CXCR4 sequences involved in coreceptor determination of human immunodeficiency virus type-1 tropism. Unmasking of activity with M-tropic Env glycoproteins.

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4 and one of a cadre of chemokine receptors triggers conformational changes in the HIV-1 envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular loop of CXCR4, which is restricted to dual tropic and T-tropic strains, was insensitive to the removal of charged residues either singly or in combinations by alanine scanning mutagenesis or to the conversion of acidic residues to lysine. Conversion of Asp-187 to a neutral residue exclusively unmasked activity with M-tropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing extracellular loop 2 of CXCR4 in a CXCR2 framework also resulted in the acquisition of M-tropic coreceptor activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the potential to be utilized by a spectrum of Env, which may be masked by charged amino acids in extracellular domains. These findings indicate that the primary structural determinants of coreceptors that program reactivity with M-, dual, and T-tropic Env are surprisingly subtle and that relatively insignificant changes in CXCR4 can dramatically alter utilization by Env of varying tropism.

Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1.

The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)-mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.

Expression of Bradyrhizobium japonicum nodulation gene in Rhizobium fredii nod mutants.

The B. japonicum USDA 110 pLAFR1 gene library was transferred to Tn5-induced Rhizobium fredii USDA 191-4 Nod- mutants with helper plasmid pRK2013. SmR TcR transconjugants occurred with a frequency of 5 x 10(-4). The transconjugants were purified and used to inoculate germinated soybean seeds. Seven nodules were obtained in the nodulation experiment. The fast-growing Nod+ SmR TcR Rhizobium fredii strain was isolated from all nodules. Each isolate had acquired a new plasmid with a molecular mass of approximately 51 kb. This recombinant plasmid was transferred to E. coli HB101 by helper pRK2013 at a frequency of 1 x 10(-4). In addition to the vector fragment, all the recombinant plasmids gave 23 kb and 6.5 kb fragments on EcoRI digestion. A DNA-DNA hybridization test with a 32P-labelled nod gene probe (prepared from pRmSL26) confirmed that the 20 kb EcoRI-BamHI DNA fragment of the plasmids exhibited sequence homology with an R. meliloti nod gene probe.

[Trial manufacture and application of soft uterine probe with reading scales (Type III) for the detection of IUD position].

PIP: The position of the IUD in the uterus is important to its contraceptive effectiveness. Pelvic x-ray examination is often imprecise and can not reveal whether the IUD is lodged in the muscles or outside of the uterus. Ultrasonic examination requires expensive equipment and is a complicated procedure. Examination of the position of an IUD inside the uterus with a flexible probe may solve the problem of uterine perforation which occurs with the use of a metal probe and may also reduce the incidence of unnecessary extraction and allow an appropriate correction of the IUD position. In August 1985, the soft silicone rubber uterine probe with a reading scale was developed and manufactured by the No. 3 Hospital of China's Medical University. The probe is inserted into the uterus together with the IUD. After the insertion procedure, clients walk about 500 meters, and a side-ways x-ray or radiography examination of the position of the IUD and the probe shows whether the IUD is properly placed or descended. A clinical study was conducted with 1000 IUD users, including 222 cases of IUD insertion after abortion and 162 cases of uterine bleeding it IUD in situ. A comparison of the post-abortion group, the irregular bleeding group and the normal group shows that a larger proportion of the former 2 groups had experienced an IUD descending. The results suggests that the large size of a post-abortion uterus makes it hard for an IUD to stay in place. Improper positioning of the IUD may be the cause of the irregular bleeding. The soft probe may also be used for uterine examinations. In cases of malformation of the uterus or of pelvic tumor, the use f the soft probe under ultrasonic examination may confirm the position of the uterus or tumor. The soft probe can also be used to clarify the association of irregular bleeding with the IUD.^ieng

General method for the identification of plasmid species in fast-growing soil microorganisms.

Using a horizontal gel electrophoresis method, we demonstrated reproducibly the presence of indigenous plasmids in different Rhizobium, Agrobacterium, and Pseudomonas strains. The method yields a large amount of plasmid DNA and is sensitive in detecting megaplasmids with molecular weights higher than 5 x 10. In two Rhizobium meliloti strains, a megaplasmid other than the low-mobility plasmid already known was detected.

[Clinical observation and preliminary study of termination of early pregnancy by administration of yellow daphne].

74 cases of early pregnancy with periods of amenorrhea of 36-69 days from the last menses were terminated by using a single dose intrauterine instillation of Wikstro-Emia Chamaedaphne Meisn Alcohol Solution (WCMAS). According to the dosages used, they were divided into 3 groups: 0.2gm, 0.4gm, and 0.6gm of WCMAS. The efficacy of the different dosages was evaluated. The most successful in the termination of early pregnancy (less than 56 days) was the 0.4gm group, the effective rate being 96.6%, and the complete abortion rate being 93.3%. The average duration of complete abortion in all was 21.3 hours, and the averge duration of vaginal bleeding was 10.9 days. Although amount of bleeding was regarded as a little greater than that of normal menstruation by some patients, yet there were no serious side effects occurring, except that some patients experienced some subjective lower abdominal pain and vomiting. Serum hCG and progesterone were determined by radioimmunoassay technique in 31 cases, and it was found that the hCG and progesterone levels both declined rapidly at 12 hours and fell to 35.5% and 46.4% respectively 24 hours after injection. Under microscopic examinations necrotic degeneration was observed in most of the chorionic villi, and marked necrosis and hemorrhage in the decidua. The granulocytes in the decidua also decreased in amount. 48 cases were followed up recently; no side effects were found and there was no interference with re-pregnancy.

Mobilization of a Sym plasmid from a fast-growing cowpea Rhizobium strain.

A large Sym plasmid from a fast-growing cowpea Rhizobium species was made mobilizable by cointegration with plasmid pSUP1011, which carries the oriT region of RP4. This mobilizable Sym plasmid was transferred to a number of Rhizobium strains, in which nodulation and nitrogen fixation functions for symbiosis with plants of the cowpea group were expressed.