[Clinical evaluation of ventricular septal defect occlusion device for the treatment of postoperative bronchopleural fistula].

Bronchopleural fistula is an abnormal sinus tract that forms between the bronchus and the thoracic cavity. It is most commonly caused by thoracic surgery. Patients often have severe pulmonary and thoracic infections, which seriously affect the quality of life and survival rate. Most of these patients do not have a second operation chance, so the bronchopleural fistula becomes a thorny problem in the clinical practice. The clinical data of 9 patients with postoperative bronchopleural fistula admitted to Anhui Provincial Chest Hospital were reviewed and analyzed. We analyzed and summarized the clinical experience of successful occlusion with a ventricular septal defect(VSD) device, which provided a potentially effective treatment for postoperative bronchopleural fistula.

Therapeutic effect of ginsenoside Rg1 on mastitis experimentally induced by lipopolysaccharide in lactating goats.

Escherichia coli is a cause of subclinical and clinical mastitis in dairy cattle and goats, and sometimes causes severe clinical disease that may result in death of the animal. Previous investigation showed that ginsenoside Rg1 extracted from Panax ginseng C.A. Meyer (Araliaceae) has an anti-inflammatory effect on the sepsis induced by E. coli lipopolysaccharide via competitive binding to toll-like receptor 4. We hypothesized that intravenous injection of Rg1 had therapeutic effect on mastitis experimentally induced by intramammary infusion of lipopolysaccharide in lactating goats. In this study, 9 lactating goats were randomly assigned to 1 of the 3 groups: (1) lipopolysaccharide intramammary infusion + saline intravenous injection, (2) lipopolysaccharide intramammary infusion + Rg1 intravenous injection, and (3) saline intramammary administration + saline intravenous injection. Because no adverse clinical signs were observed after intramammary infusion of saline and intravenous injection of Rg1 in a preliminary experiment, and available qualified goats were limited in this study, this treatment was not included in this study. One udder half of each goat received intramammary infusion of lipopolysaccharide (50 µg/kg of body weight; groups 1 and 2) or saline solution (group 3), and the other half was infused with 2 mL of saline solution at h 0. Afterward, intravenous injections of saline solution (groups 1 and 3) or Rg1 (2.5 mg/kg of body weight; group 2) were administered at h 2 and 4 post-lipopolysaccharide challenge. Blood and milk samples were collected 3, 6, 9, 12, 15, 18, 21, 24, 48, and 72 h post-lipopolysaccharide challenge, and clinical signs were monitored hourly after lipopolysaccharide challenge within the first 10 h and at the same time points as blood samples. The results showed that Rg1 treatment downregulated rectal temperature, udder skin temperature, udder girth, milk somatic cell count, and N-acetyl-ß-d-glucosaminidase and upregulated milk production, lactose, and recovered blood components, such as white blood cells, neutrophils, lymphocytes, total proteins, albumin, and globulin. Considering the positive therapeutic effect on lipopolysaccharide-induced mastitis in goats presented in this study as well as the anti-inflammatory activity found previously, the botanical Rg1 deserves further study as a therapeutic agent in the treatment of E. coli mastitis in dairy animals.

TRIM59 is a key regulator of growth and migration inrenal cell carcinoma.

Renal cell carcinoma (RCC) is the most common renal neoplasms and metastatic is common. Previous data have shown that the tripartite motif (TRIM) family proteinswere implicated in human tumoriogenesis. In this study, we aimed to investigate the role of TRIM59 in the cell growth and migration in RCC. The expression of TRIM59 in human RCC tissues was initially examined by qRT-PCR. Alentivirus-based shRNA against TRIM59 (Lv-shTRIM59) was constructed. The effects of TRIM59 knockdown on cell proliferation were examined by in vitro MTT assay, colony formation assay and in vivo a mouse xenograft model of RCC. Cell migration and invasion after knockdown of TRIM59 were also examined by transwell assay. Our data showed that the mRNA level of TRIM59 in cancerous tissues was 2-fold increased as compared with non-cancerous tissues. Knockdown of TRIM59 in a RCC cell line 786-O significantly slowed down cell proliferative rate and decreased both the colony number and sizes. In the mouse model, knockdown of TRIM59 consistently inhibited tumor growth in vivo. Moreover, it was shown that cell migration and invasion were suppressed by 68% and 50%, respectively in TRIM59-depleted 786-O cells. Our data suggest that TRIM59 may serve as a pro-oncogenic protein in promoting the progression of RCC. Knockdown of TRIM59 may be a promising strategy concerning the early detection and treatment of RCC.

Relationship between donor sperm parameters and pregnancy outcome after intrauterine insemination: analysis of 2821 cycles in 1355 couples.

The aim of this study was to investigate whether sperm parameters can affect the pregnancy outcome of artificial intrauterine insemination with cryopreserved donor spermatozoon (AID). A total of 1355 couples received 2821 AID treatment cycles in the Reproductive Medicine Center of the Tongji Medical College between January 2010 and December 2013, and the data were collected and retrospectively analysed. The relationship between pre-freezing, post-thawing as well as optimised sperm parameters and AID pregnancy outcome was investigated. Clinical pregnancy rate and cumulated pregnancy rate were also calculated. A total of 728 cycles from 2821 treatment cycles achieved pregnancies, and cumulated pregnancy rate was 25.81%. Pre-freezing progressive sperm motility in pregnant cycles was higher than that in nonpregnant cycles (P = 0.001); logistic regression analysis also indicated that pre-freezing progressive sperm motility was the only parameter affecting pregnancy outcome (P = 0.0001). Our study also showed that the cumulated pregnancy rate increased progressively and reached a plateau after the fifth cycle. In conclusion, pre-freezing progressive sperm motility should be a valuable predictor for AID pregnancy outcome. Female fertility factors should be considered, or IVF/ICSI should be recommended when couples received more than 5 AID cycles without pregnancy.

Genetic analysis of haemophilia A in Taiwan.

Haemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene. Identification of these mutations is becoming increasingly important in a variety of clinical settings. The purpose of this report is to describe our experience of FVIII gene mutation analysis in the largest cohort of patients in Taiwan including the discovery of 21 novel mutations. We tested 115 HA patients from 91 unrelated families, including 79 severe, 15 moderate and 21 mild types starting with an assay for the intron 22 inversion by long range-PCR followed if necessary by additional genetic studies. Intron 22 inversion accounted for 27.8% of the total and 36.7% of severe HA patients respectively while intron 1 inversion comprised 7.6% of severe patients. These were clearly different from the known data in caucasian populations. Of 75 patients without intron 22 or 1 inversion, 70 from 62 unrelated families revealed 56 different mutations by denaturing high-performance liquid chromatography (DHPLC), of which 21 were novel. Also, the only female patient with severe HA was found to have heterozygous non-sense mutation (c.6683G>A) of exon 24. Seven patients, including five without amplified PCR product and two without encoded DNA defect turned out to have exon(s) deletion or insertion by reverse transcript PCR (RT-PCR). In our study, the combination of various molecular techniques including LR-PCR, multiplex PCR, DHPLC and RT-PCR analysis enabled definitive detection of the causative FVIII gene defects in 112 of 113 (99%) HA patients.

Enhancement of immune responses to infectious bursal disease vaccine by supplement of an extract made from Momordica cochinchinensis (Lour.) Spreng. seeds.

The immunological effect of an extract from Momordica cochinchinensis seed (ECMS) on immune responses against infectious bursal disease (IBD) in chickens was evaluated. Fifty-two birds were equally divided into 4 groups and immunized with inactivated IBD vaccine alone (controls) or IBD vaccine emulsified with ECMS (20, 40, and 80 microg). Serum IgG antibody levels against IBD and BW were measured on 0, 7, 14, 21, 28, and 35 d after immunization. The ELISA results revealed that the chickens that received 20 microg of ECMS had significantly enhanced antibody levels on 14, 21, 28, and 35 d when compared with controls (P<0.05). A significant increase in mitogenic stimulated lymphocyte proliferation was also recorded in all ECMS groups as compared with controls (P<0.05; P<0.01). No adverse effect of ECMS was noted on growth performance, although average weight gain was significantly higher in 20 microg (7, 14, 21, 28, and 35 d) and 40 or 80 microg (14 d) of ECMS groups as compared with controls (P<0.05; P<0.01). Further studies are suggested for the investigation of immunological effects of ECMS.

Prevalence of von Willebrand disease in women with iron deficiency anaemia and menorrhagia in Taiwan.

Iron deficiency anaemia (IDA) is a frequently encountered disease, which can be attributed to menorrhagia. Most female patients with von Willebrand disease (VWD) have menorrhagia. The aim of this study was to investigate the prevalence of VWD in women with both IDA and menorrhagia in Taiwan. From January to December 2005 and November 2006 to January 2007, 56 consecutive patients with both IDA and menorrhagia were enrolled in this study. Their median age was 41 years (range 18-53). IDA was diagnosed by anaemia plus either low ferritin or transferrin saturation. Menorrhagia was evaluated by patient's menses history. Both von Willebrand factor antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) were measured for each patient. Bleeding time (BT) and platelet function analyser (PFA)-100 assay were determined as ancillary tests. The VWD diagnosis was established if: (i) both VWF:Ag (<50%) and VWF:RCo (<50%) were low; (ii) either VWF:Ag or VWF:RCo was low plus prolonged BT or prolonged PFA closure times. VWF multimer analysis was performed for subtype confirmation of VWD. Nine of the 56 (16.1%) patients were identified to have VWD. VWD patients with menorrhagia might develop IDA at younger age (34.3 vs. 39.7, P = 0.09) and had more IDA recurrence (75% vs. 16%, P = 0.03) than those patients without VWD. Of the eight VWD patients with VWF multimer analyses, all were revealed to have type I VWD. Our study demonstrates that VWD was not uncommon in women with both IDA and menorrhagia in Taiwan.

The effect of dietary l-carnitine supplementation on pulmonary hypertension syndrome mortality in broilers exposed to low temperatures.

Oxidative stress is involved in the development of pulmonary hypertension syndrome (PHS) in broilers. l-Carnitine has an antiperoxidative effect and supplemental l-carnitine has been revealed to increase broiler heart weight. The present study was conducted to evaluate the effect of an addition of 100 mg/kg l-carnitine to the basal diets on PHS mortality in cold-exposed broilers. Two-hundred and forty mixed-sex broilers were equally assigned to three groups. The control group was reared in normal temperatures throughout the experiment. Starting on day 14 continuing until the end of the experiment, the other two groups were subjected to a step-down temperature programme (by lowering the temperature 1-2 degrees C per day down to 12-14 degrees C) with or without l-carnitine added to the basal diets. Cold exposure increased the right/total ventricle ratio (RV/TV) and plasma malondialdehyde (MDA), reduced superoxide dismutase (SOD) and led to pulmonary vascular remodelling in birds without feeding additional l-carnitine. Supplemental l-carnitine reduced plasma MDA, increased SOD, inhibited remodelling and postponed the occurrence of PHS for 1 week in cold-exposed broilers; nevertheless, it did not significantly influence the cumulative PHS mortality (p > 0.05). On days 24 and 32, birds fed supplemental l-carnitine had lower RV/TV and higher total ventricle/body weight (p < 0.05) but unchanged right ventricle/body weight ratios (p > 0.05) compared to their cold-exposed counterparts, indicating an increase in left ventricle weight. However, from day 39 on, their RV/TV ratios were suddenly increased (p < 0.05). It was suggested that the l-carnitine-induced increase in left heart weight might partially account for the postponed occurrence of pulmonary hypertension in the early stage by elevating cardiac output, which might, in turn, lead to the resulting increase in pulmonary pressure. In view of its complex effects on cardiopulmonary haemodynamics, l-carnitine supplementation may be impractical for reducing PHS.

Ginsenoside Rg1 enhanced immune responses to infectious bursal disease vaccine in chickens with oxidative stress induced by cyclophosphamide.

This study was designed to evaluate the effect of oral administration of ginsenoside Rg1 on oxidative stress induced by cyclophosphamide in chickens. Ninety-six chickens were randomly divided into 4 groups, each consisting of 24 birds. Groups 2 and 3 received intramuscular injection of cyclophosphamide at 100 mg/kg body weight for 3 d to induce oxidative stress and immune suppression. Groups 1 and 4 were injected with saline in the same way as groups 2 and 3. Then chickens in group 3 were orally administrated Rg1 of 1 mg/kg body weight in drinking water for 7 d. After that, groups 1 to 3 were orally vaccinated with attenuated infectious bursal disease vaccine (Strain B87). Blood samples were collected for determination of infectious bursal disease virus-specific antibodies, cytokines, and oxidative parameters. Splenocytes were prepared for lymphocyte proliferation assay. The results showed that oral administration of ginsenoside Rg1 significantly enhanced specific antibody, IFN-γ, and IL-6 responses, and lymphocyte proliferation induced by concanavalin A and lipopolysaccharide in chickens injected with cyclophosphamide. Antioxidant activity of ginsenoside Rg1 was also observed in chickens by increased total antioxidant capacity, total superoxide dismutase, catalase, glutathione peroxidase, glutathione, ascorbic acid, and α-tocopherol, as well as decreased malondialdehyde and protein carbonyl. Therefore, oral administration of Rg1 was shown to improve the immune responses to infectious bursal disease vaccine in chickens suffering from oxidative stress.

Efficacy of nisin in treatment of clinical mastitis in lactating dairy cows.

Nisin is an antimicrobial polypeptide produced by Lactococcus lactis and is believed nontoxic to humans. The objective of this study was to evaluate a nisin-based formulation for the treatment of bovine clinical mastitis in lactating dairy cattle. A total of 92 cows with 107 clinically mastitic quarters were randomly assigned to nisin- (48 cows with 51 quarters) and gentamicin (GM)-treated (44 cows with 56 quarters) groups. In the nisin-treated group, cows received an intramammary infusion of nisin at a dose of 2,500,000 IU; in the GM-treated group, intramammary infusion of GM was administered at a dose of 0.8 g. Results indicated that nisin offered a clinical cure rate similar to GM (90.2 vs. 91.1%) and no difference in bacteriological cure rate than GM-treated group (60.8 vs. 44.6%, respectively). Proportion of the quarters with milk somatic cell counts <500,000 cells/mL was not different in the nisin-treated group (50.0 and 47.8%) compared with the GM-treated group (33.3 and 37.3%) 1 and 2 wk after treatment. Of 17 Staphylococcus aureus isolates, 82.5% were resistant to penicillin, and 35.3% to GM, but none of them to nisin. Nisin therapy eliminated 54.5% (6 of 11) of S. aureus IMI, whereas GM eliminated 33.3% (2 of 6). Nisin in milk (4.5 +/- 0.8 IU/mL) was detected only at 12 h following intramammary infusion, which was much lower than the upper limit (500 mg/mL) allowed as preservative in milk by the China authority. Because of its efficacy in the treatment of bovine clinical mastitis, especially resistant Staph. aureus-caused IMI, as well as its safety in humans, nisin deserves further study to clarify its effects on mastitis caused by different mastitis pathogens on a larger scale.

Persistence of oxytetracycline residues in milk after the intrauterine treatment of lactating cows for endometritis.

Milk samples were collected at one day intervals after the last dose from 31 cows that had received an intrauterine infusion of oxytetracycline once daily between one and five times. The tetrazolium chloride assay was used to determine whether there were significant residues of the antibiotic in the samples. A single treatment resulted in residues for between one and eight days, and the period tended to be longer in the cows that had received more than one dose. Of the 31 cows, six remained tetracycline-positive for more than five days after their last dose of the drug.

The 1.1 A crystal structure of the neuronal acetylcholine receptor antagonist, alpha-conotoxin PnIA from Conus pennaceus.

BACKGROUND: alpha-Conotoxins are peptide toxins, isolated from Conus snails, that block the nicotinic acetylcholine receptor (nAChR). The 16-residue peptides PnIA and PnIB from Conus pennaceus incorporate the same disulfide framework as other alpha-conotoxins but differ in function from most alpha-conotoxins by blocking the neuronal nAChR, rather than the skeletal muscle subtype. The crystal structure determination of PnIA was undertaken to identify structural and surface features that might be important for biological activity. RESULTS: The 1.1 A crystal structure of synthetic PnIA was determined by direct methods using the Shake-and-Bake program. The three-dimensional structure incorporates a beta turn followed by two alpha-helical turns. The conformation is stabilised by two disulfide bridges that form the interior of the molecule, with all other side chains oriented outwards. CONCLUSIONS: The compact architecture of the PnIA toxin provides a rigid framework for presentation of chemical groups that are required for activity. The structure is characterized by distinct hydrophobic and polar surfaces; a 16 A separation of the sole positive and negative charges (these two charged residues being located at opposite ends of the molecule); a hydrophobic region and a protruding tyrosine side chain. These features may be important for the specific interaction of PnIA with neuronal nAChR.

miR-760 mediates chemoresistance through inhibition of epithelial mesenchymal transition in breast cancer cells.

OBJECTIVE: Therapeutic resistance has been a great obstacle for successful treatment of breast cancer. Our study aimed to explore the role of microRNA-760 (miR-760) in chemoresistant breast cancer cells. MATERIALS AND METHODS: Real-time PCR was performed to measure the mRNA expression of miR-760 and Nanog. Western blot was used to determine the protein expression of Nanog and mesenchymal and epithelial markers. Cell viability was measured by the CCK-8 assay. RESULTS: Our results showed that the expression of miR-760 was significantly reduced the doxorubicin (DOX)-resistant MCF-7/DOX cells and chemoresistant breast cancer tissues. Moreover, up-regulation of miR-760 sensitized breast cancer cells to the anti-cancer agents. The MCF-7/DOX cells exhibited increased expression of Snail, a mesenchymal marker, and decreased levels of E-Cadherin, an epithelial marker. In addition, overexpression of miR-760 suppressed the expression of Nanog, a transcriptional factor involved in chemoresistance, and resulted in the reversal of EMT in breast cancer cells. CONCLUSIONS: Our study demonstrated that miR-760 modulated chemoresistance through the epithelial-mesenchymal transition in breast cancer cells, providing a potential therapeutic target for treatment of drug-resistant breast cancer.

Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP.

The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP have been solved at 2.9 A, 2.9 A and 2.2 A resolution, respectively. The structure of R state GPa is nearly identical to the structure of sulphate-activated R state GPb, except in the region of Ser14, where there is a covalently attached phosphate group in GPa and a non-covalently attached sulphate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer are similar to those observed previously for T state GPa. The quaternary and tertiary structural changes on the T to R transition allow these interactions to be relayed to the catalytic site in R state GPa. The transition from the T state GPb structure to the R state GPa structure results in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg10, the first residue to be located from the N terminus, in R state GPa and T state GPb is 50 A. One of the important subunit-subunit interactions in the dimer molecule involves contacts between the helix alpha 2 and the cap' (residues 35' to 45' that form a loop between the 1st and 2nd alpha helices, alpha 1' and alpha 2' of the other subunit. The prime denotes residues from the other subunit). The interactions made by the N-terminal residues induce structural changes at the cap'/alpha 2 helix interface that lead to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifts 1.2 to 2.1 A for residues 35 to 45) are partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes are between 0.5 and 1.3 A. AMP binds to R state GPb with at least 100-fold greater affinity and exhibits four additional hydrogen bonds, stronger ionic interactions and more extensive van der Waals' interactions with 116 A2 greater solvent accessible surface area buried compared with AMP bound to T state GPb.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae.

The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme. In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors. Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pili and they no longer secrete cholera toxin. TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera. The crystal structure of oxidized TcpG (refined at a resolution of 2.1 A) serves as a starting point for the rational design of such inhibitors. As expected, TcpG has the same fold as E. coli DsbA, with which it shares approximately 40% sequence identity. In addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role. While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences. For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect). Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion. Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E. coli DsbA. The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors.

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase.

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.

Three-dimensional mapping of brain neuropils in the cockroach, Diploptera punctata.

Herein, we present a complete three-dimensional (3D) map of major neuropil structures in the central brain of the cockroach Diploptera punctata. The positions of the structures have been ascertained by confocal microscopy, which, until now-for reasons of tissue opacity and nonhomogeneity-has been thought impractical in imaging fluorescently labeled structures thicker than 150 microm. In this report, however, we have used digestive enzymes and microwave-aided fixation to stain, clear, and optically section, in its entirety, an intact central brain more than 500 microm thick. The central brain from an adult female cockroach was stained thoroughly with the membrane probe NBD-ceramide and the DNA probe propidium iodide. The central brain as well as such neuropil regions as mushroom bodies, central complex, antennal glomeruli, and lobus glomerulati were individually outlined, segmented, and reconstructed in three dimensions to a spatial resolution of approximately 1 microm in the X-Y plane and 3 microm in the Z plane. The volume and surface area of each neuropil compartment were determined, and Kenyon cells of the mushroom bodies were counted. We determined that each brain hemisphere contains about 230,000 Kenyon cells, 99 antennal lobe glomeruli, and 40 lobus glomerulatus glomeruli. Segmented compartments were assigned as separate channels and merged into a single data base to reconstruct a 3D central brain containing eight different channels. This is the first 3D map at submicron resolution of an entire animal's brain that measures more than 500 microm in thickness.

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease.

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Novel microbial hydroxylation of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one.

The microbial transformation of the dl and the d-enantiomer of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one (1) were investigated. Poor yields and poor resolutions were usually obtained for the hydroxylation reactions. Transformation of 1 by Cunninghamella blakesleeana gave 6 beta-, 7 beta-, 10 beta-, 15 alpha-hydroxy derivatives 4, 5, 6, 7, and 6 beta,10 beta-dihydroxy derivative 8; transformation of 1 by Cunninghamella echinulata afforded 5, 6, and 8. Biotransformation of dl-1 by Cunninghamella species usually gave 10 beta-hydroxy product with the low enanitomeric excess or as the racemic form. However, C. echinulata was able to efficiently differentiate the two enantiomers of 1 in the course of 6 beta,10 beta-dihydroxylation reactions. The d-enantiomer of the dl-1 was the better substrate for this type hydroxylation. The 7 beta and 15 alpha-hydroxylations of 1 by microbial cultures was unusual for 19-nor type steroids, and these hydroxylation reactions were presumably due to the presence of 17 alpha-ethynyl group.

Microbial hydroxylation of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one.

The microbial transformation of the racemic mixture of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one (1) was investigated. Rhizopus nigricans (AS 3.2050), R. arrhizus (AS 3.4523), Aspergillus niger (AS 3.2744), A. ochraceus (AS 3.1408), and Curvularia lunata (NRRL 4381) transformed 1 into its 10 beta-hydroxy derivative (2) as a major metabolite. Biotransformation of 1 by Aspergillus ochraceus AS 3.1408 afforded 7 beta-hydroxy derivative (3) as the only product.