bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular basis of beta-galactosidase alpha-complementation.
Authors: Langley K E , Villarejo M R , Fowler A V , Zamenhof P J , Zabin I
Published: May 2006

In previous studies, a cyanogen bromide peptide derived from amino-acid residues 3-92 of beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) was shown to have alpha-donor activity in intracistronic alpha-complementation. We have now isolated the defective beta-galactosidase alpha-acceptor protein from the deletion mutant strain M15 of Escherichia coli and find that it lacks residues 11-41 of betal-galactosidase. This is demonstrated by the isolation and sequence determination of a cyanogen bromide peptide from the M15 protein, which is identical to the corresponding peptide from beta-galactosidase except for the missing amino acids. We conclude that the alpha-donor peptide restores the region missing in the M15 protein.
Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA and oxidized glutathione: indirect mode of action on polypeptide chain initiation.
Authors: Clemens M J , Safer B , Merrick W C , Anderson W F , London I M
Published: May 2006

In the presence of added double-stranded RNA or oxidized glutathione, protein synthesis in heminsupplemented reticulocyte lysates declines abruptly after 8-12 min of incubation at 30 degrees. The kinetics of amino-acid incorporation are very similar to those seen when lysates incorporation are very similar to those seen when lysates are incubated in the absence of added hemin. The inhibitory effects of double-stranded RNA (dsRNA) and oxidized glutathione (GSSG) are partially overcome by a homogeneous initiation factor, IF-MP, which also stimulates protein synthesis in hemin-deficient lysates. This factor is involved in the binding of Met-tRNAfmet to 40S ribosomal subunits during protein chain initiation. However, neither dsRNA alone nor GSSG alone significantly inhibits formation of [40S subunit-Met-tRNAf] complexes induced in reticulocyte lysates by dsRNA or GSSG involves one or more components present in the lysates but absent from the fractionated in vitro system. Such components may be related to the translational inhibitor that is active in hemin-deficient lysates.
Crystal structure of bovine Cu,Zn superoxide dismutase at 3 A resolution: chain tracing and metal ligands.
Authors: Richardson J , Thomas K A , Rubin B H , Richardson D C
Published: May 2006

An electron density map at 3 angstrom resolution has been calculated for Cu2+, Zn2+ superoxide dismutase from bovine erythrocytes, and the course of the main chain has been traced. The dominant structural feature is an 8-stranded barrel of antiparallel beta-pleated sheet. There is one very short helical section and two long loops of non-repetitive structure. The Cu and Zn are bound between the loops and one side of the beta barrel and are about 6 Angstrom apart, with a common histidine ligand. The Cu has four histidine ligands in a somewhat distorted square plane, and the Zn has three histidines and an aspartate in approximately tetrahedral arrangement. The two coppers of a dimer are about 34 Angstrom apart. The two subunits have essentially the same conformation and have an extensive contact area that mainly involves hydrophobic side chain interactions. The overall folding pattern of the polypeptide chain is very similar to that of an immunoglobulin domain.
Three steps in conversion of large precursor RNA into serine and proline transfer RNAs.
Authors: Seidman J G , McClain W H
Published: May 2006

Bacteriophage T4 serine and proline transfer RNAs are derived from a common precursor RNA. This precusor RNA lacks -C-C-A sequences which could provide 3' termini for the mature transfer RNAs. We have deduced part of the pathway leading to the formation of the C-C-A sequences in the transfer RNAs by characterizing incompletely matured precursor molecules which accumulate during infection of mutant hosts that lack specific enzymes associated with transfer RNA metabolism. Maturation is initiated by the addition of -C-C-AOH to the 3' terminus of the precusor RNA through the combined actionof an unidentified nuclease and tRNA nucleotidyltransferase (EC 2.7.7.25). Precursor RNA molecules terminating in -C-C-AOH is serine transfer RNA and the second product is immature proline transfer RNA. The terminal steps leading to proline transfer RNA have not been fully delineated, but are known to involve the replacement of a -C-UOH sequence by -C-C-AOH.
Radical mechanisms for 1,5-dihydroflavin reduction of carbonyl compounds.
Authors: Williams R F , Shinkai S , Bruice T C
Published: May 2006

A discussion of the expected mechanism of radical reduction of carbonyl compounds by dihydroflavin as it relates to the structure of the carbonyl compound is provided. Factors which must be taken into account are the free energies of formation of the radical anions (CR2O), THE ACIDITY OF THE CONJUGATE ACID OF THE CARBONYL FUNCTION (R2C negative charge OH), and the stability of the carbanion species of the product [(-)CR2(OH)]. It is proposed that in those instances where the product alcohol can flavin radical to CR2O or CR2OH occurs, otherwise H transfer is the terminal step for dihydroflavin reductions. The free energy of formation of the radical species CH2OH obtained by acid-catalyzed electron transfer from dihydroflavin (Fred) to formaldehyde is shown to be less than the experimentally determined free energy of activation (increment F is not equal to exp) for the reduction of formaldehyde by dihydroflavin (i.e., Fred plus CH2O leads to Fox plus CH2OH). Therefore, the radical pair composed of dihydroflavin radical species serve as an intermediate in the reduction. Our proposed mechanism is: Fred plus CH2O ka[H3O] forms k-a[H2O] Frad CH2OH leads to kc Fox plus CH3OH. The value of kaah/k-a has been obtained from the calculated standard potential Eo' for Fred plus CH2O plus H forms Frad plus CH2OH. Assuming ka to represent a two-step process (i.e., Fred plus CH2O plus H k'a forms k'-a Fred plus CH2OH kb forms k-b Frad CH2OH, where ka equals (k'akb)/(k'-ak-b), the value of k'-a will equal 10-10 M-minus 1 sec-minus 1 and kb 10-9 M-minus 1 sec-minus 1. From k'a/k'-a and kb there can be computed the expected value of increment F is not equal to calc as a function of pH. Comparison of increment F is not equal to calc to increment F is not equal to exp reveals that increment F is not equal to calc varies from increment F is not equal to exp by only about 2kcal mol-minus 1 (8.4 kJ mol-minus 1) between pH 5 and 9. Similar considerations establish that radical intermediates should serve eminently well in dihydroflavin reduction of ethyl pyruvate, pyruvic acid, etc. In these cases, 1 e transfer should compete with H transfer to yield the carbanions as the immediate products. Similar comparisons suggest that dihydronicotinamide reduction proceeds via RPyH plus C negative charge O forms RPyH plus C-0minus +H forms -H RPyH plus C-OH forms RPy plus HC-OH.
Diabetes mellitus: changes in the transport properties of isolated intestinal microvillous membranes.
Authors: Hopfer U
Published: May 2006

Isolated, small intestinal microvillous membranes from normal and acutely diabetic rats were compared with respect to D-glucose transport. D-Glucose was accumulated to a greater extent by diabetic membrane vesicles when supplied with energy in the form of a NaC1 or a NaSCN gradient across the vesicle membrane. The difference appeared to be caused by an ability of the diabetic membranes to maintain a higher driving force for active D-glucose transport and not by changes in the glucose "carrier." Increasing the glucose-independent Na-+-conductance of the membrane with monactin or gramicidin D reduced the active accumulation of D-glucose by membrane preparations from both control and diabetic groups. Concentrations of monactin and gramicidin D in the incubation medium of membrane vesicles from diabetic animals could be adjusted so that their D-glucose transport became indistinguishable from that of membranes from normal animals not treated with ionophores. These observatins suggest the microvillous membranes as one site where changes occur in acute diabetes. In addition, the change in the transport properties of the isolated membranes offer a rational explanation for the simultaneous elevation of active intestinal sugar, amino acid, and bile salt transport observed for intact intestinal tissue.
Physical basis of charge pairing in mitochondria.
Authors: Kemeny G , Singer P
Published: May 2006

The postulate of charge pairing in the mitochondrial inner membrane is justified by applying a formula due to Fuoss to calculate the probability density for the distance between a positive and a negative charge. For dielectric constants 10 or less pairing is absolute, for 20 there is some tendency towards pairing, and at 78 it is nonexistent. Pairing, partner exchange or charge substitution, inhibition, and antiport uncoupling can be rationalized within this framework.
Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild-type and mutant strains deficient in photosystem II reaction center.
Authors: Chua N H , Bennoun P
Published: May 2006

Unstacked thylakoid membrane vesicles were obtained from a homogenate of Chlamydomonas reinhardtii by flotation in a 1.8 M sucrose layer containing 5 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid)-10 mM EDTA (pH 7.5). Sodium dodecyl sulfate-gradient gel electrophoresis showed that the wildtype membranes have a total of at least 33 polypeptides ranging in molecular weights from 68,000 to less than 10,000. The wild-type and three non-photosynthetic mutant strains were studied with respect to their photosynthetic electron transport properties, their fluorescence rise kinetics, and their membrane polypeptide compositions. The results showed a strong correlation between the presence of a membrane polypeptide (molecular weight = 47,000) and the activity of the photosystem II reaction center. This polypeptide is missing from F34 (a mendelian mutant lacking Q, the primary electron acceptor of photosystem II), but is partially restored in a suppressed strain of F34 in which there is an incomplete recovery of photosystem II activity. In a thermosensitive mutant, T4, the same polypeptide is present in reduced amount only in cells grown at 35 degrees but not in those grown at 25 degrees. Evidence from fluorescence rise kinetics and partial photochemical reactions show that the cells grown at 25 degree are similar to wild-type cells but the cells grown at 35 degrees are greatly deficient in Q.
Mismatch repair in heteroduplex DNA.
Authors: Wildenberg J , Meselson M
Published: May 2006

DNA with base pair mismatches was prepared by annealing mixtures of genetically marked DNA from bacteriophage lambda. This heteroduplex DNA was used to transfect bacteria under conditions minimizing recombination. Genetic analysis of the progeny phages indicates that: (i) Mismatch repair occurs, usually giving rise to a DNA molecule with one chain with the genotype arising from repair and one parental chain. (ii) The frequency of repair of a given mismatch to wild type depends on the marker, ranging from 3 to 20%. (iii) Excision tracts may extend several hundred nucleotides but are usually shorter than about 2000 nucleotides. (iv) In Rec-mediated bacteriophage crosses, recombination of markers closer than about 10-3 nucleotide pairs frequently occurs by mismatch repair within heteroduplex DNA. (V) The average amount of heteroduplex DNA formed in a Rec-mediated recombination event is a few thousand nucleotide pairs.
Effects of alkylation of phosphodiesters and of bases of infectivity and stability of tobacco mosaic virus RNA.
Authors: Singer B , Sun L , Fraenkel-Conrat H
Published: May 2006

Upon ethyl nitrosourea treatment of RNA of tobacco mosaic virus, up to four phosphodiester groups may be alkylated per molecule without chain breakage, as shown be sucrose gradient centrifugation. This indicates that ribophosphotriesters are quite stable. However, when this alkylation reaction is of longer duration and 6 to 10 triesters are formed, then an average of 1 to 2 breaks occurs and little or no intact RNA can be isolated. Methyl nitrosourea is less effective in forming triesters (about 25% of total alkyl groups compared to about 65% for ethyl nitrosourea), and a greater number of alkyl groups can, therefore, be introduced before breaks occur. Diethyl sulfate and dimethyl sulfate, which alkylate almost only the bases of nucleic acids, do not cause significant degradation of RNA of tobacco mosaic virus, even when as many as 70 alkyl groups are bound. All types of alkylation cause similar losses in viral infectivity at low levels of alkylation. Thus, an average of two chemical events leads to one lethal event, regardless of the nature of the alkylating reaction, which, for example, is with dimethyl sulfate about 65% on the N-7 guanine while with ethyl nitrosourea it is about 65% on phosphodiesters. It is thus concluded that all alkyl groups, whether on the base or on the phosphate, have the same potential to cause inactivation and that inactivation of RNA can result from phosphotriester formation per se.
Reaction of tris(bipyridine)ruthenium(III) with hydroxide and its application in a solar energy storage system
Authors: Creutz Carol , Sutin Norman
Published: May 2006

Irradiation of Ru(bipy)
3
2+
(bipy = 2,2′-bipyridine) with light below 560 nm results in the formation of a charge-transfer excited state potentially capable of reducing water to dihydrogen with concomitant production of Ru(bipy)
3
3+
. The latter may be reduced by hydroxide [Formula: see text] to form dioxygen and regenerate the starting complex. The use of these reactions in a cell designed to bring about the photochemical decomposition of water is proposed.

The stoichiometry, kinetics, and mechanism of the Ru(bipy)
3
3+
-hydroxide reaction have been investigated by conventional and stopped-flow spectrophotometry. The dioxygen yield is a sharp function of pH, attaining its maximum value (about 80%) at pH 9. At low pH (3 and 4.8) the production of ruthenium(II) is first order with
k
obsd
= (1.41 ± 0.04) × 10
-4
sec
-1
(25°, ionic strength μ = 1.00 M with sodium sulfate). In the intermediate pH range (7.9-10.0) complex kinetics are observed. In the hydroxide range 0.01-0.50 M, ruthenium(II) production is predominantly first order with
k
obsd
=
k
a
[OH
-
] +
k
b
[OH
-
]
2
sec
-1
;
k
a
= 148 M
-1
sec
-1
and
k
b
= 138 M
-2
sec
-1
(25°, μ = 1.00 M, sodium sulfate). For the
k
a
term, the activation parameters are ΔH
‡
= 15.3 ± 1.0 kcal mol
-1
and ΔS
‡
= 7 ± 3 cal deg
-1
mol
-1
(1 cal = 4.184 J). An intermediate species (λ
max
800 nm) forms at the same rate as ruthenium(II) in this hydroxide range. It disappears with
k
obsd
= 1.2 + 1.1 × 10
2
[OH
-
] sec
-1
at 25°. Similarly absorbing (λ
max
750 to 800 nm) species are generated by the addition of hydroxyl radical to M(bipy)
3
2+/3+
[M = Fe(II), Os(II), Ru(II), Cr(III), Ru(III)] in pulse radiolysis experiments. The kinetics above pH 7 are described in terms of rate-determining nucleophilic attack by hydroxide on the bound bipyridine ring. The hydroxide adduct so generated is tentatively identified with that observed in the pulse radiolysis experiments with Ru(bipy)
3
2+
.

For reduction of Ru(bipy)
3
3+
by hydrogen peroxide ruthenium(II) production is first order with
k
obsd
=
k
c
[HO
2
-
] +
k
d
[H
2
O
2
] where
k
c
= 5.4 × 10
7
M
-1
sec
-1
and
k
d
= 8.3 M
-1
sec
-1
(25°, μ = 1.00 M, pH 3.5 to 9.7). This reaction produces dioxygen in 83 ± 15% yield at pH 6.8 and in 1.0 N sulfuric acid.
Activation volumes in enzymic catalysis: their sources and modification by low-molecular-weight solutes.
Authors: Low P S , Somero G N
Published: May 2006

Changes in enzyme conformation are often accompanied by large changes in volume. Model compound studies suggest that these volume changes may derive from two sources: (i) "hydration density" effects due to changes in the exposure to solvent of protein groups which modify water density, and (ii) "structural" contributions arising from changes in the volume of the protein itself. An experimental approach was developed to test the validity of the predictions based on model compound studies for catalytic conformational changes. By examining the effects of different solutes on the activation volumes of different enzymic reactions, we show that both sources of volume change provide significant contributions to the activation volume.
Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces.
Authors: Wong G L , Cohn D V
Published: May 2006

Six populations of bone cells (populations 1-6) were obtained by sequential digestion of mouse calvaria with collagenase and trypsin. After release from the tissue, each cell population was cultured for seven days. Parathormone, but not calcitonin, elicited an increase in intracellular cyclic AMP in the cells of populations 4, 5, and 6. In contrast, both hormones elicited increases in cyclic AMP in populations 2 and 3 but had no effect on population 1. When the cells of population 2 were exposed to a Falcontissue culture polystyrene surface for periods of time up to 5 min, many cells adhered. The nonadhering cell population contained a lesser proportion of cells responsive to calcitonin, whereas the adhering population contained a greater proportion responsive to this hormone. Conversely, when the cells of population 2 were exposed to an acid-treated nylon surface, the nonadhering cells contained a larger proportion of those responsive to calcitonin and a smaller proportion responsive to parathormone. When those cells that were enriched for calcitonin responsiveness were examined, we found an increased proportion that exhibited an asymmetric bipolar morphology. These differed from large amorphous, often binucleate, cells which predominated in those populations that responded exclusively to parathormone. These results establish that bone contains at least two types of target cells--one that responds to parathormone but not calcitonin, the other that responds predominantly to calcitonin.
Synthesis of vitellogenin in cultures of male and female frog liver regulated by estradiol treatment in vitro.
Authors: Wangh L J , Knowland J
Published: May 2006

Using the frog Xenopus laevis, we show that the addition of physiological concentrations of estradiol to cultures of liver from untreated males rapidly induces the synthesis of large amounts of vitellogenin. Sustained synthesis of vitellogenin requires continuous exposure to estradiol. A nonestrogenic steroid, dexamethasone, does not induce vitellogenin synthesis but does induce increased synthesis of a different protein in liver cultures.
Genetic complementation in heterokaryons of human fibroblasts defective in cobalamin metabolism.
Authors: Gravel R A , Mahoney M J , Ruddle F H , Rosenberg L E
Published: May 2006

Inherited methylmalonicacidemia due to deficiency of methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase; EC 5.4.99.2) activity results from at least three classes of biochemically distinct defects affecting cobalamin (Cbl: vitamin B12) metabolism (cbl A, cbl B, and cbl C mutants) and a fourth class producing a defective mutase apoenzyme. We have obtained genetic evidence in support of this biochemical heterogeneity, using heterokaryons prepared by Sendai-virus-mediated cell fusion. Nine fibroblast lines from patients with defective Cbl metabolism (4 cbl A, 3 cbl B, and 2 cbl C), two from patients with defective mutase apoenzyme, and two from controls were fused in pairwise combinations and tested for functional mutase holoenzyme using a radioautographic procedure which detects [14C]propionate incorporation into trichloroacetic-acid-precipitable material in fibroblast monolayers in situ. Each of the mutants incorporates negligible radioactivity compared to control cells. Activity is also negligible when different mutants are mixed without virus or when homokaryons are produced by self-fusion. Heterokaryons produced by fusing members of each of the four mutant classes with representatives of any other class recover the ability to incorporate [14C]propionate to levels comparable to those of control cells. However, heterokaryons produced between members of the same class fail to complement in all cases. We conclude that the mutants with defective Cbl metabolism (cbl A, cbl B, cbl C) comprise three complementation groups, that a fourth group corresponds to mutase apoenzyme deficiency, and that all four classes of mutations are recessively inherited.
Photooxidation of chlorins by quinones studied by nuclear magnetic resonance techniques
Authors: Lamola Angelo A. , Manion Marcia L. , Roth Heinz D. , Tollin Gordon
Published: May 2006

The reactions of photo-excited chlorophylls and pheophytins with quinones have been investigated by nuclear magnetic resonance techniques. In slightly alkaline solutions the quinone signals showed line broadening which is explained by degenerate electron transfer between semiquinone radical ions and their benzoquinone parents. In neutral solutions, chemically induced dynamic nuclear polarization was observed which is ascribed to the pair, chlorophyll cation-semiquinone anion. A kinetic analysis of the dependence of these effects upon the quinone concentration suggests that only the reactions of triplet-chlorophyll with the quinones give rise to chemically induced dynamic nuclear polarization effects.
Protein hydration changes during catalysis: a new mechanism of enzymic rate-enhancement and ion activation/inhibition of catalysis.
Authors: Low P S , Somero G N
Published: May 2006

There exists a linear correlation between the effect of a salt on the rate of an enzymic reaction and its effect on the activation volume (delta V++) of the reaction. Salts that increase delta V++ invariably decrease the rate of the reaction, and vice versa. The salt effects on reaction rate are, however, much larger than would be predicted solely on the basis of pressure-volume work changes deriving from the observed alterations in delta V++. Different inorganic salts affect reaction rates and activation volumes in a manner that reflects the salts' positions in the Hofmeister series. These observations, taken in conjunction with data on the effects of salts on protein functional group (aminoacid side-chains and peptide linkages) hydration, lead us to propose the following hypothesis to account for salt activation and inhibition of catalysis. Aminoacid side-chains and peptide linkages located on or near the protein surface change their exposure to water during conformational events in catalysis. These protein group transfers are accompanied by large volume and energy changes that are due largely to changes in the organization of water around these groups. When these transfer processes occur during the rate-limiting step in catalysis, these energy and volume changes can contribute to the free energy of activation (delta G++) and the activation volume of the reaction. By influencing the degree to which water can organize around transferred protein groups, salts can modify both the delta G++ (rate) and the delta V++ of a reaction.
Modification of intensity and direction of electron flow across bileaflet membranes.
Authors: Chen C H , Berns D S
Published: May 2006

The intensity and the direction of electron flow across chloroplast extract bilayer membranes are modified by the presence of biliproteins. Biliproteins including C-phycocyanin, phycoerythrin, allophycocyanin, and Chroomonas phycocyanin enhance the photosensitivity of the membranes. The photoelectric spectra of the membranes in the presence of biliproteins correlate with the absorption spectra of these biliproteins. Experiments are presented to demonstrate the specific character of the electron-directing ability of biliproteins. A possible mechanism involving the interaction between the biliproteins and the membranes is proposed.
Receptor-mediated shifts in cGMP and cAMP levels in neuroblastoma cells.
Authors: Matsuzawa H , Nirenberg M
Published: May 2006

3':5'-cGMP levels of neuroblastoma N1E-115 cells increase as much as 200-fold upon activation of muscarinic acetylcholine receptors, resulting in intracellular cGMP concentrations greater than 600 pmol/mg of protein. The cells also have receptors for adenosine which mediate an increase in 3':5'-cAMP levels. Unexpectedly, prostaglandin E1 was found to increase the concentrations of both cGMP and cAMP. Carbamylcholine, adenosine, and PGE1 were added to cells separately and in pairs to determine the effect of one compound on cell responses to another. Reciprocal inhibition, unilateral inhibition, additive, and nonadditive responses were observed with respect to cGMP and cAMP levels when different pairs of receptors were activated simultaneously.
Enhancement of immunity against murine syngeneic tumors by a fraction extracted from non-pathogenic mycobacteria.
Authors: Lamensans A , Chedid L , Lederer E , Rosselet J P , Gustafson R H , Spencer H J , Ludwig B , Berger F M
Published: May 2006

The data reported here demonstrate that a preparation extracted from nonpathogenic mycobacteria such as Mycobacterium smegmatis and hereafter referred to as interphase material protected mice against Ehrlich ascitic carcinoma, L-1210 leukemia, and another syngeneic lymphoid leukemia. Furthermore, mice treated by this preparation were much less susceptible to endotoxins than when stimulated by BCG (bacillus Calmette-Guerin) or M. smegmatis cells. Moreover, guinea pigs treated by interphase material administered in Freund's incomplete adjuvant showed an increased immune response, yet their sensitivity to tuberculin was much weaker than that of controls sensitized with Freund's complete adjuvant. Finally, resistance to Columbia SK virus infection could be demonstrated when interphase material was administered to mice prior to virus challenge.
Cellular cyclic nucleotides and enzyme secretion in the pancreatic acinar cell.
Authors: Haymovits A , Scheele G A
Published: May 2006

Cellular levels of cAMP and cGMP were measured in guinea pig pancreatic lobules incubated in vitro, during basal or stimulated secretion. Stimulation with optimal concentrations of carbamylcholine (carbachol) (10(-5) M), pancreozymin (0.1 unit/ml), and caerulein (10(-9) M) resulted within seconds in a sharp rise in cGMP levels, from five to more than 20 times that of basal levels. cAMP levels did not change significantly. cGMP increases were maximal at 2 min then subsided by 4-7 min to a plateau about two to three times that of basal level. This plateau was maintained for the duration of the secretagogue stimulus. Removal of the carbachol stimulus resulted in a rapid decrease in cGMP levels to that of the basal state. The cellular cGMP levels observed within the first 2 min of stimulation correlated closely with the dose of carbachol and the secretory response. Atropine at 10(-4) M blocked the cGMP elevation due to carbachol but not that due to pancreozymin, while carbonyl cyanide m-chlorophenyl hydrazone, an uncoupler of oxidative phosphorylation, blocked the response to both secretagogues. Similar though less extensive findings were observed using rabbit pancreatic lobules incubated in vitro. High concentrations (10(-2)-10(-3) M) of the dibutyryl and 8-bromo analogues of both nucleotides were effective, though suboptimal, secretagogues. In the case of the cAMP analogues, the secretory response was associated with a rise in endogenous cGMP levels, similar to that observed during suboptimal carbachol stimulation. These findings suggest that cGMP may be an intracellular mediator in the process of stimulus secretion coupling in the acinar cell of the exocrine pancreas.
Purkinje cell degeneration, a new neurological mutation in the mouse.
Authors: Mullen R J , Eicher E M , Sidman R L
Published: May 2006

A new autosomal recessive mouse mutation, Purkinje cell degeneration (pcd), is described. Mutants exhibit a moderate ataxia beginning at 3 to 4 weeks of age. The ataxia results from postnatal degeneration of virtually all cerebellar Purkinje cells beginning around 15 to 18 days of age and progressing rapidly over the next 2 weeks. In addition to the cerebellar disease there is slow progressive degeneration in the retina (photoreceptor cells) and olfactory bulb. Also, adult males have abnormal sperm.
Distribution of calcitonin-sensitive adenylate cyclase activity along the rabbit kidney tubule.
Authors: Chabardès D , Imbert-Teboul M , Montégut M , Clique A , Morel F
Published: May 2006

The adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] sensitivity to salmon calcitonin in 11 different segments of the rabbit nephron was investigated using a micromethod for enzyme activity measurements in samples, each containing a single piece of tubule. The required segments were isolated by microdissection from collagenase-treated rabbit kidneys. The results were expressed as femtomoles of adenosine 3':5'-cyclic monophosphate formed per mm of tubular length per 30 min of incubation time. In the presence of 0.1 mug/ml of synthetic salmon calcitonin, it was found that eight segments exhibited no hormonal sensitivity whereas maximal responses were induced in three segments, the "bright" portion of the distal convoluted tubule, the cortical and the medullary portions of the thick ascending limb of the loop of Henle (stimulated over control activity ratios were 32, 11, and27). The very high sensitivity to calcitonin of the adenylate cyclase contained in these three segments (0.01 ng/ml of salmon calcitonin inducing a 2-fold stimulation; half-miximal stimulation corresponding to about 0.3 ng/ml of salmon calcitonin) suggests that the distal convoluted tubule, as well as th cortical and medullary portions of the thick ascending limb of the loop of Henle represent physiological target structures of calcitonin action within the kidney.
Covalent attachment of metal chelates to proteins:the stability in vivo and in vitro of the conjugate of albumin with a chelate of 111indium.
Authors: Meares C F , Goodwin D A , Leung C S , Girgis A Y , Silvester D J , Nunn A D , Lavender P J
Published: May 2006

Human serum albumin has been conjugated to 1-(p-bnezenediazonium)-(ethylenedinitrilo)tetraacetic acid, a powerful chelating agent, and radioactive 111indium ions have been added specifically to the chelating groups. The product, with a specific radioactivity of about 1 mCi/mg of protein, was employed as a radiotracer in scintillation scanning studies with human volunteers. Results show that 48 hr after injection, practically all of the label remains attached to albumin. This is confirmed by electrophoresis of serum proteins; 7 days after injection, 85% of the radioactivity in the serum is still in the albumin fraction. These observations agree with in vitro studies of the labeled albumin in human serum, where loss of the metal ion from the chelating group to the protein transferrin amounts to less than 3% after 1 week and less than 5% after 2 weeks. Measurements of the distribution of label in mice up to 23 days after injection suggest that metabolism of the labeled protein does not lead to binding of indium ions by transferrin. The binding of indium and other metal ions by transferrin has previously posed a major impediment to the use of metal chelates for in vivo diagnostic procedures. Demonstration of the kinetic inertness of the chelate in these experiments suggests the use of related chelates as physical probes of biological systems.
Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry.
Authors: Mabrey S , Sturtevant J M
Published: May 2006

High sensitivity differential scanning calorimetry is applied to the study of the thermotropic behavior of mixtures of synthetic phospholipids in multilamellar aqueous suspensions. The systems dimyristoylphosphatidylcholine dipalmitoylphosphatidylcholine, and dimyristoylphosphatidylethanolamine-distearoylphosphatidylcholine, although definitely nonideal, exhibit essentially complete miscibility in both gel and liquid crystalline states, while the system dilauroylphosphatidylcholine-distearoylphosphatidylcholine is monotectic with lateral phase separation in the gel state. Comparison of the observed transition curves with theoretical curves calculated from the calorimetrically determined phase diagrams supports a literal interpretation of the phase diagrams.
Dose-dependent depletion of nuclear receptors by L-triiodothyronine: evidence for a role in induction of growth hormone synthesis in cultured GH1 cells.
Authors: Samuels H H , Stanley F , Shapiro L E
Published: May 2006

The relationship between the binding of L-triiodothyronine (T3) to nuclear receptors and the induction of growth hormone synthesis was examined in cultured GH1 cells, a rat pituitary cell line. After 24 hr, T3 induced a maximal 4-fold increase in the rate of growth hormone wynthesis; the T3 concentration that induced a half-maximal increase was 0.22 nM. The biologic dose-response curve was shifted to the left of the receptor occupancy curve (Kd = 0.5 nM) by a factor of approximately 2 when receptor binding was examined for 4 hr but showed a significantly closer agreement when examined for the same 24-hr period as the biologic response. This shift in the fractional occupancy curve is not due to further equilibration but occurs as a result of a time- and dose-dependent depletion of the nuclear receptor by T3.
A method for the modulation of membrane fluidity: homogeneous catalytic hydrogenation of phospholipids and phospholipids and phospholipid-water model biomembranes.
Authors: Chapman D , Quinn P J
Published: May 2006

The fatty acids associated with phospholipids of cell membranes, and particularly their degree of unsaturation, contribute to the fluidity of their structure and hance determine many of their biological properties. We describe a technique for modulating membrane fluidity which consists of hydrogenating the unsaturated double bonds of membrane phospholipids. This has been accomplished using a homogeneous catalyst. The process has been applied to phospholipids in organic solvents, to phospholipids dispersed as multibilayers in aqueous systems, and also to sonicated preparations of phospholipids arranged as single bilayer vesicles. Preliminary experiments have also been performed with biological membranes. These results indicate that the process of homogeneous catalysis for the modulation of lipid fluidity of biological cell membranes may have considerable future biological and biochemical application.
Changes in levels of cellular retinol- and retinoic-acid-binding proteins of liver and lung during perinatal development of rat.
Authors: Ong D E , Chytil F
Published: May 2006

Cellular retinol-binding protein and cellular retinoic-acid-binding protein, canditates for mediating the action of vitamin A, were found to be present in tissues of the fetal rat. Cellular retinol-binding proteins were still present in most tissues of the adult, but the retinoic-acid-binding protein was not detected in some, including lung, liver, intestine, and kidney. During perinatal development of lung the level of cellular retinol-binding protein remained relatively constant while the level of the cellular retinoic-acid-binding protein peaked at 10 days postnatally, then declined. It was not detectable in lung tissue from 21-day-old rats. In liver, however, the retinoic-acid-binding protein was not detectable later than 5 days postnatally, while the level of the cellular retinol-binding proteinrose sharply near birth, declining only after 21 days to the lower adult levels. The variations observed in the levels of the two binding proteins suggest different and changing requirements for retinol and retinoic acid in organ development and maturation.
Construction of plasmids carrying the cI gene of bacteriophage lambda.
Authors: Backman K , Ptashne M , Gilbert W
Published: May 2006

By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.
Protein cleavage during virus assembly: a novel specificity of assembly dependent cleavage in bacteriophage T4.
Authors: Isobe T , Black L W , Tsugita A
Published: May 2006

Cleavage of precursor proteins occurs during assembly of numerous viruses. Seven bacteriophage T4 head-related proteins areknown to be cleaved during morphogenesis. Sequences surrounding the cleavage sites in T4 head precursors P23 and IPIII are reported here. We previously determined the sequences of precursor and processed forms of IPII and IPI. Cleavage occurs at a glutamyl-alanyl bond in each protein. By comparison of sequences around five cleaved and four uncleaved Glu-Ala bonds in head precursors, it appears that cleavage is limited to the Thr or Ala, and X2 to hydrophilic residues. The results suggest the viral-induced assembly protease recognizes and cleaves an extended primary structure in the structurally dissimilar precursors.
Metabolic mapping of the primary visual system of the monkey by means of the autoradiographic [14C]deoxyglucose technique.
Authors: Kennedy C , Des Rosiers M H , Sakurada O , Shinohara M , Reivich M , Jehle J W , Sokoloff L
Published: May 2006

An autoradiographic technique that employs 2-[14-C]deoxyglucose to measure the local rates of glucose utilization within the brain has been applied to the binocular visual system of the Macaque monkey. This method, which pictorially displays the relative rates of glucose consumption in the component structures of the brain, delineates the regions of altered functional activity because of the close relationship between functional activity and energy metabolism. Bilateral retinal stimulation results in the delineation of different rates of glucose consumption in at least four cytoarchitectural layers of the striate cortex. The most intense metabolic activity appears to be in Layer IV, the locus of the termination of the geniculocortical pathway. Bilateral visual occlusion lowers the rates of glucoes consumption in striate cortex and markedly reduces the metabolic differentiation of the various layers. Unilateral visual deprivation delineates the laminae of the lateral geniculate body and the ocular dominance columns of the striate cortex. It also results in the autoradiographic visualization of regions with normally monocular input in the striate cortex, such as the rostral portions of the mushroom-like configurations in the calcarine cortex, which represent the extreme temporal crescents of the visual fields, and small regions in the most caudal part of the mushroom configurations, which are believed to represent the cortical loci of the blind spotsof the visual fields.
Interaction of integral and peripheral membrane proteins: affinity labeling of yeast cytochrome oxidase by modified yeast cytochrome c.
Authors: Birchmeier W , Kohler C E , Schatz G
Published: May 2006

To identify possible substrate-binding subunit(s) of yeast cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1-9-3-1), the purified enzyme was reacted with yeast iso-1-cytochrome c whose single free sulfhydryl group at position 107 had been activated with 5,5'-dithiobis(2-nitrobenzoate). The resulting cytochrome c derivative appeared to function as an "affinity-label" of cytochrome oxidase, since it rapidly inactivated the enzyme. Inactivation was competitively prevented by underivatized cytochrome c. When the "affinity-labeled" oxidase was analyzed by two-dimensional polyacrylamide electrophoresis in dodecyl sulfate (separation in the second dimension being carried out in the presence of excess sulfhydryl compound), it was found that the derivatized cytochrome c had specifically formed a mixed disulfide with the mitochondrially made subunit III (apparent molecular weight 24,000) of the oxidase. Similar results were obtained when underivatized iso-I-cytochrome c was crosslinked to the oxidase by oxidative disulfide bridge formation in the presence of ortho-phenanthroline and Cu++. These data indicate that the hydrophobic mitochondrially made subunit III of yeast cytochrome c oxidase is in close proximity to the cytochrome c binding site on the enzyme. Since cytochrome c and the mitochondrially made cytochrome oxidase subunit III are typical peripheral and integral membrane proteins, respectively, the present study suggests a useful approach for analyzing specific interactions between these different classes of membrane proteins.
Activation of long chain fatty acids with acyl carrier protein: demonstration of a new enzyme, acyl-acyl carrier protein synthetase, in Escherichia coli.
Authors: Ray T K , Cronan J E
Published: May 2006

A soluble enzyme activity which catalyzes the synthesis of acyl-acyl carrier protein from acyl carrier proteins, a long chain fatty acid, and ATP has been demonstrated in E. coli. The reaction requires high concentrations of both Ca++ and Mg++ for activity, and cleaves ATP to AMP and PPi. The fatty acyl product has been identified as acyl-acyl carrier protein by its solubility, thioester linkage, molecular weight, charge, and biological activity. Several criteria indicate the enzyme is distinct from acyl-CoA synthetase. The fatty acid specificity of the enzyme suggests a role of acyl-acyl carrier protein synthetase in the incorporation of fatty acids into phospholipid.
Induction of sigma factor synthesis in Escherichia coli by the N gene product of bacteriophage lambda.
Authors: Nakamura Y , Yura T
Published: May 2006

Thermoinduction of cells of E. coli carrying prophage lambdacI857 within the bfe gene brings about not only "escape synthesis" of core subunits of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2-7-7-6), but also a striking stimulation of sigma factor synthesis. The latter phenomenon, termed sigma induction, is generally observed after lambda phage infection or prophage induction. A series of experiments with various bacterial and phage strains led us to conclude that the N gene product of lambda is directly involved to the sigma induction. These and other results obtained with mutants defective in transcription termination factor rho suggest the involvement of a rho-sensitive site in the control of sigma gene expression in E. coli.
Phosphorus nuclear magnetic resonance studies on normoxic and ischemic cardiac tissue.
Authors: Gadian D G , Hoult D I , Radda G K , Seeley P J , Chance B , Barlow C
Published: May 2006

The intact heart of a young rat was excised rapidly and cooled to 0 degree C; its energy-rich compounds were examined by 31P Fourier Transform nuclear magnetic resonance. The heart showed the characteristic spectrum of sugar phosphates, inorganic phosphate, phosphocreatine, and magniesium phates, inorganic phosphate, phosphocreatine, and magnesium ATP, characteristics of the energizing state of the nonbeating tissue. Warming to 30 degrees C imposes an energy load upon the heart consistent with short-term resumption of beating, concomitant intracellular acidosis, and decomposition of all detectable energy-rich compounds. The intracellular acidity causes a shift from pH 7.0 to 6.0. The effects of possible interferences with this pH measurement are considered. The method appears to have wide usefulness in cardiac infarct models for detecting the fraction of the total volume occupied by the infarct and for studying the effect of various proposed therapies upon this infarcted volume.
Reciprocal reactivities of specific thiols when actin binds to myosin.
Authors: Duke J , Takashi R , Ue K , Morales M F
Published: May 2006

We report measurements of the reactivity (degree of labeling, as mole of ligand per mole of protein, at constant exposure time) of the reactive thiol, "SH1", of a subfragment of myosin (S-1), and of Cys-10 of F-actin under various conditions, using N-iodo-[3H]acetyl-N-(1-sulfo-5-naphthyl)ethylenediamine, a fluorescent radioactive iodoacetamide analog. When either ADP or adenyloyl imidodiphosphate (simulating unhydrolyzed ATP) is bound to the enzymatic site of S-1, the reactivity of "SH1" is slightly enhanced, but when active ATPase is going on, reactivity is reduced by about a third, presumably due to the species, (S-1) ADP,Pi. The reactivity of Cys-10 alone is very low. When the complex, (S-1)-F-actin, is formed, the reactivity of SH1 is strongly decreased, and the reactivity of Cys-10 is strongly increased. The foregoing results explain our further observation (on glycerol-treated rabbit psoas fibers) that when fibers labeled in relaxation solution are compared with fibers labeled in rigor solution, myosin is more reactive and actin is less reactive, in the former case; alpha-actinin and C-protein are also less reactive in the former case.
Structure of the iron complex in methemerythrin.
Authors: Stenkamp R E , Sieker L C , Jensen L H
Published: May 2006

The coordination of the ligands about the iron atoms in methemerythrin from Themiste dyscritum has been deduced from a 2.8 A resolution electron density map. The complex can be described in terms of two trigonal antiprisms about the pair of iron atoms in each subunit, the antiprisms having one face in common. Ligands at eight of the nine coordination positions are protein side chains, the ninth presumably being water. Comparison of the electron density map for T. dyscritum methemerythrin with the sequence of Phascolopsis gouldii hemerythrin suggests six aromatic side chain ligands (five histidine and one tyrosine) and two nonaromatic side chain ligands. The latter provide atoms at two of the three vertices of the face shared by the two antiprisms, and these along with the presumed water at the third vertex form bridges between the iron atoms of each pair.
"Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication.
Authors: Graessmann M , Graessman A
Published: May 2006

Simian virus 40 (SV40) induces tumor (T)-antigen formation, chromatin replication, and mitosis in primary mouse kidney cells arrested in G0 phase of the mitotic cycle. The temporal and quantitative relation between these early virus-specific reactions led to the hypothesis that the early SV40 mRNA contains information necessary for T-antigen formation and induction of cellular DNA synthesis. To get direct experimental evidence for this hypothesis, the early strand of SV40 DNA was transcribed in vitro by Escherichia coli DNA-dependent RNA polymerase and the SV40-specific cRNA was transferred by microinjection into epitheloid cells of confluent primary mouse kidney cultures. T-antigen formation and stimulation of DNA synthesis were investigated in the recipient cells. The experimental results obtained agree with the hypothesis that T-antigen is a virus-coded protein and that the early virus-specific mRNA contains information necessary for stimulation of cellular DNA replication in the arrested cells.
Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes.
Authors: Kasahara M , Hinkle P C
Published: May 2006

A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The uptake was time dependent and inhibited by mercuric ions or cytochalasin B. The results indicate that the uptake represents transport of the sugar into the liposomes rather than binding to the reconstituted liposomes.