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We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity.

Studies of HIV molecular evolution and pathogenesis have relied on the polymerase chain reaction (PCR) to provide sequence information from infected tissues. Until recently, studies have been constrained by the limited length of fragments that can be reliably amplified. The addition of a thermostable 3'-exonuclease activity and altered cycling profiles has increased the length of target sequences that can be amplified by more than 10-fold. We have evaluated the fidelity of long PCR (LPCR). We determined that LPCR amplification maintains the distribution of sequences found in a heterogeneous sample and introduces nucleotide misincorporations at a rate comparable to that found with routine PCR. However, a significant proportion of the LPCR-amplified DNA fragments resulted from recombination events. This result suggests that LPCR amplification may have limited utility in the production and analysis of full-length HIV clones.

This article introduced that purifying equipments were installed in the major operating departments of supply rooms in hospital, and that experimental tests were done on the packing department. In statil state, the air humidity and the number of bacteria in the room after purification was obviously lower than that before purification (P < 0.01). In trending conditions, the main technical index on 10,000 degree clean rooms were examined, all of the results were up to the national standards.

Double-stranded RNA-dependent protein kinase (PKR) has been implicated in interferon (IFN) induction, antiviral response and tumor suppression. We have generated mice devoid of functional PKR (Pkr%). Although the mice are physically normal and the induction of type I IFN genes by poly(I).poly(C) (pIC) and virus is unimpaired, the antiviral response induced by IFN-gamma and pIC was diminished. However, in embryo fibroblasts from Pkr knockout mice, the induction of type I IFN as well as the activation of NF-kappa B by pIC, were strongly impaired but restored by priming with IFN. Thus, PKR is not directly essential for responses to pIC, and a pIC-responsive system independent of PKR is induced by IFN. No evidence of the tumor suppressor activity of PKR was demonstrated.

A simple and easy technique for reconstruction of the umbilicus was devised, with emphasis on forming walls of the umbilicus and a depression in a caudal direction. A quite satisfactory result was obtained. A permanent and sufficient depression for the umbilicus can be expected as a result of three-dimensional formation of walls.

Arginine vasopress in (AVP) has been shown to have significant effects on thermoregulation. AVP-sensitive locus is located in the septal area of the limbic system. The present experiments were undertaken to investigate the effect of microinjecting AVP and AVP-antiserum into the septal area on the firing activity of temperature-sensitive neurons in preoptic-anterior hypothalamus (PO-AH) of rabbit. The results were as follows: (1) The firing rate of PO-AH warm-sensitive and cold-sensitive neurons were respectively increased and decreased by injection of AVP while that of temperature-insensitive neurons was unaffected. (2) Injection of AVP-antiserum decreased the firing rate of PO-AH warm-sensitive neurons but increased the firing rate of cold-sensitive neurons. (3) Injection of AVP increased the thermosensitivity of PO-AH warm-sensitive neurons and decreased the thermosensitivity of cold-sensitive neurons. The modulation action of AVP on PO-AH temperature-sensitive neurons was discussed.

The interactive effects of clorgyline, deprenyl and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal dopamine (DA), serotonin (5HT) and hippocampal norepinephrine (NE) were examined in the present study. Results indicated that clorgyline did not have a preventive effect against MPTP's toxicity on DA and NE, but it had a partial therapeutic effect against subchronic MPTP-induced DA depletion when given with deprenyl together. However, this effect was not significantly different from the effect of deprenyl alone. Co-administration of clorgyline and deprenyl had a satisfactory effect in preventing MPTP-induced NE depletion. On the other hand, in all clorgyline-treated groups, there was a significant increase of 5HT in both the striatum and the hippocampus.

The authors report the results of a parallel study between the three kinds of evoked potentials (EP) i.e. visual, brainstem auditory and somatosensory evoked potentials (VEP, BAEP and SEP) and psychometric tests in 30 posthepatitic cirrhotic patients. The results were compared with those from 20 peptic ulcer patients in healing stage. Psychometric tests were abnormal in 65.5% of the cirrhotic and in none of the peptic ulcer patients. The three kinds of evoked potentials were abnormal in 58.6% of the cirrhotic patients and in 5% of the peptic ulcer patients. The VEP, BAEP and SEP was abnormal in 13.3%, 10% and 50% of the cirrhotics respectively. The results of this study showed: of the three kinds of evoked potentials, SEP is the most sensitive one. The evoked potentials and the performance psychometric tests are complementary in the diagnosis of subclinical hepatic encephalopathy.

The purpose of this study was to investigate polymorphonuclear neutrophil (PMN) chemotaxis in Chinese patients with post-juvenile periodontitis (post-JP) and rapidly progressive periodontitis (RPP). Peripheral blood PMNs were isolated from nine post-JP and 13 RPP patients. Eight patients with adult periodontitis (AP) and seven clinically healthy subjects were used for comparison. Clinical data, including the plaque index and gingival index, of each subject were recorded. The "modified Boyden chamber technique" was used for PMN chemotaxis assay. Our results showed that the PMN chemotaxis index in patients with post-JP and RPP were significantly depressed compared to that in healthy subjects or patients with AP. There was no significant difference in PMN chemotaxis between post-JP and RPP, or between AP and healthy subjects. There was also no significant correlation between the plaque index, or gingival index, and PMN chemotaxis in any group. These results suggest that there is a PMN chemotaxis defect in most Chinese patients affected by post-JP or RPP, and that the PMN chemotaxis defect is not associated with clinical parameters such as the plaque index or gingival index.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.

In this paper, the synthesis and analgesic activities of some derivatives of 4-methoxycarbonyl fentanyl are reported, in which the 4-N-phenyl group or both the 4-N-phenyl group and the 1-beta-phenyl group were replaced by some nonaromatic groups. In the last case, some compounds showed very strong analgesic activity. For example, the analgesic activities of compounds 4 and 6 were shown to be 695 and 818 times more potent than that of morphine respectively. The structure-analgesic activity relationships were discussed.

Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.

Naphthylmethyl isoquinoline (NI) 1-30 mumol.L-1 inhibited the contraction of rabbit vascular smooth muscle in vitro induced by KCl, CaCl2, and norepinephrine (NE). NI 0.3-30 mumol.L-1 blocked 45Ca2+ influx process in vascular smooth muscle of aorta, mesenteric and femoral arteries by addition of KCl and NE. NI 3-10 mumol.L-1 had no effect on 45Ca2+ efflux from aorta at resting state. These results suggest that the relaxing effect of NI on rabbit blood vessels may be relevant to the inhibition of Ca2+ influx into vascular smooth muscle.

The synthesis of some 4-N-propionyl analogs of cis-3-methyl fentanyl by acyl chloride method. anhydride method, mixed anhydride method and DCC method is reported. The ethyl group in 4-N-propionyl portion of cis-3-methyl fentanyl was substituted by some groups with different electronic property. In mouse hot plate test, all compounds showed typical morphine-like action with analgesic potency corresponding to 49-1963 times that of morphine. Compound 3 was found to exhibit higher analgesic activity than cis-3-methyl fentanyl. Semiempirical INDO calculations have been undertaken for 4 typical compounds and it was found that as a result of introduction of chlorovinyl, compound 3 exhibited characteristics of electronic structure different from that of cis-3-methyl fentanyl. The relationships between analgesic activity and the electronic structure of these compounds were discussed.

This clinical study compared two occlusal registration methods (Occlusal Indicator Wax and Accufilm) with the T-Scan system for the identification of guided closure contacts. The patients were divided into two groups according to the centric relation-maximum intercuspation (CR-MIP) discrepancy and comparisons of guided closure contacts were performed with the paired methods. Wax and Accufilm materials were significantly different in their agreement on guided closure contacts. The T-Scan system demonstrated less of a disparity with both methods but more closely resembled the Occlusal Indicator Wax material. The CR-MIP slide also significantly affected the agreement between methods during identification of guided closure contacts.

In this paper, the synthesis and analgesic activities of some derivatives of N-[1-(2-phenylethyl)-4-methoxymethyl-4-piperidinyl]-N-propionylanilin e (4-methoxymethyl fentanyl) are reported. In mouse hot plate test, most compounds in this series showed strong narcotic analgesic activities but their activities were lower than those of compounds in the series of 4-methoxycarbonyl fentanyl.

Using BamHI digested and dephosphorylated pBR322 as vector, a GL-7-ACA acylase gene from Pseudomonas sp. 130 chromosomal DNA was cloned and expressed in Escherichia coli C600. Seven positive clones were detected from 3205 recombinant plasmids with 32P-labeled oligonucleotide probes in situ hybridization. Out of them, three clones which produced active GL-7-ACA acylase were identified by radio-immunological assay, chemical test and chromatographic analysis of reaction mixture. The plasmid DNAs of recombinant pMR5, pMR6 and pMR7 were extracted. Analysis of gel electrophoresis indicated that pMR5 and pMR7 contained the same 6.8kb fragment and the size of the insert in pNR6 was 5.7 kb. The effects of various E. coli hosts on the expression of cloned GL-7-ACA acylase gene is also presented.

In this paper, we report the synthesis and analgesic activities in mouse hot plate test and writhing test of some analogs of U-50488, a kappa-agonist. Results showed that compounds in which the amino group was pyrrolinyl had higher kappa-agonist activity and the substitution of two chlorine atoms in 3 and 4-positions of the benzene nucleus was very important to kappa-activity. Furthermore, all of compounds in which the amino group was piperidyl, piperazinyl or morpholinyl exhibited very weak kappa-agonist activity.

To investigate the mechanism of the changes in glucocorticoid receptor (GR) under various stress conditions, experiments were carried out on normal and partial adrenalectomized rats by utilizing radioligand binding assay and molecular hybridization. It was found that hepatic GR binding capacity and GR mRNA in intact rats decreased significantly after the scalding stress, but the changes in GR binding capacity is mainly owing to the decrease in GR mRNA levels, and other mechanisms at protein level are also involved. In partial adrenalectomized rats, hepatic GR binding capacity and GR mRNA were increased instead of decreased, whereas serum corticosterone did not rise in response to the scalding stress. Therefore, it may be concluded that the decrease in GR binding capacity and GR mRNA in intact rats after the scalding stress is mainly the result of the increased concentration of serum corticosterone. Our further studies did demonstrate that there exists downregulation of GR mRNA in rats by exogenous administration of hydrocortisone. It is also suggested that there might be factors that can upregulate GR and GR mRNA in intact rats after the scalding stress.

Receptors for dopamine have been classified into two functional types, D1 and D2. They belong to the family of receptors acting through G (or guanine nucleotide-binding) proteins. D2 receptors inhibit adenylyl cyclase, but D1 receptors stimulate adenylyl cyclase and activate cyclic AMP-dependent protein kinases. Dopamine D1 and D2 receptors are targets of drug therapy in many psychomotor disorders, including Parkinson's disease and schizophrenia, and may also have a role in drug addiction and alcoholism. D1 receptors regulate neuron growth and differentiation, influence behaviour and modify dopamine D2 receptor-mediated events. We report here the cloning of the D1 receptor gene, which resides on an intronless region on the long arm of chromosome 5, near two other members of the G-linked receptor family. The expressed protein, encoded by 446 amino acids, binds drugs with affinities identical to the native human D1 receptor. The presence of a D1 receptor gene restriction fragment length polymorphism will be helpful for future disease linkage studies.

Two-dimensional reflectance images and surface thermal distributions were recorded during argon laser induced coagulation. During laser irradiation, coagulated egg yolk formed a white lesion. The whiteness, or reflectance caused by backscattering of light from the forming lesion, would be measured after a short delay from the onset of laser irradiation. In the experiments which covered exposure time from 4.5 to 17.0 s, we found that it started slowly, the reflectance increased rapidly once the surface temperature of the lesion reached approximately 90 degrees C. After this rapid rise, the reflectance began to taper off until no change in reflectance was recorded. There was a 0.98 correlation between lesion diameter measured from a two dimensional reflectance image and the lesion diameter that was measured microscopically. There was a 0.92 correlation between reflectance at the center of the lesion and microscopically measured depth of coagulation at the same point. However, the correlation between microscopically measured coagulation depth and width was only 0.88.

The effect of naphthylmethyl isoquinoline on amplitude of contraction and automaticity of guinea pig atria were investigated. The compound was found to markedly inhibit the force of contraction and automaticity in a concentration-dependent way. Propranolol competitively antagonized the effect of isoproterenol with pA2 of 7.5. Naphthylmethyl isoquinoline and verapamil antagonized isoproterenol, but in a noncompetitive manner with the pD2' of 5.4 and 5.8, respectively. The effect of the compound on action potential and contractile force of guinea pig papillary muscles were also studied. It was shown to produce negative inotropic effects and shorten the fast action potential duration, but the maximal upstroke (Vmzx) and amplitude of action potential were not affected. Thus, an excitation-contraction uncoupling was observed. It also depressed the amplitude. Vmax and duration of Ca2(+)-mediated slow action potential induced by high K+. Elevation of the CaCl2 concentration from 2 to 5 mmol/L reversed its inhibition partially. These results indicate that naphthylmethyl isoquinoline has a blocking effect on calcium channels.

In this paper, the synthesis and analgesic activities of a series of 1-substituted derivatives of N-[1-(2-phenylethyl)-4-methoxycarbonyl-4-piperidinyl]-N-propionylanil ine (4-methoxycarbonyl fentanyl) are reported. Preliminary pharmacological results showed that most compounds in this series exhibited typical morphine-like action and that replacement of beta-phenyl group in the 1-position of piperidine ring of 4-methoxycarbonyl fentanyl by some substituents e.g., some substituted vinyl groups, heterocyclic (or alcyclic) radicals, alkyl groups or N-methyl-anilino groups, can keep strong analgesic activity. Especially, some substituted vinyl groups were found to be effective groups which could replace 1-beta-phenyl group of 4-methoxycarbonyl fentanyl. Compounds N-[1-(3, 4-dimethyl-3-pentenyl)-4-methoxycarbonyl-4-piperidinyl]-N- propionylaniline(1321) and N-[1-(4-methyl-3-pentenyl)-4-methoxycarbonyl-4-piperidinyl]-N- propionylaniline(1302) exhibited higher analgesic activity than that of 4-methoxycarbonyl fentanyl.

In the present paper, the synthesis and analgesic activities of some 4-N-propionyl analogs of 4-methoxycarbonyl fentanyl are reported. In mouse hot plate test, most of these compounds showed strong narcotic analgesic activity. The analgesic activities of compounds 1379, 1385 and 1387 were found to be more potent than that of 4-methoxycarbonyl fentanyl.

Naphthylmethyl isoquinoline (NI), a modified structure of tetrandrine, was synthesized by the China Pharmaceutical University. The effects of NI on blood pressure of animals and its possible mechanism were studied. In anesthetized rats and cats, iv NI lowered the mean arterial pressure. Meanwhile, the total peripheral vascular resistance was reduced markedly, but the heart rate, cardiac output and cardiac index showed no noticeable change. In rats its hypotensive effect was stronger than tetrandrine when equal toxic doses were compared. In conscious normotensive rats, renal hypertensive rats and SHR, oral administration of this compound resulted in a marked fall of systolic blood pressure which remained at a steady low level. It decreased the pressor response of adrenaline in rats. Intra-arterial injection of NI into the perfusing system decreased the posterior limb and renal vascular resistance immediately in constant-rate perfusion of the posterior limb and renal blood vessel of rats. It shifted the cumulative dose-response curves of noradrenaline, KCl and CaCl2 to the right in isolated rabbit aortic rings and depressed their maximal responses with IC50 9.7, 7.2 and 1.6 mumol/L, respectively, which were 2.9, 9.1 and 1.8 times stronger than that of tetrandrine, correspondingly. These results suggest that the hypotensive effect of naphthylmethyl isoquinoline is mainly attributable to its arteriolar dilatation by inhibiting calcium influx.

Administration of polyvinyl alcohol containing hydrocortisone (F-PVA) to rats twice caused a rapid increase of plasma glucocorticoids (GCs) around 30 micrograms/dl for 3 days. This rise led to decrease of glucocorticoid receptor (GR) in rat liver cytosol and spleen cells 1 h post F-PVA and remained at low level for more than 10 days. Two hours after administration of hydrocortisone 5 mg/100 g b. wt, the liver tyrosine aminotransferase (TAT) activity in normal rats increased from 13.5 +/- 2.9 to 50.7 +/- 8.9 units, while in rats injected with F-PVA 3 days ago it increased only from 13.9 +/- 2.3 to 21.0 +/- 6.3 U. The inductivity of rat liver remained low at 7, 11 and 20 days after the injection of F-PVA and recovered 30 days later. This study demonstrates the presence of down-regulation of GR in intact animal, and shows that the decrease of GR is accompanied by reduction of target organ response to GCs.

Low density lipoprotein (LDL) has been used to deliver toxic molecules to cells by receptor-mediated endocytosis. In these studies, the cholesteryl ester core of LDL was replaced with a lipophilic, toxic molecule. We now report that photoprotective azo dyes can be stably incorporated into LDL, and that this reconstituted LDL protects cells from the photosensitizing action of pyrene methanol (PM) in a receptor-dependent process. The photoprotective action of the azo dye is due to its ability to scavenge singlet oxygen that is produced by the photosensitive agent in response to UV light.

We cloned sequences of the alk (alkane utilization) operon of Pseudomonas and characterized them physically and genetically. These sequences were used to construct a DNA restriction map of the alkBAC region. We physically mapped alk::Tn7 insertions and delta alkBA deletions, and we were able to show complementation or marker rescue of alk point mutations by cloned DNA sequences. Our results confirmed the existence of an operon containing structural loci encoding activities for membrane alkane hydroxylase component (alkB), soluble alkane hydroxylase component (alkA) and membrane alcohol dehydrogenase (alkC). Physical mapping of alkC::Tn7 insertions and complementation of alkC point mutations by cloned sequences from the alkBA region showed that we were previously mistaken in inferring the existence of a separate unlinked alkC cluster. Studies with an alkB-lacZ transcription fusion construct established that the operon is transcribed in the order alkBAC and is under positive regulation by alkR regulatory functions.

We describe the determination of the nucleotide sequence of two genes (pgtB and pgtC) contained within the 3.4-kilobase DNA segment sandwiched between the transporter gene, pgtP, and the regulatory gene, pgtA. These two genes are involved in the regulation of expression of phosphoglycerate transport in Salmonella typhimurium. The sequence indicates the presence of two large open reading frames, potentially coding for two polypeptides of 397 and 593 amino acid residues. The two gene products were identified by using the bacteriophage T7 RNA polymerase-T7 promoter coupled system of Tabor and Richardson, and the observed apparent mass of 45 and 69 kilodaltons correlated well with the respective open reading frames. The cellular location of these two polypeptides was directly determined, and the polypeptides were found to be associated with the membrane. Although overall these polypeptides appear to be hydrophilic, there is one hydrophobic transmembrane segment in the smaller polypeptide and four such segments in the larger polypeptide which can account for their association with the membrane. In the accompanying paper, we present genetic evidence that pgtB and pgtC genes are involved in the induction of the pgtP expression by modulating derepressor activity.

The autofluorescence spectra of human tissues excited with the 365-nm line of pulsed xenon ion laser have been measured. The autofluorescence spectra of cancer tissues usually show characteristic peaks near 630 nm and 690 nm, which do not appear in the spectra of the corresponding normal tissues. This characteristic fluorescence can be taken as a criterion for cancer diagnosis; a consistency of 89% with the traditional biopsy method has been obtained in preliminary clinical application to the diagnosis of buccal carcinoma. Spectroscopic investigations suggest that the porphyrin compounds localized and retained in cancer tissues might be responsible for this characteristic autofluorescence.