The Decisive Step in Betaxanthin Biosynthesis Is a Spontaneous Reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris “Golden Beet”) showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris “Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.

The high spontaneous mutation rate: Is it a health risk?*

The human mutation rate for base substitutions is much higher in males than in females and increases with paternal age. This effect is mainly, if not entirely, due to the large number of cell divisions in the male germ line. The mutation-rate increase is considerably greater than expected if the mutation rate were simply proportional to the number of cell divisions. In contrast, those mutations that are small deletions or rearrangements do not show the paternal age effect. The observed increase with the age of the father in the incidence of children with different dominant mutations is variable, presumably the result of different mixtures of base substitutions and deletions. In Drosophila, the rate of mutations causing minor deleterious effects is estimated to be about one new mutation per zygote. Because of a larger number of genes and a much larger amount of DNA, the human rate is presumably higher. Recently, the Drosophila data have been reanalyzed and the mutation-rate estimate questioned, but I believe that the totality of evidence supports the original conclusion. The most reasonable way in which a species can cope with a high mutation rate is by quasi-truncation selection, whereby a number of mutant genes are eliminated by one “genetic death.”

CML66, a broadly immunogenic tumor antigen, elicits a humoral immune response associated with remission of chronic myelogenous leukemia

This report describes a tumor-associated antigen, termed CML66, initially cloned from a chronic myelogenous leukemia (CML) cDNA expression library. CML66 encodes a 583-aa protein with a molecular mass of 66 kDa and no significant homology to other known genes. CML66 gene is localized to human chromosome 8q23, but the function of this gene is unknown. CML66 is expressed in leukemias and a variety of solid tumor cell lines. When examined by Northern blot, expression in normal tissues was restricted to testis and heart, and no expression was found in hematopoietic tissues. When examined by quantitative reverse transcription–PCR, expression in CML cells was 1.5-fold higher than in normal peripheral blood mononuclear cells. The presence of CML66-specific antibody in patient serum was confirmed by Western blot and the development of high titer IgG antibody specific for CML66 correlated with immune induced remission of CML in a patient who received infusion of normal donor lymphocytes for treatment of relapse. CML66 antibody also was found in sera from 18–38% of patients with lung cancer, melanoma, and prostate cancer. These findings suggest that CML66 may be immunogenic in a wide variety of malignancies and may be a target for antigen-specific immunotherapy.

Genetic fidelity under harsh conditions: Analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius

Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75°C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S. acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in S. acidocaldarius than in other DNA-based organisms examined to date.

Spontaneous diabetes mellitus in transgenic mice expressing human islet amyloid polypeptide.

The islet in non-insulin-dependent diabetes mellitus (NIDDM) is characterized by loss of beta cells and large local deposits of amyloid derived from the 37-amino acid protein, islet amyloid polypeptide (IAPP). We have hypothesized that IAPP amyloid forms intracellularly causing beta-cell destruction under conditions of high rates of expression. To test this we developed a homozygous transgenic mouse model with high rates of expression of human IAPP. Male transgenic mice spontaneously developed diabetes mellitus by 8 weeks of age, which was associated with selective beta-cell death and impaired insulin secretion. Small intra- and extracellular amorphous IAPP aggregates were present in islets of transgenic mice during the development of diabetes mellitus. However, IAPP derived amyloid deposits were found in only a minority of islets at approximately 20 weeks of age, notably after development of diabetes mellitus in male transgenic mice. Approximately 20% of female transgenic mice spontaneously developed diabetes mellitus at 30+ weeks of age, when beta-cell degeneration and both amorphous and amyloid deposits of IAPP were present. We conclude that overexpression of human IAPP causes beta-cell death, impaired insulin secretion, and diabetes mellitus. Large deposits of IAPP derived amyloid do not appear to be important in this cytotoxicity, but early, small amorphous intra- and extracellular aggregates of human IAPP were consistently present at the time of beta-cell death and therefore may be the most cytotoxic form of IAPP.

Distribution of spontaneous plant hybrids.

Natural hybridization is a relatively common feature of vascular plant species and has been demonstrated to have played an important role in their evolution. Nonetheless, it is not clear whether spontaneous hybridization occurs as a general feature of all plant families and genera or whether certain groups are especially prone to spontaneous hybridization. Therefore, we inspected five modern biosystematic floras to survey the frequency and taxonomic distribution of spontaneous hybrids. We found spontaneous hybridization to be nonrandomly distributed among taxa, concentrated in certain families and certain genera, often at a frequency out of proportion to the size of the family or genus. Most of these groups were primarily outcrossing perennials with reproductive modes that stabilized hybridity such as agamospermy, vegetative spread, or permanent odd polyploidy. These data suggest that certain phylogenetic groups are biologically predisposed for the formation and maintenance of hybrids.

Localization of the central rhythm generator involved in spontaneous consummatory licking in rats: functional ablation and electrical brain stimulation studies.

Localization of the central rhythm generator (CRG) of spontaneous consummatory licking was studied in freely moving rats by microinjection of tetrodotoxin (TTX) into the pontine reticular formation. Maximum suppression of spontaneous water consumption was elicited by TTX (1 ng) blockade of the oral part of the nucleus reticularis gigantocellularis (NRG), whereas TTX injections into more caudal or rostral locations caused significantly weaker disruption of drinking. To verify the assumption that TTX blocked the proper CRG of licking rather than some relay in its output, spontaneously drinking thirsty rats were intracranially stimulated via electrodes chronically implanted into the oral part of the NRG. Lick-synchronized stimulation (a 100-ms train of 0.1-ms-wide rectangular pulses at 100 Hz and 25-150 microA) applied during continuous licking (after eight regular consecutive licks) caused a phase shift of licks emitted after stimulus delivery. The results suggest that the stimulation has reset the CRG of licking without changing its frequency. The reset-inducing threshold current was lowest during the tongue retraction and highest during the tongue protrusion period of the lick cycle. It is concluded that the CRG of licking is located in the oral part of NRG.

Spontaneous avoidance behavior in Drosophila null for calmodulin expression.

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.

Intracortical and corticothalamic coherency of fast spontaneous oscillations.

We report that fast (mainly 30- to 40-Hz) coherent electric field oscillations appear spontaneously during brain activation, as expressed by electroencephalogram (EEG) rhythms, and they outlast the stimulation of mesopontine cholinergic nuclei in acutely prepared cats. The fast oscillations also appear during the sleep-like EEG patterns of ketamine/xylazine anesthesia, but they are selectively suppressed during the prolonged phase of the slow (<1-Hz) sleep oscillation that is associated with hyperpolarization of cortical neurons. The fast (30- to 40-Hz) rhythms are synchronized intracortically within vertical columns, among closely located cortical foci, and through reciprocal corticothalamic networks. The fast oscillations do not reverse throughout the depth of the cortex. This aspect stands in contrast with the conventional depth profile of evoked potentials and slow sleep oscillations that display opposite polarity at the surface and midlayers. Current-source-density analyses reveal that the fast oscillations are associated with alternating microsinks and microsources across the cortex, while the evoked potentials and the slow oscillation display a massive current sink in midlayers, confined by two sources in superficial and deep layers. The synchronization of fast rhythms and their high amplitudes indicate that the term "EEG desynchronization," used to designate brain-aroused states, is incorrect and should be replaced with the original term, "EEG activation" [Moruzzi, G. & Magoun, H.W. (1949) Electroencephalogr. Clin. Neurophysiol. 1, 455-473].

Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha.

Cytokines are now recognized to play important roles in the physiology of the central nervous system (CNS) during health and disease. Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of several human CNS disorders including multiple sclerosis, AIDS dementia, and cerebral malaria. We have generated transgenic mice that constitutively express a murine TNF-alpha transgene, under the control of its own promoter, specifically in their CNS and that spontaneously develop a chronic inflammatory demyelinating disease with 100% penetrance from around 3-8 weeks of age. High-level expression of the transgene was seen in neurons distributed throughout the brain. Disease is manifested by ataxia, seizures, and paresis and leads to early death. Histopathological analysis revealed infiltration of the meninges and CNS parenchyma by CD4+ and CD8+ T lymphocytes, widespread reactive astrocytosis and microgliosis, and focal demyelination. The direct action of TNF-alpha in the pathogenesis of this disease was confirmed by peripheral administration of a neutralizing anti-murine TNF-alpha antibody. This treatment completely prevented the development of neurological symptoms, T-cell infiltration into the CNS parenchyma, astrocytosis, and demyelination, and greatly reduced the severity of reactive microgliosis. These results demonstrate that overexpression of TNF-alpha in the CNS can cause abnormalities in nervous system structure and function. The disease induced in TNF-alpha transgenic mice shows clinical and histopathological features characteristic of inflammatory demyelinating CNS disorders in humans, and these mice represent a relevant in vivo model for their further study.

Spontaneous persistent currents in a quantum spin Hall insulator

We present a mechanism for persistent charge current. Quantum spin Hall insulators hold dissipationless spin currents in their edges so that, for a given spin orientation, a net charge current flows which is exactly compensated by the counterflow of the opposite spin. Here we show that ferromagnetic order in the edge upgrades the spin currents into persistent charge currents without applied fields. For that matter, we study the Hubbard model including Haldane-Kane-Mele spin-orbit coupling in a zigzag ribbon and consider the case of graphene. We find three electronic phases with magnetic edges that carry currents reaching 0.4 nA, comparable to persistent currents in metallic rings, for the small spin-orbit coupling in graphene. One of the phases is a valley half metal.

Spontaneous Abortions and Shift Work in a Cohort of Nurses in Norway

Background: An association between spontaneous abortions and shift work has been suggested, but present research results are conflicting. The aim of the study is to evaluate the relationship between spontaneous abortions among nurses, shift schedules, and nights worked. Methods: This is a longitudinal study where we identified 914 females from a cohort of nurses in Norway who had worked the same type of shift schedule 2008-2010; either permanent day shift, three-shift rotation or permanent night shift. Information on age, work and life-style factors, as well as spontaneous abortions during lifetime and the past three years (2008-2010) was obtained by annual questionnaires. Results: A higher prevalence of experienced spontaneous abortions before study start (2008) was found among nurses working permanent night shift compared to other nurses. In a linear regression analysis, a risk of 1.3 was found for experienced spontaneous abortions before study start among permanent night shift nurses, with day shift as reference, when adjusting for age, smoking, caffeine and job strain, but the finding was not statistical significant (95 per cent confidence interval 0.8-2.1). Permanent night shift workers had a risk of 1.5 experiencing spontaneous abortions in 2008-2010 compared to day shift nurses, although not statistical significant (95 per cent confidence interval 0.7-3.5). The number of night shifts the past three years was not associated with experiencing spontaneous abortions 2008-2010, but associated with a reduced risk of experiencing spontaneous abortions during lifetime. The results must be interpreted in the light of a possible selection bias; both selections into the occupation of nursing and into the different shift types of the more healthy persons may have occurred in this population. Conclusion: No significant increased risk of spontaneous abortion among permanent night shift nurses compared to day-time nurses was found in this study, and no association was found between spontaneous abortions and the number of worked night shifts.

Water-triggered spontaneous surface patterning in thin films of mexylaminotriazine molecular glasses

Surface patterning that occurs spontaneously during the formation of a thin film is a powerful tool for controlling film morphology at the nanoscale level because it avoids the need for further processing. However, one must first learn under which conditions these patterning phenomena occur or not, and how to achieve control over the surface morphologies that are generated. Mexylaminotriazine-based molecular glasses are small molecules that can readily form amorphous thin films. It was discovered that this class of materials can either form smooth films, or films exhibiting either dome or pore patterns. Depending on the conditions, these patterns can be selectively obtained during film deposition by spin-coating. It was determined that this behavior is controlled by the presence of water or, more generally, of a solvent in which the compounds are insoluble, and that the relative amount and volatility of this poor solvent determines which type of surface relief is obtained. Moreover, AFM and FT-IR spectroscopy have revealed that the thin films are amorphous independently of surface morphology, and no difference was observed at the molecular or supramolecular level. These findings make this class of materials and this patterning approach in general extremely appealing for the control of surface morphology with organic nanostructures.