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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (8): 901-912.doi: 10.3736/jcim20110813

• Original Experimental Research • Previous Articles     Next Articles

Analysis of the capability of ultra-highly diluted glucose to increase glucose uptake in arsenite-stressed bacteria Escherichia coli

Anisur Rahman Khuda-Bukhsh1(), Arnab De1, Durba Das1, Suman Dutta1, Naoual Boujedaini2#br#   

  • Received:2011-02-25 Accepted:2011-04-21 Online:2011-08-20 Published:2011-08-15

Objective: Whether ultra-highly diluted homeopathic remedies can affect living systems is questionable. Therefore, this study sees value in the analysis of whether homeopathically diluted glucose 30C has any effect on Escherichia coli exposed to arsenite stress.
Methods: E. coli were cultured to their log phase in standard Luria-Bertani medium and then treated with either 1 mmol/L or 2 mmol/L sodium arsenite, with or without supplementation of either 1% or 3% glucose, an ultra-highly diluted and agitated ethanolic solution (70%) of glucose (diluted 1060 times), glucose 30C or 70% ethanol (placebo) in the medium. Glucose uptake, specific activities of hexokinase and glucokinase, membrane potential, intracellular adenosine triphosphate (ATP) and expression of glucose permease in E. coli were analyzed at two different time intervals. Arsenic content in E. coli (intracellular) and in the spent medium (extracellular) was also determined.
Results: In arsenite-exposed E. coli, the glucose uptake increased along with decreases in the specific activities of hexokinase and glucokinase, intracellular ATP and membrane potential and an increase in the gene expression level of glucose permease. Glucose uptake increased further by addition of 1%, 3% or ultra-highly diluted glucose in the medium, but not by the placebo.
Conclusion: The results demonstrated the efficacy of the ultra-highly diluted and agitated glucose in mimicking the action of actual glucose supplementation and its ability to modulate expressions of hexokinase and glucokinase enzymes and glucose permease genes, thereby validating the efficacy of ultra-high dilutions used in homeopathy.

Key words: arsenites, glucose uptake, Escherichia coli, hexokinase, glucokinase, adenosine triphosphate, membrane potentials, homeopathy

Figure 1

E. coli growth curve under different doses of sodium arsenite"

"

Primer name Primer sequences (5′-3′)
G-3-PDH (house-keeping gene) Forward: CCCACTAACATCAAATGGGG
Reverse: CCTTCCACAATGCAAAGTT
ptsG (glucose permease) Forward: CGGCGCTGACCTGGTTCCTG
Reverse: ACGGAACCGCCTGCTTCTGC

"

Group n Glucose uptake
45 min 90 min
Normal control 9 4.17±0.70 0.53±0.04
Normal control+1% glucose 9 114.50±4.50* 27.00±5.00*
Normal control+3% glucose 9 115.00±3.50* 29.50±2.00*
Normal control+placebo 30C 9 1.74±0.60 0.29±0.08
Normal control+glucose 30C 9 4.46±0.40* 0.62±0.07*
1 mmol/L arsenite 9 7.56±0.90* 2.87±0.50*
1 mmol/L arsenite+placebo 30C 9 7.47±1.00 2.90±0.59
1 mmol/L arsenite+glucose 30C 9 8.20±1.00 3.12±0.40
2 mmol/L arsenite 9 8.21±0.90* 3.10±0.25*
2 mmol/L arsenite+placebo 30C 9 8.12±1.00 2.98±0.50
2 mmol/L arsenite+glucose 30C 9 8.80±1.20 3.45±0.50

"

Group n Activity of hexokinase
45 min 90 min
Normal control 9 0.058±0.006 0.045±0.004
Normal control+glucose 30C 9 0.054±0.004* 0.036±0.002*
1 mmol/L arsenite 9 0.023±0.003* 0.021±0.001*
1 mmol/L arsenite+placebo 30C 9 0.020±0.004 0.020±0.003
1 mmol/L arsenite+glucose 30C 9 0.017±0.003 0.015±0.005
2 mmol/L arsenite 9 0.020±0.004* 0.017±0.002*
2 mmol/L arsenite+placebo 30C 9 0.020±0.002 0.016±0.002
2 mmol/L arsenite+glucose 30C 9 0.013±0.002 0.013±0.003

"

Group n Activity of glucokinase
45 min 90 min
Normal control 9 0.068±0.005 0.042±0.005
Normal control+glucose 30C 9 0.060±0.002* 0.038±0.002*
1 mmol/L arsenite 9 0.009±0.001* 0.007±0.001*
1 mmol/L arsenite+placebo 30C 9 0.009±0.002 0.007±0.001
1 mmol/L arsenite+glucose 30C 9 0.008±0.002△ 0.003±0.001△
2 mmol/L arsenite 9 0.009±0.002* 0.006±0.001*
2 mmol/L arsenite+placebo 30C 9 0.009±0.002 0.006±0.001
2 mmol/L arsenite+glucose 30C 9 0.007±0.001 0.003±0.002

"

Group n ATP measurement
45 min 90 min
Normal control 9 4.750±0.113 1.930±0.111
Normal control+glucose 30C 9 4.807±0.118* 1.995±0.115*
1 mmol/L arsenite 9 0.834±0.111* 0.579±0.115*
1 mmol/L arsenite+placebo 30C 9 0.834±0.115 0.575±0.113
1 mmol/L arsenite+glucose 30C 9 1.202±0.115△ 0.779±0.112△
2 mmol/L arsenite 9 0.819±0.115* 0.559±0.113*
2 mmol/L arsenite+placebo 30C 9 0.817±0.118 0.584±0.119
2 mmol/L arsenite+glucose 30C 9 1.776±0.115▲ 0.834±0.111▲

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Group n Membrane potential
45 min 90 min
Normal control 9 68.80±2.00 94.10±2.65
Normal control+glucose 30C 9 54.50±1.45* 88.70±2.40*
1 mmol/L arsenite 9 33.50±1.25* 65.80±1.55*
1 mmol/L arsenite+placebo 30C 9 31.90±1.50 63.70±1.80
1 mmol/L arsenite+glucose 30C 9 15.80±1.60 48.80±1.69
2 mmol/L arsenite 9 30.20±1.50* 62.70±1.90*
2 mmol/L arsenite+placebo 30C 9 28.70±1.95 61.10±2.00
2 mmol/L arsenite+glucose 30C 9 10.30±1.60 44.70±1.60

Figure 2

Analysis of membrane potential by flow-cytometry Cell membrane potential was measured by a flow-cytometer after two different doses of sodium arsenite insult. A: Normal control cells; B: Glucose 30C-treated control cells; C: 1 mmol/L arsenite-treated cells; D: 2 mmol/L arsenite-treated cells; E: 1 mmol/L arsenite and placebo-treated cells; F: 2 mmol/L arsenite and placebo-treated cells; G: 1 mmol/L arsenite and glucose 30C-treated cells; H: 2 mmol/L arsenite and glucose 30C-treated cells."

Figure 3

Gene expression analysis by reverse transcriptase-polymerase chain reaction Arbitrary band intensities of ptsG (glucose permease gene) were analysed against the house keeping gene (G-3-PDH) under different conditions. (a) and (d) represent the expression of house keeping gene after 1 mmol/L and 2 mmol/L of sodium arsenite addition, respectively; (b) Expression of ptsG at 45 min after the addition of 1 mmol/L arsenite; (c) Expression of ptsG at 90 min after the addition of 1 mmol/L arsenite; (e) Expression of ptsG at 45 min after the addition of 2 mmol/L arsenite; (f) Expression of ptsG at 90 min after the addition of 2 mmol/L arsenite. The components of different lanes are: Ln1: Normal control cells; Ln2: Sodium arsenite-treated cells; Ln3: Sodium arsenite plus placebo-treated cells; Ln4: Sodium arsenite plus glucose 30C-treated cells; Ln5: Normal control plus glucose 30C-treated cells."

"

Group n Band intensity
45 min 90 min
Normal control 9 1.00±0.10 1.00±0.09
Normal control+glucose 30C 9 1.19±0.06* 1.31±0.09*
1 mmol/L arsenite 9 1.14±0.04* 1.16±0.07*
1 mmol/L arsenite+placebo 30C 9 1.04±0.08 1.03±0.06
1 mmol/L arsenite+glucose 30C 9 1.22±0.05 1.28±0.09
2 mmol/L arsenite 9 1.12±0.10* 1.27±0.12*
2 mmol/L arsenite+placebo 30C 9 1.09±0.20 1.07±0.12
2 mmol/L arsenite+glucose 30C 9 1.41±0.12 1.42±0.15

"

Group n Gene expression
45 min 90 min
Normal control 9 1.00±0.15 1.00±0.18
Normal control+glucose 30C 9 1.15±0.25* 1.56±0.88*
1 mmol/L arsenite 9 1.67±0.49* 4.11±0.89*
1 mmol/L arsenite+placebo 30C 9 1.50±0.49 3.18±1.25
1 mmol/L arsenite+glucose 30C 9 2.53±0.50 5.02±0.65
2 mmol/L arsenite 9 3.83±0.20* 6.72±0.45*
2 mmol/L arsenite+placebo 30C 9 2.28±0.19 6.20±0.44
2 mmol/L arsenite+glucose 30C 9 4.75±0.50 7.84±0.68

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Group n Arsenic measurement
45 min 90 min
Normal control 9 196.67±17.0 227.32±20.0
Normal control+
glucose 30C
9 218.90±21.0* 253.00±25.0*
1 mmol/L arsenite 9 295.79±23.0* 321.24±26.0*
1 mmol/L arsenite+
placebo 30C
9 289.93±22.0 314.71±28.0
1 mmol/L arsenite+
glucose 30C
9 337.50±22.0△ 356.80±27.5△
2 mmol/L arsenite 9 368.18±26.0* 419.05±32.0*
2 mmol/L arsenite+
placebo 30C
9 364.92±25.0 413.83±30.5
2 mmol/L arsenite+
glucose 30C
9 398.00±24.6▲ 445.80±31.8▲

"

Group n Arsenic measurement
45 min 90 min
Normal control 9 1.54±0.12 0.35±0.02
Normal control+
glucose 30C
9 1.32±0.15* 0.22±0.05*
1 mmol/L arsenite 9 19.24±1.50* 2.79±0.80*
1 mmol/L arsenite+
placebo 30C
9 19.62±1.80 2.81±0.70
1 mmol/L arsenite+
glucose 30C
9 16.90±1.80△ 2.54±0.90△
2 mmol/L arsenite 9 22.04±2.34* 3.44±1.10*
2 mmol/L arsenite+
placebo 30C
9 21.82±2.00 3.50±0.85
2 mmol/L arsenite+
glucose 30C
9 20.40±2.12▲ 2.80±1.20▲
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[9] Bing Hu , Hong-mei An , Ke-ping Shen , Qin Du. Senescence-inducing effects of Chinese herbal medicine Tenglong Buzhong Decoction on human colon carcinoma LS-174-T cells and the mechanism. Journal of Chinese Integrative Medicine, 2010, 8(11): 1048-1052
[10] Ya-bing Zhou, Jian-er Yu, Jie Wu, Li Bai, Li-li Huo, Xin-guang Zhang, Li-qing Li . Effects of Chinese herbal medicine Bushen Gubiao Recipe on toll-like receptor 4 and CD4 +CD25 +foxp3 +regulatory T cells in mice with recurrent respiratory tract infections. Journal of Chinese Integrative Medicine, 2010, 8(11): 1053-1059