A STUDY OF THE GLUCOCORTICOID RECEPTOR OF THE CYTOSOL AND NUCLEI IN NONLETHALLY AND LETHALLY SCALDED RATS

The glucocorticoid receptor (GCR) of the cytosol of liver and brain was studied by radioligand binding assay, using [~3H] dexamethasone (Dex) as the ligand in lethally and nonlethally scalded rats. As compared with controls, the binding capacity (R_0) was decreased and the apparent K_d of [~3H] Dex specific binding was increased in both scalded groups. In the lethal group, the R_u is much lower than that in the nonlethal group. In order to determine whether the decrease of R_0 was due to the translocation of [~3H] Dex-GCR complex into the cell nuclei, the [~3H] corticosterone (B) specific binding of the hepatic nuclei was measured by the exchange assay. There was no significant difference between the control and scalded groups. The possible mechanisms and the clinical significance of these changes were discussed.     

SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION

We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.     

ENHANCEMENT OF THE SALT EFFECT ON THE CONFORMATION OF M. L. DNA MOLECULES BY METHYLATION

Methylated Micrococcus luteus DNA (M. L. DNA) in which the DNA methylation level was 2.39% was prepared by using Hhal DNA methylase. (The natural M. L. DNA is completely unmethylated.) The [θ]_(275) value of M. L. DNA was decreased and the —[θ]_(220-250) value was increased with increasing concentration of MgC1_2. The UV spectra of M. L. DNA at different concentration of MgCl_2 showed that a hypochromisity happens at 260nm with increasing concentration of MgC1_2. The heat-releasing of M. L. DNA also increased with increasing concentration of MgC1_2. A11 these results showed that the M. L. DNA molecules tend to shrink in space, The difference between methylated DNA and unmethylated DNA was compared. The sensitivity of methylated DNA to MgC1_2 is 40—70 fold higher than that of the unmethylated M. L. DNA.     

INTERACTION BETWEEN ADENOSINE-5′-MONOPHOSPHATE AND FRUCTOSE 1,6-BISPHOSPHATASE AND MECHANISM OF ALLOSTERIC INHIBITION——ULTRAVIOLET SPECTROSCOPIC STUDIES

Ultraviolet difference spectroscopic studies of AMP and dAMP upon binding to snake muscle fructose 1, 6-bisphosphatase showed that both of the ligands interacting with the enzyme generated three positive peaks located at 270 nm, 280 nm and 288 nm. The binding constants calculated from the titration curve of difference spactra by the successive addition of nueleotides were 2.0×10~6M~(-1) and 1.2×10~6M~(-1) respectively. The fact of the same binding constants for the two ligands indicated that the unique inhibition behavior of dAMP differing from AMP was not due to the difference of binding between AMP and dAMP, but due to the absence of 2'-OH group on the ribose of dAMP. In contrast to the difference spectra of AMP binding, the U. V. difference spectra produced by tryptic digestion of fructose 1, 6-bisphosphatase gave two negative bands at 280 nm and 288 nm and had a close resemblance to that of the enzyme in urea. The opposite signs of the two kinds of difference U. V. spectra produced by AMP binding     

STUDY ON THE EFFECT OF POLYSTYRENE SULFONIC ACID MACROMOLECULES ON THE POLYMERIZATION OF ACRYLAMIDE

The effect of polystyrene sulfonic acid (PSSA) macromolecules on the polymerization of acrylamide (AM) has been studied. It was found that the rates of polymerization of AM were greatly increased in the presence of PSSA in the polymerization system.The maximum value of the rate of polymerization of AM was obtained when the ratio of [—SO_3H]: [AM] reached 3:1. When the insoluble crosslinked PSSA was used instead of the soluble one, this effect decreased considerably. The interaction between molecules of PSSA and AM was determined by infrared spectroscopy, elementary analysis and X-ray photoelectron spectroscopy. The combination form, —SO_3-NH_3~ CO—, formed between sulfonic group and amide group was found to be existed since the infrared absorption band of —NH_2 shifted from 3400cm~(-1) to 3150 cm~(-1), the binding energy of electron N_(18) changed from 399.7 eV to 401.3 eV, and the atomic ratio of N to S of the products was similar to the ratio of reagents. Based on these experimental results, the mechanism of AM polymerization in the presence of PSSA is proposed. The initial step is the combination of AM with sulfonic group to form —CONH_3~ , then followed by polymerization on the PSSA macromolecule. The role of PSSA on the polymerization of AM is discussed.     

THE POSSIBLE MECHANISM OF BINDING INTERACTION OF INSULIN MOLECULE WITH ITS RECEPTOR

Detailed structural comparisons and investigation of DPI, 2Zn insulin and some other derivatives of insulin were performed by the least-squares superimposition technique and the graphics technique. It is pointed out in this paper that the binding interaction with the receptor molecule should take place mainly on an amphipathic surface of the insulin molecule. In the middle, there is a hydrophobic surface with an area of about 150 consisting of many hydrophobic residues; while the polar or charged groups distributing around the hydro. phobic surface construct a hydrophilic zone. The hydrophobic surface is usually covered by the extended B-chain C-terminal peptides with great mobility and protected from the solvent molecules. The angle between the amphipathic surface and the surface of dimerization is about 20 degrees. The results from the detailed structural comparison between A1-(L-Trp) insulin and A1-(D-Trp) insulin have provided a very good explanation to their great difference in biological activity,     

THE SOLID SOLUTION PHASE DIAGRAM OF THE MIXTURE OF ORTHO- AND PARACHLOROTOLUENES

The complete miscible solid solution with a minimum freezing point (-50.75℃; 26.88% para-) of the ortho- and para-chlorotoluene has been proved in this paper by many methods as the thermal analysis method, the X-ray matter phase analysis, the fractional crystallized separation method and the solidified separation method of solid solution, and by the Fel'dman's experiential productivity formula compared with the theoretical productivity formula which was obtained from the solidified separation according to the solid solution phase diagram. Therefore, the international official conclusion of simple eutectic type phase diagram of ortho- and para-chlorotoluene mixture does not agree with the experimental facts.The powder diffraction files of ortho- and para-chlorotoluenes are given in this paper.     

REGULATION OF GLUCOCORTICOID RECEPTOR BY GLUCOCORTICOIDS(Ⅱ) ——THE STUDIES ON RAT LIVER, BRAIN AND SPLEEN

The concentration of glucocorticoids (GC) in plasma was maintained at stress level, 20--40μg/dl, for 3 days by subcutaneous injection of hydrocortisone (F) in polyvinyl alcohol (PVA) into rats, and the specific binding of [~3H]Dexamethasone (Dex) in liver, spleen and brain was determined before and after injection. The binding capacity of glucocorticoid receptor (GR) in liver and spleen was decreased significantly 1 h after injection and maintained at low level for several days after the concentration of GC in plasma had returned to the normal level. The K_d was not altered. The changes of GR in brain was not significant. Thus it may be concluded that GC can down-regulate GR in rats, but with different characteristics in various target organs.     

STUDIES ON TRANSPLANTATION OF THE LAMININ RECEPTOR AND ITS ROLES IN EXPERIMENTAL METASTASIS OF MURINE LEWIS LUNG CARCINOMA CELLS

The isolated laminin receptor (LN-R) labeled by ~(125)I was reconstituted into liposomes.~(125)I-LN-R-liposomes and free ~(125)I-LN-R were separated by Sepharose 4B column chromatogra-phy. The LN-R-liposomes showed affinity for laminin (LN) and were capable of binding toimmobilized LN substrate. In order to make transplantation of LN-R, LN-R-liposomes werefused with cultured murine Lewis lung carcinoma cells with the help of polyethylene glycol(PEG) induction. The radiation with the fused cells was not removed by salt sclution. Thebinding of the fused cells enriched in foreign LN-R to LN substrate increased by 87.5%.Furthermore, the murine Lewis lung carcinoma cells with and without transplanted LN-Rwere injected into C_(57)BL/6J mice through tail veins (5×10~5 cells/each mouse)respectively.The mice in the test group died earlier than those in the control group. The total weight oflung tumor in the test group remarkably increased in comparison with those in the controlgroup. The results taken together     

THE BIPHASIC EFFECTS OF SOME OPIATES AND OPIOID PEPTIDES ON RAT VAS DEFERENS

It has been found that the effects of opiates and opioid peptides on RVD are of two types. Compounds of the first type such as Etor, Eton, fentanyl and prodinc derivatives, Met-and Leu-Enk and DADL are biphasic in nature. At lower concentrations (10~(-8)-10~(-5)M) they have inhibitory effects and at higher concentrations (10~(-5)-10~(-4)M) they are excitatory. Compounds of the second type, such as Mor, benzomorphan derivatives, Cyc and SKF and the antagonist Nx have virtually no or only weakly inhibitory effects but may antagonize the inhibitory effect produced by the first group of compounds at lower concontrations. MC belongs to the second type, but it cannot antagonize the inhibitory effect of β-End. The results are discussed in terms of multiple receptors, the high and low affinity binding sites, and the interrelationship between receptors.     

DIPOLE POLARIZATION RELAXATION AND MOLECULAR STRUCTURE OF MAIN-AND SIDE-CHAIN LIQUID-CRYSTALLINE POLYMERS

Molecular mobility in thermotropic polyesters and side-chain polymers with different struc-ture of mesogens and spacers has been studied by dielectrical method in dilutesolutions. The results made it possible to establish the multiplicity of dielectric relaxationtransitions which reflects the small- and large-scale types of molecular motion. It was shownthat dielectric relaxation processes occurring in accordance with local mechanism (relaxationtimes 10~(-9)--10~(-7)s. and the activation energy 10--50kJ/mol) are due to the mobility of kineticchain elements of different length within a monomer units. It was found that the dielectricrelaxation process connected with a large-scale form of molecular motion (relaxation times10~(-5)--10~(-6)s. and the activation energy 100kJ/mol) did not depend on the molecular massbut was infiuenced by factors changing the conformational state of the macromolecule. It isestablished tha the cooperative reorientation mobility of associated mesogenic fragments isthe source of the large-scale process.     

Celestine blue as a new indicator in electrochemical DNA biosensors

Celestine blue(CB)was introduced as a new electroactive indicator in DNA biosensors.The interaction of CB with DNA was investigated by electrochemical and spectroscopic methods.The effect of buffer kind and p H on the electrochemical behavior of CB was studied.The peak currents of CB were linearly related to DNA concentration in the range of 5.0×10~(-9) to 1.0×10~(-7)mol/L.The detection limit of this approach was 4.76×10~(-10) mol/L.Based on spectrometry data a hypochromic effect was observed in UV-Vis spectra of CB with increasing DNA concentration.The results illustrate the possible interaction mode between CB and DNA is electrostatic binding.     

MOLECULAR STRUCTURE AND ABSOLUTE CONFIGURATION OF SUBEROGORGIN

In the present paper it is reported that the molecular structure and absolute configuration of poisonous suberogorgin are determined by using X-ray diffraction method. The crystal of suberogorgin belongs to orthogonal system with space group D_2~4-P2_12_12_1. The crystallographic parameters are: a=16.135, b=13.189, c=12.901, Z=8. The initial model of the crystal structure was solved by the direct method. The refinement of the strueture parameters was carried out by using the least square method and led to a final R-factor of 0.056. In accordance with the molecular structure of suberogorgin mentioned above, the solvent effect of NMR has been further discussed and the relationship between the molecular structure of suberogorgin and its toxicity has also been preliminarily investigated.     

THE INTERACTION OF ETHYL BENZOATE AND TRIFLUOROACETIC ACID IN EXCITED STATE

This paper is concerned with the interaction of ethyl benzoate (EBA) and trifluoroaceticacid (TFA) molecules in the excited state by measurements of steady--state and transient fluo-rescence spectra at room and low temperatures. The results show that not only the 1:1 exci-plex but also the 2:1 triplex between EBA and TFA was formed. The formation pathway ofthe triplex is that the exciplex formed at first, then by the interaction with the EBA mole-cule it transformed into triplex. But it could not be formed by the dimer of EBA by interac-tion with TFA. The dimer of EBA is confirmed in the concentrated solution. So there arefour components in EBA/TFA--concentrated solution and their fluorescence lifetimes aremeasured respectively.     

STUDIES ON THE PROPERTY OF SULFHYDRYL BINDING SITE ON THE LUNG NORMAL AND CANCER CELL MEMBRANE OF CHINESE HAMSTER WITH MALEIMIDE SPIN LABELS

In this paper, five maleimide spin labels with different chain lengths were used to studythe properties of binding sites of sulfhydryl groups on the meanbrane proteins of normal cellV_79 and cancer cells V_79-B_1 in the lung of Chinese hamster. The ratio of the strongly immobi-lized component to the weakly immobilized eomponent (s/w) and the rotational correlation time(τ_e) were calculated on the basis of ESR spectra. From the varying of the s/w and τ_c withthe ehain lengths of spilt labels it was deducted that the binding sites of sulfhydrylgroups on V_79 and V_79-B_1 membrane proteins were of eonical shape and that the binding sitesof sulfhydryl groups on V_79 membrane protein were deeper and narrower than those on V_79-B_1.     

AN INVESTIGATION ON THE ab initio CALCULATION OF INTERMOLECULAR INTERACTION——NON-BONDED INTERACTION IN THE HYDROGEN BOND COMPLEX (HF)_2

An ab initio calculation of the hydrogen bond complex (HF)_2 is given with the 6-311 G~(**) basis set, according to which the potential surface around the balance point of the distancès and the orientations between two HF molecules is obtained. The atomic charges in the system are calculated with the PD/LSF method (potential-derived/least-square-fitting method) and then an analysis of the hydrogen bond interaction between two HF molecules is given with the (exp-6-1) potential function, by means of which it is shown that the main interaction between them is not an electro-static but a charge transfer one. The potential curve between two HF molecules is like a Morse function.     

THE STRUCTURE BASIS OF THE POOR FIBRIN SPECIFICITY OF UROKINASE(Ⅱ)——THE INHIBITION OF UROKINASE A CHAIN 149--157 ON THE FIBRIN STIMULATED ACTIVATION OF PLASMINOGEN BY TISSUE TYPE PLASMINOGEN ACTIVATOR

In view of the similarity of the charge distribution between fibrin A_α148--161 and Achain 149--157 of urokinase,the latter might compete with fibrin A_α148--161 when singlechain pro-urokinase is converted to double chain urokinase.To test this, the stretch of uro-kinase A chain 135--157 was separated from the low molecular weight urokinase, a competi-tive binding between this stretch and fibrin to tPA kringle-2 was shown by radio-bindingassay. The inhibition of the stretch on the fibrin stimulated activation of plasminogen wasdemonstrated in the caseinolytic system. The synthesized novapeptide urokinase A chain 149--157 (R-peptide) showed a significant inhibition on the activation of plasminogen in the pres-ence of fibrin. By contrasting finely with R-peptide, a synthesized novapeptide in which Arg154and Arg156 were replaced by Asp (D-peptide) did not show any inhibition effect on the fi-brin stimulated activation of plasminogen by tPA. These results suggest that the positivelycharged residues in the     

THE STUDY OF PHYCOBILIPROTEINS IN Porphyra yezoensis——ASSOCIATION AND DISSOCIATION OF R-PHYCOERYTHRIN

In fresh Porphyra yezoensis, R-phycoerythrin exists mainly as octamer (αβ)_8 with only a small percentage of dimer (αβ)_2.The octamer could be dissociated into dimer in the presence of oxygen and visible light.The dimer was ultracentrifuged in a sucrose linear gradient to obtain the monomer,The monomer could be associated again into dimer.This reversible change between the monomer and dimer of R-phycoerythrin may be attributed to the shift of hydrogen bonds between forms of phycoerythrin molecules.     

THE STUDY OF ADAPTABILITY OF Drepanopus bispinosus TO TEMPERATURE AND SALINITY IN THE ANTARCTIC BURTON LAKE

The tolerance to temperautre and salinity of a Calanoida copepod: Drepanopus bispinosus from the Burton Lake in the Vestfold Hills, Antarctica, was tested in each season. The results obtained show that the copepod has a marked physiological property of tolerance to low temperature and high salinity, and this property could be strengthened after winter. The response to different temperature and salinity of the copepod evidently expressed the tardy characteristics contrasted with seasons. This means that for a long term in early winter(lower temperature and higher salinity) the animal still has higher tolerance to lower salinity and higher temperature; and it is opposite in early summer. These physiological features are possibly caused by natural acclimatization in the lake. The experimental data were processed and plotted by computer. The correlation of animal's survival rate with the changes of temperature and salinity presented an approximately smooth surface of a three-degree elliptic sphere.     

~1H NMR INVESTIGATION OF THE BINDING OF METHYL-β-D-GALACTOSES WITH SIALIC ACID RESIDUES ON HEPATIC BINDING PROTEIN——THE EFFECT OF DIVALENT CALCIUM ION

The present study is a proton NMR investigation of the influence of Ca~(2+) on the interaction between the intact sialic acid residues on HBP (Hepatic Binding Protein) and methyl-β-D-galactoses. The proton NMR spectra of HBP and methyl-β-D-galactose mixing solution at different Ca~(2+) concentratiou have been measured. The analysis of the experimental results indicates that Ca~(2+) participates in the binding of sialic acid residues on HBP with methyl-β-D-galactoses and this enables the galactose molecules to, be in stable bound state. The proton NMR spectra of the samples containing EDTA have confirmed this conclusion.