Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development

We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.     

Phase I clinical and pharmacokinetic study of carzelesin (U-80244) given daily for five consecutive days

Carzelesin (U-80244), one of the synthetic DNA minor groove binding cyclopropylpyrroloindole analogues, was selected for clinical development because of its high potency, promising antitumor activity in murine solid tumors and leukemia, and significant therapeutic efficacy against colon and rhabdomyosarcoma xenografts. In this Phase I study, carzelesin was given daily for 5 consecutive days to (a) determine the maximum tolerable dose (MTD) and the pattern of toxicity of this schedule; (b) define the pharmacokinetic profile of the parent, as was done for the intermediate compound U-76073 and the DNA-reactive agent U-76074; and (c) document any antitumor activity observed. Carzelesin was given as a 10-min infusion with a constant-rate infusion pump. Treatment was repeated every 4 weeks or when blood counts had recovered to normal values. The starting dose of 12 microgram/m2/day was escalated by 20-30% increments until the MTD (defined as the dose leading to grade 4 hematological or grade 3 nonhematological toxicity in at least two of six patients) was reached. Pharmacokinetic studies were planned on days 1 and 5 of the first cycle in at least two patients per dose level. Plasma levels of carzelesin, U-76073, and U-76074 were determined by high-performance liquid chromatography with UV detection and a detection limit of 0.5 ng/ml. Twenty-five patients were entered in the study, and 56 cycles were evaluable for hematological toxicity. Subsequent dose levels evaluated were 24, 30, 35, and 40 microgram/m2. Both neutropenia and thrombocytopenia were dose limiting and cumulative, with a high interpatient variability. Neutropenia occurred earlier (median time to neutrophil nadir and recovery, 15 and 29 days, respectively) than thrombocytopenia (median time to platelet nadir and recovery, 25 and >/=26 days, respectively); there were delays of treatment because of persisting thrombocytopenia in all patients treated at the MTD. At the MTD, the peak plasma concentrations of carzelesin were achieved at the end of the infusion and were higher than those found cytotoxic in vitro against tumor cell lines. Carzelesin was detectable up to a maximum of 1 h after the infusion. Smaller amounts of U-76073 were detectable for a maximum of 30 min only at the MTD, whereas U-76074 was never found. An 8-month partial remission was reported in one previously untreated patient with hepatocellular carcinoma at 40 microgram/m2. The MTD was fixed at 40 microgram/m2 daily; 35 and 30 microgram/m2 are the daily doses recommended for Phase II studies in good- and poor-risk patients. The daily regimen for 5 days seems to offer no advantage over the single intermittent schedule that has been selected for the Phase II program in Europe.     

Virion incorporation of human immunodeficiency virus type-1 Vif is determined by intracellular expression level and may not be necessary for function

OBJECTIVE: To define the role of laparoscopic ultrasound (LUS) in the staging of pancreatic tumors. SUMMARY BACKGROUND DATA: Laparoscopy has recently been established as a valuable tool in the staging of pancreatic cancer. It has been suggested that the addition of LUS to standard laparoscopy could improve the accuracy of this procedure. METHODS: A prospective evaluation of 90 patients with pancreatic tumors undergoing laparoscopy and LUS was performed over a 27-month period. LUS equipped with an articulated curved and linear array transducer (6 to 10 MHz) was used. All patients underwent rigorous laparoscopic examination. Clinical, surgical, and pathologic data were collected. RESULTS: The median age was 65 years (range 43 to 85 years). Sixty-four patients had tumors in the head, 19 in the body, and 3 in the tail of the pancreas. Four patients had ampullary tumors. LUS was able to image the primary tumor (98%), portal vein (97%), superior mesenteric vein (94%), hepatic artery (93%), and superior mesenteric artery (93%) in these patients. LUS was particularly helpful in determining venous involvement (42%) and arterial involvement (38%) by the tumor. This resulted in a change in surgical treatment for 13 (14%) of the 90 patients in whom standard laparoscopic examination was equivocal. CONCLUSIONS: LUS is useful in evaluating the primary tumor and peripancreatic vascular anatomy. When standard laparoscopic findings are equivocal, LUS allowed accurate determination of resectability. Supplementing laparoscopy with LUS offers improved assessment and preoperative staging of pancreatic cancer.     

Primary structure and high expression of human agrin in basement membranes of adult lung and kidney

In previous in vivo studies we have reported that atrial natriuretic factor enhanced induced salivary secretion and increased isoproterenol-induced amylase release in the rat suggesting that, ANF effect could be mediated by phosphatidylinositol hydrolysis. In the present work, the effect of ANF on rat parotid tissue incubated in vitro was investigated with the aim to assess whether the phosphoinositol pathway was involved in ANF intracellular signaling in the parotid gland. Results showed that ANF induced a dose dependent increase in amylase fractional release, which was lower than that evoked by any concentration of isoproterenol. Furthermore 100 nM ANF enhanced isoproterenol-evoked amylase release. The effect of ANF was not affected in the presence of propranolol suggesting the noninvolvement of the beta adrenergic receptor, which is the main stimulus for the output of the enzyme in the parotid gland. However, ANF increased phosphatidylinositol hydrolysis, which implies an increase in intracellular calcium, which is necessary for the achievement of maximal response in amylase release. This effect was abolished in the presence of neomycin supporting ANF direct stimulation of phospholipase C. These results suggest the involvement of the C type natriuretic peptide receptor coupled to phospholipase C in ANF evoked amylase release and ANF enhancement of the isoproterenol-induced output of the enzyme.     

Detection of resistance to amphotericin B among Cryptococcus neoformans clinical isolates: performances of three different media assessed by using E-test and National Committee for Clinical Laboratory Standards M27-A methodologies

Although reliable detection of resistance in vitro is critical to the overall performance of any susceptibility testing method, the recently released National Committee for Clinical Laboratory Standards M27-A methodology for susceptibility testing of yeasts discriminates poorly between resistant and susceptible isolates of Candida spp. We have previously shown that both substitution of antibiotic medium 3 for RPMI 1640 medium in the microdilution variant of the M27-A method and use of the E-test agar diffusion methodology permit detection of amphotericin B-resistant Candida isolates. To determine the relevance of these observations to Cryptococcus neoformans, we have evaluated the performances of both the M27-A and the E-test methodologies with this yeast using three different media (RPMI 1640 medium, antibiotic medium 3, and yeast nitrogen base). As with Candida, we found that only antibiotic medium 3 permitted consistent detection of resistant isolates when testing was performed in broth by the M27-A method. When testing was performed by the E-test agar diffusion method, both RPMI 1640 medium and antibiotic medium 3 agar permitted ready detection of the resistant isolates. Reading of the results after 48 h of incubation was required for testing in broth by the M27-A method, while the MIC could be determined after either 48 or 72 h when the agar diffusion method was used.     

Effects of antimicrobial treatment at the end of lactation on milk yield, somatic cell count, and incidence of clinical mastitis during the subsequent lactation in a dairy herd with a low prevalence of contagious mastitis

Calcium-binding proteins (CaBPs) have been described as involved in the stimulus-secretion coupling mechanisms in secretory glands. CaBPs were revealed with 45Ca, after electrophoresis in SDS-PAGE and transference to Zeta probe membranes, in Duvernoy's or venom gland homogenates from three families of South American snakes: Viperidae (Bothrops jararaca and Crotalus durissus terrificus); Elapidae (Micrurus corallinus), and Colubridae (Phylodrias patagoniensis and Oxyrhopus trigeminus). A band with an estimated molecular weight of 12 KDa was found in all glands studied. Bands with 17, 28, and 67 KDa were found in all glands, except in O. trigeminus Duvernoy's gland. A 18 KDa band was found in Viperidae and Elapidae venom glands, and a 88 KDa band was observed only in Viperidae venom gland homogenates. Some of these CaBPs were identified by Western blotting or by immunohistochemistry, as parvalbumin (12 KDa) and calbindin (28 KDa). When the secretion of these glands were analyzed, CaBPs were detected only in B. jararaca venom, with bands of 14, 35, 42, and 72 KDa. The profile of CaBPs was not modified at different phases of the secretory cycle of the glands, as well as after isoproterenol treatment.     

Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity

In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein.