The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3delta, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10(-8) M) or a higher concentration of spermine (10(-6) M).

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase-deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.

The polyamines putrescine, spermidine, and spermine and ornithine decarboxylase (ODC), the rate-limiting enzyme in their biosynthetic pathway, play an important role in cell proliferation, differentiation, and transformation. In the present study, we have analyzed polyamine concentrations and ODC activity in samples from benign breast diseases (n = 36), benign breast tissue adjacent to the primary carcinoma (n = 19), and breast carcinoma (n = 104). ODC activity in primary carcinoma was significantly higher (2.42 +/- 0.22 nmol CO2/h g; P < 0.001) than that found in benign breast (0.62 +/- 0.15 nmol CO2/h g) or in breast tissue adjacent to the primary carcinoma (0.52 +/- 0.16 nmol CO2/h g). The total polyamine content of breast cancer tissues was higher than in benign breast diseases (704.3 +/- 38.3 nmol/g wet weight versus 295.8 +/- 27.4 nmol/g wet weight) and correlated well with ODC activity (Pearson, r = 0.42; P < 0.001). ODC activity correlated with histological grade, peritumoral lymphatic or blood vessel invasion, S-phase fraction, and cathepsin D. Total polyamine concentration increased with S-phase fraction, cathepsin D, and aneuploidy. No significant correlation was found between ODC or polyamines and tumor size, lymph node involvement, or steroid receptor status. A major finding in our study was that ODC activity was an independent prognostic factor for recurrence and death. The results indicate that the estimation of ODC activity and polyamines in human breast carcinoma might be useful to determine tumor aggressiveness and suggest that ODC may have a potential value as both a prognostic factor and a chemoprevention target in human breast cancer.

Chenopodium quinoa (Willd.) is an Andean plant showing a remarkable tolerance to abiotic stresses. In Chile, quinoa populations display a high degree of genetic distancing, and variable tolerance to salinity. To investigate which tolerance mechanisms might account for these differences, four genotypes from coastal central and southern regions were compared for their growth, physiological, and molecular responses to NaCl at seedling stage. Seeds were sown on agar plates supplemented with 0, 150 or 300mM NaCl. Germination was significantly reduced by NaCl only in accession BO78. Shoot length was reduced by 150mM NaCl in three out of four genotypes, and by over 60% at 300mM (except BO78 which remained more similar to controls). Root length was hardly affected or even enhanced at 150mM in all four genotypes, but inhibited, especially in BO78, by 300mM NaCl. Thus, the root/shoot ratio was differentially affected by salt, with the highest values in PRJ, and the lowest in BO78. Biomass was also less affected in PRJ than in the other accessions, the genotype with the highest increment in proline concentration upon salt treatment. Free putrescine declined dramatically in all genotypes under 300mM NaCl; however (spermidine+spermine)/putrescine ratios were higher in PRJ than BO78. Quantitative RT-PCR analyses of two sodium transporter genes, CqSOS1 and CqNHX, revealed that their expression was differentially induced at the shoot and root level, and between genotypes, by 300mM NaCl. Expression data are discussed in relation to the degree of salt tolerance in the different accessions.

Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS affect the intracellular Ca(2+) dynamics by activating a range of nonselective Ca(2+)-permeable channels in plasma membrane (PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents and net K(+) and Ca(2+) fluxes induced by hydroxyl radicals (OH(•)) in pea (Pisum sativum) roots. OH(•), but not hydrogen peroxide, activated a rapid Ca(2+) efflux and a more slowly developing net Ca(2+) influx concurrent with a net K(+) efflux. In isolated protoplasts, OH(•) evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K(+) efflux in intact roots. This current displayed a low ionic selectivity and was permeable to Ca(2+). Active OH(•)-induced Ca(2+) efflux in roots was suppressed by the PM Ca(2+) pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine, and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH(•)-induced ionic currents in root protoplasts and K(+) efflux and Ca(2+) influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit the OH(•)-induced cation fluxes. The net OH(•)-induced Ca(2+) efflux was largely prolonged in the presence of spermine, and all PAs tested (spermine, spermidine, and putrescine) accelerated and augmented the OH(•)-induced net K(+) efflux from roots. The latter effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter intracellular Ca(2+) homeostasis by modulating both Ca(2+) influx and efflux transport systems at the root cell PM.

UNASSIGNED

Spermidine administration is linked to increased survival in several animal models.

UNASSIGNED

The aim of this study was to test the potential association between spermidine content in diet and mortality in humans.

UNASSIGNED

This prospective community-based cohort study included 829 participants aged 45-84 y, 49.9% of whom were male. Diet was assessed by repeated dietitian-administered validated food-frequency questionnaires (2540 assessments) in 1995, 2000, 2005, and 2010. During follow-up between 1995 and 2015, 341 deaths occurred.

UNASSIGNED

All-cause mortality (deaths per 1000 person-years) decreased across thirds of increasing spermidine intake from 40.5 (95% CI: 36.1, 44.7) to 23.7 (95% CI: 20.0, 27.0) and 15.1 (95% CI: 12.6, 17.8), corresponding to an age-, sex- and caloric intake-adjusted 20-y cumulative mortality incidence of 0.48 (95% CI: 0.45, 0.51), 0.41 (95% CI: 0.38, 0.45), and 0.38 (95% CI: 0.34, 0.41), respectively. The age-, sex- and caloric ratio-adjusted HR for all-cause death per 1-SD higher spermidine intake was 0.74 (95% CI: 0.66, 0.83; P < 0.001). Further adjustment for lifestyle factors, established predictors of mortality, and other dietary features yielded an HR of 0.76 (95% CI: 0.67, 0.86; P < 0.001). The association was consistent in subgroups, robust against unmeasured confounding, and independently validated in the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) Study (age-, sex-, and caloric ratio-adjusted HR per 1-SD higher spermidine intake: 0.71; 95% CI: 0.53, 0.95; P = 0.019). The difference in mortality risk between the top and bottom third of spermidine intakes was similar to that associated with a 5.7-y (95% CI: 3.6, 8.1 y) younger age.

UNASSIGNED

Our findings lend epidemiologic support to the concept that nutrition rich in spermidine is linked to increased survival in humans. This trial was registered at www.clinicaltrials.gov as NCT03378843.

Occludin is an integral membrane protein that forms the sealing element of tight junctions and is critical for epithelial barrier function. Polyamines are implicated in multiple signaling pathways driving different biological functions of intestinal epithelial cells (IEC). The present study determined whether polyamines are involved in expression of occludin and play a role in intestinal epithelial barrier function. Studies were conducted in stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) associated with a highly differentiated phenotype. Polyamine depletion by alpha-difluoromethylornithine (DFMO) decreased levels of occludin protein but failed to affect expression of its mRNA. Other tight junction proteins, zonula occludens (ZO)-1, ZO-2, claudin-2, and claudin-3, were also decreased in polyamine-deficient cells. Decreased levels of tight junction proteins in DFMO-treated cells were associated with dysfunction of the epithelial barrier, which was overcome by exogenous polyamine spermidine. Decreased levels of occludin in polyamine-deficient cells was not due to the reduction of intracellular-free Ca(2+) concentration ([Ca(2+)](cyt)), because either increased or decreased [Ca(2+)](cyt) did not alter levels of occludin in the presence or absence of polyamines. The level of newly synthesized occludin protein was decreased by approximately 70% following polyamine depletion, whereas its protein half-life was reduced from approximately 120 min in control cells to approximately 75 min in polyamine-deficient cells. These findings indicate that polyamines are necessary for the synthesis and stability of occludin protein and that polyamine depletion disrupts the epithelial barrier function, at least partially, by decreasing occludin.

Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1(G93A) contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Na v ) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1(G93A) and ACM-SOD1(G86R)) or TDP43 (ACM-TDP43(A315T)) mutants; we show that such exposure rapidly (within 30-60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Na v channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Na v channel activity.

Deoxyhypusine synthase is the first enzyme involved in the post-translational formation of hypusine, a unique amino acid that occurs at one position in a single cellular protein, eukaryotic translation initiation factor 5A (eIF-5A). This NAD-dependent enzyme catalyzes the formation of deoxyhypusine by transfer of the butylamine portion of spermidine to the epsilon-amino group of a specific lysine residue in the eIF-5A precursor. Its purification from rat testis was accomplished by ammonium sulfate fractionation and successive ion-exchange chromatographic steps, followed by chromatofocusing on a hydrophilic resin (Mono P). A pI of 4.7 was determined by isoelectric focusing. Amino acid sequences of five tryptic peptides of the pure enzyme did not correspond to any sequences in the protein data banks. The enzyme migrates as a single band on SDS-polyacrylamide gel electrophoresis with an apparent monomer molecular mass of approximately 42,000 Da. Matrix-assisted laser desorption mass spectrometry gave a monomer mass of 40,800 Da. There is evidence, however, that the active enzyme exists as a tetramer of this subunit. Rabbit polyclonal antibodies to the 42-kDa protein precipitated deoxyhypusine synthase activity. The enzyme shows a strict specificity for NAD. Purified deoxyhypusine synthase catalyzes the overall synthesis of deoxyhypusine and, in the absence of the eIF-5A precursor, catalyzes the cleavage of spermidine.

Expression of spermidine/spermine N(1)-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G(2) arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H(2)O(2) due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR ->> Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G(2) arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G(2)/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf ->> MEK ->> ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.

The superinduction of spermidine/spermine N1-acetyltransferase (SSAT) gene has been associated with a cytotoxic response to a new class of antineoplastic polyamine analogues. The initial mechanism of SSAT superinduction is an increase in transcription in response to analogue exposure. This increased transcription appears to be modulated through the association between a nuclear protein factor and a cis-element described here as the polyamine-responsive element (PRE). The PRE was identified as a 9-base pair sequence, 5'-TATGACTAA-3', in the context of a 31-base pair stretch from -1522 to -1492 base pairs with respect to the SSAT transcriptional start site. This element binds a nuclear factor from polyamine analogue-responsive cells, but not from polyamine analogue-insensitive cells. The labeled PRE was used to clone and identify the transcription factor, Nrf-2, that binds constitutively to the PRE sequence. Although the PRE sequence shares homology to the originally identified Nrf-2 recognition sequence, the two sequences are not identical. The Nrf-2 transcription factor appears only to be present in cell types that are capable of expressing high amounts of SSAT. The results of these studies suggest that Nrf-2, bound to the PRE, plays an important regulatory role of expression of the human SSAT gene.

Schizosaccharomyces pombe cells that cannot synthesize spermidine or spermine because of a deletion-insertion in the gene coding for S-adenosylmethionine decarboxylase (Deltaspe2) have an absolute requirement for spermidine for growth. Flow cytometry studies show that in the absence of spermidine an overall delay of the cell cycle progression occurs with some accumulation of cells in the G(1) phase; as little as 10(-6) M spermidine is sufficient to maintain normal cell cycle distribution and normal growth. Morphologically some of the spermidine-deprived cells become spherical at an early stage with little evidence of cell division. On further incubation in the spermidine-deprived medium, growth occurs in most of the cells, not by cell division but rather by cell elongation, with an abnormal distribution of the actin cytoskeleton, DNA (4', 6-diamidino-2-phenylindole staining), and calcofluor-staining moieties. More prolonged incubation in the spermidine-deficient medium leads to profound morphological changes including nuclear degeneration.

Although treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) leads to depletion of intracellular polyamines and to related growth inhibition in vitro, its cytostatic effects in vivo are disappointing. This may be due to abolition of DFMO-induced growth inhibition by polyamines released during normal body cell turnover, to dietary polyamines, or to putrescine synthesized by the microbial flora in the GI tract. We studied selectively (aerobic) and totally (aerobic + anaerobic) GI tract-decontaminated LI210-bearing mice fed with 3 types of diet differing in their polyamine and carbohydrate residue contents and treated with combinations of intraperitoneal DFMO and oral deuterium-labelled putrescine. Our data show that, irrespective of diet type, total decontamination markedly potentiates the moderate tumor growth inhibition that is caused by DFMO alone. During total decontamination, growth-inhibited L1210 cells accumulate in the G0/G1 phase of the cell cycle. Although orally administered deuterium-labelled putrescine gave rise to deuterium labelling of L1210 putrescine, spermidine and spermine, the polyamine levels in our diets played only a minor role.

Approximately 30 to 40% of human leukemic cell lines are completely deficient in the purine catabolic enzyme 5'-methylthioadenosine phosphorylase. Using two 5'-methylthioadenosine phosphorylase-negative leukemias, we have for the first time been able to measure the synthesis and biological effects of 5'-methylthioadenosine (MTA) in intact mammalian tumor cells. Malignant cells lacking this enzyme, unlike enzyme-positive cells, excreted MTA into the culture medium at a rate of 0.58 to 0.70 nmol/hr/mg protein. The production of the nucleoside was inhibited effectively by nontoxic concentrations of methylglyoxal bis(guanylhydrazone), a putrescine-depending S-adenosylmethionine decarboxylase inhibitor, and also by spermidine and spermine but was enhanced by putrescine. In a reciprocal fashion, MTA at low concentrations progressively increased both the synthesis and concentration of putrescine but suppressed spermine production. The unique alterations in polyamine metabolism induced by elevated MTA levels could offer a selective growth advantage to the 5'-methylthioadenosine phosphorylase-deficient cells and thus may be related to the high frequency of this enzyme deficiency among human leukemic cell lines.

Infravesical outflow obstruction of a duration of 3 days, 10 days and 6 weeks was induced in female rats by a standardized degree of urethral obstruction. A striking ability of the detrusor to respond to an acute obstruction with both smooth muscle cell hypertrophy and hyperplasia leading to an approximately 10-fold increase of the total muscle mass of the bladder wall after 6 weeks of obstruction was found. The maximum relative growth rate was greatest in the bladders subjected to obstruction for only 3 days, and this was also reflected by the concentrations of ornithine decarboxylase and the polyamines spermidine and spermine. The total amount of DNA in the detrusor was already significantly increased after 3 days, while a 9-fold increase was observed in the group subjected to obstruction for 6 weeks. At this time the smooth muscle cell nucleus volume also showed a considerable increase, and a comparison of the nucleus density and the DNA concentration suggested an increased mean DNA content per muscle cell nucleus. The concentration of RNA in the detrusor had already increased significantly after 3 days and also remained so after 10 days and 6 weeks of obstruction, a finding that coincided with the abundant appearance of nucleoli seen at electron microscopic investigation. The previously reported decreased ability to pressure production at small volumes in the rat urinary bladder subjected to an acute infravesical outflow obstruction might thus, at least in part, be due to changed contractile properties of the hypertrophic cells, and/or to an inefficient incorporation of the newly formed smooth muscle cells.

The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.

Sequence-specific DNA-protein interactions are basic to DNA function. To better understand these interactions, we studied the effect of position on cleavage of DNA by the type II restriction enzyme (EC 3.1.21.4) Nae I. We discovered two classes of Nae I restriction sites: sites susceptible and sites resistant to cleavage. Kinetic analysis showed that Nae I was activated by DNA containing cleavable Nae I sites to rapidly cleave resistant Nae I sites by a noncompetitive mechanism with a Km for substrate DNA of about 2 nM and a KA for activating DNA of about 6 nM; activation increased catalysis but not substrate binding. Deletion mutagenesis in vitro showed that sequences flanking the Nae I recognition site were responsible for the differences between activating and nonactivating Nae I sites. The polyamine spermidine had a dramatic effect on the interaction of Nae I with DNA; in the presence of 1 mM spermidine, resistant sites were cleaved rapidly and cleavable DNA inhibited cleavage. The direct regulation of enzymatic activity by DNA sequences in trans, and the modulation of this regulation by a polyamine that is sensitive to the cell cycle, provides a regulatory switch mechanism. The implications of this switch for biological control functions are discussed.

Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N(1)-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6-24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6-24 h), then increased by approximately 50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6-72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24-72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.

Gentle lysis of E. coli cells in the presence of a DNA counterion (either 1.0 M NaCl or 5 mM spermidine) permits the isolation of the folded intact bacterial chromosome associated with membrane fragments. Most of the proteins in these chromosomes are also found in purified membrane preparations, and they can be identified as belonging to either the inner or the outer bacterial membrane. Ultraviolet irradiation of the membrane-attached chromosomes causes the formation of a stable complex between two inner membrane proteins (molecular weight 80,000 and 56,000 daltons) and 5-bromodeoxyuridine (BrdU)-substituted DNA. The photochemical attachment of BrdU-substituted DNA to specific membrane proteins suggests that these proteins may be bound to the DNA in vivo. Such DNA-membrane-binding proteins may have a role in the attachment of the folded chromosome to the bacterial envelope.

Members of the Brassicaceae accumulate complex patterns of sinapate esters, as shown in this communication with seeds of oilseed rape (Brassica napus). Fifteen seed constituents were isolated and identified by a combination of high-field NMR spectroscopy and high resolution electrospray ionisation mass spectrometry. These include glucose, gentiobiose and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate and an unusual cyclic spermidine amide. One of the glucose esters (1,6-di-O-sinapoylglucose), two gentiobiose esters (1-O-caffeoylgentiobiose and 1,2,6'-tri-O-sinapoylgentiobiose) and two kaempferol conjugates [4'-(6-O-sinapoylglucoside)-3,7-di-O-glucoside and 3-O-sophoroside-7-O-(2-O-sinapoylglucoside)] seem to be new plant products. Serine carboxypeptidase-like (SCPL) acyltransferases catalyze the formation of sinapine and sinapoylmalate accepting 1-O-beta-acetal esters (1-O-beta-glucose esters) as acyl donors. To address the question whether the formation of other components of the complex pattern of the sinapate esters in B. napus seeds is catalyzed via 1-O-sinapoyl-beta-glucose, we performed a seed-specific dsRNAi-based suppression of the sinapate glucosyltransferase gene (BnSGT1) expression. In seeds of BnSGT1-suppressing plants the amount of sinapoylglucose decreased below the HPLC detection limit resulting in turn in the disappearance or marked decrease of all the other sinapate esters, indicating that formation of the complex pattern of these esters in B. napus seeds is dependent on sinapoylglucose. This gives rise to the assumption that enzymes of an SCPL acyltransferase family catalyze the appropriate transfer reactions to synthesize the accumulating esters.

OBJECTIVE

Polyamines are ubiquitous intracellular polycationic molecules essential for cell growth and differentiation. Polyamine analogs down-regulate ornithine decarboxylase, induce spermidine/spermine N1-acetyltransferase, deplete natural polyamine pools, inhibit growth, and induce programmed cell death in breast cancer models. This study evaluated the activity of the first-generation analog DENSpm in women with metastatic breast cancer.

METHODS

The overall accrual goal was 34 patients (30 evaluable) in a two-stage design. The second stage of accrual was to proceed if>> or =2 among first 15 evaluable patients were progression free at 4 months. The primary objective was to determine whether>> or =20% of metastatic breast cancer patients treated with DENSpm as second- or third-line therapy remained progression free after 4 months.

RESULTS

Sixteen patients (median age, 52 years; range, 34-65; median performance status, 1; range, 0-1) enrolled in the first stage received 43 cycles (median, 2; range, 1-6) of 100 mg/m2 DENSpm as a 15-min infusion i.v. on days 1-5 every 21 days. All 16 patients were evaluable for toxicity; 15 were evaluable for response. All patients had disease progression by 4 months, and the study closed after the first stage of accrual. The main toxicities included grade 1-2 abdominal pain, transient perioral numbness, nausea, and grade 1 thrombocytopenia. Two patients had grade 3 abdominal pain during cycle 2 infusion: one was hospitalized, and another was subsequently retreated at 80% dose without pain recurrence.

CONCLUSIONS

Although this dose and administration schedule of DENSpm was quite tolerable, no evidence of clinical activity was detected. Encouraging preclinical activity of polyamine analogs alone and in combination with cytotoxic drugs supports the continued evaluation of newer-generation polyamine analogs for the treatment and prevention of breast cancer.

The in vitro conversion of single-stranded DNA from bacteriophage fd to duplex structures depends on E. coli RNA polymerase, DNA polymerase III, riboand deoxyribonucleoside triphosphates, Mg(+2), spermidine or DNA-unwinding protein of E. coli, and two additional protein factors, referred to here as Factors I and II. These two factors are also essential for dTMP incorporation catalyzed by DNA polymerase III and dependent on poly(dA).oligo(dT) primer-template. In the latter reaction, there is an absolute dependency on ATP or dATP.