Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics.

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.

Combination therapy with zidovudine prevents selection of human immunodeficiency virus type 1 variants expressing high-level resistance to L-697,661, a nonnucleoside reverse transcriptase inhibitor.

L-697,661 is a human immunodeficiency virus type 1 (HIV-1)-specific nonnucleoside reverse transcriptase (RT) inhibitor. Its tolerability and activity in combination with zidovudine were evaluated in a 48-week double-blind study. One hundred nineteen zidovudine-naive HIV-1-infected patients with CD4 cell counts of 200-500/mm3 received either combination therapy, L-697,661 alone, or zidovudine alone. Activity was assessed by CD4 cell count changes. Selection for L-697,661-resistant virus was monitored by susceptibility testing of RT expressed by circulating viral RNA. Therapy was generally well tolerated. All groups receiving zidovudine exhibited transient increases in CD4 cell counts, while the L-697,661 monotherapy group showed a significant decline and yielded RT > 100-fold resistant to L-697,661 and associated with substitutions at RT residue 181. The RT from patients receiving combination therapy was maximally 15-fold less susceptible to L-697,661. Hence, cotreatment with zidovudine prevents selection of HIV-1 variants that are highly resistant to L-697,661 in patients naive to both compounds.

Giant splenic artery aneurysm.

Giant aneurysms of the splenic artery are uncommon. The mean size of splenic artery aneurysms is reported to be 2.1 cm; they are rarely larger than 3 cm. We present two cases in which the splenic artery aneurysm was larger than 8 cm, discuss the management and operative approach to these infrequently encountered entities, and provide a brief review of the relevant literature.

A new frontier in understanding the mechanisms of developmental abnormalities.

Recent advancements in molecular developmental biology afford an opportunity to apply newly developed tools for understanding the mechanisms of both normal and abnormal development. Although a number of agents have been identified as causing developmental abnormalities, our knowledge of the mechanisms by which these alterations occur is minimal. This paper reviews some of the important issues in this area that may lead to understanding the basic developmental processes and mechanisms by which toxic agents may interfere with these processes. Approximately 70% of developmental defects are of unknown etiology. Historically, it has been assumed that these defects were most likely to be induced by exposure to chemical or physical agents during organogenesis. There is now convincing evidence that exposure during preorganogenesis developmental stages to certain agents can also lead to fetal abnormalities as a result of direct damage to the exposed early conceptus. Thus, pre- or postimplantation exposure of the developing conceptus to toxicants may result in a "derailment" in the genetic control of development and the coordinated cascade of events that occur during normal development. For example, developmental abnormalities may be induced by disrupting the coordinated expression of developmental genes involved in genomic imprinting, cell lineage specification, cell mixing and recognition, cell-cell interaction, cell migration and differentiation, and segmentation, depending on the time of exposure. Because of our lack of knowledge about the molecular and cellular bases of chemically induced abnormal development, a number of assumptions are currently used in the process of evaluating and interpreting data for developmental toxicity studies. The study of mechanisms of normal and abnormal development and the pharmacokinetic-pharmacodynamic relationships in humans and experimental animals are key to the development of appropriate risk assessment assumptions and dose-response models for characterizing the risk for developmental toxicity in the human population. This article summarizes the discussions of the workshop on developmental abnormalities organized by the Committee on Toxicology of the National Research Council.

Operative color Doppler imaging for vascular surgery.

Operative color Doppler imaging (CDI) was performed during 57 vascular operations (24 carotid, 24 lower extremity, six renal artery, and three other operations), and its benefits were assessed in comparison to B-mode imaging. Pre-reconstruction operative CDI was used selectively in 18 operations, and post-reconstruction operative CDI was used routinely in all operations. In 16 operations (28.6%), post-reconstruction CDI diagnosed vascular defects such as intimal flaps (n = 4), anastomotic stenoses (n = 7), and in situ bypass arteriovenous fistulas (n = 4). Vascular defects at eight operations required immediate repair. Operative CDI had advantages over B-mode imaging in (1) detection of preoperatively unknown vascular abnormalities before reconstruction, (2) faster recognition of vascular defects after reconstruction, (3) unique ability to detect problems (i.e., arteriovenous fistulas) that were unidentifiable by B-mode imaging, and (4) provision of supplemental blood flow information.

Gram-negative bacterial infection of aortic aneurysms.

Widespread use of antibiotics and change in pathogenesis altered the bacteriology of infected aortic aneurysms. In the past, bacterial endocarditis was the major source of emboli infecting the aorta. Now, gram-negative sepsis in elderly patients is often the initiating event of infection in atherosclerotic aneurysms. Four cases of gram-negative infection in aortic aneurysms were treated. The etiology, presentation, and surgical management are reviewed. Three abdominal aortic aneurysms were infected during urinary tract sepsis and one infection occurred with Salmonella septicemia. The clinical triad of fever, abdominal pain, and a pulsatile abdominal mass led to a preoperative diagnosis in three of four patients. Debridement of infected tissue and bypass through non-infected tissue planes remain the cornerstones of modern surgical management. Despite prompt diagnosis and proper surgical management, the mortality of gram-negative aortic infection remains high because of early rupture and extensive atherosclerotic disease.

Abdominal aortic aneurysms infected by Escherichia coli.

Three cases of atherosclerotic abdominal aneurysms infected by Escherichia coli urinary tract sepsis are presented together with a review of four additional cases of E. coli-infected aneurysms. Pathophysiology and a current system of classification of aortic infection are discussed. Important clinical features of gram-negative aortic infection include a diagnostic triad and the tendency to early rupture. Resection of infected tissue and extra-anatomic bypass for revascularization are the cornerstones of operative management. The mortality rate of E. coli aortic infection is high, with one known survivor. Death is contributed to by the high frequency of preoperative rupture, the age of the patient, and the extent of atherosclerotic disease.

Evaluation of the mutagenic potential of corn (Zea mays L.) grown in untreated and atrazine (AAtrex) treated soil in the field.

Bacterial assays using extracts from field corn plants (harvested at one month, silage and mature stages) do not indicate that soil treatment with atrazine, at its maximum use rate, alters the endogenous mutagens present in these extracts, nor that atrazine itself is degraded to mutagenic products. Extracts of corn grown in soil treated with AAtrex were equally mutagenic with those of corn grown in untreated soil when tested in Salmonella typhimurium TA-100 by a reversion assay or in Salmonella typhimurium TM-677 in a forward mutation assay. Higher concentrations of histidine in corn grown in AAtrex treated soil may interfere with the reversion assay, but do not affect the forward mutation assay. The nature of the agent(s) responsible for the positive response was not determined. The mutagenicity may be due to natural plant constituents, an artifact of the sample preparation, or mycotoxins from some unrecognized plant infection. The experimental results in these field studies do not show that atrazine is degraded or metabolized by corn plants to mutagens in this sensitive bacterial assay.

Diet, nutrition, and cancer: interim dietary guidelines.

The Committee on Diet, Nutrition, and Cancer of the National Academy of Sciences recently evaluated the role of diet in carcinogenesis. Both epidemiological and laboratory evidence suggests that a high intake of total fat increases susceptibility to cancer of different sites, particularly the breast and colon. In epidemiological studies frequent consumption of certain fruits and vegetables and in laboratory experiments some components of fruits and vegetables, especially cruciferous vegetables, appear to decrease the incidence of cancers at various sites. In contrast, frequent consumption of salt-cured, salt-pickled, or smoked foods, possibly because they may contain nitrosamines or polycyclic aromatic hydrocarbons, appears to increase the risk of esophageal or stomach cancer. Excessive alcohol consumption among smokers appears to be associated with an elevated risk of cancers of the oral cavity, esophagus, larynx, and respiratory tract. Interim dietary guidelines to reduce the risk of cancer were proposed in accordance with these conclusions. No definitive conclusions were reached for other dietary factors, including total calories, cholesterol, fiber, and selenium, nor could the quantitative contribution of diet to overall cancer risk be estimated.

Bioactivation of dimethylnitrosamine in intrasanguinous host-mediated assay and its association with in vitro mutagenesis assays.

These studies have revealed the usefulness of in vivo intrasanguine host-mediated assay (HMA) to detect point mutations. Mutations were found to occur at a significant rate in Salmonella typhimurium G-46 employed as indicator organisms recovered from liver, lung, kidney and spleen of DMN-treated animals compared to negative control animals. These differences were true for both male and female animals. The number of Salmonella typhimurium G-46 recovered from the testes was not large enough to make a valid judgment about mutations occurring in testes. The results from in vitro studies do not match with the in vivo host-mediated assay results for mutants occurring in spleen from the male and the female mice. The results also do not correlate for in vitro and in vivo studies involving female kidneys. These results suggest there may be no one-to-one correlation between the organ bioactivation in vitro and in vivo, and predictions of in vivo target organ cannot always be made from in vitro studies with isolated microsomal enzymes.

Regulation of dimethylnitrosamine metabolism by androgenic hormones.

Hormonal regulation of dimethylnitrosamine (DMN) metabolism by mouse kidney microsomes was investigated using an in vitro mutagenesis system that detects bioactive metabolites of this procarcinogen by measuring reverse mutation in Salmonella typhimurium his- auxotrophs. Induction of microsomal DMN metabolizing enzymes was androgen-specific. Testosterone and medroxyprogesterone acetate were active inducers, d/1 norgestrel was less active, while epitestosterone, estradiol, and progesterone were ineffective. THe response to testosterone and medroxyprogesterone acetate was time-and dose-dependent. Eleven strains of inbred mice were screened for their response to exogenous testosterone. DMN metabolism was stimulated in all mouse strains except for RF/J mice. Other androgenic end points were responsive in the latter strain, however. These observations suggest that induction of renal microsomal DMN metabolising enzymes is androgen-specific and probably mediated via the androgen receptor. The insensitivity of RF/J mice may be due to a mutation affecting a key step in the enzymatic activation of DMN.