Stimulus-Specific Hierarchy of Enhancer Elements Within the Rat Prodynorphin Promoter

Abstract: Induction of the prodynorphin gene occurs in a tissue-specific manner following different physiological stimuli. Using electrophoretic mobility shift assays, we studied the relative activity of the five major regulatory sites in the rat prodynorphin promoter. Prodynorphin cyclic AMP-responsive element 2 (DynCRE2), DynCRE3, and the noncanonical prodynorphin AP-1 (ncDynAP-1) regulatory sites control, in a coordinated manner, prodynorphin induction in the spinal cord after noxious stimulation, whereas prodynorphin up-regulation in supraoptic neurons is regulated predominantly by the ncDynAP-1. Conversely, prodynorphin transactivation in the ovaries upon gonadotrophin stimulation is controlled by DynCRE1 and DynCRE3. Our results support the idea that stimulus-specific changes in nuclear proteins establish a functional hierarchy among regulatory sites in the prodynorphin promoter and provide further insight in the molecular mechanisms that govern prodynorphin gene regulation.     

The ms2io6A37 Modification of tRNA in Salmonella typhimurium Regulates Growth on Citric Acid Cycle Intermediates

The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms²i⁶A) is present in position 37 (adjacent to and 3′ of the anticodon) of tRNAs that read codons beginning with U except tRNA _I,V^Ser in Escherichia coli. In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms²io⁶A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms²i⁶A in E. coli. Mutants (miaE) of S. typhimurium in which ms²i⁶A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms²io⁶A37 and is not due to a second cellular function of tRNA (ms²io⁶A37)hydroxylase, the miaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.     

Percutaneous Radio-Frequency Rhizotomy in the Treatment of Trigeminal Neuralgia

A percutaneous technique of selective partial trigeminal root coagulation was evaluated in the treatment of 38 patients suffering from trigeminal neuralgia, 1 patient with pain secondary to oral carcinoma and 1 patient with atypical facial pain. The pain of trigeminal neuralgia was relieved in 94.7 percent of patients. Pain was relieved in the patient with oral carcinoma, but not in the patient with atypical facial pain. There was no mortality and no permanent morbidity outside of the trigeminal nerve lesion. The procedure requires only a brief hospital stay without the time, expense and hazards of open cranial surgical procedures.     

Abortion after PGF2α in heifers with multiple pregnancies following superovulation with FSH and PGF2α

Multiple ovulations were induced with follicle stimulating hormone and estrus was synchronized with prostaglandin F_2α (PGF_2α) in 23 Holstein heifers. In 19 heifers which responded to the treatments, an average of 1.8 corpora lutea were formed after the induced estrus and 6 of 19 heifers conceived (total of ten fetuses at 39 days gestation) to artificial insemination at 60 and 84 hr after the PGF_2α injection. Injection of 33 mg PGF_2α Tham salt into the six pregnant heifers on day 40 of gestation caused abortion between 54 and 66 hr after treatment in all heifers.     

Human cytomegalovirus induces inhibition of macrophage differentiation by binding to human aminopeptidase N/CD13

Human CMV (HCMV) infection in immunocompromised patients is frequently associated with impaired immunological functions. We have recently found that HCMV inhibits cytokine-induced differentiation of monocytes into macrophages. In this study, we demonstrate that HCMV-induced inhibition of macrophage differentiation was dependent on binding of virus particles to the cell surface molecule CD13/aminopeptidase N, which involved Ca2+ -dependent intracellular signaling pathways. We found that treatment of cells with the CD13-specific mAbs My7 and WM15 inhibited macrophage differentiation, and that My7 and WM15 induced a rise in intracellular Ca2+ in similar ways as HCMV. In contrast, binding of the CD13-specific Ab clone SJ1D1 blocked the ability of HCMV to inhibit macrophage differentiation, and blocked the HCMV-induced intracellular Ca2+ response. In addition, the Ca2+ modulator thapsigargin partially blocked the ability of HCMV to inhibit cellular differentiation. Furthermore, we demonstrate that recombinant viral glycoprotein gB was able to inhibit macrophage differentiation in similar ways as the virus. Thus, these results suggest that binding of HCMV to monocytes induces an intracellular rise of Ca2+, of which one result is a block in the ability of the cells to differentiate into macrophages. These observations suggest an efficient viral strategy to interfere with cellular differentiation pathways, and may also in part explain the generalized immunosuppression that is often observed in HCMV-infected patients.     

Characterization of the primary photointermediates of Drosophila rhodopsin

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.     

Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.     

Complex formation by human cytomegalovirus glycoproteins M (gpUL100) and N (gpUL73)

The envelope glycoproteins of human cytomegalovirus (HCMV) virions are incompletely characterized. We have analyzed complex formation between glycoprotein M (gM or gpUL100) and a second glycoprotein. gM-homologous proteins are conserved throughout the herpesvirus family and represent type III membrane proteins containing multiple hydrophobic sequences. In extracellular HCMV particles, gM was found to be complexed through disulfide bonds to a second protein with an apparent molecular mass of 50 to 60 kDa. The 50- to 60-kDa protein was found to be derived from reading frame UL73 of HCMV strain AD169. UL73-homologous genes are also conserved within herpesviruses. When transiently expressed by itself, the UL73 gene product consisted of a protein of 18 kDa. However, in the presence of gM, the UL73 gene product was posttranslationally modified to the 50- to 60-kDa species. Thus, gM and the UL73 gene product, which represents the gN homolog of herpesviruses, form a disulfide-linked complex in HCMV virions. Transient expression of gM and gN followed by fluorescence imaging with monoclonal antibodies against either protein demonstrated that complex formation was required for transport of the proteins from the endoplasmic reticulum to the Golgi and trans-Golgi compartments. Finally, we tested the gM-gN complex for reactivity with sera from HCMV-seropositive donors. Whereas most sera failed to react with either gM or gN when expressed alone, 62% of sera were positive for the gM-gN complex. Because a murine monoclonal antibody reactive with gN in the gM-gN complex efficiently neutralizes infectious virus, the gM-gN complex may represent a major antigenic target of antiviral antibody responses.     

Ionizing radiation-dependent gamma-H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related

The histone variant H2AX is rapidly phosphorylated at the sites of DNA double-strand breaks (DSBs). This phosphorylated H2AX (gamma-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PI3K-related protein kinases (in mammals, ataxia telangiectasia mutated and Rad3-related [ATR], ataxia telangiectasia mutated [ATM], and DNA-PKCs) has been a subject of debate; it has been suggested that ATM is required for the induction of foci at DSBs, whereas ATR is involved in the recognition of stalled replication forks. In this study, using Arabidopsis as a model system, we investigated the ATR and ATM dependency of the formation of gamma-H2AX foci in M-phase cells exposed to ionizing radiation (IR). We find that although the majority of these foci are ATM-dependent, approximately 10% of IR-induced gamma-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR-induced damage is recognized by ATR. In addition, we find that in plants, gamma-H2AX foci are induced at only one-third the rate observed in yeasts and mammals. This result may partly account for the relatively high radioresistance of plants versus yeast and mammals.