by conventional polyvalent antibodies.
Serotonin modulates a specific potassium current in the sensory neurons that show presynaptic facilitation in Aplysia.
Authors: Klein M , Camardo J , Kandel E R
Published: May 2006

Serotonin exerts a long-lasting excitatory action on sensory neurons of Aplysia californica by decreasing outward K+ current. The depression of outward current delays repolarization of the action potential and extends the duration of Ca2+ influx into the presynaptic terminals, thereby contributing to the facilitation of transmitter release that underlies behavioral sensitization. We have extended the analysis of serotonin's action and find that it acts on a specific serotonin-sensitive K+ current (S current), which is different from the early K+ current (IA), the delayed K+ current (IK), the Ca2+-dependent K+ current (IC), and the muscarine-sensitive M current. The serotonin-sensitive current in these cells persists when IA and IK are reduced by conditioning depolarization or channel-blocking agents. The S current is not activated by intracellular injection of Ca2+, nor is it affected by substitution of Ba2+ for Ca2+, a treatment that reduces IC. Moreover, intracellular injection of cyclic AMP exerts an effect indistinguishable from that of serotonin. This observation and the insensitivity of the current to Ba2+ distinguishes the S current from M current. S current is activated at the resting potential and does not inactivate with steady-state depolarization. It is active sufficiently early during an action potential to contribute to the repolarization of the action potential and therefore accounts for the physiological effects of serotonin.
Photodissociation of water by
p
- and
n
-type polycrystalline iron oxides by using visible light (≤2.7 eV) in the absence of external potential
Authors: Leygraf C. , Hendewerk M. , Somorjai G. A.
Published: May 2006

A polycrystalline
p
/
n
diode assembly, consisting of pressed magnesium- and silicon-doped iron oxide powders, has been shown to photodissociate water by using visible light in the absence of any external potential. In the investigated pH range (8-14) the device produces hydrogen catalytically in amounts that are readily detectable by gas chromatography. The relatively low-power conversion efficiency of 0.05% is believed to be due to the poor change-transfer properties of the
p
-type iron oxide used.
Chemical mechanisms for cytochrome P-450 oxidation: spectral and catalytic properties of a manganese-substituted protein.
Authors: Gelb M H , Toscano W A , Sligar S G
Published: May 2006

Bacterial cytochrome P-450 induced by camphor (P-450cam) is reconstituted with manganese-protoporphyrin IX, yielding an enzyme that displays unique spectral properties relative to previously characterized manganese-porphyrin systems. The nitric oxide complex of the manganese(II)-protein shows a hyper-metalloporphyrin spectrum suggestive of thiolate ligation to the porphyrin-bound manganese ion. In the presence of iodosobenzene as a source of active oxygen, manganese-substituted cytochrome P-450cam serves as a catalyst for the epoxidation of an enzyme-bound olefin substrate. This reactivity proceeds through a spectrally detectable intermediate that resembles the manganese(V)-oxo complexes that have been well documented with model systems employing artificial manganese-metalloporphyrins in organic solution. Interestingly, manganese-substituted cytochrome-P-450cam shows no hydroxylation activity either in the reconstituted camphor hydroxylase system with pyridine nucleotide or in the presence of iodosobenzene and the Mn(III) form of the protein.
Site-specific DNA condensation and pairing mediated by the int protein of bacteriophage lambda.
Authors: Better M , Lu C , Williams R C , Echols H
Published: May 2006

The int protein of bacteriophage lambda catalyzes the site-specific integrative recombination that inserts lambda DNA into the host chromosome. The attachment site region of lambda DNA required for this reaction spans 230 base pairs and includes four separable binding sites for int protein. We have used the electron microscope to determine the functional consequences of the interaction of int with its multiple binding sites. We find that int condenses a 230-base pair segment of DNA into a compact structure about 14 nm in diameter; the condensed region includes all of the four binding sites for int. Condensed segments will form paired structures between attachments sites. We suggest that a sequential cooperative interaction between bound int molecules provides for a distinct reactive DNA conformation and for pairing between substrate sites.
Enhancement of membrane-fusing activity of sendai virus by exposure of the virus to basic pH is correlated with a conformational change in the fusion protein.
Authors: Hsu M C , Scheid A , Choppin P W
Published: May 2006

The effect of pH on the membrane-fusion activity of Sendai virus was examined (pH 5.0-9.5) by using, as assays of activity, hemolysis of chicken erythrocytes and the fusion of baby hamster kidney (BHK-21) cells. Exposure of virus to basic pH increased fusion activity; the optimum pH was found to be approximately equal to 9.0. All assays were carried out at pH 7.0, and the virus retained enhanced fusion activity after it was exposed to basic pH and returned to neutral pH. The enhanced fusion activity was correlated with an irreversible conformational change in the fusion protein (F protein) of the virus, as demonstrated by a change in the circular dichroism spectrum of the protein.
Platelet-derived growth factor binds specifically to receptors on vascular smooth muscle cells and the binding becomes nondissociable.
Authors: Williams L T , Tremble P , Antoniades H N
Published: May 2006

Radioiodinated platelet-derived growth factor (125I-PDGF) was used in studies of PDGF binding sites on vascular smooth muscle cells. There was an excellent correlation between the ability of 125I-PDGF to stimulate cell proliferation and to bind specifically to cultured vascular smooth muscle cells. The half-maximal concentration for both processes was 0.1 nM. There were 50,000 binding sites per cell. Reduced PDGF, prepared by treatment of PDGF with 20 mM dithiothreitol, had neither the ability to bind to smooth muscle cells nor to stimulate cellular proliferation. Epidermal growth factor, nerve growth factor, fibroblast growth factor, and histone B did not compete for the binding sites at a concentration of 10 nM. 125I-PDGF binding was slowly reversible at 4 degrees C and was rapidly and totally reversible after a 1-min incubation at 37 degrees C. After continued incubation at 37 degrees C, the binding became irreversible. The half-time for formation of the nondissociable state of 125I-PDGF binding was approximately equal to 5 min at 37 degrees C. The nondissociable state of binding was not formed at 4 degrees C even after 1 hr of incubation. These data suggest that the sites we labeled are the PDGF receptors that mediate PDGF's mitogenic action and that a nondissociable state of PDGF binding is formed at 37 degrees C. It is likely that nondissociable PDGF represents internalized ligand or binding to sites that are converted to a high-affinity state after the ligand binds.
Mortality among health-conscious elderly Californians.
Authors: Enstrom J E , Pauling L
Published: May 2006

In an attempt to obtain epidemiologic evidence regarding the mortality rate among vitamin supplement users, a prospective study was made of 479 elderly Californian respondents to a 1974 questionnaire carried in Prevention, a health magazine that advocates vitamin supplement usage. Based on self-reported questionnaire data obtained in 1974 and 1977, this cohort does indeed consume large quantities of vitamin and mineral supplements. In addition, the cohort is quite health conscious and appears to have taken up a "Prevention life-style" in recent years. For instance, these individuals are primarily nonsmokers, although about 50% formerly smoked cigarettes. Most of them eat meat, poultry, or fish but do so in moderation; and they consume only modest amounts of alcohol, whole milk, white bread, salt, and sugar. Their socioeconomic status is somewhat higher than the national average. Because this group differs from the general population in many ways it is difficult to separate the various factors that might influence their health. During 6 years of follow-up, a total of 107 deaths occurred. Based on comparison with 1977 United States whites, the standardized mortality ratio (SMR) is 78% for the males, 54% for the females, and 68% for both sexes combined. All three SMR values are significantly less than 100% (P less than 0.05). For both sexes combined, the SMR is 86% for cancer, 62% for total cardiovascular diseases, and 73% for all other causes. Only the cardiovascular SMR is significantly lower than 100%. The death rate for the males is approximately the same as that reported among other healthy nonsmoking questionnaire respondents. but the death rate for females is significantly less (P less than 0.01). The only notable relationships between questions asked in 1974 and subsequent mortality are those indicating a higher mortality rate associated with inactivity, heart trouble, and very low and very high levels of vitamin E intake. For this highly selected cohort, the overall "Prevention life-style" appears to be a healthy one, but the cohort experiences no clear reduction in total mortality because of high levels of vitamin intake per se.
Kinetics of interaction between beta-receptors, GTP protein, and the catalytic unit of turkey erythrocyte adenylate cyclase.
Authors: Tolkovsky A M , Braun S , Levitzki A
Published: May 2006

The kinetics of turkey erythrocyte membrane adenylate cyclase activation by beta-agonists and guanyl-5'-yl imidodiphosphate is explored as a function of the concentration of the GTP regulatory protein and of the catalytic unit. It was found that the overall kinetics of activation is first order and is independent of the concentration of the GTP regulatory unit N, the catalytic unit C, and of hormone over a very wide concentration range. It was established that the rate-limiting step does not involve GDP dissociation from the inactive N unit or the association between activated N' and C. Also, it was found that guanyl-5'-yl imidodiphosphate binding occurs in a random fashion and is not hormone dependent. These results enable us to exclude models of the sequential type in which N in its inactive form is bound to receptor R, is released in an active form N' upon hormone activation, and then binds to C, activating the latter. An acceptable model that accounts for all of the data conforms to the original formulation of "collision coupling" in which N is tightly associated to C at all times.
Cytosolic malic dehydrogenase activity is associated with a putative substrate for the transforming gene product of Rous sarcoma virus.
Authors: Rübsamen H , Saltenberger K , Friis R R , Eigenbrodt E
Published: May 2006

A cellular protein of apparent Mr 34,000--36,000 was suggested as a possible physiological substrate for the protein kinase (EC 2.7.1.37) activity associated with the transforming gene product of Rous sarcoma virus. We find this protein to migrate with an apparent Mr of 38,000 in NaDodSO4/polyacrylamide gels. It was not separable from cytosolic malic dehydrogenase activity when purified by chromatography on DEAE-Sephacel, hydroxylapatite, poly(A)-Sepharose, and blue Sepharose, by gel filtration, and by isoelectric focusing. The Mr 38,000 protein as well as cytosolic malic dehydrogenase activity focused with a pI of 7.5. In gel filtration experiments, both displayed an apparent native Mr of 68,000. The male dehydrogenase activity contained in homogeneous preparations of the Mr 38,000 protein had a specific activity of up to 130 units/mg of protein. The recovery of the enzyme was 5--10% of the activity in the extract. Antiserum against the Mr 38,000 protein inactivated the malic dehydrogenase activity associated with the Mr 38,000 protein.
Delimitation of far upstream sequences required for maximal in vitro transcription of an H2A histone gene.
Authors: Grosschedl R , Birnstiel M L
Published: May 2006

Sea urchin (psammechinus miliaris) H2A histone genes shown to be promoter mutants from oocyte injection experiments were tested for their ability to initiate transcription in vitro. Circular templates were transcribed with HeLa cell extracts, and the transcripts were assayed by mung bean or S1 nuclease mapping of the 5' ends. The transcripts of the H2A mutants produced in vitro were qualitatively similar and, in most cases, identical to those seen in oocyte injection experiments, but quite large quantitative differences were observed for some H2A mutant genes. Both the T-A-T-A box and far upstream sequences residing in the modulator segment E [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 7102--7106] were found to be essential for maximal transcription in vitro. Deletion of either of these sequence elements reduced transcription to 20%. A similar reduction in the amount of H2a transcripts was found when a T-A-T-A-to-T-A-G-A point mutant was tested in vitro. Essential far upstream sequences were mapped between nucleotides -139 and -111, 5' to the initiation site of transcription. In the standard run-off transcription test using restriction fragments, the effects of these sequences could be mimicked by free DNA ends, suggesting that the function of this in vitro upstream sequence might be to provide an entry side for RNA polymerase II.
Territorial limits and functional anatomy of the simian virus 40 replication origin.
Authors: Bergsma D J , Olive D M , Hartzell S W , Subramanian K N
Published: May 2006

The region at and near the simian virus 40 (SV40) DNA replication origin contains a series of palindromes, a 17-base pair (bp) A + T-rich sequence, three copies of a 21-bp repeat, and two copies of a 72-bp repeat. We have constructed a series of recombinant plasmids containing sequential deletions at the region of SV40 DNA replication origin starting from the end near the repeats. These deletions were introduced by using in vitro and in vivo techniques. The relative replication efficiency of these recombinant plasmids were directly assayed in COS-1 monkey kidney cells capable of providing the tumor antigen necessary for the replication of these molecules. Recombinants lacking both copies of the 72-bp repeat did not exhibit any reduction in replication efficiency. Recombinants lacking the 21-bp repeats showed decreased replication efficiency; the reduction in replication efficiency was proportional to the number of copies of the 21-bp repeat deleted in these recombinants. A recombinant retaining the palindromes at the region of SV40 DNA replication but lacking the A + T-rich sequence and the repeats failed to replicate. Based on these results, the SV40 DNA replication origin is subdivided into two regions, and their boundaries are defined. One of these two regions is a core region containing the 17-bp, 15-bp, and 27-bp palindromes and, quite likely, the 17-bp A + T-rich sequence which are necessary for replication. The other is an auxiliary region that consists of the 21-bp repeats and has a dose-dependent enhancement effect on replication efficiency.
Specific cellular stimulation in the primary immune response: a quantized model.
Authors: Vogelstein B , Dintzis R Z , Dintzis H M
Published: May 2006

A general theory for the initial phase of T cell independent immune response is derived from elementary physical-chemical considerations and from the premise that response entails a quantized linkage of cell surface receptors. The theory leads to the construction of explicit antigen dose--response and antigen dose--suppression curves, to the calculation of intrinsic affinities for receptors, and to the deduction that receptors are divalent in character. The theory may be applicable to other cell surface phenomena wherein requirements for stimulation and suppression are equivalent to those found in the immune system.
Intracellular stability of diphtheria toxin fragment A in the presence and absence of anti-fragment A antibody.
Authors: Yamaizumi M , Uchida T , Takamatsu K , Okada Y
Published: May 2006

The erythrocyte ghost function method was used to introduce 125I-labeled diphtheria toxin, its fragments A and B, and two labeled crossreacting material (CRM), mutant proteins CRM176 and CRM197, into cultured mouse cells. Fragment A was relatively stable in mouse cytoplasm at 37 degrees C and at least 80% was recovered from cells after 24 hr of incubation. In contrast, wild-type fragment B and A fragments from CRM176 and CRM197 were unstable and were degraded with half-lives of about 2.5 hr under similar conditions. When a rabbit anti-fragment A IgG fraction was introduced with wild-type A, the rate of degradation of A was accelerated, whereas the rates of degradation of A176 and A197 were retarded by the same antibody. In every instance the degradation rate appeared to be that of the IgG fraction itself with a half-life of about 7.5 hr.
Developmentally regulated mRNAs in mouse liver.
Authors: Barth R K , Gross K W , Gremke L C , Hastie N D
Published: May 2006

The mouse liver contains a group of 10--12 different tissue-specific mRNAs, each present at an average concentration of 12,000--15,000 copies per cell [Hastie, N. D. & Bishop, J. O. (1976) Cell 9, 761--774]. We have determined, by translation in vitro, that these mRNAs are developmentally regulated in the liver. We have also used specific cloned probes to quantitate the developmental time course of expression of five different abundant liver mRNAs. We have found that there are at least three periods during liver development when specific abundant mRNAs are first detectable: prior to 14 days postconception, at birth, and during the onset of sexual maturity. These results indicate that all the members of this mRNA group are not under common developmental regulation. One of the abundant liver mRNAs (p54 mRNA) increases more than 1000-fold in the liver 1 day before birth. We discuss factors that may be involved in the developmental regulation of expression of the genes encoding these mRNAs.
Polymerization of the ninth component of complement (C9): formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement.
Authors: Podack E R , Tschopp J
Published: May 2006

The ninth component of complement (C9) has a marked propensity to polymerize. C9 polymers [poly(C9)] formed spontaneously in Veronal-buffered saline upon incubation of purified C9 for 64 hr at 37 degrees C or within 2 hr at 46--56 degrees C. Poly(C9) formed at 37 degrees C was visualized by electron microscopy as a tubular structure with an internal diameter of 110 A and a length of 160 A. Its ultrastructure suggested a dodecameric composition and resembled that of the membrane attack complex of complement. The wider end of the tubular structure was formed by an approximately 30-A-thick torus with inner and outer diameters of 110 A and 220 A, respectively. Because the dimensions of C9 within poly(C9) were 160 x 55 A (maximal) and 20 A (minimal) and because monomeric C9 has dimensions of approximately 80 x 55 A, it is proposed that monomeric C9 unfolds during polymerization into tubules. Polymerization also occurred upon treatment of C9 for 1 hr at 37 degrees C with 0.6 M guanidine . HCl, 0.1 M octyl glucoside, or 1.5% sodium deoxycholate. Guanidine . HCl-induced C9 polymers consisted of elongated highly curved strands 55--80 A wide, suggesting that these polymers were formed by globular C9 that had not unfolded.
Biochemical signal transmitted by Fc gamma receptors: phospholipase A2 activity of Fc gamma 2b receptor of murine macrophage cell line P388D1.
Authors: Suzuki T , Saito-Taki T , Sadasivan R , Nitta T
Published: May 2006

The detergent lysate of the P388D1 macrophage cell line was subjected to affinity chromatography on two different media, Sepharose coupled to heat-aggregated human IgG (IgG-Sepharose) and Sepharose coupled to the phosphatidylcholine analog rac-1-(9-carboxyl)nonyl-2-hexadecylglycero-3-phosphocholine (PC-Sepharose). Both IgG- and phosphatidylcholine-binding proteins were further purified by Sephadex G-100 gel filtration and isoelectric focusing in the presence of 6 M urea. The isolated IgG-binding proteins specifically bound to IgG2a, but not to IgG2b, whereas the isolated phosphatidylcholine-binding proteins specifically bound to IgG2b but not to IgG2a. Phosphatidylcholine-binding proteins possessed a typical phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4), which was maximal (10 mumol/min per mg of protein) at pH 9.5, depended on Ca2+, and was specific for cleavage of fatty acid from the C-2 position of the glycerol backbone of phosphatidylcholine. The noted enzymatic activity was augmented 4-fold by preincubating phosphatidylcholine-binding proteins with heat-aggregated murine IgG2b but not with IgG2a. IgG-binding proteins, on the other hand, are devoid of any detectable phospholipase A2 activity. Thus, the functional significance of Fc gamma 2b receptor of P388D1 macrophage cell line would be the generation of phospholipase A2 activity at the cell surface upon specific binding to Fc gamma 2b fragment.
Cell surface properties of HLA antigens on Epstein-Barr virus-transformed cell lines.
Authors: Smith L M , Petty H R , Parham P , McConnell H M
Published: May 2006

A number of monoclonal antibodies have been used to investigate the distributions and rates of lateral motion of the HLA-A,B, and-DR antigens on several Epstein--Barr virus-transformed B-cell lines. The lateral diffusion coefficients (D) of fluorescein conjugates of the monoclonal antibodies bound to the cell surface were determined by fluorescence recovery after pattern photobleaching. Ds of HLA-A and-B were found to be comparable and of the order of 10(-9) to 10(-10) cm2/sec for each of the seven monoclonal antibodies and four cell lines examined. The HLA antigens appear to be monomeric on the cell surface based on experiments using mixtures of arsanilic acid-conjugated and fluorescein-conjugated antibodies. Four monoclonal antibodies against DR antigens were examined. Two of these, Genox 3.53 and L243, labeled the cell surface uniformly and gave Ds comparable to those obtained for the HLA-A and -B antigens. The other two, DA2 and 2.06, rapidly patched on the cell surface and were immobile. The DA2, L243, and Genox 3.53 antibodies bound outside of the caps formed with the arsanilic acid-conjugated 2.06 antibody and a second-step rhodamine-conjugated rabbit anti-arsanilate antibody. This is consistent with recent biochemical evidence that there are multiple distinct antigens coded for by the HLA-DR region.
Biological activities of antitrinitrophenyl and antidinitrophenyl mouse monoclonal antibodies.
Authors: Hirayama N , Hirano T , Köhler G , Kurata A , Okumura K , Ovary Z
Published: May 2006

The biological activities of mouse IgG1, IgG2a, IgG2b, and IgE monoclonal were investigated. As has been shown with conventional antibodies, monoclonal IgG1 and IgE are the two classes capable of sensitizing the mouse for anaphylactic reactions, IgE sensitizes the rat, and IgG2a sensitizes the guinea pig. What has not been seen with conventional antibodies is that large amounts of monoclonal IgG2a or IgG2b can sensitize the mouse, very large amounts of monoclonal IgE can sensitize the guinea pig, and very large amounts of IgG1 and IgG2a can sensitize the rat. One of the IgG1 monoclonal antibodies was capable of fixing complement by the classical pathway.
Photochemical generation of carbon monoxide and hydrogen by reduction of carbon dioxide and water under visible light irradiation
Authors: Lehn Jean-Marie , Ziessel Raymond
Published: May 2006

Visible light irradiation of solutions of Ru(2,2′-bipyridine)
3
2+
, cobalt(II) chloride, and carbon dioxide in acetonitrile/water/triethylamine generates simultaneously carbon monoxide and hydrogen. The reaction involves photoinduced reduction of CO
2
and H
2
O, triethylamine serving as electron donor in the Ru(2,2′-bipyridine)
3
2+
/Co
2+
system. The amount of gas (CO + H
2
) produced and the selectivity ratio CO/H
2
depend markedly on the composition of the system. Addition of free bipyridine strongly decreases CO generation but increases H
2
production. With different tertiary amines, NR
3
, both the quantity (CO + H
2
) and the ratio CO/H
2
increase markedly along the sequence R = methyl, ethyl, propyl. Higher selectivity for CO
2
reduction to CO in preference to water reduction occurs when triethanolamine is used instead of triethylamine. CoCl
2
is the most efficient mediator for both CO and H
2
generation and specifically promotes CO formation, whereas salts of other cations studied only yield H
2
. The mechanism of the reaction may involve intermediate formation of Co(I) species. These processes represent an abiotic photosynthetic system allowing simultaneous generation of CO and H
2
and regulation of the CO/H
2
ratio. Mechanistic studies and explorations of other components that may increase efficiency and product selectivity should be carried out. The results obtained are also of significance for solar energy conversion with consumption of a pollutant, CO
2
.
Energetics of plasmid-mediated arsenate resistance in Escherichia coli.
Authors: Mobley H L , Rosen B P
Published: May 2006

Plasmid R773, which codes for resistances to arsenate, arsenite, and antimony, was introduced into Escherichia coli strain AN120, a mutant deficient in the H+-translocating ATPase of oxidative phosphorylation. Cultures depleted of endogenous energy reserves were loaded with 74AsO3-4, and arsenate efflux was measured after dilution into medium containing various energy sources and inhibitors. Rapid extrusion of arsenate occurred when glucose was added. Arsenate was extruded both against and down a concentration gradient. In this strain glucose allows formation of both ATP via substrate-level phosphorylation and an electrochemical proton gradient (or protonmotive force) via oxidation of the products of glycolysis. When oxidation was inhibited by cyanide, glucose metabolism still produced arsenate efflux. Energy sources such as succinate, which supplies a protonmotive force but not ATP, did not result in efflux. Measurement of intracellular ATP concentration under each set of conditions demonstrated a direct correlation between the rate of efflux and ATP levels. Osmotically shocked cells lost the ability to extrude arsenate; however, no arsenate-binding activity was detected in osmotic shock fluid from induced cells. These results suggest that the arsenate efflux system is coupled to cellular ATP rather than an electrochemical proton gradient, possibly by an arsenate-translocating ATPase.
Nucleotide sequence of the ilvB promoter-regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP.
Authors: Friden P , Newman T , Freundlich M
Published: May 2006

The DNA sequence of the promoter-regulatory region of the ilvB operon of Escherichia coli was determined. This region encodes a potential leader polypeptide containing 32 amino acids, 12 of which are the regulatory amino acids valine and leucine. Approximately 50 residues downstream from the coding region for the potential leader peptide is a site for terminating transcription. In vitro transcription experiments show that transcription terminates at this site and produces a leader mRNA of approximately equal to 188 nucleotides. A model for the multivalent regulation of this operon by valyl- and leucyl-tRNA is proposed on the basis of the mutually exclusive formation of five strong stem-and-loop structures in the leader mRNA. In addition, the -35 and -70 regions of this sequence show close structural homologies to areas in cyclic AMP receptor protein (CRP)-dependent promoters reported to be important for CRP function. In vitro transcription from the ilvB promoter was greatly increased by cyclic AMP and CRP. Taken together, these data strongly suggest that the ilvB biosynthetic operon is negatively controlled by multivalent transcription termination and is positively regulated by cyclic AMP and CRP.
Expression of the alpha subunit of human chorionic gonadotropin is specifically correlated with tumorigenic expression in human cell hybrids.
Authors: Stanbridge E J , Rosen S W , Sussman H H
Published: May 2006

The expression of HeLa parent phenotype protein markers, the alpha subunit of human chorionic gonadotropin and placental alkaline phosphatase isoenzymes, has been evaluated in paired tumorigenic and nontumorigenic HeLa-fibroblast human cell hybrids. Both of these proteins have been used clinically as markers of malignancy. The results showed that both are expressed in the hybrids. Expression of the gonadotropin subunit in the hybrids is specifically correlated with tumorigenicity; the placental alkaline phosphatase isoenzyme showed no such correlation and was expressed in both tumorigenic and nontumorigenic hybrids.
Intracellular cysteine delivery system that protects against toxicity by promoting glutathione synthesis.
Authors: Williamson J M , Boettcher B , Meister A
Published: May 2006

Depletion of glutathione by inhibition of its synthesis by buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, leads to increased sensitivity to (i) irradiation and (ii) oxidative stress. In the present work, an intracellular cysteine delivery system was used to promote glutathione synthesis, and this was found to protect against toxicity. Thus, administration of L-2-oxothiazolidine-4-carboxylate protected against acetaminophen toxicity in mice; the thiazolidine, which is converted to L-cysteine by the enzyme 5-oxo-L-prolinase (present in many animal tissues and in plants) promotes the synthesis of glutathione, which is the actual protectant. The effect of this thiazolidine in increasing the level of glutathione is prevented by administration of buthionine sulfoximine. This thiazolidine may be useful in the treatment of other toxicities and in the treatment of certain diseases. It may also be valuable as a component of amino acid mixtures used in therapy and as a safener in agriculture.
Difference in saturable binding of low density lipoprotein to liver membranes from normocholesterolemic subjects and patients with heterozygous familial hypercholesterolemia.
Authors: Harders-Spengel K , Wood C B , Thompson G R , Myant N B , Soutar A K
Published: May 2006

To investigate the possible role of the low density lipoprotein (LDL) receptor in the catabolism of LDL by the human liver, the binding of 125I-labeled LDL to membrane fractions prepared from human liver biopsies was determined. Biopsy samples taken for routine histology were obtained from seven patients with heterozygous familial hypercholesterolemia, one with non-familial hypercholesterolemia, and seven normocholesterolemic subjects. LDL was bound by the membranes from normal subjects in a saturable manner that was inhibited by 56% in the presence of excess LDL. Binding of LDL was also inhibited by modification of the lipoproteins with 1,2-cyclohexanedione. The amount of 125I-labeled LDL bound to membranes from familial hypercholesterolemic livers that could be displaced with excess LDL was significantly less than that bound by normocholesterolemic membranes. These observations suggest that LDL receptors are expressed in normal human liver and are defective in the livers of familial hypercholesterolemic patients.
Human noninsulin-dependent diabetes: identification of a defect in plasma cholesterol transport normalized in vivo by insulin and in vitro by selective immunoadsorption of apolipoprotein E.
Authors: Fielding C J , Reaven G M , Fielding P E
Published: May 2006

Plasma cholesterol metabolism in patients with poorly controlled noninsulin-dependent diabetes was characterized by inhibition of cholesterol net transport between cultured cells (fibroblasts) and plasma, inhibition of cholesterol esterification, and inhibition of cholesteryl ester transfer to low and very low density lipoproteins, relative to a normal control group. Plasma from these patients also contained a 2-fold higher level of apolipoprotein E (apo E). Effective control of hyperglycemia with insulin normalized both the parameters of plasma cholesterol metabolism and plasma levels of apo E. Removal of apo E by immunoaffinity chromatography normalized cell-to-plasma cholesterol transport but was without effect on the rate of cholesterol esterification or of cholesteryl ester transfer. These findings suggest that an inhibition in the chain of reactions by which cellular cholesterol is transferred in esterified form to low and very low density lipoproteins is associated with the appearance of an apo E-dependent "shunt" pathway, returning cholesterol from plasma back to the cells and so nullifying the normal cell-to-plasma transport pathway.
An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.
Authors: Leis J F , Kaplan N O
Published: May 2006

The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.
Characterization of a cloned repetitive DNA sequence concentrated on the human X chromosome.
Authors: Yang T P , Hansen S K , Oishi K K , Ryder O A , Hamkalo B A
Published: May 2006

A tandemly repeated DNA sequence organized predominantly, if not entirely, in a specific manner on the human X chromosome has been cloned in pBR322 and characterized. The sequence was detected as a 2-kilobase band in ethidium bromide-stained agarose gels of BamHI-digested total human nuclear DNA. Although in situ hybridization of the cloned sequence to human metaphase chromosomes showed a single major site of hybridization at the centromere region of the X chromosome and minor sites of hybridization at several autosomal centromeres, Southern blot analysis of restricted total human DNA indicated that the cloned probe is related to other repeated DNAs, particularly the human alphoid DNAs. Restriction enzyme analysis of the cloned fragment revealed an internal repeat structure based upon multiples of 170 base pairs, confirming this relatedness. All available data, however, suggest that the 2-kilobase spacing of BamHI sites within the repeat may be specific to the X chromosome.
Sequence of the gene coding for the β-subunit of dinitrogenase from the blue-green alga
Anabaena
Authors: Mazur Barbara J. , Chui Chok-Fun
Published: May 2006

The nitrogen fixation
nif K
gene of the blue-green alga
Anabaena
, which codes for the β-subunit of dinitrogenase, has been subjected to sequence analysis. The
nif K
protein is predicted to be 512 amino acids long, to have a
M
r
or 57,583, and to contain six cysteine residues. Three of these cysteines are within peptides homologous to FeS cluster-binding cysteinyl peptides from ferredoxins and from a high potential iron protein and, thus, may be ligands to which FeS clusters bind in dinitrogenase. The sequences surrounding the cysteine residues are 70% homologous to the corresponding cysteinyl tryptic peptides of the
Azotobacter vinelandii
dinitrogenase, although the positions of the cysteine residues are not always conserved between the two proteins. A 15-amino acid coding sequence precedes
nif K
on its transcript. Amino acid codon usage is highly asymmetric and parallels that found for the
Anabaena
dinitrogenase reductase gene (
nif H
). Putative promoter and ribosome binding site sequences were identified for
nif K
. These regulatory sequences are homologous to sequences preceding
nif D; nif D
codes for the α-subunit of dinitrogenase but is separated from
nif K
on the chromosome by 11,000 nucleotides. The
nif K
promoter also is virtually identical to a promoter-like sequence that immediately precedes the start of the transcript for the large subunit of ribulosebisphosphate carboxylase from maize chloroplasts. This homology appears to support the theory that chloroplasts evolved from blue-green algae.