• Molecules and Cells
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• 제20권1호
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• pp.127-135
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• 2005

The uptake of putrescine, spermidine and spermine by Fortner's hamster amelanocytic melanoma AMEL-3 cells was observed in this study to be time-dependent, temperature-sensitive, pH-dependent and saturable. Metabolic poisons nullified polyamine uptake, an indication that this is an energy-requiring mechanism. The presence of $Na^+$ ions was found to be requisite to full activity. Valinomycin, gramicidin, monensin and the calcium ionophore calcimycin were also observed to inhibit the process substantially. The transporter active site would seem to contain sulfhydryl groups. Other diamines and polyamine analogues, as well as cationic diamidines, suppressed putrescine uptake. The presence of the ornithine decarboxylase inhibitor DFMO in the culture medium induced putrescine inflows. Putrescine, in turn, induced the negative expression of the carrier, thus suggesting that this influx mechanism is governed by up/down regulation. The cationic diamidine CGP 40215A and its analogue CGP039937A competitively inhibited putrescine transport, with Ki values of 1.9 and $15{\mu}M$, respectively. The role of polyamine uptake in these cultures is discussed.

• The Korean Journal of Physiology and Pharmacology
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• 제1권4호
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• pp.457-466
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• 1997

The effects of DFMO or/and putrescine on the dexamethasone-induced apoptosis of CEM cells were studied to investigate the role of polyamines in anti-leukemic glucocorticoid action. Dexamethasone- induced apoptosis was preceded by significant decreases of cellular polyamine contents and putrescine uptake activity. But DFMO produced decreases of putrescine and spermidine contents and marked increase of putrescine uptake activity, but did not induce apoptosis. However, dexamethasone and DFMO, respectively, induced $G_1-arrest$ in cell cycle and hypophosphorylation of pRb, resulting in the increase of $G_1$ to S ratio and decrease of CEM cell count. DFMO enhanced the dexamethasone-induced apoptosis and $G_1-arrest$. On the other hand, putrescine little affected the apoptotic and $G_1-arresting$ activities of dexamethasone, but almost suppress the effects of DFMO and also the DFMO-dependent enhancement of dexamethasone effects. These results suggested that the dexamethasone-induced apoptosis to be associated with pRb hypophosphorylation and $G_1-arrest$ in CEM cells might be ascribed to the concomitant decreases of cellular polyamine contents and putrescine uptake activity.

• BMB Reports
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• 제39권4호
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• pp.394-399
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• 2006

The transport of putrescine into a moderately salt tolerant cyanobacterium Synechocystis sp. PCC 6803 was characterized by measuring the uptake of radioactively-labeled putrescine. Putrescine transport showed saturation kinetics with an apparent $K_m$ of $92{\pm}10\;{\mu}M$ and $V_{max}$ of $0.33{\pm}0.05\;nmol/min/mg$ protein. The transport of putrescine was pH-dependent with highest activity at pH 7.0. Strong inhibition of putrescine transport was caused by spermine and spermidine whereas only slight inhibition was observed by the addition of various amino acids. These results suggest that the transport system in Synechocystis sp. PCC 6803 is highly specific for polyamines. Putrescine transport is energy-dependent as evidenced by the inhibition by various metabolic inhibitors and ionophores. Slow growth was observed in cells grown under salt stress. Addition of low concentration of putrescine could restore growth almost to the level observed in the absence of salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased putrescine transport with an optimum 2-fold increase at 20 mosmol/kg. The stimulation of putrescine transport mediated by osmotic upshift was abolished in chloramphenicol-treated cells, suggesting possible involvement of an inducible transport system.

• Journal of Microbiology
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• 제43권5호
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• pp.398-405
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• 2005

Polyamines such as putrescine are small, ubiquitous polycationic molecules that are required for optimal growth of eukaryotic and prokaryotic cells. These molecules have diverse effects on cell physiology and their intracellular content is regulated by de novo synthesis and uptake from the environment. The studies presented here examined the structure of a putative polyamine transporter (Pot) operon in Streptococcus pneumoniae (pneumococcus) and growth of pneumococci in medium containing putrescine substituted for choline. RT-PCR experiments demonstrated that the four genes encoding the Pot system are co-transcribed with murB, a gene involved in an intermediary step of peptidoglycan synthesis. Pneumococci grown in chemically-defined media (CDM) containing putrescine without choline enter logarithmic phase growth after 36-48 hs. However, culture density at stationary phase eventually reaches that of choline-containing medium. Cells grown in CDM-putrescine formed abnormally elongated chains in which the daughter cells failed to separate and the choline-binding protein PspA was no longer cell-associated. Experiments with CDM containing radiolabeled putrescine demonstrated that pneumococci concentrate this polyamine in cell walls. These data suggest that pneumococci can replicate without choline if putrescine is available and this polyamine may substitute for aminoalcohols in the cell wall teichoic acids.

• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.137-146
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• 1999

Interactions among dexamethasone, dehydroepiandrosterone (DHEA), lipopolysaccharide (LPS), and antimycin A on the glutamate uptake and the polyamine uptake were investigated in primary cultures of rat cerebral cortical astrocytes to examine the effects of dexamethasone and DHEA on the regulatory role of astrocytes in conditions of increased extracellular concentrations of glutamate or polyamines. 1. $[^3H]Glutamate$ uptake: LPS and antimycin A decreased $V_{max},$ but both drugs had little effect on $K_m.$ Dexamethasone also decreased basal $V_{max}$ without any significant effect on $K_m.$ And dexamethasone further decreased the antimycin A-induced decrease of $V_{max}.$ DHEA did not affect the kinetics of basal glutamate uptake and the change by LPS or antimycin A. 2. $[^{14}C]Putrescine$ uptake: LPS increased $V_{max},$ and antimycin A decreased $V_{max}.$ They showed little effect on $K_m.$ Dexamethasone decreased $V_{max}$ of basal uptake and further decreased the antimycin A-induced decrease of $V_{max},$ and also decreased $V_{max}$ to less than control in LPS-treated astrocytes. DHEA did not affect $K_m$ and the change of $V_{max}$ by LPS or antimycin A. 3. $[^{14}C]Spermine$ uptake: Antimycin A decreased $V_{max},$ and LPS might increase $V_{max}.\;K_m$ was little affected by the drugs. Dexamethasone decreased basal $V_{max}$ and might further decrease the antimycin A-induced decrease of $V_{max}.$ And dexamethasone also decreased $V_{max}$ to less than control in LPS-treated astrocytes. DHEA might increase basal $V_{max}$ and $V_{max}$ of LPS-treated astrocytes. 4. $V_{max}$ of glutamate uptake by astrocytes was increased by putrescine (1000 ${\mu}M$ & 2000 ${\mu}M$) and spermidine (200 ${\mu}M,$ 500 ${\mu}M$ & 2000 ${\mu}M$). Spermine, 200 ${\mu}M$ (and 100 ${\mu}M$), also increased $V_{max},$ but a higher dose of 2000 ${\mu}M$ decreased $V_{max}.\;K_m$ of glutamate uptake was not significantly changed by these polyamines, except that higher doses of spermine showed tendency to decrease $K_m$ of glutamate uptake. In astrocytes, dexamethasone inhibited the glutamate uptake and the polyamine uptake in normal or hypoxic conditions, and the polyamine uptake might be stimulated by LPS and DHEA. Polyamines could aid astrocytes to uptake glutamate.

• Journal of Microbiology and Biotechnology
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• 제19권5호
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• pp.447-454
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• 2009

The transport of spermidine into a cyanobacterium, Synechocystis sp. pec 6803, was characterized by measuring the uptake of $^{14}C$-spermidine. Spermidine transport was shown to be saturable with an apparent affinity constant ($K_m$) value of $67{\mu}M$ and a maximal velocity ($V_{max}$) value of 0.45 nmol/min/mg protein. Spermidine uptake was pH-dependent with the pH optimum being 8.0. The competition experiment showed strong inhibition of spermidine uptake by putrescine and spermine, whereas amino acids were hardly inhibitory. The inhibition kinetics of spermidine transport by putrescine and spermine was found to be noncompetitive with $K_i$ values of 292 and $432{\mu}M$, respectively. The inhibition of spermidine transport by various metabolic inhibitors and ionophores suggests that spermidine uptake is energy-dependent. The diminution of cell growth was observed in cells grown at a high concentration of NaCl. Addition of a low concentration of spermidine at 0.5 mM relieved growth inhibition by salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased spermidine transport with about 30-40% increase at 10 mosmol/kg upshift.

• Biomolecules & Therapeutics
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• 제5권3호
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• pp.265-271
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• 1997

Since the advent of chemotherapy, certain types of cancer have been particularly resistant to chemotherapeutic treatment. One of the most well-studied types of resistance is resistance to multiple struc-turally dissimialr hydrophobic chemotherapeutic agents, or multidrug resistance (MDR). We found that MDR cells (KBV20C, KB7D) being highly resistant to colchicine, etoposide, and vincristine were found to have very low level of putrescine and low level of spermidine than the drug sensitive parental cells (KB) but they had almost same level of spermine as the drug sensitive cells. Although both MDR and drug sensitive cells had almost same rate of polyamine uptake, MDR cells were much more sensitive to an inhibitor of polyamine synthesis, methylglyoxal-bis guanylhydrazone (MGBG), suggesting that MDR cells might be defective in polyamine synthesis. These results also suggest that HGBG can be used for treatment of MDR in vivo.

• 대한약리학회지
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• 제32권1호
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• pp.113-123
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• 1996

세포내 polyamine은 DNA 구조 뿐 아니라 전사과정, 세포의 성장, 분화, 및 증식 등에 간여하는 바, 배양 흉선세포의 apoptosis 을 억제한다고 한다. 따라서 dexamethasone에 의한 생쥐 흉선세포의 apoptosis 반응에 대한 polyamine의 억제작용을, polyamine 생성과 대사억제제들로 처치한 흉선세포의 일차배양실험에서 관찰하여, 그 결과를 A23187과 DHEA의 작용과 비교하였다. 1) 흉선세포 생존율이 dexamethasone, DHEA, A23187, DFMO, MGBG들에 의하여 직접 현저히 억제되며, aminoguanidine, putrescine, spermidine, 및 spermine들에 의해서는 영향을 받지 않았다. 2) 흉선세포 DNA의 분절화가 dexamethasone과 A2318T에 의하여 유의하게 증강되어 있으며 DHEA에 의하여도 다소 증가되었으나, DFMO, MGBG, aminoguanidine, putrescine, spermidine, 및 spermine들에 의하여는 크게 영향을 받지 않았다. 3) Dexamethasone에 의한 흉선세포의 apoptosis는 DHEA에 의하여 억제된 반면, DFMO, MGBG, 및 aminoguanidine에 의하여는 영향을 받지 않았다. Spermine은 dexamethasone과 A23187에 의한 세포생존율 감소를 유의하게 억제하였으며, A23187에 의한 세포생존율 감소는 putrescine과 spermidine에 의하여도 억제되는 경향을 보였다. 4) DFMO 및 MGBG에 의한 흉선세포 생존율 감소는 spermine에 의해 현저히 억제되었으나, putrescine과 spermidine에 의하여는 영향을 받지 않았다. 5) Dexamethasone을 DFMO 또는 MGBG와 병합처치하여 나타나는 흉선세포 생존율 감소는 각각 spermine과 putrescine에 의하여 유의하게 억제되었으나, aminoguanidine 또는 DHEA와 dexamethasone의 병합처치에 의한 생존율 감소는 polyamine 전처치에 의해 감소되지 않았다. 이상의 결과는 polyamine이 흉선세포의 apoptosis 반응을 억제할 수 있고, 이같은 억제효과의일부가 $[Ca^{2+}]_i$ 증가에 관련되는 신호전달과정과 연관될 뿐 아니라, 세포막의 polyamine transporter를 통한 polyamine 섭취가 이들의 생합성 또는 유리기능과 함께 세포내 polyamine 함량을 조정하므로, 흉선세포의 apoptosis에 억제적으로 작용할 수 있음을 시사하는 것으로 사료된다.

• Journal of Dairy Science and Biotechnology
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• 제32권2호
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• pp.77-92
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• 2014

발효식품은 식료품 중에서 생체 아민(biogenic amine, BA) 중독을 일으키는 문제점을 가지고 있는데, 우유 발효식품 중에서는 치즈가 티라민(tyramine), 히스타민(histamine), 푸트레신(putrescine) 등이 유해한 수치의 BA를 함유하고 있는 생산품이라고 추측되고 있다. 다시 말해서, 생체 아민(BA)은 생물학적 활동을 가진 유기 및 염기성, 질소 화합물로 주로 아미노산이 탈카르복실화 반응(decarboxylation)을 거쳐서 생성된다. 다양한 종류의 식품(예: 유제품) 은 높은 수치의 BA를 함유하고 있다. 치즈에서는 1 kg 당 1,000 mg 이상의 BA가 검출되기도 한다. BA 함유량이 높은 치즈를 섭취할수록 체내에 독성이 축적된다. 때문에 유제품 내의 BA 함유에 관한 특정한 규정이 없더라도 유제품 내에 BA가 축적되는 현상을 방지해야 한다. 높은 수치의 BA를 함유한 식품을 섭취하였을 경우에 발생하는 위험에 대한 인식이 점점 높아지고 있다. 유제품 제조 및 가공 시에 BA의 생합성 및 축적에 영향을 주는 요소를 과학기술적 측면에서 연구가 요구된다. 따라서 BA의 합성 및 축적에 영향을 미치는 요소에 대한 이해도가 높을수록 유제품의 BA 함유량을 감소시킬 수 있다. 이러한 BA는 3 가지의 조건이 만족될 때 합성이 이루어진다. (i) 이용 가능한 기질 아미노산(substrate amino acid)가 있어야 하며, (ii) 이화작용경로가 활성화된 미생물이 존재해야 하며, (iii) 탈카르복실화 반응활성 (decarboxylation activity)이 일어나기에 적합한 환경조건을 필요로 한다. 이 3가지의 조건들은 저온살균, starter culture의 사용, NaCl 농도, 시간, 숙성 및 보존 온도, pH, 온도, 숙성 후 기술적인 과정 등 여러 가지 요소에 영향을 받으며, 이 요소들에 대한 과학적인 이해가 요구된다. 또한 BA 생산과 관련된 요소들, 특히 환경 조건, BA 생산 미생물, 유전자 구성, BA 생산에 관여하는 생합성경로, 유제품 내에 존재하는 BA 및 BA 생산 미생물 검출 방법 등에 대한 연구가 집중적으로 향후 진행되어야 할 것이다.