Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes.

Xenopus eggs contain two distinct cdc2 homologs of 34 and 32 kd. We show that the 32 kd cdc2 protein, like the 34 kd protein, is a kinase. However, unlike the 34 kd homolog, the 32 kd cdc2 kinase activity does not decrease dramatically at the end of mitosis. The 32 kd protein does not associate with mitotic cyclins B1 and B2 but does associate with cyclin A and a novel doublet of proteins of 54 kd that may regulate its activity. We also show that depletion of the 32 kd cdc2 homolog from a Xenopus extract blocks DNA replication, but does not inhibit entry into mitosis. By contrast, depletion of the 34 kd cdc2 homolog or absence of mitotic cyclins from an extract does not inhibit replication, but does block entry into mitosis. Our results indicate that in higher eukaryotes, DNA replication (G1-S) and mitosis (G2-M) may be controlled by distinctly different cdc2 proteins.     

Measurement of PGH synthase activity using a pan-specific monoclonal antibody

A pan-specific monoclonal antibody that recognizes a variety of prostaglandin moieties and does not recognize arachidonic acid or the hydroxy-eicosatetraenoic acids was used to assess the general ability of a tissue to produce prostaglandins. Although this assay does not give a quantitative measure of PGH synthase activity, it does provide a sensitive and convenient means of screening a large number of samples for prostaglandin production.     

Determination of the physicochemical constants and spectrophotometric characteristics of the metallochromic Zincon and its potential use in biological systems

In this work we report a detailed characterization of the metallochromic Zincon. Zincon forms complexes with Zn2+ and Cu2+, producing change in colour; the complexes with Fe2+, Mn2+ and Ca2+ cause the bleaching of the Zincon solutions. Mg2+ does not interact with Zincon nor does it change its spectral characteristics. The presence of Ca2+ and Mg2+ does not interfere with the spectral characteristics of the Zn-Zincon complex. The Kd, Ks and delta epsilon values for the complexes were determined. The delta epsilon values were very high, making this spectrophotometric method very sensitive. The complex Zn-Zincon is fully reversible; however, the complex Cu-Zincon is only partially reversible. The free Zincon, and the complexes Zn-Zincon and Cu-Zincon, does not partition into organic solvents, does not permeate liposome membrane, and neither does it interact with biological membranes. All these characteristics make the metallochromic Zincon useful in biological systems.     

Inhibitory Effect of Bacteriophage P22 Infection on Host Cell Deoxyribonuclease Activity

Infection of Salmonella typhimurium with phage P22 causes a decrease in the activity of host deoxyribonuclease which degrades single-stranded deoxyribonucleic acid (DNA). This decrease is reversed when the infecting phage is P22c(+); it is not reversed if the infecting phage kills the cell. The decrease does not occur in infections with P22ts25.1 (which only adsorbs and injects DNA) or in infections of a lysogen by a nonvirulent phage. It does occur, however, after infections with other phages which are blocked in phage DNA synthesis. Inhibiting protein synthesis with chloramphenicol does not in itself cause the decrease in uninfected cells, but it does prevent infected cells from showing this effect.     

Serum, bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts

The mitogens serum, vasopressin and bradykinin stimulate a significant rise in the inositol phosphate content of cultured human fibroblasts within 10 seconds, while serum- and bradykinin-stimulated arachidonic acid release does not occur until after 30 seconds. The release of inositol phosphates is not secondary to a rise in Ca activity since the Ca ionophore ionomycin does not stimulate release of inositol phosphates. Moreover, we show that phospholipase C in human fibroblasts is activated by these mitogens at resting Ca levels since TMB-8, which blocks the mitogen-induced rise in Ca activity, does not affect the serum-stimulated accumulation of inositol phosphates.     

Leptin does not directly affect CNS serotonin neurons to influence appetite

Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sert(cre)) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sert(cre) promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sert(cre)-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function.     

Fertility control in female white-tailed deer

Silastic rods containing either melengestrol acetate (MGA) or levonorgestrel (LN) were placed in anestrous white-tailed deer (Odocoileus virginianus borealis) does to evaluate the contraceptive efficacy of the implants over a 2 yr period. Implants of MGA were placed in five does during mid-pregnancy to evaluate the effect of this treatment on pregnancy, parturition and lactation. Pregnancies were not observed in the five animals implanted with MGA during anestrus. Three of five does implanted with LN became pregnant in the first season. Pregnancy was not interrupted in the five pregnant does implanted with MGA and it was necessary to remove the implants and treat the does with an estrogen to achieve parturition. One of five fawns was delivered alive and was raised by the doe. MGA was effective for 2 yr as a contraceptive in white-tailed deer, LN was ineffective as used, and MGA placed in pregnant does delayed or prevented normal parturition and thus should not be used in pregnant deer.     

A COMPARATIVE STUDY OF PINEAL ACETYL-CoA HYDROLASE AND N-ACETYLTRANSFERASE ACTIVITIES

pineal acetyl-CoA hydrolase is measurable at 4 days before birth. It increases rapidly to a maximum of 0.37 nmol/min/0.1 mg protein during the first week after birth, thereafter gradually decreasing and stabilizing at adult levels (0.27 nmol/min/0.1 mg protein) 3-4 weeks after birth. Unlike A/-acetyltransferase, the activity of acetyl-CoA hydrolase does not increase following treatment with isoproterenol, does not exhibit a circadian rhythm and is not inactivated on exposure of the animals to light at night. In addition, denervation of the pineal gland does not alter acetyl-CoA hydrolase activity.     

BSF1 induces membrane protein phosphorylation but not phosphoinositide metabolism, Ca2+ mobilization, protein kinase C translocation, or membrane depolarization in resting murine B lymphocytes

The findings presented in this study provide evidence that BSF1 receptors and mIg transmit signals via dissimilar transduction mechanisms that result in a common biologic response, hyper-Ia expression. Specifically, BSF1-containing supernatant does not induce PtdInsP2 hydrolysis as determined by measurement of PtdOH and InsP3. Additionally, BSF1 does not stimulate Ca2+ mobilization, PKC translocation from cytosol to membrane, or membrane depolarization. All of these metabolic events appear to play a central role in hyper-Ia expression mediated by mIg and are initiated after treatment of resting B cells with anti-Ig antibodies. In vitro phosphorylation studies with partially purified plasma membranes from resting B cells revealed that BSF1 interaction with membrane receptors stimulates a membrane-associated protein kinase that phosphorylates an endogenous protein of 44 KDa. Anti-Ig does not stimulate phosphorylation of the 44 KDa protein, suggesting that it does not activate the membrane-associated protein kinase. This observation provides the first evidence of a signal transduction mechanism associated with BSF1-receptor ligation. It indicates that although BSF1 does not modulate events associated with PKC activation, it may function via activation of a membrane-associated protein kinase. This provides a focal point for further studies directed at elucidating signal transduction resulting from BSF1-receptor interaction.     

Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.     

Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair

The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3' flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3' flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.     

Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil domains of ATRIP

ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA.     

Inhibition of aldosterone synthesis by atrial natriuretic factor

Atrial natriuretic factor (ANF) inhibits basal and stimulated aldosterone synthesis in adrenal glomerulosa cells. ANF probably acts through specific membrane receptors. Alterations in cyclic GMP and cyclic AMP levels do not account for ANF's inhibitory effect. ANF does not block angiotensin II (AngII) receptors nor does it interfere with phosphoinositide metabolism or calcium movements stimulated by adrenal agonists. ANF does not inhibit protein synthesis nor does it work by inhibiting NA+,K+-ATPase or depleting cell potassium. ANF decreases conversion of endogenous cholesterol to pregnenolone, the step stimulated by adrenocorticotropin and AngII. ANF does not affect the conversion of 20-alpha-hydroxycholesterol, which easily penetrates mitochondrial membranes to the site of the cholesterol side-chain cleavage enzyme. These results suggest that ANF inhibits the ability of endogenous cholesterol to reach or interact with the side-chain cleavage enzyme. ANF does not act like a calcium channel-blocking agent. However, ANF is less effective at high-calcium concentrations, which suggests that it may inhibit a step that calcium stimulates. Understanding ANF action will probably require identification of the specific biochemical changes (mediators) that it induces. Parallel efforts to understand how other agents stimulate steroidogenesis (particularly in the areas of protein synthesis, protein phosphorylation, and cholesterol movements) will further this understanding.     

Mechanism of 3-phenylpyruvate-induced insulin release from isolated pancreatic islets.

3-Phenylpyruvate evoked a monophasic insulin release from perifused mouse islets. L-Phenylalanine was not an insulin secretagogue and was oxidized by islets at a very low rate, suggesting that 3-phenylpyruvate does not trigger insulin release by enhancing production of reducing equivalents. Moreover, allosteric activation of glutamate dehydrogenase does not play a role in 3-phenylpyruvate-induced insulin secretion.     

Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein

Forskolin, a novel diterpene activator of adenylate cyclase in membranes and intact cells, activates the enzyme in membranes from mutant cyc-S49 murine lymphoma cells and the soluble enzyme from rat testes. Each of these enzymes consists only of the catalytic subunit and does not have a functional guanine nucleotide-binding protein. In both cases forskolin converts the manganese-dependent enzymes to a form which does not require manganese for activity. Forskolin can also stimulate a detergent-solubilized preparation of adenylate cyclase from rat cerebral cortex. Activation of adenylate cyclase by forskolin is therefore not dependent on a perturbation of membrane structure nor does it require a functional guanine nucleotide-binding subunit.     

Effect of chemical modification on the binding of gossypol by gossypin (11S protein) and congossypin (7S protein) of cottonseed

Binding of gossypol by gossypin and congossypin and their succinylated and sulfhydryl group-blocked derivatives has been measured. The binding by gossypin and congossypin is characterized by weak interaction. Succinylation of gossypin decreases the binding affinity whereas that of congossypin increases it. Blocking of sulfhydryl groups of both the proteins does not significantly affect gossypol binding, Succinylation dissociates gossypin and causes conformational changes whereas it does not dissociate congossypin but causes conformational changes. Sulfhydryl group blocking does not dissociate gossypin or congossypin, nor does it cause any conformational changes.     

The impairment argument for the immorality of abortion: A reply

In his recent article Perry Hendricks presents what he calls the impairment argument to show that abortion is immoral. To do so, he argues that to give a fetus fetal alcohol syndrome is immoral. Because killing the fetus impairs it more than giving it fetal alcohol syndrome, Hendricks concludes that killing the fetus must also be immoral. Here, I claim that killing a fetus does not impair it in the way that giving it fetal alcohol syndrome does. By examining the reason why giving a fetus this condition is wrong, I conclude that the same reasoning, on common pro‐choice accounts, does not apply to killing the fetus. Accordingly, Hendricks's argument does not succeed in showing abortion is immoral.     

Genomic imprinting: normal complementation of murine chromosome 16

Parental imprinting effects for chromosome 16 were investigated using disomic animals which were obtained by mating (Rb32Lub x Rb2H) F1 mice. Two allelic forms of the enzyme CuZn-superoxide dismutase, Sod-1a and Sod-1c, were used to identify maternally or paternally disomic animals. Both types of disomic animals were found with the expected frequencies and did not visibly differ from one another or from non-disomic animals. These results indicate that the genomic imprinting mechanism either does not act on chromosome 16, or, if it does, does not do so in a manner which affects normal development.     

Construction and evaluation of a coronary catheter for chronic implantation in dogs

Construction of a Silastic catheter and the procedure for chronic implantation in a coronary artery in dogs is described. In addition, studies designed to evaluate whether chronic coronary artery catheterization altered coronary vascular reactivity and myocardial function are presented. The results of these studies indicate that chronic implantation of the catheter in a coronary artery of conscious dogs does not significantly interfere with the normal reactivity of the coronary vascular bed, does not compromise regional or global left ventricular function, and does not induce collateral vessel development. This technique will be useful in studies involving the neural and metabolic regulation of the coronary circulation in animals subjected to exercise and/or exercise training.     

Characterization of the Chondrocyte Actin Cytoskeleton in Living Three-Dimensional Culture: Response to Anabolic and Catabolic Stimuli

The actin cytoskeleton is a dynamic network required for intracellular transport, signal transduction, movement, attachment to the extracellular matrix, cellular stiffness and cell shape. Cell shape and the actin cytoskeletal configuration are linked to chondrocyte phenotype with regard to gene expression and matrix synthesis. Historically, the chondrocyte actin cytoskeleton has been studied after formaldehyde fixation - precluding real-time measurements of actin dynamics, or in monolayer cultured cells. Here we characterize the actin cytoskeleton of living low-passage human chondrocytes grown in three-dimensional culture using a stably expressed actin-GFP construct. GFP-actin expression does not substantially alter the production of endogenous actin at the protein level. GFP-actin incorporates into all actin structures stained by fluorescent phalloidin, and does not affect the actin cytoskeleton as seen by fluorescence microscopy. GFP-actin expression does not significantly change the chondrocyte cytosolic stiffness. GFP-actin does not alter the gene expression response to cytokines and growth factors such as IL-1band TGF-b. Finally, GFP-actin does not alter production of extracellular matrix as measured by radiosulfate incorporation. Having established that GFP-actin does not measurably affect the chondrocyte phenotype, we tested the hypothesis that IL-1band TGF-bdifferentially alter the actin cytoskeleton using time-lapse microscopy. TGF-bincreases actin extensions and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1bcauses cellular contraction indicative of RhoA activation. The ability to visualize GFP-actin in living chondrocytes in 3D culture without disrupting the organization or function of the cytoskeleton is an advance in chondrocyte cell biology and provides a powerful tool for future studies in actin-dependent chondrocyte differentiation and mechanotransduction pathways.