Xanthan: enzymatic degradation and novel perspectives of applications.

The extracellular heteropolysaccharide xanthan, synthesized by bacteria of the genus Xanthomonas, is widely used as a thickening and stabilizing agent across the food, cosmetic, and pharmaceutical sectors. Expanding the scope of its application, current efforts target the use of xanthan to develop innovative functional materials and products, such as edible films, eco-friendly oil surfactants, and biocompatible composites for tissue engineering. Xanthan-derived oligosaccharides are useful as nutritional supplements and plant defense elicitors. Development and processing of such new functional materials and products often necessitate tuning of xanthan properties through targeted structural modification. This task can be effectively carried out with the help of xanthan-specific enzymes. However, the complex molecular structure and intricate conformational behavior of xanthan create problems with its enzymatic hydrolysis or modification. This review summarizes and analyzes data concerning xanthan-degrading enzymes originating from microorganisms and microbial consortia, with a particular focus on the dependence of enzymatic activity on the structure and conformation of xanthan. Through a comparative study of xanthan-degrading pathways found within various bacterial classes, different microbial enzyme systems for xanthan utilization have been identified. The characterization of these new enzymes opens new perspectives for modifying xanthan structure and developing innovative xanthan-based applications. KEY POINTS: • The structure and conformation of xanthan affect enzymatic degradation. • Microorganisms use diverse multienzyme systems for xanthan degradation. • Xanthan-specific enzymes can be used to develop xanthan variants for novel applications.

Activation of Subcutaneous Mast Cells in Acupuncture Points Triggers Analgesia.

This review summarizes experimental evidence indicating that subcutaneous mast cells are involved in the trigger mechanism of analgesia induced by acupuncture, a traditional oriental therapy, which has gradually become accepted worldwide. The results are essentially based on work from our laboratories. Skin mast cells are present at a high density in acupuncture points where fine needles are inserted and manipulated during acupuncture intervention. Mast cells are sensitive to mechanical stimulation because they express multiple types of mechanosensitive channels, including TRPV1, TRPV2, TRPV4, receptors and chloride channels. Acupuncture manipulation generates force and torque that indirectly activate the mast cells via the collagen network. Subsequently, various mediators, for example, histamine, serotonin, adenosine triphosphate and adenosine, are released from activated mast cells to the interstitial space; they or their downstream products activate the corresponding receptors situated at local nerve terminals of sensory neurons in peripheral ganglia. The analgesic effects are thought to be generated via the reduced electrical activities of the primary sensory neurons. Alternatively, these neurons project such signals to pain-relevant regions in spinal cord and/or higher centers of the brain.

Modulation of extracellular ATP content of mast cells and DRG neurons by irradiation: studies on underlying mechanism of low-level-laser therapy.

Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca(2+)]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

Genistein as antiviral drug against HIV ion channel.

Various drugs found in Chinese herbs are well known for their antiviral potency. We have tested several flavonoids with respect to their potency to block the viral protein U of the human immunodeficiency type 1 virus, which is believed to form a cation-permeable ion channel in the infected cell. We used Xenopus oocytes with heterologously expressed viral protein U as model system to test the efficacy of the drugs in voltage-clamp experiments. This method had been demonstrated in the past as a useful tool to screen drugs for their potency in inhibition of ion channel activity. The viral protein U-mediated current could be inhibited by Ba(2+) with a K1/2 value of 1.6 mM. Therefore, we determined viral protein U-mediated current as current component blocked by 10 mM Ba(2+). We screened several flavonoids with respect to their effects on this current. The flavonols quercetin and kaempferol, and the flavanols (-)epigallochatechin and (-)epichatechin were ineffective. The flavanone naringenin showed at 20 µM slight (about 10%) inhibition. The most potent drug was the isoflavon genistein which exhibited at 20 µM significant inhibition of about 40% with a K1/2 value of 81 ± 4 µM. We suggest that viral ion channels, in general, may be a good target for development of antiviral agents, and that, in particular, isoflavons may be candidates for development of drugs targeting viral protein U.

Kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus.

The protein coded by the open-reading-frame 3a of SARS coronavirus has been demonstrated to form a cation-selective channel that may become expressed in the infected cell. The activity of the channel is involved in the mechanism of virus release. Drugs that inhibit the ion channel can, therefore, inhibit virus release, and they could be a source for development of novel therapeutic antiviral agents. Various drugs found in Chinese herbs that are well known as anticancer agents also have an antiviral potency. Here we tested the flavonols kaempferol, kaempferol glycosides, and acylated kaempferol glucoside derivatives with respect to their potency to block the 3a channel. We used the Xenopus oocyte with a heterologously expressed 3a protein as a model system to test the efficacy of the flavonols. Some of these drugs turned out to be potent inhibitors of the 3a channel. The most effective one was the glycoside juglanin (carrying an arabinose residue) with an IC50 value of 2.3 µM for inhibition of the 3a-mediated current. Kaempferol derivatives with rhamnose residue also seem to be quite effective. We suggest that viral ion channels, in general, may be a good target for the development of antiviral agents, and that, in particular, kaempferol glycosides are good candidates for 3a channel proteins of coronaviruses.

ATP release from mast cells by physical stimulation: a putative early step in activation of acupuncture points.

In Chinese medicine acupuncture points are treated by physical stimuli to counteract various diseases. These stimuli include mechanical stress as applied during the needle manipulation or tuina, high temperatures as applied during moxibustion, and red laser light applied during laser acupuncture. This study aimed to investigate cellular responses to stimuli that might occur in the tissue of acupuncture points. Since they have a characteristically high density of mast cells that degranulate in response to acupuncture, we asked whether these processes lead to ATP release. We tested in in vitro experiments on mast cells of the human mast-cell line HMC-1 the effects of the physical stimuli; mechanical stress was applied by superfusion of the cells with hypotonic solution, heat was applied by incubation of the cells at 52°C, and red laser light of 657 nm was used for irradiation. We demonstrate that all the stimuli induce ATP release from model human mast HMC-1 cells, and this release is associated with an intracellular free Ca(2+) rise. We hypothesize that ATP released from mast cells supplements the already known release of ATP from keratinocytes and, by acting on P2X receptors, it may serve as initial mediator of acupuncture-induced analgesia.

Regulation of the cardiovascular function by CO2 laser stimulation in anesthetized rats.

Physical stimulation of body surface points is known to affect various organ functions. In traditional Chinese medicine, so-called acupoints were defined. These points can be physically stimulated to effectively treat various diseases. Here we describe for the first time the effect of CO(2) laser stimulation at the acupoints Neiguan (PC-6), Quchi (LI-11), Zusanli (ST-36), and Taichong (LR-3) on heart rate and mean arterial blood pressure in anesthetized rats. CO(2) laser stimulation increased the skin surface temperature to 54°C. Our results revealed that the laser stimulation at the left or right PC-6 and LR-3 increased heart rate and mean arterial pressure. There was no response of heart rate and mean arterial pressure during and after stimulation of the left LI-11, but laser stimulation at the right LI-11 slightly increased heart rate and mean arterial pressure. On the other hand, laser stimulation at the left and right ST-36 decreased heart rate and mean arterial pressure. The effects on mean arterial pressure were more pronounced than those on heart rate. After full spinal cord transection, all heart-rate and mean-arterial-pressure responses were attenuated or completely abolished. These results suggest that CO(2) laser stimulation at either the left or right PC-6, ST-36, and LR-3, as well as at the right LI-11 can modulate the cardiovascular functions in anesthetized rats, and its modulatory site might be supraspinal.

Inhibition of Activity of GABA Transporter GAT1 by δ-Opioid Receptor.

Analgesia is a well-documented effect of acupuncture. A critical role in pain sensation plays the nervous system, including the GABAergic system and opioid receptor (OR) activation. Here we investigated regulation of GABA transporter GAT1 by δOR in rats and in Xenopus oocytes. Synaptosomes of brain from rats chronically exposed to opiates exhibited reduced GABA uptake, indicating that GABA transport might be regulated by opioid receptors. For further investigation we have expressed GAT1 of mouse brain together with mouse δOR and µOR in Xenopus oocytes. The function of GAT1 was analyzed in terms of Na(+)-dependent [(3)H]GABA uptake as well as GAT1-mediated currents. Coexpression of δOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. Activation of δOR further reduced the rate of GABA uptake as well as GAT1-mediated current. Coexpression of µOR, as well as µOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. Inhibition of GAT1-mediated current by activation of δOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia.

Stimulation of TRPV1 by Green Laser Light.

Low-level laser irradiation of visible light had been introduced as a medical treatment already more than 40 years ago, but its medical application still remains controversial. Laser stimulation of acupuncture points has also been introduced, and mast-cells degranulation has been suggested. Activation of TRPV ion channels may be involved in the degranulation. Here, we investigated whether TRPV1 could serve as candidate for laser-induced mast cell activation. Activation of TRPV1 by capsaicin resulted in degranulation. To investigate the effect of laser irradiation on TRPV1, we used the Xenopus oocyte as expression and model system. We show that TRPV1 can functionally be expressed in the oocyte by (a) activation by capsaicin (K(1/2) = 1.1 µM), (b) activation by temperatures exceeding 42°C, (c) activation by reduced pH (from 7.4 to 6.2), and (d) inhibition by ruthenium red. Red (637 nm) as well as blue (406 nm) light neither affected membrane currents in oocytes nor did it modulate capsaicin-induced current. In contrast, green laser light (532 nm) produced power-dependent activation of TRPV1. In conclusion, we could show that green light is effective at the cellular level to activate TRPV1. To which extend green light is of medical relevance needs further investigation.

Effects of alpha-asarone on the glutamate transporter EAAC1 in Xenopus oocytes.

The major excitatory neurotransmitter transporter EAAC1 in the mammalian central nervous system is considered a possible target for Chinese herbal medicine. Extracts of Acorus tatarinowii (Schott) were tested for their effects on EAAC1 activity. XENOPUS oocytes with heterologously expressed EAAC1 were used as the model system. Rate of glutamate uptake was determined by means of the isotopic tracer technique. Glutamate-induced current was recorded under a two-electrode voltage clamp. As a highly effective component, alpha-asarone was identified. The rate of glutamate uptake was stimulated by 200 microM of alpha-asarone by about 15 %. In contrast, the same concentration reduced the EAAC1-mediated current by about 35 % at a holding potential of - 60 mV; half maximum inhibition was obtained at about 60 microM. Our experimental data suggest that both stimulation of glutamate uptake and inhibition of EAAC1-mediated current by alpha-asarone could contribute to reduced excitatory activity.

[Skin slice used as a model for investigating acupuncture effects].

OBJECTIVE: To establish an acupoint-connective tissue model for studying the mechanism of acupuncture by using in vitro patch clamp technique in the rat skin slices. METHODS: The local connective tissue under the corium of "Housanli" (ST 36) area from SD rat was acutely and bluntly separated and fixed in a chamber filled with artificial incubation solution. Mast cells in the prepared connective tissue slice were labeled by toluidine blue (TB) or neutral red (NR). The whole-cell current of mast cells responding to pressure stimulation applied through a patch pipette was recorded in rat slices derived from acupoint ST 36 area by using in vitro patch-clamp technique. RESULTS: 1) After staining with TB and NR, the labeled mast cells were found to distribute in the extracellular matrix of the connective tissue samples, and their degranulation phenomenon could be seen clearly. 2) The whole-cell current of mast cells in response to mechanical stress stimulation was successfully recorded in the connective tissue slices of the rat acupoint ST 36 area. The cellular membrane currents increased evidently when pressure gradients of -30, -60 or -90 cmH2O were applied to the recorded mast cells. CONCLUSION: The connective tissue slice from the rat ST 36 area may be used as a model for investigating the peripheral mechanism of acupuncture by combining the microtechniques and electrophysiological techniques. The results obtained in this model prove for the first time by electrophysiology that the mast cells in the connective tissue are probably involved in the transduction process of the mechanical signal from acupuncture stimulation. This new model provides a base for investigating the characters of the cells, collagen fibers, proteoglycans, etc. and their interactions in the acupoint connective tissue in the future.

[Electroacupuncture alleviates complete Freund's adjuvant peripheral chronic inflammatory pain in mice].

OBJECTIVE: To probe into the law of acupuncture analgesic effect and specificities of acupoint action. METHODS: Adult male Kunming mice were randomly divided into a control group, a model group, a model plus electroacupuncture (EA) group and a model plus sham EA group. Chronic inflammatory pain model was prepared by injection of complete Freund's adjuvant (CFA) into right posterior foot, and paw withdrawing latency (PWL) induced by radiation heat was used as pain threshold index, and changes of PWL in all the groups were investigated. RESULTS: After modeling, PWL significantly shortened on the inflammatory side (P < 0.05); EA at bilateral "Zusanli" (ST 36) and "Kunlun" (BL 60) could significantly reverse the shortened PWL (P < 0.05), and this effect was prolonged along with increase of EA times, but in the sham EA group PWL did not significantly change; EA at bilateral "Shousanli" (LI 10) and Neiguan" (PC 6) could not reverse the shortened PWL (P > 0.05). CONCLUSION: Analgesic effect of EA is strengthened along with increase of EA times and shows specificity of acupoints to a certain extent.

Interaction of δ-opioid Receptor with Membrane Transporters: Possible Mechanisms in Pain Suppression by Acupuncture / 针灸推拿医学（英文版）

Objective: To investigate the possible mechanisms in acupuncture analgesia by interaction of &opioid receptor with neurotransmitter transport proteins or the Na+-K+pump. Methods: Microinjection of respective heterologous cRNA into the Xenopus oocytes as a model system, and measurement of steady-state currents under two-electrode voltage clamp. Results: The co-expression of the δ-opioid receptor with GAT1, EAAC1 or the sodium pump resulted in reducing activity of the respective transporter. Opioid receptor activation affected transporter activity in different ways: 1) GAT1 was further inhibited; 2) EAAC1 was stimulated; 3) Na+-K+ pump activity interfered with agonist sensitivity of DOR. Pump inhibition led to higher sensitivity for DPDPE. Conclusion: GABA transporter inhibition and glutamate transporter stimulation may counteract pain sensation by affecting the neurotransmitter concentration in the synaptic cleft and, therefore, may contribute synergistically to pain suppression by acupuncture. Sodium pump inhibition by endogenous ouabain may amplify these effects. These synergistic effects may be the molecular mechanism of inhibiting pain sense and/or acupuncture analgesia.

Investigation of the Role of Mast Cells in Acupuncture Effects and Their Sensitivity to Physical Stimuli During TCM Treatment / 针灸推拿医学（英文版）

Objective: To better understand the function of mast cells in acupuncture points (acupoints) inacupuncture-induced analgesia. The author tested their sensitivity to mechanical, thermo and light stimulation.Methods: The tail flick model was applied to measure analgesia in rats, and the author determined the density of mast cells in tissue slices and their degranulation ratio before/after acupuncture. The author also applied the patch-clamp technique to investigate activation of human mast cells (HMC 1 cell line) by mechanical stress or noxious heat, and the author optically observed degranulation phenomena of mast cell in response to red laser light.Results: Manual stimulation by acupuncture at Zusanli (ST 36) of the rat resulted in analgesia and the effect was more pronounced than after stimulation of a sham point nearby the acupuncture point. A higher density of mast cells was found at Zusanli (ST 36) than at the sham point, and acupuncture caused a remarkable increase in degranulation. Pretreatment with disodinm chromoglycate (DSCG, a mast cell stabilizer) injected into Zusanli (ST 36) impaired the analgesic effect of acupuncture. In whole-cell patch-clamp, increasing hydrostatic pressure induced a current that could be inhibited by 10 ～tM of ruthenium red (RuR), an inhibitor of TRPV2. Temperatures above 50～C, which are reached during moxibustion, induced a similar RuR-sensitive current. Laser light of 640 nm (48 mW) applied to acupoints had been shown previously to be highly effective in analgesia. 20 min of light application induced pronounced degranulation in HMC 1 that could be blocked by 0.02 g/mL DSCG as well as by RuR. Conclusion: The author found that mechanical stimulation of acupoints is associated with mast cell degranulation and mast-cell degranulation can be correlated with acupuncture-induced analgesia. The author suggest that TRPV2 plays a key role in mast-ceU degranulation in response to mechanical, thermal (moxibustion) as well as laser-light stimulation forming a facilitating step in acupuncture-induced analgesia.

Skin Slice as a Model for the Investigation of the Role of Mast Cells in Acupuncture Effects / 针灸推拿医学（英文版）

Obiective:To establish a new and better model to investigate the properties of mast cells that couldbe involved in acupuncture process mechanisms.Methods:Connective tissue under the conum at the area of acupuncture point Zusanli(ST 36)from rat was acutely bluntly separated with forceps and scissors。And incubated in bath solution up to several hours.Mast cells in slices of that tissue were irradiated with laser light of 650 mn,and changes in the appearance were observed under mlcroscope. In addition.patch.clamp technique in whole-cell configuration was employed to induce mechano-sensitive currents by pressure applied through the patch pipette.Results:1)A high density of mast cells embedded in the extracellular matrix was detected in the tissue slices using toluidine blue staining.The mast cells survived for up to several hours;2)Laser irradiation for 10 min(40 mW) resulted in fusion of intracellular vesicles with the plasma membrane of the mast-cell surface;3)Whole.cell currents increased when pressure gradients of-30 cm,-60 cm or-90 cm H20 were applied,the response was attenuated in the presence of 1 0 u M Ruthenium Red(a common blocker of channels 0f the vallinoid.sensitive transient receptor potential family TRPV).Conclusion:The skin tissue slice of rat Zusanli(ST 36)may be used as a model to investigate the role of mast cells in acupuncture.t he results obtained in this model support the suggestion that skin mast cells play a role In laser as well as mechanical acupuncture and TRPV channels may be involved in acupuncture effects.

Effects of α-Asarone on the Neuronal Glutamate Transporter EAAC1 / 针灸推拿医学（英文版）

Objective: α-asarone is a major effective component that can be isolated from Acorus tatarinowii Schott,a Chinese herbal medicine. Clinical investigations have shown that α-asarone has strong sedative and anti-convulsive action in the central nervous system. In recent years, several medicines containing a-asarone were applied in treatment of asthma, bronchitis, expectorant, or epilepsy. However, the underlying cellular mechanism of ct-asarone is still unknown. Here the authors considered EAAC1, the transporter for the excitatory glutamate, as a possible target. Methods: Supercritical CO2 fluid extraction and silica gel column chromatography were used to obtain ct-asarone from the rhizomes of Acorus tatarinowii Schott. Xenopus oocytes with heterologously expressed EAAC 1 were used as a model system. Rate of glutamate uptake was measured by means of isotopic tracer technique. Glutamate-induced current was recorded under two-electrode voltage clamp. 40μg/mL of ct-asarone was used for testing its effect on EAAC1 activity. Results: ct-asarone induced a slight, but still significant stimulation of rate of glutamate uptake by 15%. In contrast, EAACl-mediated current became reduced (by 30% at -100 mV). Since EAAC 1 can operate in transport and also in an ion-channel mode, the result indicates strong inhibition of the channel mode. This inhibition is voltage-dependent becoming larger at more negative potentials. Conclusion: The stimulation of glutamate uptake reduces glutamate concentration in the synaptic cleft and, hence, reduces excitatory synaptic activity. The inhibition on the ion-channel mode stabilizes the membrane potential, and therefore, also contributes to reduced excitatory activity.