bluuebunny/crossref_metadata_2025 huggingface dataset:
A possible folding pathway of bovine pancreatic RNase.
Authors: Némethy G , Scheraga H A
Published: May 2006

A theoretical pathway for the folding of RNase into its native conformation is derived from the contact map computed from crystallographic coordinates. The pathway is based on the hypothesis of Tanaka and Scheraga, according to which localized conformations stabilized by short- and medium-range interactions form before those conformational features that are stabilized primarily by long-range interactions. The pathway deduced from the contact map agrees with experimental information on intermediates detected in the thermal unfolding of RNase and in immunochemical studies on the formation of stable antigenic sites when deduced RNase is oxidized with glutathione. Ambiguities in the interpretation of the contact map are resolved by the combination of structural information contained in the contact map and experimental information.
Origins of phage T4 DNA replication as revealed by hybridization to cloned genes.
Authors: Halpern M E , Mattson T , Kozinski A W
Published: May 2006

[3H]Thymidine-labeled progeny DNA was isolated after infection of Escherichia coli with two different bacteriophage T4 mutants. These strands were isolated shortly after the initiation of DNA replication and hybridized to 15 different (EcoRI) T4 restriction fragments cloned in plasmid vectors. Uniformly labeled T4 [32P]DNA extracted from phage particles was cohybridized as a normalizing reference. The results obtained lead to the conclusions that, among the loci tested, initiation occurs predominantly in the area of genes 50-5 and less prominently in the area of genes 25-29. However, our data do not support the idea of initiation in the area of genes 40-43. In contrast, this area displays the least replication among the genes tested.
Synthesis and processing of maize storage proteins in
Xenopus laevis
oocytes
Authors: Larkins Brian A. , Pedersen Karl , Handa Avtar K. , Hurkman William J. , Smith L. D.
Published: May 2006

Xenopus
oocytes injected with zein mRNAs efficiently synthesized maize storage proteins for prolonged periods. Under optimal conditions, zein was synthesized at 3 ng/hr and represented approximately 10% of the total protein synthesized in the oocyte. The mRNA from the normal maize inbred line directed synthesis of all the major zein components; however, products of mRNA from the
opaque
-2 mutant did not contain the largest zein component. Zein proteins synthesized in the oocyte were 2000 daltons smaller than proteins synthesized by cell-free translation of mRNAs in the wheat germ and reticulocyte systems. This result, which suggested that the oocyte processed prezein polypeptides into native zein proteins, was confirmed by amino-terminal sequence analysis of zein proteins from the oocytes. Cyanogen bromide cleavage of translation products from oocytes and the wheat germ system confirmed the existence of several proteins within each of the major zein components. However, we were unable to detect the presence of signal sequences on zein peptides by this technique.
α- and β-adrenergic stimulation of arachidonic acid metabolism in cells in culture
Authors: Levine Lawrence , Moskowitz Michael A.
Published: May 2006

Madin—Darby canine kidney cells (MDCK) synthesize prostaglandin (PG) F
2α
, PGI
2
(measured as 6-keto-PGE
1α
), PGE
2
, PGD
2
, and thromboxane A
2
(measured as thromboxane B
2
). When incubated in the presence of norepinephrine (6 μM), the syntheses of these arachidonic acid metabolites are stimulated 3-fold. Norepinephrine's effect can be antagonized by the addition of α-adrenergic receptor blocking agents (phenoxybenzamine>phentolamine>yohimbine>dibenamine>tolazoline) but not by the β-adrenergic blocking drug propranolol. Norepinephrine's stimulation is also inhibited by low concentrations of dihydroergotamine, bromocryptine, ergocryptine, and ergotamine. The stimulation of PG synthesis by norepinephrine is reversible, continues during the 24 hr of incubation, and requires the presence of norepinephrine at the receptor site but it is not blocked by the addition of colchicine, cytochalasin B, or cycloheximide. Neither phenoxybenzamine nor ergotamine at concentrations that block norepinephrine's stimulation of PG biosynthesis suppresses the increase in PG synthesis induced by exogenous arachidonic acid, suggesting that the α-adrenergic regulation is not occurring primarily at the cyclooxygenase step in the metabolism of arachidonic acid. In mouse lymphoma cells (WEHI-5), low concentrations of isoproterenol or norepinephrine stimulate the synthesis of thromboxane, an effect that can be blocked by the addition of propranolol but not by relatively high concentrations of phenoxybenzamine or ergotamine. Taken together, these results suggest that α-adrenergic receptor stimulation promotes the deacylation of phospholipids by MDCK cells whereas β-adrenergic mechanisms lead to activation of similar pathways in WEHI-5 cells.
Purification and primary structure of the neuropeptide egg-laying hormone of
Aplysia californica
Authors: Chiu A. Y. , Hunkapiller M. W. , Heller E. , Stuart D. K. , Hood L. E. , Strumwasser F.
Published: May 2006

Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in
Aplysia californica
. In the present study, ELH has been purified to homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a
M
r
of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: H-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln- Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH. Enzyme data indicate that the COOH-terminal lysine may be modified but its exact nature remains to be determined. There is no similarity between the amino acid sequence of ELH and that of presently known vertebrate neuropeptides. The two-step purification procedure, starting with a homogenate of bag cell clusters, consisted of cation exchange chromatography on SP C25 (Sephadex) followed by gel filtration on Bio-Gel P-6. Our purification results in a 100-fold enrichment of ELH from bag cell homogenates and a 36% recovery of purified radiolabeled marker ELH. Analysis of purified ELH radiolabeled with [
35
S]methionine or [
3
H]leucine on isoelectric focusing gels and on 8 M urea/sodium dodecyl sulfate gels showed only a single peak containing 90% of the radiolabel. Radiolabeled ELH migrated with a pI of 9.0-9.2 and an apparent
M
r
of 3500-5700. ELH retained egg-laying bioactivity when eluted from this segment of the gel. We find that 2.5 nmol of pure ELH consistently induces egg laying at 20°C.
Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin
Authors: Pedersen Cort A. , Prange Arthur J.
Published: May 2006

Oxytocin produces uterine contractions and milk ejection, functions related to parturition and nuturing. Studies were conducted to determine if this peptide, native to the brain and the posterior pituitary gland, plays a role in the induction of maternal behavior. Intact virgin female rats were given 0.4 μg of oxytocin, 0.4 μg of [Arg
8
]vasopressin, or saline through lateral ventricular cannulae. Forty-two percent of intact rats receiving oxytocin displayed full maternal behavior towards foster pups. None of the saline- or vasopressin-treated animals displayed full maternal behavior. Criteria in five behavioral categories had to be fulfilled by an animal within 2 hr of injection for its behavior to be considered fully maternal. When partial maternal responses were considered, oxytocin was significantly more effective than saline and marginally more effective than vasopressin. Five animals responding fully maternally after oxytocin injection were allowed to stay with pups for 10 days. All five continued to display full maternal behavior during this time. Nearly all animals that responded fully maternally to oxytocin injection were in the last day of diestrus or in proestrus or estrus. This suggested that elevated or recently elevated levels of estrogen may be necessary for the induction of full maternal behavior by oxytocin. Twenty-seven virgin female rats were ovariectomized and given either 100 μg of estradiol benzoate per kg in oil subcutaneously or oil alone immediately after operation. Forty-eight hours later, all animals received 0.4 μg of oxytocin intracerebroventricularly. Eleven of 13 estrogen-primed animals became fully maternal; none of 14 nonprimed animals became fully maternal.
Stimulation of benzodiazepine receptor binding by gamma-aminobutyric acid.
Authors: Karobath M , Sperk G
Published: May 2006

The effect of the neurotransmitter gamma-aminobutyric acid (GABA) on high-affinity binding of benzodiazepines to brain membranes has been investigated. GABA stimulated [3H]diazepam binding by more than 100% when extensively washed membranes from brain tissue were used. This GABA-stimulated benzodiazepine binding occurred in all brain regions examined. The stimulation was specific for GABA agonist. It was inhibited by the GABA receptor blocker bicuculline methiodide. A large number of compounds structurally closely related to GABA but without direct effect on the GABA receptor failed to enhance [3H]diazepam binding. The stimulation of benzodiazepine binding was caused by an increase in affinity; the number of binding sites remained unchanged. Half-maximal activation of [3H]diazepam binding occurred in the presence of 300 nM muscimol or 900 nM GABA. beta-Guanidinopropionic acid and imidazoleacetic acid were much weaker activators. It is suggested that the described stimulation of benzodiazepine high-affinity binding is mediated by a receptor for GABA. This site of GABA action exhibits different properties when compared to GABA receptors, as characterized by high-affinity binding of GABA agonists.
Sodium ascorbate potentiates the growth inhibitory effect of certain agents on neuroblastoma cells in culture.
Authors: Prasad K N , Sinha P K , Ramanujam M , Sakamoto A
Published: May 2006

Mouse neuroblastoma (NB) cells in culture were more sensitive to sodium L-ascorbate than were rat glioma cells by the criterion of growth inhibition (due to cell death and reduction in cell division). Sodium L-ascorbate at nonlethal concentrations potentiated the effect of 5-fluorouracil (FUra), x-irradiation, bleomycin, RO20-1724, prostaglandin E1, and sodium butyrate on NB cells but did not produce such an effect on glioma cells. Sodium L-ascorbate did not enhance the effect of vincristine, 6-thioguanine, or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) except at higher drug doses and it reduced the cytotoxic effect of methotrexate and 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) on NB cells. Sodium D-ascorbate produced effects similar to those produced by sodium L-ascorbate on NB cells. L-Ascorbic acid-2-sulfate (barium salt) affected neither the growth rate nor the effect of 5-FUra on NB cells. Glutathione, a reducing agent, was more toxic to NB cells in comparison to D- OR L-ascorbate; however, at a similar concentration it failed to potentiate the effect of 5-FUra on NB cells.
src
gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus
Authors: Rübsamen Helga , Friis Robert R. , Bauer Heinz
Published: May 2006

Sera from certain rabbits bearing Schmidt-Ruppin strain Rous sarcoma virus (RSV)-induced tumors precipitated p60

src

from chicken cells transformed by the homologous virus as well as by other strains [Prague strain RSV, Bryan high-titer strain RSV, and Bratislava 77 strain of avain sarcoma virus (ASV)], the molecular weights (
M
r
s) ranging from 60,000 to 64,000. The p60

src

immunoprecipitated from cells transformed by each of these strains incorporated [γ-
32
P]ATP into the
M
r
53,000 subunit of IgG, though with differing activities. No such protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) was observed when the following immunoprecipitates were used: from uninfected cells, from untransformed cells infected by Rous-associated virus, or from cells transformed by acute leukosis viruses, avian erythroblastosis virus, or myelocytoma virus 29. The kinase reaction had a pH optimum at pH 5.9 and an apparent
K
m
for ATP of 4.9 ± 2 μM, and was dependent on Mg
2+
(
K
b
= 46 ± 12 mM), for which Ca
2+
was no substitute. The kinase was cyclic AMP independent.

In order to test whether the protein kinase reaction is directly catalyzed by p60

src

, we compared the
in vitro
temperature sensitivities of the kinase activities from cells infected by transformation-temperature-sensitive mutant and parental wild-type virus. The first-order rate constant for the inactivation of the kinase from extracts of cells infected by the mutant virus was 2-fold greater than that from cells infected by wild-type virus. This result implicates the protein kinase as an enzymatic activity of the
src
gene product, the p60

src

. Concomitant with the loss of the kinase activity by heat inactivation, p60

src

loses 60-70% of its phosphate content. The kinetics of dephosphorylation exactly parallel those for the inactivation of the kinase activity, suggesting that the p60

src

kinase is itself dependent on phosphorylation for its activity.
Differentiation of a rat mammary cell line in vitro.
Authors: Dulbecco R , Bologna M , Unger M
Published: May 2006

We have studied the development of fusiform (probably related to myoepithelial) cells in Rama 25 cultures [Bennett, D. C. Peachey, L. A., Durbin, H. & Rudland, P. S. (1978) Cell 15, 283--298]; we show that they are generated from special differentiated structures (projections) that contain a rapidly differentiating cell type (F-precursor cells). Clonal sublines isolated from projections develop in several directions under both environmental and genetic control. Some types of differentiation are reversible; others are irreversible. The various cell occurring in vitro may correspond to specific cell types in vivo.
Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures.
Authors: Thompson W C , Deanin G G , Gordon M W
Published: May 2006

In cultured muscle cells the carboxyl-terminal tyrosine of alpha-tubulin was shown to exchange rapidly with free tyrosine. The rapid turnover of this residue was dependent upon the presence of intact microtubules. Half-life determinations were made by two methods: (i) the cells were pulse-labeled in hypertonic medium, in which the major tyrosine incorporation was post-translational, and then chased with isotonic medium; and (ii) the cells were pulsed and chased in isotonic medium, and the post-translational component of the radioactivity of purified alpha-tubulin was calculated. Both methods yielded a half-life of 37 min or less for the terminal tyrosine residue, whereas the half-life of tubulin itself was shown to be greater than 48 hr.
Fc-receptor variants of a mouse macrophage cell line
Authors: Unkeless Jay C. , Kaplan Gilla , Plutner Helen , Cohn Zanvil A.
Published: May 2006

Variants of the J774 mouse macrophage cell line that lack immunologically important membrane receptors were isolated. After mutagenesis, variants were selected in a metrizamide gradient that separated cells heavily rosetted with sheep erythrocytes (E) coated with rabbit anti-E IgG (EIgG) from poorly rosetted cells. Stable variants that exhibited altered binding were found with a frequency of <10
-7
, and five clones were studied in detail. The variants failed to bind E opsonized with a monoclonal mouse IgG2b anti-E antibody but bound monomeric IgG2a normally when compared to the parental J774 line (
K
a
4°C=≈1×10
8
M
-1
; ≈2×10
5
sites per cell). This demonstrates the independence of the receptor for mouse IgG2b complexes (FcRII) from the trypsin-sensitive receptor for mouse IgG2a monomer (FcRI). The variants bound an average of 10-15 EIgG per cell, compared to >20 per cell for J774. After trypsinization, three variants bound only three to five EIgG per cell; the J774 line was not affected by this treatment. Monomeric IgG2a could inhibit the binding of soluble rabbit IgG—antigen complexes to the variants but not to the parent line. Finally, E coated with IgM and complement (EIgMC) were bound poorly by all the variants, relative to the J774 parent. These results show that rabbit IgG complexes are bound by both FcRI and FcRII on mouse macrophages. The impairment of EIgMC rosetting in the variants suggests that the C3b receptor and FcRII are related.
Amino acid sequence of ATP phosphoribosyltransferase of Salmonella typhimurium.
Authors: Piszkiewicz D , Tilley B E , Rand-Meir T , Parsons S M
Published: May 2006

The amino acid sequence of ATP phosphoribosyltransferase [1-(5'-phosphoribosyl)-ATP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.17] of Salmonella typhimurium has been determined. The amino acid sequence analysis was carried out with a combination of manual and automated methods. It was complemented by DNA sequence analysis (done in another laboratory) of the hisG gene, which codes for it. The subunit polypeptide chain contains 299 amino acid residues and has a molecular weight of 33,216. The amino-terminal segment of the protein is relatively basic in character and has limited sequence homologies with the lac repressor and histidinol dehydrogenase. In addition, the protein contains a 40-residue segment that has 13 residues identical with the sequence surrounding the active-site cysteine of glyceraldehyde-3-phosphate dehydrogenase.
Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity.
Authors: Hathaway D R , Adelstein R S
Published: May 2006

In an actomyosin fraction isolated from human platelets, phosphorylation of the 20,000-dalton light chain of myosin is stimulated by calcium and the calcium-binding protein calmodulin. The enzyme catalyzing this phosphorylation has been isolated by using calmodulin-affinity chromatography. Platelet myosin light chain kinase activity was monitored throughout the isolation procedures by using the 20,000-dalton smooth muscle myosin light chain purified from turkey gizzards as substrate. The partially purified myosin kinase requires both calcium and calmodulin for activity and has a specific activity of 3.1 mumol of phosphate transferred to the 20,000-dalton light chain per mg of kinase per min under optimal assay conditions. Km values determined for ATP and myosin light chains are 121 microM and 18 microM, respectively. Of several substrates surveyed as phosphate acceptors (alpha-casein, histone II-A, phosphorylase b, protamine, histone V-S, and phosvitin), only the 20,000-dalton myosin light chain is phosphorylated at a significant rate. These results suggest that platelet myosin light chain kinase is a calcium-dependent enzyme and that the requirement for calcium is mediated by the calcium-binding protein calmodulin.
Conditions for inhibiting and enhancing effects of the protease inhibitor antipain on x-ray-induced neoplastic transformation in hamster and mouse cells.
Authors: Borek C , Miller R , Pain C , Troll W
Published: May 2006

Using cultured normal hamster embryo cells and the heterploid mouse C3H cell line 10T1/2, clone 8, we have studied the effect of the protease inhibitor antipain on x-ray-induced neoplastic transformation. We found in both cell systems that, while there was no effect on cell survival as compared to irradiated controls, the addition of antipain at a concentration of 6 microgram/ml to the cultures 24 hr prior to irradiation resulted in enhanced transformation as compared to the frequency in cultures exposed to radiation alone. Yet the addition of antipain to cultures 10 min after irradiation resulted in a decreased transformation rate. This decrease was not found when antipain was added to the mouse cells 24 hr after irradiation or to the hamster cells 48 hr after irradiation. These results suggest that the protease inhibitor antipain has more than one mechanism of action in modulating the fixation and expression of transformation by x-irradiation, possibly by the modification of DNA repair.
Selective growth of some rodent epithelial cells in a medium containing citrulline.
Authors: Sun N C , Sun C R , Tennant R W , Hsie A W
Published: May 2006

We have defined a medium (called Sun's modified Waymouth medium) that selectively cultures some rodent epithelial cells that are capable of using citrulline in place of arginine. A growth-response study of the ability of 47 different mammalian cell cultures (of mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, monkey, and human origin) to use arginine or its biosynthetic precursors, ornithine, citrulline, or argininosuccinate, showed that all epithelial cells and some fibroblasts are capable of growing in citrulline medium; however, primary embryo fibroblasts and 12 established fibroblast cell lines derived from Syrian hamsters failed to grow. The citrulline medium also allowed selective outgrowth of epithelial cells, without contaminating fibroblasts, from Syrian hamster tracheal explants. This absolute nutritional difference between Syrian hamster epithelial and fibroblast cells allows citrulline medium to be used for selective cultivation of epithelial cells, which should be valuable for study of growth, differentiation, and malignant transformation of mammalian epithelial cells.
Cytotoxic T cells: Lyt phenotype and blocking of killing activity by Lyt antisera
Authors: Nakayama Eiichi , Shiku Hiroshi , Stockert Elisabeth , Oettgen Herbert F. , Old Lloyd J.
Published: May 2006

We reexamined two questions concerning Lyt antigens of cytotoxic T cells of the mouse: is Lyt-1 antigen expressed on cytotoxic effector cells and can cytotoxicity be blocked by antibody to Lyt antigens in the absence of added complement? A 3-hr
51
Cr-release assay with splenic effector cells and leukemia or myeloma target cells was used to measure cell-mediated cytotoxicity. The cytotoxic activity of effector cells against allogeneic targets was abolished by exposure to Lyt-1, Lyt-2, or Lyt-3 antiserum and complement. Specificity was established by tests with C57BL/6 Lyt congenic mice and absorption studies with thymocytes. Similarly, the cytotoxicity of effector cells directed against semisyngeneic myeloma targets was reduced by Lyt-1, -2, or -3 antiserum and complement. Effector cell cytotoxicity against another semisyngeneic target was only marginally affected by Lyt-1 antiserum and complement, but was abolished by Lyt-2 or -3 antiserum and complement. It appears likely that cytotoxic T cells are a heterogeneous population with regard to Lyt-1 expression and that past studies indicating an apparent absence of Lyt-1 on cytotoxic T cells revealed a quantitative, not qualitative, feature of these cells. With regard to the activity of Lyt antisera in the absence of added complement, selective blocking of effector cell cytotoxicity for allogeneic and semisyngeneic targets was found with Lyt-2 and Lyt-3 antisera but not with Lyt-1 antiserum. The specificity of blocking was established by tests with Lyt congenic mice and absorption studies with thymocytes. With the exception of blocking by antisera to the
H-2
haplotype expressed by the target cell, no effector cell blocking was observed with alloantisera or heteroantisera to a range of other cell surface molecules present on mouse lymphoid cells. One possibility to account for the selective blocking by Lyt-2 and Lyt-3 antisera is that Lyt-2,3 determinants on the surface of cytotoxic T cells have a close spatial relation to the T cell receptor.
Opioid peptides and alpha-melanocyte-stimulating hormone in genetically obese (ob/ob) mice during development.
Authors: Rossier J , Rogers J , Shibasaki T , Guillemin R , Bloom F E
Published: May 2006

Compared to littermate controls (C57BL/6J ob/?), body weights of genetically obese (ob/ob) mice are significantly higher at 1-6 months of age; the greatest percentage weight gain of the ob/ob group occurs during the first 3 months of life. Levels of pituitary immunoreactive beta-endorphin and immunoreactive alpha-melanocyte-stimulating hormone are also significantly elevated in ob/ob animals compared to controls. However, these pharmacological differences only emerge at 4-6 months of age--3 months after the appearance of obesity. High levels of immunoreactive endorphin in the pituitary are, therefore, more likely to be a consequence than a cause of obesity. Furthermore, numerous other neurologic abnormalities, which may or may not play a role in the obesity syndrome, are evident in ob/ob mice. Compared to controls, ob/ob total brain, hypothalamus, and pituitary weights are 11%, 16%, and 23% less, respectively. Levels of immunoreactive Leu5-enkephalin in pars nervous are also 200% higher in ob/ob mice; this increase is apparent at 1-6 months of age and is highly correlated with changes in body weight.
Plasmin-mediated proteolysis of casein in bovine milk.
Authors: Eigel W N , Hofmann C J , Chibber B A , Tomich J M , Keenan T W , Mertz E T
Published: May 2006

Plasminogen was found to be present in bovine milk by crossreactivity between rabbit antiserum to plasminogen and casein prepared from milk by acid precipitation. This result was further supported by recovery of intact 125I-labeled plasminogen from rabbit milk after its intravenous injection. Freshly isolated whole bovine casein was observed to undergo slow autoproteolysis at 37 degrees C. Polyacrylamide gel electrophoresis revealed gradual disappearance of major caseins accompanied by appearance and increase in intensity of numerous electrophoretic bands. This autoproteolysis was inhibited by low concentrations of epsilon-aminocaproic acid (0.1 mM) and diisopropyl fluorophosphate (1 mM); catalytic amounts of urokinase accelerated the process. Autoproteolysis of isolated bovine beta-casein was shown by both urea and sodium dodecyl sulfate gel electrophoresis to result in formation of gamma 1- and gamma 2-caseins. Similar electrophoretic bands were formed when beta-casein was degraded by plasmin prepared from bovine blood serum. These results support the hypothesis that bovine plasmin occurs in milk and is identical to alkaline milk protease.
Relation between leaf senescence and stomatal closure: Senescence in light
Authors: Thimann Kenneth V. , Satler Sergio O.
Published: May 2006

Senescence of isolated oat seedling leaves, floating on water or solutions in white light, has been followed by the disappearance of chlorophyll and the liberation of free amino nitrogen. In parallel, measurements of stomatal aperture were made with a diffusion resistance porometer, and borne out also by changes in fresh weight. The stomata open as expected in the light but slowly begin to close after the first day; correspondingly, in the dark they close at once but gradually begin to open on successive days. Abscisic acid causes closure and this is accompanied by senescence. Phenylmercuric nitrate also causes closure and again the reaction is closely paralleled by senescence. Kinetin maintains stomatal opening even more than does light alone, and this is accompanied by complete prevention of chlorophyll loss for at least 5 days. Covering the leaf surface with a film of Vaseline, especially when detergents are added, accelerates senescence. Merely floating the leaf segments on hypertonic solutions of sucrose or mannitol suffices to bring the rate of senescence in light up to the rate in darkness. It is concluded that the effect of light in delaying senescence is primarily due to its effect on the stomatal aperture, and, more generally, that stomatal aperture is the principal controlling agent in leaf senescence.
Mutation generating a fragment of the major heat shock-inducible polypeptide in
Drosophila melanogaster
Authors: Caggese C. , Caizzi R. , Morea M. , Scalenghe F. , Ritossa F.
Published: May 2006

Drosophila melanogaster
tissues carrying a third chromosome with the deletion
Df
(
3R
)
Kar
D2
make a 40,000-dalton (Dal) heat shock protein not made by wild type. The unusual polypeptide was inducible in every tissue examined. Tryptic peptide fingerprints showed it to include part of the 70,000-Dal major heat shock protein. Mapping experiments placed the mutation responsible for the 40,000-Dal protein at or close to the
kar
D2
deletion. One break point of the deletion is in subdivision 87A, close to or at a heat shock locus that codes for the 70,000-Dal protein. The results are consistent with the possibility that this break point is within a gene for the 70,000-Dal protein, leaving only the initial portion of its coding sequence. This would specify the direction of transcription of the mutant gene as proximal to distal on the normal chromosome. The 87A heat shock locus should contain at least two genes for the 70,000-Dal protein, because embryos homozygous for the
kar
D2
deletion and lacking the heat shock locus at 87C, which also codes for the 70,000-Dal protein, nevertheless produced both the 40,000-Dal and the 70,000-Dal proteins upon temperature elevation. Using the presence of the 40,000-Dal protein to monitor chromosome segregation, we found that embryos homozygous for deletions of the heat shock puff site at 93D exhibited a normal electrophoretic pattern of heat shock proteins.
Staphylococcal nuclease: Proposed mechanism of action based on structure of enzyme—thymidine 3′,5′-bisphosphate—calcium ion complex at 1.5-Å resolution
Authors: Cotton F. Albert , Hazen Edward E. , Legg Margaret J.
Published: May 2006

The structure of the staphylococcal nuclease (EC 3.1.4.7)—thymidine 3′,5′-bisphosphate—Ca
2+
(enzyme—inhibitor) complex has been extended to 1.5-Å resolution by using much additional data and a phase refinement scheme based on an electron-density map modification procedure. By correlating this structure with the known properties of the enzyme, a mechanism of action is proposed that involves nucleophilic attack on phosphorus by a water molecule, which is bound to Glu-43, in line with the 5′-CH
2
O(H) leaving group. The carboxylate of Glu-43 promotes this attack by acting as a general base for the abstraction of a proton from the attacking water molecule. Nucleophilic attack is further facilitated by polarization of the phosphodiester by an ionic interaction between a Ca
2+
ion and a phosphate oxygen atom and by four hydrogen bonds to phosphate oxygen atoms from guanidinium ions of Arg-35 and Arg-87. These interactions may also catalyze the reaction by lowering the energy of a trigonal bipyramidal transition state. The hydrolysis of nucleic acid substrate proceeds by cleavage of the 5′—P—O bond to yield a free 5′-hydroxyl group and a terminal, 3′-phosphate monoester group. In the inhibitor complex the only general acid group found in a position to donate a proton to the leaving 5′-oxygen is the guanidinium ion of Arg-87. Alternative proton donors, presently lacking direct structural support, could be the phenolic hydroxyl group of Tyr-113 or a water molecule. The precision and rigidity of the location of the reactants at the active site and the probable dual binding and catalytic roles of the guanidinium ions of Arg-35 and Arg-87 are especially noteworthy.
Mechanism of penicillin action: penicillin and substrate bind covalently to the same active site serine in two bacterial D-alanine carboxypeptidases.
Authors: Yocum R R , Waxman D J , Rasmussen J R , Strominger J L
Published: May 2006

It has been hypothesized that penicillin acts as a structural analog of the acyl-D-alanyl-D-alanine terminus of nascent bacterial cell wall and that it consequently binds to and acylates the active site of the enzyme(s) that crosslinks the cell wall to form an inactive penicilloyl enzyme [Tipper, D.J. & Strominger, J.L. (1965) Proc. Natl. Acad. Sci. USA 64, 1133-1138]. This study directly proves that penicillin acylates the active site of two penicillin-sensitive enzymes, D-alanine carboxypeptidases from Bacillus stearothermophilus and Bacillus subtilis. Active site peptides were generated by chemical or enzymatic cleavage of these carboxypeptidases after covalently labeling with [14C]penicillin G or after trapping an acyl-enzyme intermediate derived from the depsipeptide substrate. [14C]diacetyl-L-lysyl-D-alanyl-D-lactate. The amino acid sequences of the penicillin- and substrate-labeled peptides were identical. Both penicillin and substrate were covalently bound via an ester linkage to the same active site residue, a serine at position 36 of the B. stearothermophilus carboxypeptidase and the corresponding serine in the B. subtilis carboxypeptidase. The two D-alanine carboxypeptidases showed significant homology around the active site. Moreover, homology between these two enzymes and four beta-lactamases of known sequence suggests that these two groups of enzymes are evolutionally related.
Relation between senescence and stomatal opening: Senescence in darkness
Authors: Thimann Kenneth V. , Satler Sergio
Published: May 2006

The senescence (proteolysis and loss of chlorophyll) of isolated leaves of oat seedlings in the dark is inhibited or delayed by compounds of six different types: phenazine methosulfate, fusicoccin, α,α′-dipyridyl, cycloheximide, spermidine, and two cytokinins. In every case but the last, these compounds in optimum concentration caused the stomata to open and remain partly or completely open throughout the 72- or 96-hr experimental period. The cytokinins caused only a partial opening, which is ascribed to their exerting two different effects. Taken together with the previous report that five different treatments that accelerated or promoted senescence in the light caused stomatal closure or occlusion, these data establish a general parallel between stomatal aperture and senescence, with strong indication that the stomatal aperture is the causal factor. A possible explanation of the relationship is proposed.
Cell-free translation and thyroxine induction of carbamyl phosphate synthetase I messenger RNA in tadpole liver.
Authors: Mori M , Morris S M , Cohen P P
Published: May 2006

Total RNA of tadpole and frog (Rana catesbeiana) liver was isolated by either 7 or 8 M guanidine . HCl extraction and translated in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The identity of carbamyl phosphate synthetase I[carbamoyl-phosphate synthase (ammonia); ATP:carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5] synthesized in vitro with the purified enzyme was established as follows: (i) immunoprecipitation by a specific antibody; (i) comigration with purified carrier enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis; (iii) copurification with carrier enzyme by affinity chromatography on Cibacron Blue F3GA-coupled agarose; and (iv) formation of identical proteolytic cleavage products. Inclusion of protease inhibitors in the system resulted in no apparent change in the polypeptide molecular weight. These results indicate that carbamyl phosphate synthetase I is synthesized as a polypeptide that is indistinguishable from the mature enzyme by the analytical methods used and that it is not grossly modified during its transport into mitochondria. The level of translatable mRNA for carbamyl phosphate synthetase-I in tadpole liver was increased about 2-fold 1 day after thyroxine treatment and did not change significantly through 4 subsequent days of treatment. Thus the thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver is at least partly due to an increase of translatable mRNA for this enzyme.
Membrane damage caused by irradiation of fluorescent concanavalin A.
Authors: Sheetz M P , Koppel D E
Published: May 2006

Visible light irradiation of fluoresceinated concanavalin A (f-Con A) bound to the outside of resealed erythrocyte membranes caused crosslinking of as much as 50% of the membrane proteins. Crosslinking was absent in controls in which equivalent amounts of f-Con A were added to the membranes but prevented from binding by the presence of 10 mM alpha-methylmannoside. The photodamage was not accompanied by a change in the membrane permeability barrier or membrane shape. Although fluorescein bleaching accompanies the formation of protein aggregates, the amount of aggregated protein is not simply a function of the number of fluoresceins bleached. The percentage of aggregated protein decreases when the same dose of light is given in a shorter time. Although certain antioxidants and free-radical scavengers had no detected effect on the crosslinking, reducing agents such as cysteamine and reduced glutathione either blocked or reversed the protein crosslinking. The mechanism of photoinduced oxidation and the implications of these results for fluorescence studies of cell membranes are discussed.
Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.
Authors: Kosower N S , Kosower E M , Newton G L , Ranney H M
Published: May 2006

The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivatives of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently label hemoglobin (reactive thiol groups), membrane proteins, and glutathione of normal human red cells under physiological conditions. Monobromobimane and dibromobimane are effective on intact cells while red cell membranes may be impermeable to the positively charged monobromotrimethylammoniobimane, the latter being effective only on lysed cells. These bimane labels provide a class of labeling agents that may have wide applicability in biological materials.
Chromosomal location of the genes for human immunoglobulin heavy chains.
Authors: Croce C M , Shander M , Martinis J , Cicurel L , D'Ancona G G , Dolby T W , Koprowski H
Published: May 2006

We have studied somatic cell hybrids between P3x63Ag8 mouse myeloma cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and either human peripheral lymphocytes or human lymphoblastoid or myeloma cells for the production of human immunoglobulin chains and for the expression of enzyme markers assigned to each of the different human chromosomes. Human chromosome 14 was the only human chromosome present in all independent hybrids producing mu, gamma, and alpha human heavy chains. In two of the independent hybrids that produced human heavy chains, human chromosome 14 was the only human chromosome present in the hybrid cells. Loss of human chromosome 14 from these hybrids resulted in the concomitant loss of their ability to produce human immunoglobulin heavy chains. In view of these results, we conclude that the genes for human immunoglobulin heavy chains are located on human chromosome 14 in immunoglobulin-producing human cells.
Conditioned responses in courtship behavior of normal and mutant
Drosophila
Authors: Siegel Richard W. , Hall Jeffrey C.
Published: May 2006

Male courtship behaviour in
Drosophila melanogaster
is modified by prior sexual experience. Whereas naive males nearly always court virgin females persistently, males previously paired with unreceptive fertilized females subsequently court virgin females in an abbreviated manner, if at all. The probability of diminished male courtship is directly related to the duration of the prior “conditioning” period with a fertilized female. Naive males court fertilized females less vigorously than they court virgins; this depression of male behavior occurs even if the male is blind or if the fertilized female cannot actively reject his courtship. These results suggest that fertilized females are a source of both courtship-provoking and courtship-inhibiting olfactory cues and that the central association of these cues in males is sufficient to bring about the retention of modified courtship behavior. Mutant “amnesiac” males, selected as memory-deficient in a learning test unrelated to courtship [Quinn, W. G., Sziber, P. P. & Booker, R. (1979)
Nature (London)
277, 212-214], are trainable by exposure to fertilized females, but the experience-dependent behavior—diminished courtship performance—wanes abnormally rapidly—i.e., less than 1 hour, compared to 2-3 hr for wild-type flies.
Occurrence of crossed strand-exchange forms in yeast DNA during meiosis.
Authors: Bell L , Byers B
Published: May 2006

The crossed strand-exchange form (Holliday structure, half chiasma) has been predicted as an intermediate in the genetic recombination of eukaryotes. We report here the detection of this form in the yeast plasmid, 2-micron DNA, isolated during meiosis. Physical mapping has previously suggested that two forms of 2-micron DNA arise because of recombination between inverted repeat regions. After appropriate digestion with restriction endonuclease, a crossed strand-exchange form intermediate in this recombination would yield an X-shaped form resistant to loss by branch migration because of nonhomology in sequences flanking the region of homology. We first generated this X-shaped form artificially by reannealing melted restriction fragments of 2-micron DNA. This enabled us to develop a procedure for the physical separation of the X-shaped form by agarose gel electrophoresis. We then used this electrophoretic procedure to isolate a naturally occurring form of identical structure from the 2-micron DNA of meiotic cells. Electron microscopy demonstrated that the exchange junction had the expected configuration of strands and indicated that the junction occurred within the region of homology.
Biological effects of allogeneic effect factor on T lymphocytes: in vitro induction of cytotoxic T lymphocytes manifesting preferential lytic activity against H-2-identical tumor cells.
Authors: Altman A , Bechtold T E , Cardenas J M , Katz D H
Published: May 2006

The studies reported herein were designed to determine the effects of allogeneic effect factor (AEF), a soluble mediator generated in the course of allogeneic cell interactions, on the differentiation of cytotoxic T lymphocytes in vitro. Normal, unprimed spleen cells from various strains of mice cultured with AEF for 5 days, in the absence of any stimulator cells, developed into cytotoxic lymphocytes capable of lysing target cells in a short-term 51Cr release assay. T lymphocyte-depleted spleen cells did not become cytotoxic when cultured with AEF, and the cytotoxic cells themselves were found to be T lymphocytes. AEF-induced cytotoxic T lymphocytes preferentially lysed H-2-identical target cells. Thus, AEF, as opposed to similar "helper" factors derived from mixed lymphocyte cultures, appears to be unique in its ability to trigger normal, unprimed T lymphocytes to differentiate into cytotoxic T lymphocytes in the absence of specific antigenic stimulation.
Definition of a unique cell surface antigen of mouse leukemia RL male 1 by cell-mediated cytotoxicity.
Authors: Nakayama E , Shiku H , Takahashi T , Oettgen H F , Old L J
Published: May 2006

BALB/c x-ray-induced leukemia RL male 1 is strongly immunogenic for (BALB/c x C57BL/6)F1 mice. Transplants of RL male 1 regressed after initial growth, and after tumor regression mice could resist repeated inocula of 10(7) RL male 1 cells. Spleen cells from immunized mice after in vitro stimulation with RL male 1 were cytotoxic for RL male 1 cells in 3-hr 51Cr assays. Pretreatment of immune spleen cells with Thy-1, Lyt-2, or Lyt-3 antisera and complement eliminated cytotoxic activity, indicating that effector cells for RL male 1 lysis are T cells. Tests with other target cells showed little or no cytotoxicity. Analysis of the specificity of T-cell killing of RL male 1 by competitive inhibition assays with unlabeled cells indicated that only RL male 1 could inhibit killing; other BALB/c tumors (13 x-ray or murine leukemia virus-induced leukemias and three myelomas) failed to inhibit lysis of RL male 1. A range of alloantisera and heteroantisera were tested for their capacity to block lytic activity in the absence of added complement. H-2d antisera and Lyt-2 and -3 antisera blocked lysis, the latter at the level of the effector cell. Antisera to other cell surface alloantigens, murine leukemia virus-related antigens, and immunoglobulins did not block RL male 1 lysis. Thus, T cells from mice immunized against RL male 1 recognize an individually distinct or unique antigen that does not appear to be related to any of the serologically defined cell surface determinants of RL male 1. In its restriction to a single leukemia, the RL male 1 antigen resembles the individually distinct antigens of chemically induced tumors and other tumor types of rodents.
Characterization of parallel subtraction
Authors: Anderson W. N. , Morley T. D. , Trapp G. E.
Published: May 2006

Parallel subtraction is an operation defined on pairs of positive operators. In terms of electrical networks, one may pose the following problem: Given an electrical network, represented by a specified positive operator, determine the set of positive operators which when connected in parallel with the specified operator yield another prescribed operator. The set of solutions of this electrical network problem is shown to have a minimum. The minimum is termed “the parallel difference of the fixed operators,” and the operation is termed “parallel subtraction.” The parallel difference is used to obtain explicit error estimates for an iteration procedure which approximates the geometric mean of positive operators. This concept of the geometric mean reduces to the square root of the product of the operators if the operators commute. Finally, by using the geometric mean, an operator version of the Gaussian mean is presented.
Receptors for IgA on a subpopulation of human B lymphocytes.
Authors: Gupta S , Platsoucas C D , Good R A
Published: May 2006

Human B lymphocytes from peripheral blood and tonsils and leukemic B cells from patients with chronic lymphocytic leukemia (CLL) were examined for the presence of receptors for IgA. Receptors for IgA were present on 5-20% of B cells from peripheral blood of normal persons, 6-21% of B cells from tonsils removed because of chronic enlargement, and 4-10% of B cells from patients with CLL. Prior incubation of B lymphocytes in medium containing fetal calf serum was normally essential for expression of receptors for IgA. IgM and IgG receptors were also examined on B cells from tonsils and CLL. Receptors for IgM were present on a large proportion of B cells from tonsils and B cells from patients with CLL. Their detection also required incubation of B cells in medium containing fetal calf serum for 2-3 days. In one patient with CLL, IgM receptors were present on only a small proportion of B cells. No significant change in IgG receptors on B cells from tonsils or CLL was observed when freshly isolated B cells and cultured receptors on B cells progressively decreased as a consequence of shedding of free Fc receptors into the supernates.
Separation of functional subsets of human T cells by a monoclonal antibody.
Authors: Reinherz E L , Kung P C , Goldstein G , Schlossman S F
Published: May 2006

A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55-60% of the peripheral blood T cell population (OKT4+) and unreactive with normal B cells, null cells, and macrophages. The OKT4- T cell population contained the previously described TH2+ subset that has been shown to contain cytotoxic/suppressor cells. With cell-sorter separation of OKT4+ and OKT4- cells, it was shown that these T cell subsets were functionally discrete. Both gave proliferative responses with concanavalin A, alloantigens, and phytohemagglutinin although OKT4+ cells were much more responsive to the latter. OKT4+ cells alone responded to soluble antigens whereas OKT4- cells alone were cytotoxic after alloantigenic sensitization of unfractionated T cells. However, both OKT4+ and OKT4- cells were required during sensitization for optimal development of cytotoxicity. These data suggest that the OKT4+ subset represents a helper population and that the OKT4- subset contains the cytotoxic effector population. OKT4 could be a valuable reagent for determining alterations of these functional subsets in human diseases.