In vitro inhibition of proliferation of HL-60 cells by tetrandrine and coriolus versicolor peptide derived from Chinese medicinal herbs.

Coriolus versicolor polysaccharide peptide (CVP) and the bis-benzylisoquinoline alkaloids, tetrandrine (TET) and berbamine (BER), the active ingredients isolated from Chinese medicinal herbs known to possess antitumor activities, concentration-dependently inhibited the proliferation of human leukemic HL-60 cells. CVP did not affect the growth of normal human peripheral blood lymphocytes (PBL), whereas TET elicited concentration-dependent cytotoxic effects. Morphological observation and DNA analysis revealed that CVP elicited no effect on the morphological features of HL-60 cells and did not cause DNA fragmentation, but TET and BER caused cell shrinkage with the formation of apoptotic bodies, and showed clear evidence of DNA fragmentation. These findings indicate that TET and BER, but not CVP, inhibited the proliferation of HL-60 cells via induction of apoptosis.

Electroacupuncture and laser stimulation treatment: evaluated by somatosensory evoked potential in conscious rabbits.

Tooth pulp generated somatosensory evoked potential (TPSEP) recordings were used to evaluate analgesic responses to Electroacupuncture (EA) and laser stimulation (Ls) treatments in Dutch-hybrid male rabbits. TPSEP recorded 100 trials at 30 minute intervals for 120 minutes. Three groups, EA, Ls and control were analyzed. The EA group received intermittent 4 Hz, intensity 2-10 volts electro-stimulation for twenty minutes. In the Ls group, a gallium aluminium arsenide (GaAlAs) laser diode with wavelength 780 nm, switched pulse frequency 9720 Hz, energy density 0.6 J/point was employed. Both EA and Ls groups received stimulation of the points Ho-ku (LI-4) and Tzu-San-Li (ST-36). Results showed tooth pulp generated noxious stimulation produced a consistent late near-field SEP waveform, which is similar to those recorded in humans and correlated to the sensation of pains, thus confirming that tooth pulp stimulation is a reliable dolormetric index. EA and Ls TPSEP recordings showed decreased peak-wave amplitude in late near-field components. These decreases correlate to analgesia. This technique provides an objective and reliable dolormetric index.

Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant.

The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (lambda max = 510 nm) when excited with ultraviolet (UV) or blue light (lambda max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802-805; Stearns, Curr. Biol. 5 (1995) 262-264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501-12504] in a process involving cyclization of a Tyr66 aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151-154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with red-shifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (lambda max = 505 nm), but has a single excitation peak (lambda max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389-396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.

Red-shifted excitation mutants of the green fluorescent protein.

Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP. Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins. Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus. These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism. In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.

The anti-tumor effect of a small polypeptide from Coriolus versicolor (SPCV).

A new small polypeptide was isolated from the crude extraction of polysaccharide peptide of Coriolus versicolor (Cov-1) by HPLC and CIEF. It has a smaller molecular weight (10K) compared with that of PSP (100K) and was named small peptide of Coriolus versicolor, SPCV. It was found that SPCV possesses potent cytotoxic effect on human tumor cell lines of HL-60, LS174-T, SMMU-7721, and SCG-7901. The IC50 of SPCV on HL-60 was 30 micrograms/ml. The inhibition rates of leukemia cells and SCG-7901 were significantly higher in SPCV treated group than that in PSP and PSK groups. SPCV also has immunopotentiating effect as it increased WBC and IgG levels. Pretreatment of SPCV for two weeks decreased the incidence of tumor mass in nude mice inoculated with tumor cells.

Effect of acupuncture on immunoglobulins of serum, saliva and gingival sulcus fluid.

The present research is a preliminary investigation of the changes of immunoglobulins in serum, saliva and gingival sulcus fluid in response to acute and chronic acupuncture stimulation in 70 volunteers. The acute effect of acupuncture showed that 30 minutes and 24 hours after acupuncture treatment saliva IgA levels were significantly increased in those who had previous low levels of IgA, but decreased in those who had previous high levels. When acupuncture was given daily for 2 weeks, the saliva IgA level was significantly increased, about 20% higher than that of the initial value. The IgG levels in serum and gingival sulcus fluid were decreased at 30 minutes after acupuncture treatment. On the other hand, chronic acupuncture stimulation (daily for 7-10 days) significantly increased IgG levels in both serum and gingival sulcus fluid. The regulatory effect of acupuncture on the immunological function of body defense system is discussed.

Evaluation on the treatment of morphine addiction by acupuncture Chinese herbs and opioid peptides.

Experimental studies on the effects of acupuncture, combined Chinese herbs, and opioid peptides on morphine withdrawal symptoms were carried out in 119 addicted rats. Electroacupuncture was found to be the most effective method as it reduced the morphine withdrawal scores to -85%. The combined herbs, Qiang Huo, Gou Teng, Chuan Xion, Fu Zi and Yan Hu Suo suppressed the withdrawal scores of -68%. The opioid peptides, endorphin, enkephalin, and dynorphin, produced marked sedative effect and alleviated the withdrawal symptoms, reducing the scores from -28% to -74%. It is suggested that acupuncture and herbs, being non-opiate and having less side effect, might be used as alternative or supplementary treatment on morphine addiction.

Further study of the neurohumoral factor, endorphin, in the mechanism of acupuncture analgesia.

Cross circulation was performed between the femoral arteries and veins of paired rabbits that were entirely conscious. Jaw opening reflex by dental pulp stimulation was used as pain index. When acupuncture was applied to the donor rabbit the pain thresholds of both the donor and recipient were elevated from 30 minutes to one hour after acupuncture stimulation and lasted for another 30 minutes after withdrawal of acupuncture. The analgesic effect in both the donor and recipient was abolished by the pretreatment of naloxone. The increase in the pain threshold of the non-acupunctured recipient was due to an opiate-like, humoral substance generated from the acupuncture donor. The possible role of endorphins in the mechanism of acupuncture analgesia is discussed.

Endorphin release: a possible mechanism of acupuncture analgesia.

The action of acupuncture stimulation on analgesia has been investigated. The brain and serum extracts of acupunctured rabbits injected into rabbits produced a marked analgesic effect on the recipient, as shown by a great increase of their pain threshold. This effect is counteracted by a specific opiate anatagonist, naloxone. The data suggest that the release of the endogenous substances with morphine-like biological properties, endorphins, is increased by acupuncture stimulation, thus inhibiting pain perception.