The Earth's Angular Momentum Budget on Subseasonal Time Scales.

Irregular length of day (LOD) fluctuations on time scales of less than a few years are largely produced by atmospheric torques on the underlying planet. Significant coherence is found between the respective time series of LOD and atmospheric angular momentum (AAM) determinations at periods down to 8 days, with lack of coherence at shorter periods caused by the declining signal-to-measurement noise ratios of both data types. Refinements to the currently accepted model of tidal Earth rotation variations are required, incorporating in particular the nonequilibrium effect of the oceans. The remaining discrepancies between LOD and AAM in the 100- to 10-day period range may be due to either a common error in the AAM data sets from different meteorological centers, or another component of the angular momentum budget.

Stimulation of Rauscher leukemia virus DNA polymerase DNA-directed DNA synthesis by cationic trypanocides and polyamines.

Activated DNA-directed DNA synthesis catalyzed by Rauscher leukemia virus (RLV) and other type C mammalian retroviral DNA polymerases is uniquely stimulated by biologically active polyamines. Cationic trypanocides may act as antagonists of polyamine function. As described here, several cationic trypanocides stimulate RLV polymerase-catalyzed DNA-directed DNA synthesis at concentrations significantly inhibiting eukaryotic DNA polymerases. Such stimulation is negated by polyamines. Kinetic analysis of the stimulation of RLV DNA polymerase by three structurally dissimilar cationic trypanocides (Antrycide, Burroughs-Wellcome Compound 64A, and Bayer Compound 1694) suggests that such stimulation is, in part, due to a drug:DNA structural interaction resembling the polyamine:DNA structural complex recognized by the RLV DNA polymerase.

Severe hypovitaminosis C occurring as the result of adoptive immunotherapy with high-dose interleukin 2 and lymphokine-activated killer cells.

Adoptive immunotherapy of human cancer was investigated in our institution as part of a National Cancer Institute extramural group study. This treatment, for patients with metastatic malignant melanoma, hypernephroma, and colon carcinoma, consisted of three phases: (a) 5 days of i.v. high-dose (10(5) units/kg every 8 h) interleukin 2, (b) 6 1/2 days of rest plus leukapheresis; and (c) 4 days of high-dose interleukin 2 plus three infusions of autologous lymphokine-activated killer cells. Toxicities included fever, chills, tachycardia, hypotension, vomiting, diarrhea, and fluid retention. Ascorbic acid is known to be important to cell-mediated immunity, and it has been reported to be depleted during physiologically stressful events. Therefore, we determined plasma ascorbic acid levels in patients (n = 11) before adoptive immunotherapy and before and after Phases 1, 2, and 3 of treatment. Patients entering the trial were not malnourished. Mean plasma ascorbic acid levels were normal (0.64 +/- 0.25 mg/dl) before therapy. Mean levels dropped by 80% after the first phase of treatment with high-dose interleukin 2 alone (0.13 +/- 0.08 mg/dl). Mean plasma ascorbic acid levels remained severely depleted (0.08 to 0.13 mg/dl) throughout the remainder of the treatment, becoming undetectable (less than 0.05 mg/dl) in eight of 11 patients during this time. Values obtained from 24-h urine collections on two of two patients indicated that ascorbate was not excreted in the urine. Plasma ascorbic acid normalized in three of three patients tested 1 mo after the completion of treatment. Unlike the results for ascorbic acid, blood pantothenate and plasma vitamin E remained within normal limits in all 11 patients throughout the phases of therapy. Responders (n = 3) differed from nonresponders (n = 8) in that plasma ascorbate levels in the former recovered to at least 0.1 mg/dl (frank clinical scurvy) during Phases 2 and 3, whereas levels in the latter fell below this level.

Template-specific requirements for DNA synthesis by the Mason-Pfizer monkey virus DNA polymerase: unique aspects.

The biochemical properties of DNA polymerase purified from Mason-Pfizer monkey virus were studied, with respect to synthetic and natural template-primer utilization. Thes studies revealed the following new information about the Mason-Pfizer monkey virus enzyme: (a) Mason-Pfizer monkey virus polymerase was found to prefer template: primer molar nucleotide ratios of 2.5-5: 1 for optimal rates of synthesis with poly(C) .(dG)12-18 as template-primer. (b) Poly(A)-directed synthesis was stimulated by the addition of low concentrations of inorganic phosphate to the reaction mixture. (c) Poly(2' -O-methyl-cytidylate), poly(rCm), was the only template studied for which Mn2+ proved the preferred divalent cation. Combinations of divalent cations stimulated rather than inhibited poly(rCm)-directed poly(dG) synthesis by the Mason-Pfizer monkey virus enzyme. (d) Heteropolymeric regions of rabbit globin mRNA and avian myeloblastosis virus 70 S RNA could be copied by the Mason-Pfizer monkey virus polymerase with oligo(dT), oligo(U) or in the case of avian myeloblastosis virus RNA, endogenous primers. In all such studies, Mg2+ was the preferred divalent cation and a distinct preference for the DNA primer in the reverse transcription of natural RNAs was observed. These new findings necessitated comparative studies with the DNA polymerases from Rauscher murine leukemia virus and murine mammary tumor virus, as representative type C and type B retroviruses. Although the Mason-Pfizer monkey virus enzyme was found to share some properties in common with both type C and type B mammalian viral enzymes, certain of the above properties rendered it unique among the polymerases examined.

The peroxisome proliferator-activated receptor interacts with the retinoid X receptor in vivo.

The peroxisome proliferator-activated receptor (PPAR) binds cooperatively to cognate peroxisome proliferator-responsive elements (PPRE) in vitro through heterodimerization with retinoid X receptors (RXR). We used the yeast two-hybrid system to determine whether these two nuclear receptors physically interact in vivo. Mouse (m) PPAR and human (h) RXR alpha were synthesized as fusion proteins to either the DNA-binding domain (GBD) or the transactivation domain (GAD) of the yeast GAL4 transcription-activator protein, and were tested for their ability to activate expression of a GAL1::lacZ reporter gene. Strong activation was observed only in yeast transformed with combinations of GBD::mPPAR and GAD::hRXR alpha or with GAD::mPPAR and GBD::hRXR alpha. Homodimeric interaction by mPPAR was not detected. These results provide evidence for the interaction of PPAR and RXR alpha in vivo in the absence of a PPRE target site or exogenously added ligands.

A synaptojanin-homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation.

The Ser-Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5' phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a DeltasigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.

Salmonella pathogenicity islands: big virulence in small packages.

Reflecting a complex set of interactions with its host, Salmonella spp. require multiple genes for full virulence. Many of these genes are found in 'pathogenicity islands' in the chromosome. Salmonella typhimurium possesses at least five such pathogenicity islands (SPI), which confer specific virulence traits and may have been acquired by horizontal transfer from other organisms. We highlight recent progress in characterizing these SPIs and the function of some of their genes. The role of virulence genes found on a highly conserved plasmid is also discussed. Collectively, these packages of virulence cassettes are essential for Salmonella pathogenesis.

Hypovitaminosis C in patients treated with high-dose interleukin 2 and lymphokine-activated killer cells.

Patients (n = 15) with metastatic malignant melanoma, hypernephroma, and colon carcinoma received a three-phase adoptive immunotherapy protocol: phase 1, 10(5) units (high-dose) interleukin-2 (IL-2) iv every 8 h or 1 mg/m2 continuous intravenous infusion; phase 2, 6.5 d rest + leukapheresis; phase 3, 4 d of high-dose IL-2 plus three infusions of autologous lymphokine-activated killer cells. Toxicities of treatment included fever, chills, tachycardia, hypotension, vomiting, diarrhea, and fluid retention. Patients entering the trial were not malnourished, and mean plasma ascorbic acid concentrations before therapy were normal (36.3 +/- 14.2 mumol/L). Mean concentrations dropped by 80% after the first phase of treatment with high-dose IL-2 alone (to 7.4 +/- 4.5 mumol/L). Mean plasma ascorbic acid concentrations remained severely depleted (between 4.5 and 7.4 mumol/L) throughout the remainder of the 15-d treatment. Ascorbic acid concentrations became undetectable (less than 2.8 mumol/L) in 12/15 patients during this time. Blood pantothenate and plasma vitamin E concentrations remained within normal limits in all patients tested throughout the phases of therapy.

Photodynamic therapy.

Photodynamic therapy (PDT) has been developed over the past decade into a useful treatment for several types of solid cancers in man. This unique therapy requires a photosensitiser accumulated in tumours and local activation by visible light generally delivered from lasers and delivered to the patient through various types of fibers and endoscopes. PDT appears to be most effective in treating certain superficial, difficult to treat cancers such as carcinoma in situ of the urinary bladder (here complete control is the intent), but also is effectively used in bulkier tumours obstructing bronchi or the oesophagus where palliation can be achieved. The primary mechanism of action is the in situ generation of an active form of molecular oxygen (singlet oxygen) which causes the rapid, local onset of vascular stasis and eventual vascular haemorrhage and tumour wall destruction. This process appears to be mediated through various cytokines such as prostaglandin, lymphokines and thromboxanes. The ultimate clinical value of PDT will be seen over the next few years following health agency approval worldwide.

Effects of exogenous polyamine and trypanocides on the DNA polymerase activities from Trypanosoma brucei brucei, mouse thymus and murine leukemia virus.

The effects of exogenously added spermine on activated (gapped) DNA-directed and poly(dC) . (dG)12-18-directed DNA synthesis were tested on the chromatographically separated DNA polymerase activities of Trypanosoma brucei brucei. Activated DNA-directed DNA synthesis by the Peak I (eluting from DNA-agarose at 0.15 M KCl) and Peak II (eluting at 0.3 M KCl) polymerase was consistently inhibited or stimulated, respectively, by exogenous spermine. Kinetic analysis revealed that inhibition of the Peak I enzyme with respect to template DNA occurred by a mixed mechanism, while a major factor in the stimulation of the Peak II enzyme by spermine appeared to be the polyamine-mediated reversal of "substrate inhibition' by DNA at concentrations above 10 micrograms/ml. The apparent Km values of Peak I and Peak II DNA polymerase for activated DNA were determined to be 5 and 0.5 microgram/ml, respectively. In contrast to the results observed with activated DNA, activation of Peak II-enzyme-catalyzed poly(dC)-directed DNA synthesis was similar at all template-primer concentrations. Peak I enzyme-catalyzed poly(dG) synthesis was either inhibited or slightly stimulated by spermine, depending upon the presence or absence of heteropolymeric DNA, respectively. Dose-dependent inhibition of DNA-directed DNA synthesis catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase alpha, and Rauscher murine leukemia virus reverse transcriptase by trypanocides was examined to determine a possible mechanism of selective toxicity by such agents. The drugs Antrycide (quinapyramine), pentamidine, imidocarb, Berenil (diminazene aceturate), WR-199-385-[2,5-bis(4-guanylphenyl)furan . 2HCl] and isometamidium inhibited DNA polymerases of the eucaryotic cells at approximately the same degree, and at similar concentrations. The presence of spermine in reaction mixtures did not spare any drug inhibition. Stimulation of reverse transcriptase activity was observed in the presence of Antrycide and imidocarb, however, this could be negated by stimulatory amounts of spermine present in the reaction mixture. The results, obtained using an activated DNA-directed assay system, suggest that trypanosomal DNA polymerases are not the selective target of trypanocidal drugs currently available.

Identification of COUP-TFII as a peroxisome proliferator response element binding factor using genetic selection in yeast: COUP-TFII activates transcription in yeast but antagonizes PPAR signaling in mammalian cells.

Peroxisome proliferator-response elements (PPRE) are cis-acting regulatory elements that confer responsiveness to peroxisome proliferators and various fatty acids by serving as target sites for ligand-activated peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. Other cellular factors, including additional nuclear hormone receptors, also interact with PPREs and modulate PPAR function. We have developed a positive selection strategy in yeast to identify mammalian factors that functionally interact with PPREs. Saccharomyces cerevisiae containing an integrated copy of the HIS3 gene under transcriptional control of a minimal CYC1 promoter and two copies of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase PPRE was constructed and transformed with a rat liver cDNA yeast expression library. Plasmids were isolated from his + transformants. One plasmid contained a cDNA encoding the complete rat chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily. COUP-TFII potently activated PPRE-linked reporter gene expression in yeast, and COUP-TFII synthesized in yeast or in vitro formed specific protein/DNA complexes with this PPRE. Significantly, COUP-TFII did not activate transcription of PPRE-linked reporter genes in mammalian cells but rather strongly inhibited induction mediated by PPAR/RXR. Our findings demonstrate the utility of using genetic screening in yeast to identify sequence-specific DNA binding transcription factors.

Calreticulin modulates the in vitro DNA binding but not the in vivo transcriptional activation by peroxisome proliferator-activated receptor/retinoid X receptor heterodimers.

Calreticulin is a ubiquitous calcium binding/storage protein found primarily in the endoplasmic reticulum. Calreticulin has been shown to inhibit DNA binding and transcriptional activation by glucocorticoid and androgen hormone receptors by binding to the conserved sequence KXFF(K/R)R, present in the DNA-binding domains of all known members of the steroid/nuclear hormone receptor superfamily. To determine whether calreticulin might be a general regulator of hormone-responsive pathways, we examined its effect on DNA binding in vitro and transcriptional activation in vivo by heterodimers of the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR alpha). We show here that purified calreticulin inhibits the binding of PPAR/RXR alpha heterodimers and of other nuclear hormone receptors, to peroxisome proliferator-responsive DNA elements in vitro. However, overexpression of calreticulin in transiently transfected cultured cells had little or no effect on transactivation mediated by PPAR/RXR alpha. Therefore, while calreticulin inhibits the binding of both nuclear and steroid hormone receptors to cognate response elements in vitro, our findings suggest that calreticulin does not necessarily play an important role in the regulation of all classes of hormone receptors in vivo.

Effect of daily administration of 0.5 mg. of chlormadinone acetate on plasma levels of follicle-stimulating hormone, luteinizing hormone, and progesterone during the menstrual cycle.

PIP: Investigators at Cornell University Medical College and New York Medical College in New York City studied the effects of chlormadinone acetate administration on hormone levels in an effort to better understand the contraceptive mode of action of this drug. 9 healthy women had 2 consecutive cycles studied. While the first cycle was a control cycle, the second one involved chlormadinone acetate administration, .5 mg/day from Day 1 to Day 28 or until the occurrence of spontaneous menstruation if this delayed until after Day 28. Basal body temperature was recorded each morning. Chlormadinone tended to suppress the mean luteinizing hormone and follicle stimulating hormone peaks and the plasma progesterone levels. 3 patients are believed to have ovulated during the experimental cycle, but probably in 2 of them the luteal phase was less pronounced than a normal luteal phase. However, 1 of the remaining 6 patients had failed to ovulate in the control cycle. Though limitations exist in the study of the parameters investigated here, such study is necessary since direct evidence of ovulation (e.g., pregnancy, observation of corpus luteum) is usually unobtainable.^ieng

Transactivation by PPAR/RXR heterodimers in yeast is potentiated by exogenous fatty acid via a pathway requiring intact peroxisomes.

Peroxisome proliferator-activated receptors (PPARs) are orphan members of the nuclear hormone receptor superfamily. PPARs bind to cognate response elements through heterodimerization with retinoid X receptors (RXRs). Together PPAR/RXR regulate the transcription of genes for which products are involved in lipid homeostasis, cell growth, and differentiation. PPARs are activated by fatty acids and by nongenotoxic rodent hepatocarcinogens called peroxisome proliferators through as of yet undefined signal transduction pathways. In an effort to elucidate the requirements for PPAR function and the pathways of its activation, we expressed mouse PPAR alpha and human RXR alpha in the yeast Saccharomyces cerevisiae. Mouse PPAR alpha and human RXR alpha had little activity individually in yeast; however, when cosynthesized, they were able to synergistically activate transcription via cognate response elements. Transactivation was independent of exogenously added activators of either receptor but was potentiated by the addition of petroselinic acid, a fatty acid shown to activate PPARs in mammalian cells. Similar experiments were carried out in a mutant yeast strain lacking peroxisomes entirely or in a mutant strain deficient for 3-ketoacyl-CoA thiolase, the final enzyme of the peroxisomal beta-oxidation cascade. The findings showed that constitutive transactivation by PPAR/RXR did not require the complete beta-oxidation pathway or intact peroxisomes but required intact peroxisomes for potentiation by exogenously added petroselinic acid. This study demonstrates that at least part of the mammalian peroxisome proliferator-signaling pathway can be faithfully reconstituted in yeast and that activation of PPAR by at least one particular fatty acid requires the integrity of peroxisomes.

Transactivation of the peroxisome proliferator-activated receptor is differentially modulated by hepatocyte nuclear factor-4.

Peroxisome proliferator-activated receptors (PPARs) stimulate the expression of several genes involved in lipid metabolism by binding to specific cis-acting peroxisome proliferator-responsive elements (PPREs) via cooper-ativity with retinoid X receptors. We demonstrate here that hepatocyte nuclear factor-4 (HNF-4), another member of the nuclear hormone receptor superfamily, bound with differing affinities to the PPREs from the genes encoding rat acyl-CoA oxidase and hydratase-dehydrogenase, the first two enzymes of the peroxisomal beta-oxidation pathway. In cotransfection assays, HNF-4 repressed rat PPAR-dependent activation of a reporter gene linked to the acyl-CoA oxidase PPRE, either in the absence or presence of the peroxisome proliferator, Wy-14,643. Rat PPAR-dependent activation of a reporter gene linked to the hydratase-dehydrogenase PPRE was less efficiently repressed by HNF-4 in the absence of Wy-14,643 than was activation from the acyl-CoA oxidase PPRE. However, in the presence of Wy-14,643, HNF-4 functioned cooperatively with PPAR to significantly enhanced induction from the hydratase-dehydrogenase PPRE. These results suggest that the genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway are subject to differential regulation by the interplay of multiple members of the steroid/nuclear hormone receptor superfamily, mitigated in part by the structures of the PPREs and by the presence of activators of PPARs.

Photodynamic endometrial ablation in the nonhuman primate.

OBJECTIVE: To test the effectiveness of 5-aminolevulinic acid (ALA)-induced photodynamic endometrial ablation in the rhesus monkey under varying conditions of light delivery (fractionated versus continuous) and steroid priming. METHODS: Photodynamic endometrial ablation was carried out in 17 rhesus monkeys that were either postmenopausal or in the early proliferative phase. Four hours after intralumenal injection of ALA (250 mg in 1 mL hyskon), a quartz fiber with a diffusing tip was inserted. A KTP tunable dye laser delivered 300 mW of light (635 nm) for 60 minutes in either continuous or fractionated fashion (20 minutes on, 5 minutes off, and 40 minutes on). In some experiments, thermistors were used to monitor temperature in the lumen and myometrium during light treatment. Hysterectomy was performed 3 or 4 days after treatment, and endometrial damage was assessed histologically. Two additional monkeys (one rhesus and one cynomolgus monkey) were exposed to the same protocol, except hyskon was substituted for ALA to control for potential ablative effects due to light treatment alone. RESULTS: Endometrial ablation was evident in all ALA-photosensitized specimens. The degree of ablation around the light fiber ranged from moderate to complete. The depth of ablation ranged from 1.14 +/- 0.54 to 2.15 +/- 1.62 mm (mean +/- standard deviation). Ablation was most complete in uteri of menopausal monkeys. Light treatment after ALA increased lumenal temperature from 36 C to 50 C, whereas temperature was not significantly increased by light treatment in the controls. CONCLUSION: This is the first report of endometrial destruction in the primate using a photodynamic approach. Whereas clinical application of photodynamic therapy (PDT) requires complete endometrial ablation to produce long-lasting amenorrhea, our results suggest that PDT may offer a simple office-based approach to endometrial ablation.

Tubal and uterine motility in vivo in the rhesus monkey.

Previous data indicate that there are definite qualitative and quantitative patterns of tubal activity during the various phases of the menstrual cycle. In the present investigation, the simultaneous activity of the uterus and Fallopian tube was recorded in vivo in mature rhesus monkeys throughout the menstrual cycle. The observations indicate that there are characteristic patterns of both tubal and uterine activity, depending on the various phases of the menstrual cycle. The simultaneous recordings of tubal and uterine activity show that the tubal and uterine contractions are not synchronous or even have a similar pattern. The presence of a transmetrial catheter for recording of uterine activity seems to modify the patterns of tubal activity, particularly during the ovulatory phase. It seems possible that the transmetrial catheter has a similar effect as an intrauterine contraceptive device in this species.