Exopolysaccharides in plant-bacterial interactions.

Rhizobial plant symbionts and bacterial plant pathogens produce exopolysaccharides that often play essential roles in the plant interaction. Many of these exopolysaccharides are acidic heteropolysaccharides that have repeating subunit structures with carbohydrate and noncarbohydrate substituents, while others are homopolysaccharides such as alginate, levan, cellulose, and glucan. While the homopolysaccharides are synthesized by mechanisms that vary with the particular polysaccharide, the heteropolysaccharides as a rule are synthesized by subunit assembly from nucleotide diphosphate-sugar precursors on a membrane-bound lipid carrier followed by polymerization and secretion. Many mutants in exopolysaccharide synthesis have been isolated, and in several cases this has led to the identification of genes that function in particular steps of biosynthesis, as well as in regulation of exopolysaccharide biosynthesis. The genetic regulation of exopolysaccharide synthesis in many plant pathogens is complex, perhaps reflecting the various niches, free living and in planta, in which exopolysaccharides function. In some cases, exopolysaccharide synthesis is regulated coordinately with other virulence factors, and in other cases separately. Regulatory genes that have homology to the two-component sensor and transcriptional effector systems are a common motif. In Rhizobium species, exopolysaccharide synthesis is regulated by transcriptional as well as posttranslational mechanisms. Exopolysaccharides function differently in the root-nodule symbiosis versus plant pathogenesis. Specific Rhizobium exopolysaccharide structures promote nodule development and invasion in legumes that form indeterminate nodules. In plant pathogenesis, less specific mechanisms of pathogenesis occur: exopolysaccharides cause wilting by blocking xylem vessels, are partly responsible for water-soaked lesions, and may also aid in invasion, growth, and survival in plant tissues.


Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae.

Yeast RNA viruses include L-A (and its toxin-encoding satellites M1, M2, ...) and L-BC dsRNA viruses and the single-stranded replicons 20S RNA and 23S RNA. L-A has a single-segment 4.6-kb linear genome encoding a major coat protein (gag) and its RNA-dependent RNA polymerase (pol), the latter expressed as a gag-pol fusion protein formed by a -1 ribosomal frameshift. In vitro replication, transcription, and binding systems for L-A have been used to define cis sites necessary for packaging and replication of viral RNA. Cellular functions that promote viral replication include the MAK3-encoded N-acetyltransferase whose modification of the gag N terminus is necessary for L-A virus assembly. The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes. 20S RNA is an apparently naked circular RNA replicon (with a dsRNA form called W) encoding a RNA polymerase-like molecule. Its copy number is induced 10,000-fold in 1% potassium acetate, and it is subject to the same SKI antiviral system that represses L-A, L-BC, and M dsRNA copy number.


Genetics of Campylobacter and Helicobacter.

This article reviews the current state of genetic analysis of Campylobacter and Helicobacter. Chromosomal genes cloned from Campylobacter and Helicobacter species are listed along with the method used to identify the cloned gene. Campylobacter plasmid genes that have been cloned and expressed in Escherichia coli and that specify resistance to tetracycline, kanamycin, or chloramphenicol are presented. This review also examines our current knowledge of genetic exchange in Campylobacter, including conjugative plasmid transfer, natural transformation, electrotransformation, and bacteriophage transduction. In Helicobacter, natural transformation has been described and both plasmids and bacteriophages have been observed. Plasmid cloning vectors have been constructed for Campylobacter. Available vectors are discussed and restriction maps of some useful vectors that we have constructed are included. The genome sizes of C. jejuni and C. coli are approximately 1.7 megabases (Mb), whereas the genome size of H. pylori ranges from 1.60 to 1.73 Mb. The positions of various genes on the C. jejuni and C. coli genome maps have been determined using both homologous and heterologous DNA probes. Genomic maps of these organisms are presented.


Autoregulatory factors and communication in actinomycetes.

The ability to produce a wide variety of secondary metabolites and a mycelial form of growth that develops into spores are two biological aspects characteristic of the gram-positive bacterial genus Streptomyces. Secondary metabolism and cell differentiation are controlled by diffusible low-molecular-weight chemical substances called autoregulators. A-factor, the representative of the autoregulators, triggers streptomycin production and aerial-mycelium formation in Streptomyces griseus. A-factor exerts its regulatory function with the aid of a receptor protein that itself acts as a repressor-type regulator. The A-factor signal via the A-factor-receptor protein is transferred to downstream genes, such as streptomycin-production genes and sporulation genes, through multiple regulatory genes in a complex regulatory cascade. Thus, A-factor can be termed a "microbial hormone." This review deals with the A-factor-regulatory cascade as a model system for other autoregulators. The biosynthesis of A-factor, the structures and characteristics of other autoregulators, and the importance of these autoregulators in the ecosystem are also included.


Genetics of competition for nodulation of legumes.

An economically important problem in microbial ecology concerns the efficacy of rhizobial inoculants for the formation of nitrogen-fixing root nodules on legume crop plants such as soybean, alfalfa, and clover. Some strains of rhizobia can increase symbiotic nitrogen fixation under controlled conditions. However, attempts to improve nitrogen fixation under agricultural conditions with such strains often fail, usually as a result of the presence of indigenous rhizobia limiting nodulation by the inoculum strains. This problem is referred to as the Rhizobium competition problem, and molecular genetics is being used to address the problem from two perspectives. First, the host specificity of rhizobia is being characterized with the long term goal of developing strains that can nodulate a very strain-specific host-legume genotype. Second, the genetic basis of competitiveness in several strains is being examined. Genetic determinants of nodulation competitiveness have been isolated and mechanisms for their stable integration into the genome of superior nitrogen-fixing strains have been developed. Several phenotypes have been identified as playing an important role in nodulation competitiveness including antibiosis, motility, speed of nodulation, cell-surface characteristics, and nodulation efficiency. Several solutions to this problem are likely to result from these strategies and will be useful for certain legumes in specific locations.


Positive regulation in the gram-positive bacterium: Bacillus subtilis.

Temporally and environmentally regulated gene expression in prokaryotes occurs primarily at the level of transcription initiation. Two main modes of regulation have been described, including either the binding of a repressor that blocks transcription or the interaction of a positive regulator with the transcription complex, leading to transcription initiation. Several classes can be distinguished among positive regulators according to their mechanisms of action. This review describes the different types of positive regulators identified in Bacillus subtilis, a gram-positive bacterium. These include accessory regulatory polypeptides, classical positive regulators that bind to target sites located just upstream from the promoter, ambiactive regulators that can act both positively and negatively, antiterminators, two-component signal transduction systems, and positive regulators associated with specific secondary sigma factors.


Penicillin and cephalosporin biosynthetic genes: structure, organization, regulation, and evolution.

Penicillins and cephalosporins are produced by a wide variety of microorganisms, including some filamentous fungi, many gram-positive streptomycetes, and a few gram-negative unicellular bacteria. All produce these beta-lactam antibiotics by essentially the same biosynthetic pathway. Recently, most of the penicillin and cephalosporin biosynthetic genes have been cloned, sequenced, and expressed. The biosynthetic genes code for enzymes that possess multifunctional peptide synthetase, cyclase, epimerase, expandase, hydroxylase, lysine aminotransferase, and acetyltransferase activities and are organized in chromosomal gene clusters and coordinately expressed. DNA hybridization screens of streptomycetes demonstrate that beta-lactam biosynthetic genes may be more widespread in nature than is indicated by conventional antibiotic screens. They offer the possibility of expanding the search for organisms with potential to make new beta-lactam antibiotics. Attempts to improve current yields of beta-lactams in production strains by introducing into them additional copies of biosynthetic genes have been partially successful. Comparative sequence analysis of bacterial and fungal beta-lactam biosynthetic genes show they share very high sequence identity. A model that explains the similarity of biosynthetic genes from an evolutionary standpoint assumes horizontal gene-transfer between the two groups of organisms. Indirect evidence suggests the transfer occurred from the bacteria to the fungi.


Signaling and host range variation in nodulation.

Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.


The natural history and pathogenesis of HIV infection.

Infection with human immunodeficiency virus (HIV) results in progressive deterioration of the cell-mediated immune system characterized by T-helper-cell dysfunction and loss in the face of signs of generalized immune-system activation. The final stage of HIV disease, AIDS, has a myriad of opportunistic infections and malignancies as its hallmarks. The causal relationship between HIV and this complex disease pattern is clear but the mechanisms by which it occurs are not well understood. There are a number of new developments in our understanding of the natural history of HIV infection from a laboratory standpoint. Our review of this information raises further questions as to the validity of the conventional "cytopathic" model and all its direct descendants. In response to these conflicts, we have developed and present an alternative hypothesis in which AIDS pathogenesis, in all its manifestations, is seen as the outcome of one central process, excess immune activation generated by the interaction of virus with the CD4 receptor. The implications of this hypothesis on therapy of HIV infections are discussed.


Functional and evolutionary relationships among diverse oxygenases.

Oxygenases that incorporate one or two atoms of dioxygen into substrates are found in many metabolic pathways. In this article, representative oxygenases, principally those found in bacterial pathways for the degradation of hydrocarbons, are reviewed. Monooxygenases, discussed in this chapter, incorporate one hydroxyl group into substrates. In this reaction, two atoms of dioxygen are reduced to one hydroxyl group and one H2O molecule by the concomitant oxidation of NAD(P)H. Dioxygenases catalyze the incorporation of two atoms of dioxygen into substrates. Two types of dioxygenases, aromatic-ring dioxygenases and aromatic-ring-cleavage dioxygenases, are discussed. The aromatic-ring dioxygenases incorporate two hydroxyl groups into aromatic substrates, and cis-diols are formed. This reaction also requires NAD(P)H as an electron donor. Aromatic-ring-cleavage dioxygenases incorporate two atoms of dioxygen into aromatic substrates, and the aromatic ring is cleaved. This reaction does not require an external reductant. All the oxygenases possess a cofactor, a transition metal, flavin or pteridine, that interacts with dioxygen. The concerted reactions between dioxygen and carbon in organic compounds are spin forbidden. The cofactor is used to overcome this restriction. For the oxygenases that require the NAD(P)H cofactor, the enzyme reaction is separated into two steps, the oxidation of NAD(P)H to generate two reducing equivalents, and the hydroxylation of substrates. Flavoprotein hydroxylases that catalyze the monohydroxylation of the aromatic ring carry out these two reactions on a single polypeptide chain. In other oxygenases, the NAD(P)H oxidation and a hydroxylation reaction are catalyzed by two separate polypeptides that are linked by a short electron-transport chain. Two reducing equivalents generated by the oxidation of NAD(P)H are transferred through the electron-transport chain to the cofactor on a hydroxylase component that they reduce. Dioxygen couples with the reduced cofactor and subsequently hydroxylates substrates. The electron-transport chains associated with oxygenases contain at least two redox centers. The first redox center is usually a flavin, while the second is an iron-sulfur cluster. The electron transport is initiated by a single two-electron transfer from NAD(P)H to a flavin, followed by two single-electron transfers from the flavin to an iron-sulfur cluster. The primary sequences of many oxygenases have been determined, and according to their sequence similarities, the oxygenases can be grouped into several protein families. Among proteins of the same family, the sequences in regions involved in cofactor binding are strongly conserved. Local sequence similarities are also observed among oxygenases from different families, primarily in regions involved in cofactor binding.


Arrest of bacterial DNA replication.

The chromosomes of both gram-positive and gram-negative bacteria contain sites that arrest the progression of DNA replication forks. These replication-arrest sites limit the end of the replication cycle to a particular region of the chromosome, called the terminus region. Replication arrest is mediated by protein-DNA complexes that show polarity of function: they arrest DNA replication from one direction only. This paper reviews our current knowledge of the replication-arrest complexes of Bacillus subtilis and Escherichia coli and examines possibilities for the function and mechanism of action of these complexes within the bacterial cell.


The lipophosphoglycan of Leishmania parasites.

Protozoan parasites of the genus Leishmania have the remarkable ability to avoid destruction in the hostile environments they encounter throughout their life cycle. The molecular details of how these pathogens persevere with impunity under harsh conditions are beginning to be understood. The fact that Leishmania parasites have adapted to not only survive, but to proliferate probably is due to the protection conferred by specialized molecules on the parasite's cell surface. One such macromolecule is a novel glycoconjugate called lipophosphoglycan. This heterogeneous, lipid-containing polysaccharide is the major surface molecule of the parasite and has been implicated in a surprisingly large number of functions that may contribute the the parasite's pathogenesis. This review emphasizes the structural aspects of lipophosphoglycan and its possible functions and biosynthesis.


Human immunodeficiency virus and the central nervous system.

Neurological disease frequently complicates HIV-1 infection. In addition to opportunistic infections, a syndrome of combined cognitive and motor impairment, referred to as the AIDS dementia complex, has been recognized. While presumed to relate to HIV-1 itself, the pathogenesis of this syndrome remains uncertain. Because of the limited extent of productive brain HIV-1 infection in many cases, and because such infection involves macrophages and microglia rather than cells of neuroectodermal origin, current speculation centers on indirect mechanisms of brain injury including virus- or cell-coded neurotoxins. We review clinical and laboratory studies and also describe models of the interaction of HIV-1 and immune responses that might account for brain injury.


Metabolism and functions of trypanothione in the Kinetoplastida.

Trypanosomatids differ from all other organisms in their ability to conjugate the sulfur-containing tripeptide, glutathione, and the polyamine, spermidine, to form trypanothione [N1,N8-bis(glutathionyl)spermidine]. Together with the NADPH-dependent flavoprotein, trypanothione reductase, the dithiol form of trypanothione provides an intracellular reducing environment in these parasites, substituting for glutathione and glutathione reductase found in the mammalian host. Trypanothione and its related enzymes are involved in defense against damage by oxidants, certain heavy metals, and possibly xenobiotics. Trypanothione and its metabolic precursor, glutathionylspermidine, are also implicated in the modulation of spermidine levels during growth. Several existing trypanocidal drugs interact with the trypanothione system, suggesting that trypanothione metabolism may be a good target for the development of new drugs. The purification and properties of three key enzymes (glutathionylspermidine synthetase, trypanothione synthetase, and trypanothione reductase) are discussed, and the catalytic mechanism, substrate-specificity, and the three-dimensional structure of trypanothione reductase are compared to that of glutathione reductase.


Replication cycle of Bacillus subtilis hydroxymethyluracil-containing phages.

The present review focuses on phage 2C, a member of a family of virulent phages that multiply in Bacillus subtilis. The best known members of this group are SPO1, phi e, H1, 2C, SP8, and SP82, the genomes of which are made of double-stranded DNA of about 150 kilobase pairs (kbp). The two DNA strands have different buoyant densities. Moreover, thymine (T) is completely replaced by hydroxymethyluracil (hmUra). Comparison of the phage DNAs has shown that both base substitutions and deletions have contributed to the evolution of their genomes. In addition, all of the hmUra-phage genomes contain colinear redundant ends, amounting to 10% of total bases. Two lines of evidence suggest that the redundant ends of 2C DNA, in spite of extensive homology, contain unique sequences. Further studies focused on DNA replication during the lytic cycle. The semiconservative replication of the infecting viral genome is followed by extensive recombination. At the level of replication forks, viral DNA synthesis is discontinuous on both strands during the whole cycle. Deoxythymidinetriphosphate, required for viral DNA synthesis in permeabilized infected bacteria, was incorporated in small amounts into phage DNA. The putative primary origin of replication has been cloned and localized on the viral genome. Some viral promoters have been successfully cloned in Escherichia coli. These sequences, however, did not promote transcription in B. subtilis. The abnormal base might be required for promoter activity in the natural host.


The anthropological evidence for change through Romanisation of the Poundbury population.

Poundbury Camp cemetery was in use for about 500 Years and was the burial ground for an Iron Age Durotrigian group, a rural Roman settlement and an urban Romano-British community. Low variance of metrical characters and persistence of familial traits in the three groups suggest a continuity of the population and in situ growth. However evidence for an anthropological response to the cultural romanisation of the population has been found in a number of skeletal traits including squatting which was most often adopted by Durotrigian females. Dietary changes are indicted by variation in concentrations of trace elements, including lead, in the bones.


Ancient skeletal remains of the Canary Islands: bone histology and chemical analysis.

Prehispanic burials from the Canary Islands are often well preserved. Many of the bodies are mummified, most of them were not interred, but deposited in caves. Bone histological and trace element analysis of 117 skeletons of the prehispanic period of the Canary Islands was performed. In some of the islands we have found a high prevalence of osteoporosis, whereas in others, histomorphometrically assessed trabecular bone mass (TBM) (in undecalcified iliac crest specimens) was in the normal range. Bone trace elements analysis have shown high bone S(r), Mg and Mn, and low Fe, Zn and Cu in those skeletons with a more reduced TBM. These facts speak for a relative protein-calorie malnutrition and a consumption of a mainly vegetarian diet. This is especially marked in the skeletons from Gran Canaria.


Anthropometrical study of the Asmat (Irian Jaya).

An anthropometric study in the Asmat population, a coastal group of Papua living in the south-western part of Irian Jaya (Indonesian New Guinea) covered 318 subjects of both sexes, belonging to four different villages (Basim, Senggo, Ewer and Piramanak) of the Asmat region. The dimensions and derived indices, describing body, head and face morphology as well as body composition show the Asmat to be slender and muscular, with shoulders, chest and pelvis of medium dimensions and with long legs in relation to the trunk. They are dolichocephalic with narrow faces and rather large noses. Comparisons of the four Asmat groups indicate the importance of geographic position on size of the subjects and the similarities detected between the groups by discriminant and principal component analysis.


[The color and structure of the human iris. 2. Studies of 200 twins].

Subject of the present report is an investigation of the heredity of 30 iris characteristics. The material basis of this sample of twins (100 monozygotic and 100 dizygotic pairs), the largest ever taken as a basis for the purpose of iris research, consists of standardized colour photographs of the examined persons' irises and iris-microscopic observations. The pairs of twins are compared to 100 non-related casual pairs in order to render a better estimation of the degree of heredity of the features possible. On every occasion several classes were constituted for the concordance verification. These classes result from the number of the classes of the forms of markedness and present different levels of similarity. The different distribution of the twin pairs and the control pairs on the concordance classes are always examined for their statistical significance, and for each group of pairs a concordance value is computed. The intervals between the concordance values of the monozygotic and dizygotic pairs and the control pairs indicate the degree of heredity of the investigated feature. Taking all the compiled factors and conditions into consideration, an evaluation of the features as heredity features is made. Though a hereditary component can be observed for all iris characteristics, only about a third could be called heredity features. Quantity of pigment and the hitherto undescribed limiting layer folds were evaluated as "very good", whereas pigment colour in the fine analysis, quality of the anterior stroma leaf, frequency of iris crypts and iris frill position were judged as "good". Tone of blue, markedness of Woelfflin nodules, quantity and markedness of contractional rings could be evaluated as "moderately good" as to their heredity. On the basis of the prior concordance investigations for each of the 300 pairs, a "similarity index" and a "concordance index" were computed in order to arrive at evidence as to the degree of similarity of the single pairs; i.e. in order to detect the limits of the similarity between non-identical twins and the dissimilarity between identical twins. This concluding investigations lead to the question of the possibility of a diagnosis of genetic identicalness solely on the basis of the comparison of irises.


Fighting back: promoting emancipatory nursing actions.

Poverty, education, and social problems are inextricably linked to health concerns and cannot be addressed in isolation from each other. Nurses are being challenged to care for clients who are socially, politically, and economically disadvantaged. The model of emancipatory nursing actions is derived from the work of Freire, Habermas, and Katz and presented as a practice model in guiding nurses to begin choosing actions that seek to help people fight back from the depths of their despair, rather than helping people cope and adapt to their oppression. Emancipatory interventions are provided to help nurses launch a new direction toward freeing their clients, rather than herding them through an uncaring and disjointed health and social service system.


Conceptual and methodological issues in nurse case management research.

Conceptual and methodological issues facing researchers who are studying the process and outcomes of nurse case management are identified. The evolution of a research program designed to understand and evaluate one model of nurse case management is described as an example of responding to these issues. The development of a body of knowledge around nurse case management is essential to assure the expansion of this role and to justify reimbursement through current and evolving insurance mechanisms.


Nursing care partnerships at the Denver Nursing Project in Human Caring: an application and extension of caring theory in practice.

Jean Watson's theory of nursing as the art and science of human caring provides the framework for practice at the Denver Nursing Project in Human Caring, a nurse-managed center for people living with HIV/AIDS. The purpose of this article is to describe the development, implementation, and evaluation of a new model of nursing practice at the Caring Center, called nursing care partnerships. Client and nurse narrative accounts are presented as a means of grounding the reader in the care partners' relationships that are formed during the journey of HIV/AIDS.


Components of nurse innovation: a model from acute care hospitals.

Components that promote nurse innovation in acute care hospitals are explicated in the Acute Care Nursing Innovation Model. Grounded in nursing care delivery systems and excellent management-organizations perspectives, nurse executives and 30 nurse "intrapreneurs" from 10 innovative hospitals spanning the United States shared their experiences and insights through semistructured, tape-recorded telephone interviews. Guided by interpretive interactionist strategies, the essential components, characteristics, and interrelationships are conceptualized and described so that others may be successful in their innovative endeavors. Successful innovation is dependent on the fit between and among the components; the better the fit, the more likely the innovation will succeed.


Parse's theory as a model for practice: the cutting edge.

If nursing practice is the performing art of the science of nursing, then practice is guided by nursing theory and cannot be considered separately from it. Nursing theory based practice is the cutting edge of the discipline. Practice methods reflect the beliefs and values of the theories from which they evolve. Nurses' values and beliefs about human beings and health are incarnated in the way they practice. This article presents Parse's theory of human becoming as a model for practice; explicates the beliefs, values, and ethic of the practice method; analyzes the logical coherence of theory and method; illustrates the pragmatics of the method, and gives evidence of its soundness. The theory-research-practice triad developed by Parse articulates a paradigmatic view of the discipline as a whole with a view of the human being as a living unity freely choosing personal meanings in life situations. Parse's practice methodology provides nurses with a way of practicing with a focus on the quality of life as it is humanly lived. It is presented here as the practice component of an emergent theory-research-practice tradition on the cutting edge of nursing science.


Family nursing practice paradigm perspectives and diagnostic approaches.

Family nursing practice reflects different belief systems about the nature of the family and family health. Competing belief systems (paradigms) are examined as they relate to the concepts of concern to family nursing. Using a family case study, divergent diagnostic approaches emerging from the paradigms are described and demonstrated. Implications are delineated for the further development of family nursing practice.


Nursing practice model for maternal role sufficiency.

In 1975 Meleis set forth a conceptual framework for nursing practice centered on the concepts of role insufficiency and role supplementation. Later, Millor introduced a parental role sufficiency model for nursing research in child abuse and neglect. Based on the works of Meleis and Millor, a nursing practice model is proposed that focuses on maternal role sufficiency. It includes assessment of prenatal characteristics, measurement of developmental and health-illness outcomes, and preventive role supplementation intervention.


Pharmacokinetic-interaction study of didanosine and ranitidine in patients seropositive for human immunodeficiency virus.

The potential pharmacokinetic interactions between didanosine, an acid-labile antiretroviral agent, and ranitidine, an H2-receptor antagonist, were evaluated by a crossover study of 12 male patients seropositive for the human immunodeficiency virus. Single oral doses of 375 mg of didanosine, formulated as a citrate-phosphate-buffered sachet, or of 150 mg of ranitidine were administered alone or in combination (ranitidine was given 2 h prior to didanosine). Serial blood samples and total urinary output were collected after each treatment and analyzed for didanosine and/or ranitidine by validated high-performance liquid chromatography-UV assay methods. Pharmacokinetic parameters were calculated by noncompartmental methods. There were significant increases in mean area under the curve from time zero to infinity and mean urinary recovery for didanosine given in combination with ranitidine compared with those for didanosine alone. There were no significant differences between didanosine coadministered with ranitidine and didanosine alone in the respective mean peak concentrations in plasma, times to peak, elimination half-lives, or renal clearances. The mean area under the curve for ranitidine given with didanosine was significantly less than that for ranitidine given alone. There were no significant differences between the mean peak concentrations in plasma, times to peak, elimination half-lives, renal clearances, or urinary recovery values for ranitidine coadministered with didanosine and values for ranitidine given alone. These data demonstrate that administration of didanosine 2 h after ranitidine will result in a minor increase in the bioavailability of didanosine. A modification in the dose of didanosine or ranitidine is not necessary if the dose of ranitidine precedes that of didanosine by 2 h.


CP-72,588, a semisynthetic analog of the polyether ionophore UK-58,582 with increased anticoccidial potency.

We have employed semisynthesis to enhance the anticoccidial potency of a polyether ionophore. CP-72,588 is the alpha-methyl analog of the fermentation-derived polyether ionophore UK-58,852. The parent ionophore required a dose of 15 ppm to achieve anticoccidial efficacy in chickens equivalent to that of salinomycin at 60 ppm. CP-72,588 demonstrated substantially improved potency, with efficacy at 5 to 7.5 ppm. The intrinsic antimicrobial potencies of the two ionophores are similar; however, CP-72,588 was found in chicken tissues at higher levels than those of the parent ionophore when each was administered at the same dose (8 ppm). The enhanced potency of CP-72,588 may be partially due to enhanced uptake into tissues.


Comparative study of pharmacokinetics and serum bactericidal activities of cefpirome, ceftazidime, ceftriaxone, imipenem, and ciprofloxacin.

We compared the pharmacokinetics and the serum bactericidal activities of cefpirome, ceftazidime, ceftriaxone, imipenem, and ciprofloxacin. Fifteen healthy volunteers received 1 g of cefpirome, ceftazidime, and ceftriaxone intravenously, 500 mg of imipenem-cilastatin intravenously, and 500 mg of ciprofloxacin orally. High-performance liquid chromatographic assays were used to quantitate unchanged antibiotic in plasma and urine. Serum bactericidal activities were determined against six clinical isolates each of Staphylococcus aureus, Enterobacter cloacae, and Pseudomonas aeruginosa by using a modified microdilution method of Reller and Stratton (L. B. Reller and C. W. Stratton, J. Infect. Dis. 136:196-204, 1977). Overall, cefpirome exhibited pharmacokinetics similar to those of ceftazidime: half-life (t1/2), 1.95 h; concentration at 1 h (C1h), 47 to 49 micrograms/ml for both antibiotics. Ceftriaxone displayed the longest t1/2 (7.65 h) and the highest C1h (137.8 micrograms/ml), while we observed the shortest t1/2 (1.05 h) and the lowest C1h (19.85 micrograms/ml) with imipenem. At 1 h, cefpirome and, even more so, imipenem showed significantly better serum bactericidal activities against S. aureus (1:273 and 1:80) than did the other antibiotics (P less than 0.0005; analysis of variance with randomized block design and Bonferroni correction). Against E. cloacae, we observed the highest serum bactericidal titers at 1 h with cefpirome, and this superiority vis-à-vis the other antibiotics tested was maintained for up to 8 h after dosing. Ceftazidime remained the most active agent tested against P. aeruginosa (serum bactericidal activity titers, 1:43 at 1 h) up to 8 h. In summary, the study showed that cefpirome and imipenem provide more potent serum bactericidal activities than do broad-spectrum cephalosporins against S. aureus; thus, both of these antibiotics should be adequate against serious S. aureus infections. In addition, cefpirome appears to be a promising alternative for treatment of infections caused by E. cloacae and P. aeruginosa.


Effects of sub-MICs of antibiotics on cell surface characteristics and virulence of Pasteurella multocida.

The effects of sub-MICs of certain antibiotics, namely, penicillin G, tetracycline, and trimethoprim-sulfamethoxazole, on the cell surface characteristics and the virulences of two toxigenic isolates of Pasteurella multocida representing capsular types A and D were evaluated. Expression of proteins, in particular, outer membrane proteins and iron-regulated proteins, was not affected by exposure of bacterial cells to low concentrations of antibiotics. However, exposition of surface antigens was modified by sub-MICs of the antibiotics tested. The lipopolysaccharide profile of one isolate (capsular type D) was altered by penicillin G. Sub-MICs of penicillin G and tetracycline diminished the virulence of the capsular type A isolate and adherence to porcine tracheal rings of the capsular type D isolate. Production of dermonecrotic toxin was not affected by sub-MICs of the antibiotics tested. Our results indicate that growth of P. multocida in the presence of low concentrations of antibiotics seems to have, depending on the isolate, profound effects on cell surface characteristics, with concomitant effects on adherence or virulence. Our results also indicate that production of dermonecrotic toxin, an important virulence factor of P. multocida isolates associated with porcine atrophic rhinitis, was not affected by sub-MICs of the antibiotics studied.


Penetration of cefpodoxime proxetil in lung parenchyma and epithelial lining fluid of noninfected patients.

The pulmonary disposition of cefpodoxime was studied in 12 patients with pulmonary opacities after a single oral dose of 260 mg of cefpodoxime-proxetil, which is equivalent to 200 mg of cefpodoxime. Blood and lung tissue samples were collected during surgery, and bronchoalveolar lavage was carried out 3 h (group A) or 6 h (group B) after drug administration. Urea was used as an endogenous marker for measurement of the volume of epithelial lining fluid (ELF). Concentrations were measured by using a microbiological assay. The mean concentrations of cefpodoxime in plasma, ELF, and lung tissue were, respectively, 1.85 +/- 0.82 mg/liter, 0.22 +/- 0.13 mg/liter, and 0.89 +/- 0.80 mg/kg of body weight in group A and 1.40 +/- 1.25 mg/liter, 0.12 +/- 0.14 mg/liter, and 0.84 +/- 0.61 mg/kg in group B. Concentrations in lung parenchyma 6 h after dosing were at least equal to or above the MICs for 90% of the strains of most organisms commonly found in respiratory tract infections, whereas data for ELF suggest levels of drug insufficient to inhibit bacteria.


Microbiological efficacy and pharmacokinetics of prophylactic antibiotics in liver transplant patients.

The pharmacokinetics of perioperative systemic antibiotics and the microbiological effectiveness of oral nonabsorbable antibiotics started immediately prior to surgery were studied in 18 adult patients undergoing liver transplantation. All patients received cefotaxime, 2 g intravenously, at 6-h intervals during surgery and then at 8-h intervals thereafter for 48 h; eight patients also received ampicillin at the same dose and schedule. This regimen produced levels of antibiotics in blood that appeared appropriate for prophylaxis. The first dose peak (68 +/- 18 micrograms/ml) and trough (6.9 +/- 4.7 micrograms/ml) levels of cefotaxime in serum and the first dose peak (73 +/- 22 micrograms/ml) and trough (4.1 +/- 2.3 micrograms/ml) levels of ampicillin in serum, which were assayed by high-performance liquid chromatography, were similar to levels reported in normal volunteers, despite mean intraoperative blood loss of 3.3 liters and fluid replacement of 21 liters. On postoperative days 1 and 2, the levels of cefotaxime and ampicillin were maintained at or above 0.9 and 1.3 micrograms/ml, respectively, with little accumulation. By random assignment, 8 patients received systemic antibiotics alone and 10 patients received systemic antibiotics plus a 3-week regimen of oral nonabsorbable antibiotics (gentamicin, polymyxin E, and nystatin) beginning when a donor liver was procured. Pre- and postoperative cultures of rectum, throat, and gastric aspirate samples showed persistence of aerobic gram-negative bacilli for the first 2 postoperative weeks in about half of the patients in each group. Failure of the regimen of oral nonabsorbable antibiotics to supplement cefotaxime in eradicating aerobic gram-negative bacilli from stools probably results from impaired peristalsis during and after surgery and warrants earlier initiation of the regimen.


Endotoxin concentration in neutropenic patients with suspected gram-negative sepsis: correlation with clinical outcome and determination of anti-endotoxin core antibodies during therapy with polyclonal immunoglobulin M-enriched immunoglobulins.

We carried out a study in patients with severe neutropenia from hematologic malignancy and suspected gram-negative sepsis to evaluate the clinical significance of endotoxin concentrations in plasma before and during a therapeutic intervention with a human polyclonal immunoglobulin M (IgM)-enriched immunoglobulin preparation (Pentaglobin; Biotest, Dreieich, Germany). Twenty-one patients with acute leukemia or non-Hodgkin's lymphoma entered the study upon the development of clinical signs of gram-negative sepsis and received the IgM-enriched immunoglobulin preparation every 6 h for 3 days (total dose, 1.3 liter with 7.8 g of IgM, 7.8 g of IgA, and 49.4 g of IgG), in addition to standardized antibiotic treatment. Concentrations of endotoxin and IgM and IgG antibodies against lipid A and Re lipopolysaccharide (LPS) in plasma were determined by a modified chromogenic Limulus amebocyte lysate test and semiquantitative enzyme linked immunosorbent assay, respectively, before each immunoglobulin infusion and during the following 25 days. Seventeen patients were endotoxin positive; in five of these patients, gram-negative infection was confirmed by microbiologic findings. Prior to therapy, endotoxemia correlated significantly with the occurrence of fever, and a quantitative correlation between the endotoxin concentration and body temperature was found during the individual course of infection in 8 of the 17 patients. Overall mortality from endotoxin-positive sepsis was 41% (7 of 17) and 64% (7 of 11) in patients with symptoms of septic shock. Nonsurvivors had significantly higher maximum concentration of endotoxin in plasma compared with those of survivors at the first study day (median of 126 versus 34 pg/ml; P < 0.05) and during the whole septic episode (median of 126 versus 61 pg/ml; P < 0.05). In survivors, immunoglobulin therapy resulted in a significant decrease in endotoxin levels in plasma within the initial 18-h treatment period, from a pretreatment median value of 28 pg/ml to a value of 8 pg/ml (P< 0.05). In the seven patients who died from uncontrollable infection, no effect of therapy on endotoxin levels in plasma was observed. IgM and IgG antibodies against lipid A and Re LPS increased significantly under immunoglobulin treatment, with significant correlations between antibodies against lipid A and Re LPS. These data strongly suggest a prognostic significance of the endotoxin levels in plasma and a potential effect of treatment with a polyclonal IgM-enriched immunoglobulin preparation. Further studies are needed to substantiate these findings and to assess the impact on the clinical course by way of a prospective placebo-controlled clinical trial.


Effect of beta-lactams on peptidoglycan metabolism of Haemophilus influenzae grown in animals.

We have examined bacterial determinants that influence beta-lactam activity in Haemophilus influenzae cells cultivated in a system that reproduces in vivo growth conditions. Bacteria grown in diffusion chambers were recovered from the peritoneal cavities of rats, and their cell properties were compared with those of bacteria grown in broth cultures by various tests performed in vitro. The rate of peptidoglycan synthesis was measured as the incorporation of [14C]alanine into cell wall material in the presence of chloramphenicol. The total incorporation of [14C]alanine into peptidoglycan was markedly increased in cells grown in rats prior to the assay but was efficiently reduced by the beta-lactams. The extent of cross-linking was lower in the peptidoglycan of in vivo-grown bacteria, as estimated by sodium dodecyl sulfate- to trichloroacetic acid-insoluble radioactive cell wall material ratios. A whole-cell labeling assay with 125I-penicillin was used to characterize the penicillin-binding proteins (PBPs). Four PBPs showed a striking reduction in the binding of the labeled penicillin in cells grown in rats. Such changes resembled the PBP alterations seen in beta-lactamase-negative clinical strains that were resistant to the beta-lactams. Although ampicillin and moxalactam showed delayed inhibitory activities in vitro for cells collected from rats, cells recovered from beta-lactam-treated rats showed evidence of antibiotic effectiveness (binding of the beta-lactams to PBPs in vivo and altered morphology), and the killing of cells exposed to antibiotics in broth or in peritoneal fluid was equally good. Finally, the frequencies of spontaneous resistance or tolerance to ampicillin or moxalactam were estimated, and there was no significant difference for in vitro- or in vivo-grown cells. These data demonstrated that the cultivation of H. influenzae in animals created changes in PBPs and the overall peptidoglycan metabolism. Such alterations did not impair the bactericidal activities of the beta-lactams, although they resulted in delayed bacterial inhibition, a phenomenon that may have important consequences in antibiotherapy.


Central nervous system targeting of 2',3'-dideoxyinosine via adenosine deaminase-activated 6-halo-dideoxypurine prodrugs.

AIDS dementia complex is a neurologic disorder, characterized by increasingly severe cognitive, behavioral, and motor impairment, which is associated with human immunodeficiency virus (HIV) infection in the central nervous system (CNS). Many of the dideoxynucleosides effective systemically in the treatment of HIV infections, such as 2',3'-dideoxyinosine (ddI), exhibit limited penetration into the CNS and limited or variable effectiveness in reversing the symptoms of AIDS dementia. Thus, approaches for increasing the CNS uptake of ddI and other dideoxynucleosides are needed. The CNS uptake of a series of 6-halo-2',3'-dideoxypurine ribofuranosides (6-halo-ddPs) previously shown to be active against HIV because of their conversion to ddI through the action of adenosine deaminase was examined in rats. In vitro studies in rat blood and brain tissue homogenate suggested a favorable selectivity for bioconversion in brain tissue, but with bioconversion half-lives varying widely within the series. In vivo infusions of 6-chloro-ddP (6-Cl-ddP), 6-bromo-ddP (6-Br-ddP), and 6-iodo-ddP (6-I-ddP) resulted in significant increases (20- to 34-fold) in the ddI concentration ratios in brain parenchyma/plasma when compared with those after an infusion of ddI alone. Absolute concentrations of ddI in brain parenchyma were increased 10- and 4-fold, respectively, following 30-min infusions of 6-Cl-ddP or 6-Br-ddP, but were 2.4-fold lower after an infusion of 6-I-ddP relative to that after a control infusion of ddI. Detailed studies of the plasma pharmacokinetics, CNS uptake kinetics, and bioconversion of 6-Cl-ddP were conducted to compare in vivo transport and bioconversion parameters with those predicted from in vitro measurements and to rationalize the efficiency of CNS delivery of ddI from 6-Cl-ddP. The results show that increased lipophilicity alone does not ensure that a given prodrug will deliver higher levels of a parent compound to the CNS. Both the selectivity and absolute rate of bioconversion in the brain are important factors.


Modes of action and inhibitory activities of new siderophore-beta-lactam conjugates that use specific iron uptake pathways for entry into bacteria.

We describe here the mechanism of inhibition of two new siderophore-beta-lactam conjugates against Escherichia coli X580. One conjugate is a spermidine-based catechol siderophore-carbacephalosporin (JAM-2-263), and the other is an N5-acetyl-N5-hydroxy-L-ornithine tripeptide hydroxamate siderophore-carbacephalosporin (EKD-3-88). In an agar diffusion test, both conjugates produced large inhibitory zones against strain X580. Resistant strains (i.e., JAMR and EKDR) could be isolated after exposure of X580 to the conjugates JAM-2-263 and EKD-3-88, respectively. No cross-resistance was observed in these individual isolates. JAMR and EKDR were studied further to elucidate the mechanism of inhibition of each conjugated drug. The affinities of JAM-2-263 and EKD-3-88 for penicillin-binding proteins (PBPs) of isolated inner membranes were determined by a competition assay with 125I-penicillin V. JAM-2-263 targeted primarily PBPs 1A/B and 5/6, while EKD-3-88 targeted PBPs 1A/B and 3. Strains X580, JAMR, and EKDR showed similar PBP affinities for the conjugates. However, marked changes were observed in the iron-regulated outer membrane proteins of resistant isolates grown on agar plates depleted of iron. EKDR lost the expression of FhuA (78 kDa) and its sensitivity to phages T1 and T5, whereas JAMR lost the expression of Cir (74 kDa) and its sensitivity to colicin Ia. These results revealed the requirement of FhuA and Cir for the inhibitory activities of EKD-3-88 and JAM-2-263, respectively. In an antibiotic diffusion assay, ferrichrome (1 microM) strongly antagonized the activities of both conjugates against X580 and JAMR, including the residual activity of JAM-2-263 against JAMR. However, the susceptibility of strain EKDR lacking the ferrichrome receptor (FhuA-) to the two conjugates remained the same in the presence of ferrichrome. The antagonistic effect of ferrichrome on the activity of JAM-2-263 may also indicate a role for FhuA in the activity of this beta-lactam conjugate. A FhuA- Cir- double mutant confirmed this hypothesis, since it showed a higher level of resistance to JAM-2-263. To reproduce iron-restricted in vivo growth conditions, we grew X580 and EKDR cells in diffusion chambers implanted in the peritoneal cavities of rats. Strain EKDR showed impaired growth in such a cultivation system. This is the first report of beta-lactam drug transport into E. coli cells that involves the FhuA outer membrane protein.


Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States.

Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with increasing frequency in the United States. We analyzed the penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) genotypes, and ribotypes of 22 multiresistant serotype 23F isolates of S. pneumoniae from the United States and 1 isolate each from Spain and South Africa. Also included were seven multiresistant isolates of other serotypes, three penicillin-resistant but chloramphenicol-susceptible serotype 23F isolates, and two penicillin-susceptible isolates (one penicillin-susceptible isolate was serotype 23F). Fifteen of the 22 multiresistant isolates from the United States and the isolates from Spain and South Africa had identical PBP patterns, MLEE profiles, and ribotypes. Six of the remaining seven multiresistant isolates were related by PBP pattern, but demonstrated slightly different MLEE and/or ribotype profiles, possibly because of acquisition of additional resistance markers (four of the six isolates were also resistant to erythromycin). The remaining multiresistant serotype 23F isolate had a unique PBP pattern and ribotype and was only distantly related to the other pneumococcal isolates by MLEE analysis. The PBP patterns, MLEE profiles, and ribotypes of the multiresistant serotype 23F isolates were easily distinguished from those of six multiresistant isolates of other serotypes; three other penicillin-resistant, chloramphenicol-susceptible, serotype 23F isolates; and two penicillin-susceptible isolates. One exception was a multiresistant serotype 19A isolate that was highly related to the clonal group by PBP pattern and MLEE analysis and that had a ribotype similar to those of the other erythromycin-resistant serotype 23F isolates. MLEE analysis and ribotyping were more discriminating than were the PBP patterns in discerning strain differences. These data strongly suggest that a multiresistant clone of S. pneumoniae serotype 23F that is related to multiresistant isolates from Spain and South Africa has become disseminated in the United States. Clinicians should be alerted to the spread of these multiresistant strains in the United States.


Prospective study of oral teicoplanin versus oral vancomycin for therapy of pseudomembranous colitis and Clostridium difficile-associated diarrhea.

A prospective, randomized study comparing oral teicoplanin with oral vancomycin in the treatment of pseudomembranous colitis (PMC) and Clostridium difficile-associated diarrhea (CDAD) was performed. Teicoplanin was administered at a dosage of 100 mg twice a day for 10 days, and vancomycin was administered at a dosage of 500 mg four times a day for 10 days. CDAD was diagnosed by demonstrating both C. difficile and cytotoxin in the feces of symptomatic patients (more than three loose stools per day). The diagnosis of PMC was also based on colonoscopy. Cytotoxin assay and cultures were checked in all patients 7 to 10 days after discontinuation of therapy and 25 to 30 days thereafter. Of the 51 patients enrolled, 46 were judged to be assessable. Among these, 26 received teicoplanin and 20 received vancomycin. At enrollment, both groups were comparable in terms of age, sex, occurrence of PMC or CDAD, and previous antibiotic treatment. Eighteen of the 20 patients in the vancomycin group and 10 of the 26 patients in the teicoplanin group had previously undergone surgery (P = 0.0004). Treatment resulted in the clinical cure of 20 (100%) vancomycin and 25 (96.2%) teicoplanin patients (P = 0.56). After discontinuation of therapy, clinical symptoms recurred in four (20%) vancomycin patients and two (7.7%) teicoplanin patients (P = 0.21). Posttherapy asymptomatic C. difficile carriage (positive follow-up cultures without any clinical symptoms) occurred in five (25%) vancomycin patients and two (7.7%) teicoplanin patients (P = 0.11).Overall, 9 of 20 (45%) vancomycin patients and 5 of 26 (19.2%) teicoplanin patients (P=0.059) appeared not to be cleared of C. difficile after treatment. No adverse effects related to vancomycin or teicoplanin therapy were observed.


Antimicrobial susceptibilities and beta-lactamase characterization of Capnocytophaga species.

Capnocytophaga species have been associated with a wide variety of infections in both immunocompetent and immunocompromised patients. On the basis of data from antimicrobial susceptibility studies, beta-lactam antibiotics have been considered efficacious therapy. Six of 19 isolates from primarily clinical sources across Canada demonstrated beta-lactamase production, and agar dilution susceptibility testing showed broad resistance to beta-lactam antibiotics. For the beta-lactamase producing isolates, clavulanate reduced the MIC of amoxicillin for 90% of the strains tested by 64-fold. Isolates were highly susceptible to clindamycin, imipenem, and ciprofloxacin. Characterization of the beta-lactamases produced by two of these isolates (Van1 and Van2) was performed. Isoelectric focusing revealed an identical isoelectric point of 5.6 for both enzymes, but they had markedly different relative hydrolysis efficiencies, and different conditions were required to extract the enzymes. This study demonstrates the production of different types of beta-lactamases by Capnocytophaga spp. and suggests the need to screen all clinical isolates of Capnocytophaga spp. for the presence of beta-lactamases.


Absence of synergistic activity between ampicillin and vancomycin against highly vancomycin-resistant enterococci.

The emergence of clinical enterococcal isolates resistant to both ampicillin and vancomycin is a cause of great concern, as there are few therapeutic alternatives for treatment of infections caused by such organisms. We evaluated the effects of the combination of ampicillin with vancomycin against vancomycin-resistant clinical enterococcal isolates. Using both the checkerboard technique and time-kill curves, we examined 28 strains of enterococci (17 Enterococcus faecalis and 11 Enterococcus faecium strains) with different levels of resistance to vancomycin. Of these, 15 strains were also highly gentamicin resistant, and 9 demonstrated resistance to ampicillin. Only seven strains of E. faecalis were inhibited synergistically by the combination of vancomycin with ampicillin, and even then, the concentrations of vancomycin at which synergism was demonstrated were above levels achievable in serum. None of the ampicillin-resistant isolates (all E. faecium) were inhibited synergistically at any concentration of the drugs. In no instance was bactericidal synergism observed, and in most cases the combination resulted in less killing than with ampicillin alone. Antagonism was not observed at clinically relevant concentrations. The results of this study suggest that the combination of vancomycin with ampicillin has little to offer against these emerging pathogens.


Subcellular localization of tobramycin and vancomycin given alone and in combination in proximal tubular cells, determined by immunogold labeling.

The subcellular localization of tobramycin and vancomycin in the renal cortices of rats was determined with ultrathin sections by immunogold labeling. Four groups of four rats each were treated for 10 days with saline (NaCl, 0.9%), tobramycin at dosages of 20 mg/kg of body weight per 12 h intraperitoneally, vancomycin at dosages of 25 mg/kg/12 h subcutaneously, or the combination tobramycin-vancomycin. On day 11, the animals were killed, and cubes of renal cortex were fixed overnight in phosphate-buffered glutaraldehyde (0.5%), dehydrated in ethanol, and embedded in Araldite 502 resin. Ultrathin sections were made and incubated with sheep antitobramycin antibody followed by protein A-gold (15-nm diameter) complex or rabbit antivancomycin antibody followed by gold (30-nm diameter)-labeled goat anti-rabbit antibody. For the double labeling, incubations were made on opposite sides of the grid. Tobramycin was detected over the lysosomes of proximal tubular cells, but the labeling was concentrated into small areas in the matrix of the lysosomes. Vancomycin was seen over the lysosomes of proximal tubular cells and was distributed uniformly throughout the matrix of the lysosomes. In rats treated with tobramycin-vancomycin, both drugs were still detected in lysosomes of proximal tubular cells. It is concluded that tobramycin and vancomycin accumulate in lysosomes of proximal tubular cells throughout 10 days of treatment and that vancomycin has no effect on the subcellular distribution of tobramycin.


Pharmacodynamic interaction between RP 59500 and gram-positive bacteria infecting fibrin clots.

The fibrin clot penetration and in vivo bactericidal activity of RP 59500, a new semisynthetic streptogramin, for two Staphylococcus aureus strains (one methicillin resistant and the other methicillin susceptible), two Staphylococcus epidermidis strains (one methicillin resistant and the other methicillin susceptible), and one Enterococcus faecalis strain were evaluated. The clots, inserted subcutaneously, were infected with a mean of 10(8) CFU of the pathogen per g. For each strain, groups of four rabbits received a single intravenous injection of 50 mg of RP 59500 per kg of body weight over 30 min. The mean peak level of RP 59500 in serum in the infected rabbits was 61.9 +/- 6.3 micrograms/ml. The drug was detectable in serum at a level of 0.8 micrograms/ml up to 4 h after administration. The mean peak fibrin clot drug level at 1 h was 3.3 +/- 0.1 micrograms/g. At 6 h, the level in clots was 1.2 +/- 0.1 micrograms/g. The mean half-life in serum in infected rabbits was 0.34 +/- 0.01 h, while in clots the drug exhibited a longer half-life of 3.8 +/- 0.4 h. In vivo, this new streptogramin sterilized the clots infected with the two S. aureus strains studied in less than 1 h and induced a marked reduction in colony counts of the two S. epidermidis strains studied for up to 24 h. The activity of the streptogramin against E. faecalis was limited. These results suggest that RP 59500 should be further evaluated for the treatment of infection with methicillin-resistant staphylococci.


Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens.

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Bräu, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.


Serum and blister fluid pharmacokinetics and bactericidal activities of ampicillin-sulbactam, cefotetan, cefoxitin, ceftizoxime, and ticarcillin-clavulanate.

Ampicillin-sulbactam, ticarcillin-clavulanate, cefoxitin, cefotetan, and ceftizoxime are promoted for the treatment of mixed aerobic-anaerobic bacterial infections. Their activities have been compared in vitro but not in vivo. In order to assess the in vivo activities of these agents in serum and interstitial fluid, we administered single, intravenous doses of these antimicrobial agents to healthy subjects. Concentrations of the antimicrobial agents in serum and suction-induced blister fluid and bactericidal activity were measured by high-pressure liquid chromatography and the standard methodology of the National Committee for Clinical Laboratory Standards, respectively. The organisms used for bactericidal activity tests were one isolate each of Staphylococcus aureus, Klebsiella pneumoniae, and Bacteroides fragilis. Pharmacokinetic parameters in serum and blister fluid were similar to those derived in other investigations. Of note were the high and prolonged concentrations of ticarcillin and cefotetan in blister fluid, despite high-level serum protein binding. The bactericidal activities in serum and blister fluid reflected the relative in vitro activities and kinetic dispositions of the various antimicrobial agents except for the bactericidal activity of cefotetan, which was substantially lower in blister fluid than serum, despite a blister fluid:serum area under the concentration-time curve ratio of 1.5. Similarly, the activity of ticarcillin-clavulanate in blister fluid was also substantially less than would have been predicted by the blister fluid:serum ratio of the area under the concentration-time curve of 1.1, possibly because of the low concentrations of clavulanate in blister fluid. The rankings of the in vivo bactericidal activities of the five drugs were as follows: for S. aureus, ampicillin-sulbactam > ticarcillin-clavulanate > ceftizoxime > cefoxitin > cefotetan; for K. pneumoniae, ceftizoxime > cefotetan > ampicillin-sulbactam = ticarcillin-clavulanate > cefoxitin; and for B.fragilis, ticarcillin-clavulanate > cefotetan > ceftizoxime > ampicillin-sulbactam = cefoxitin.


Modulation of interactions of Candida albicans and endothelial cells by fluconazole and amphotericin B.

Using an in vitro model of intravascular infection, we examined the effects of exposure to subinhibitory concentrations of fluconazole and amphotericin B on the ability of Candida albicans to adhere to and damage human umbilical vein endothelial cells. Incubation of the organisms for 18 h in 0.5x the MICs of fluconazole and amphotericin B inhibited endothelial cell adherence by 22 and 91%, respectively (P less than 0.001 for each drug). Candida-induced endothelial cell injury was also decreased by exposing the organisms to the antifungal drugs while in contact with the endothelial cells. Fluconazole inhibited damage by approximately 50% at concentrations ranging from 0.25x to 5x the MIC (P less than 0.01 for each concentration). Exposure to amphotericin B at 0.5x the MIC completely blocked the ability of the organisms to injure endothelial cells. The capacities of the antifungal agents to inhibit endothelial cell injury paralleled their abilities to suppress candidal germination. Organisms exposed to up to 5x the MIC of fluconazole had diminished, but still detectable, germ tube production and elongation, whereas incubation in 0.5x the MIC of amphotericin B completely abrogated germination. In addition to their direct effects on the growth of C. albicans, fluconazole and amphotericin B may decrease the ability of the fungus to disseminate hematogenously by inhibiting the organisms' capacity to adhere to and injure endothelial cells.


Intravenous and oral zidovudine pharmacokinetics and coagulation effects in asymptomatic human immunodeficiency virus-infected hemophilia patients.

Pharmacokinetic and coagulation studies were carried out over a 12-week period with 11 asymptomatic hemophilia patients with human immunodeficiency virus infection receiving zidovudine (ZDV). The patients received 300 mg every 4 h while awake (the accepted dose at the time of this study); consecutive 24-h intravenous (i.v.) and 12-h oral pharmacokinetic studies were conducted at weeks 1, 6, and 12. Coagulation studies were conducted at weeks 0, 4, 8, and 12. The numbers of units of factors VIII and IX and cryoprecipitate transfused during the 12-week periods before, during, and after ZDV treatment were recorded. Following i.v. and oral ZDV administration, the concentration in plasma declined rapidly over the first 4 h, and in some patients, ZDV was still detectable at 4 to 10 h. The i.v. total clearances (means +/- standard deviations) were 14.9 +/- 7.3, 11.2 +/- 3.7, and 15.1 +/- 4.7 ml/min/kg of body weight. The i.v. distribution volumes were 1.08 +/- 0.5, 1.0 +/- 0.4, and 1.65 +/- 1.4 liters/kg. The bioavailabilities were 0.54 +/- 0.22, 0.46 +/- 0.19, and 0.59 +/- 0.13 at weeks 1, 6, and 12, respectively. The pattern of ZDV-glucuronide (GZDV) disposition was similar to that of ZDV, and the peak plasma GZDV-to-ZDV ratio was higher after oral dosing, consistent with first-pass metabolism. In some individuals, up to 33% of an i.v. dose was excreted unchanged. At weeks 6 and 12, greater than 300 mg of total ZDV (GZDV plus ZDV) was recovered in the urine of some patients, suggesting tissue redistribution. Concentration in plasma after oral ZDV administration were variable, both within and between patients. The von Willebrand antigen level consistently decreased throughout the study but was not accompanied by a parallel change in ristocetin cofactor A activity, and no clinical adverse effects on coagulation were noted. This study demonstrates that ZDV can be used in hemophilia patients without worsening of their bleeding tendencies. The clinical significance of decreased ZDV clearance and the prolonged terminal elimination phase of ZDV will require further study with patients receiving chronic ZDV.


Sequence analysis of the beta-lactamase repressor from Staphylococcus aureus and hybridization studies with two beta-lactamase-producing isolates of Enterococcus faecalis.

The putative beta-lactamase (Bla) repressor gene, blaI, from the staphylococcal plasmid pI524 was isolated, and the DNA sequence was determined. The sequence of blaI was found to be identical to the blaI sequence from pI9789 (blaI blaZ seg-1), a plasmid related to pI524. A blaI probe from pI524 was hybridized with plasmid and genomic DNA from Bla+ Enterococcus faecalis isolates HH22 and PA. The Bla structural gene of HH22 has been previously shown to be of staphylococcal origin, but DNA homologous to the staphylococcal Bla repressor was not found, indicating that the constitutive production of beta-lactamase in these E. faecalis isolates may be the result of a missing repressor protein.


Effects of antacids, ferrous sulfate, and ranitidine on absorption of DR-3355 in humans.

This study examined the effects of widely used antacids (aluminum hydroxide, magnesium oxide, and calcium carbonate), ferrous sulfate, and ranitidine on the absorption of a fluorinated quinolone, (-)-(S)-9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro- 7H- pyrido-[1,2,3,-de][1,4]benzoxazine-6-carboxylic acid hemihydrate (DR-3355), in healthy male volunteers enrolled in three separate randomized crossover studies. Study 1 used 100-mg doses of DR-3355 and concurrent doses of aluminum hydroxide (1 g) or magnesium oxide (500 mg), while study 2 used DR-3355 (100 mg) and concurrent ferrous sulfate (160 mg) or calcium carbonate (1 g). Study 3 used DR-3355 (100 mg) and concurrent ranitidine (150 mg). Each study included control doses of DR-3355 (100 mg) alone. When aluminum hydroxide, ferrous sulfate, and magnesium oxide were coadministered with DR-3355, the relative bioavailability of DR-3355 was decreased to 56, 81, and 78%, respectively, of that for DR-3355 (100 mg) alone. Urinary excretion of DR-3355 was also significantly decreased by coadministration of these drugs. Thus, the magnitude of the decrease in the area under the concentration-time curve for DR-3355 varied among antacids, and the ranking of their inhibitory effects correlated with previously reported rankings of stability constants for chelate formation. DR-3355 bioavailability was not influenced by the concurrent administration of calcium carbonate and ranitidine, indicating that changes in gastric pH do not affect DR-3355 absorption.


Dehydroepiandrosterone protects mice from endotoxin toxicity and reduces tumor necrosis factor production.

Recent reports have demonstrated an immunomodulating activity of dehydroepiandrosterone (DHEA) different from that described for glucocorticoids. The present study was designed to test DHEA's activity in endotoxic shock and to investigate its effect on endotoxin-induced production of tumor necrosis factor (TNF). Mortality of CD-1 mice exposed to a lethal dose of lipopolysaccharide (LPS; 800 micrograms per mouse) was reduced from 95 to 24% by treatment with a single dose of DHEA, given 5 min before LPS. LPS administration resulted in high levels of TNF, a response that was significantly blocked by DHEA, both in vivo and in vitro. DHEA treatment also reduced LPS-induced increments in serum corticosterone levels, a parameter considered not to be mediated by TNF. In another experimental model, mice sensitized with D-galactosamine, followed by administration of recombinant human TNF, were subjected to 89% mortality rate, which was reduced to 55% in DHEA-treated mice. These data show that DHEA protects mice from endotoxin lethality. The protective effect is probably mediated by reduction of TNF production as well as by effecting both TNF-induced and non-TNF-induced phenomena.


Pharmacokinetics of 18F-labeled fleroxacin in rabbits with Escherichia coli infections, studied with positron emission tomography.

18F-labeled fleroxacin was used to measure the pharmacokinetics of fleroxacin in healthy and infected animals by positron emission tomography (PET) and tissue radioactivity measurements. In all experiments, a pharmacological dose of unlabeled drug (10 mg/kg) was coinjected with the tracer. The pharmacokinetics of [18F]fleroxacin was measured in groups of healthy mice (n = six per group) at 10, 30, 60, and 120 min after injection and in groups of rats with Escherichia coli thigh infections (n = six per group) at 60 and 120 min after injection by radioactivity measurements in excised tissues. In healthy rabbits (n = 4) and in rabbits with E. coli thigh infections (n = 4), tissue concentrations of drug were determined by serial PET imaging over 2 h; after the final image was acquired, animals were sacrificed and concentrations measured by PET were compared with the results of tissue radioactivity measurements. In all three species, there was rapid equilibration of [18F]fleroxacin to significant concentrations in most peripheral organs; low concentrations of drug were detected in the brain. Accumulations of radiolabeled drug in infected and healthy thigh muscles were similar. Peak concentrations of drug of more than three times the MIC for 90% of members of the family Enterobacteriaceae (greater than 100-fold for most organisms) were achieved in all tissues except brain and remained above this level for more than 2 h. Especially high peak concentrations were achieved in the kidney (greater than 75 micrograms/g), liver (greater than 50 micrograms/g), blood (greater than 25 micrograms/g), and bone and lung (greater than 10 micrograms/g). Since the MICs for 90% of all Enterobacteriaceae are <2 micrograms/ml, fleroxacin should be particularly useful in treating gram-negative infections affecting these tissues. In contrast, the low concentration of drug delivered to the brain should limit the toxicity of the drug for the central nervous system.


Effects of the aggregation state of amphotericin B on its toxicity to mice.

Amphotericin B (AmB) is a very effective antifungal agent for most systemic fungal infections. However, the relatively high toxicity of this drug imposes limits on its clinical usefulness. Most of the current work in this field is devoted to the search for less-toxic formulations of the drug. Here we describe the effects of three surfactants, one anionic and the other two nonionic, on the aggregation state of AmB in solutions which were injected intravenously into mice. The degree of aggregation of AmB was monitored spectroscopically and by light scattering. The toxicity was expressed as percentage of survivors. These results were compared with those obtained with doses of AmB the same as those present in a commercial formulation of AmB, Fungizone. Two surfactants, lauryl sucrose and sodium deoxycholate, used at concentrations which induced monomerization of AmB, substantially decreased the acute toxicity of AmB to mice. Conversely, the third surfactant, Tween 80, showed a synergistic potentiation of the toxicity of the antibiotic. A good correlation was found between the in vivo toxicity and the aggregation state of AmB in injected solutions. Solutions in which AmB was almost entirely monomeric were half as toxic after 24 h and about six times less toxic after 1 week than the corresponding solutions of Fungizone.


Use of Tn5lac to study expression of genes required for production of the antibiotic TA.

The beta-galactosidase activities arising from Tn5lac insertions in several genes required for antibiotic TA production were measured under different growth conditions. In all of the non-TA-producing mutants, the beta-galactosidase specific activity was higher when the cells were grown in nutrient-limited 0.5CTS medium (0.5% Casitone plus alanine, serine, and glucose) than in rich 2CT medium (2% Casitone). One of the mutants, 420, had low beta-galactosidase specific activity in both media. The other seven mutants containing inserts in genes essential for TA production had specific activities of 139 to 367 U/mg of protein in 0.5CTS medium and 11 to 48 U/mg of protein in 2CT medium. The beta-galactosidase specific activities of two strains, 1030 and 420, increased during exponential growth in 0.5CTS medium. The beta-galactosidase specific activities of both strains increased greatly when the cells were grown in the presence of magnesium phosphate, which traps ammonium ions. The Tn5lac insertions in 1030 and 420 were used to screen for mutants with increased levels of transcription. An N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in 1030 that mapped 17 kb from the omega 1010 insert increased the specific activity of beta-galactosidase 21 times in 2CT medium. The regulatory mutation appears to release the repression caused by 2CT medium. A UV-induced mutation in 420 increased the beta-galactosidase specific activity 1.4 to 2.4 times. Medium conditions that affect the transcription of TA genes are discussed in terms of enhanced antibiotic TA production.


Low-dose trimethoprim-sulfamethoxazole alone and in association with zidovudine for prevention and treatment of murine Pneumocystis carinii pneumonia.

Low-dose trimethoprim-sulfamethoxazole (TMP-SMX) alone was found to be as effective as low-dose TMP-SMX plus zidovudine and standard-dose TMP-SMX alone in preventing and treating Pneumocystis carinii pneumonia (PCP) in an immunosuppressed-rat model. Zidovudine alone had no preventive or curative effect on PCP. We conclude that the initially reported reduced incidence of PCP in human immunodeficiency virus-infected patients treated with zidovudine alone is not due to anti-P. carinii activity of zidovudine. Furthermore, the clinical efficacy of low-dose TMP-SMX for the prevention and treatment of PCP should be further investigated.


Effects of diclofenac on ceftriaxone pharmacokinetics in humans.

The effects of diclofenac, a nonsteroidal antiinflammatory drug, on the biliary and urinary excretion of ceftriaxone were evaluated in subjects with a T drain in the common bile duct. The kinetic study was carried out on the sixth postoperative day of treatment with ceftriaxone alone (2 g intravenously; group 1) or ceftriaxone combined with diclofenac (50 mg every 12 h orally from postoperative days 3 to 6; group 2). A significant increase in the elimination half-life of ceftriaxone was observed in group 2 patients. Diclofenac caused a significant rise in ceftriaxone biliary excretion. This increase was not sufficient to balance the significant deficit of urinary excretion of ceftriaxone.


In vivo expression of in vitro anticoccidial activity.

Large-scale screening has led to the identification of several experimental compounds that have very potent intrinsic activity against coccidia, but the lack of translation to in vivo efficacy has been a major hurdle in developing such leads into effective new drugs. We developed methods to explore the impact of oral availability and appropriate distribution in tissue, both of which are potentially important factors in the expression of activity in vivo. For the compounds that we examined, neither oral absorption nor distribution to the site of infection appeared to be the critical barrier to in vivo expression of intrinsic anticoccidial activity. Elucidation of the nature of additional factors that might be involved could assist greatly in the identification of useful new anticoccidial agents.


Bactericidal activity of ramoplanin against antibiotic-resistant enterococci.

Ramoplanin, a new lipoglycodepsipeptide antibiotic, was uniformly active against 65 strains of enterococci, including strains highly resistant to vancomycin, penicillin G, and gentamicin. MBCs were usually within a fourfold dilution of the MICs. In time-kill studies, ramoplanin alone demonstrated dose-dependent bactericidal activity against enterococcal strains that resisted killing by vancomycin or penicillin in combination with gentamicin.


Activities of newer fluoroquinolones against Shigella sonnei.

The activities of six fluoroquinolones were determined for 117 separate strains of Shigella sonnei. The order of increasing activity (MICs for 90% of strains tested) was enoxacin (0.25 micrograms/ml), temafloxacin (0.032 micrograms/ml), sparfloxacin (0.016 micrograms/ml), CI-960 (0.008 micrograms/ml), ciprofloxacin (0.008 micrograms/ml), and PD-131628-2 (0.008 micrograms/ml). These data, along with results of killing and mutational rate studies, showed that all six fluoroquinolones were highly inhibitory against S. sonnei and five fluoroquinolones were rapidly and persistently bactericidal.


Phase I study of antilipopolysaccharide human monoclonal antibody MAB-T88.

Monoclonal antibody MAB-T88 is a human monoclonal immunoglobulin M antibody directed at the lipopolysaccharide of gram-negative bacteria. In this study, nine patients who were expected to become neutropenic from antineoplastic chemotherapy received an infusion of MAB-T88, three patients at each of three doses: 1, 4, and 8 mg/kg of body weight. MAB-T88 was shown to be safe, with an effective half-life in plasma of 25.4 h, and no patient developed immunoglobulin G antibody to MAB-T88.


Novel nucleoside strategies for anti-HIV and anti-HSV therapy.

This mini-review describes the structure-activity relationships of several series of new nucleoside analogues. It also points to the possibilities of finding new molecular modifications which could lead to antivirals with activity both against human immunodeficiency viruses (HIV) and herpesviruses.


Anti-HIV-1 activity of 3'-azido-3'-deoxythymidine (AZT) in primary mononuclear phagocytes.

Conflicting data have been reported on ability of 3'-azido-3'-deoxythymidine (AZT) to protect mononuclear phagocytes from HIV-1 infection. We compared the antiviral potency of AZT in three types of primary human mononuclear phagocytes: peripheral blood monocytes, monocyte-derived macrophages (in vitro differentiated) and alveolar macrophages (in vivo differentiated). To establish highly-productive virus infection, purified cells (greater than 99%) from healthy donors were challenged with the macrophage-tropic HTLV-IIIBa-L strain at input multiplicities ranging from 0.05 to 20 TCID50 per cell. AZT (0.1 nM-10 microM) was added immediately after infection and either continued for the duration of the experiment or stopped 1-7 days after infection. The kinetics of HIV-1Ba-L replication were assessed by measuring p24 antigen production on days 4-28 post-infection. Continuous treatment with AZT reproducibly inhibited viral replication in a concentration-dependent manner in all three cell types. The IC90 of AZT was 0.04 microM in blood monocytes, 0.009 microM in monocyte-derived macrophages, and 0.0001 microM in alveolar macrophages (mean of 3-4 donors for each cell type). AZT was not cytotoxic at less than 10 microM as assessed by cell viability, cell protein, and interferon-gamma-activated H2O2-release. In experiments in which AZT treatment was stopped after infection, viral replication resumed after a lag of 7-14 days and increased exponentially toward control levels. This occurred despite initial inhibition of virus production to below the limit of p24 detection (approximately 50 pg/ml). These results indicate that AZT is a potent inhibitor of HIV-1 replication in primary mononuclear phagocytes regardless of the stage of cell differentiation, and that AZT is most active in tissue (alveolar) macrophages. AZT does not irreversibly block infection of mononuclear phagocytes, however, as viral replication resumes after removal of AZT.


Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication.

A cell culture system for the evaluation of compounds which inhibit HBV replication (Korba and Milman, Antiviral Res. 15:217, 1991) has been developed into a standardized assay. Toxicity of test compounds was assessed by the uptake of neutral red dye under culture and treatment conditions which were identical to those used for the antiviral assays. A total of 667 separate cultures of 2.2.15 cells were evaluated for this study. In 86 untreated cell cultures, representing 15 experiments over a 24-month period, the levels of extracellular HBV virion DNA and intracellular HBV DNA forms were found to vary by less than 2.5-fold overall. Virion DNA in serum and intracellular viral DNA replication intermediates [RI] are the two most reliable and commonly followed markers of hepadnavirus replication in patients and experimental animals. In these assays, levels of extracellular HBV virion DNA and intracellular HBV RI were well correlated in 2.2.15 cells. Less correlation was observed between the levels of HBV virion DNA and the 3.2-kb episomal HBV genomes present in the cells. A threshold level of 22-37 intracellular replicating HBV genomes appeared to be required before virions were detected in the culture medium. The activities of several 2'-substituted and 3'-substituted deoxynucleoside analogs against HBV replication were compared using this standardized assay. Dideoxycytosine [ddC] and dideoxyguanosine [ddG] were the most selective 2',3'-dideoxynucleosides against HBV in 2.2.15 cells. Substitution of fluorine at the 2' position abolished the antiviral activity of ddC, but enhanced the selective antiviral activities of dideoxythymidine and dideoxyuracil. Several 2'-fluorinated pyrimidine arabinosyl furanosides, reported to be potent (but toxic) inhibitors of hepadnaviruses in vivo, demonstrated relatively low selective antiviral activities in 2.2.15 cells. The current data base allows for validation of any given set of test evaluations through statistical analysis of both the positive and the negative treatment controls present in each experiment; thus, relevant comparisons of the selectivity of anti-HBV activities for different compounds examined in future experiments can be made.


Zidovudine inhibits hepatitis B virus replication.

Hepatitis B virus DNA polymerase is a viral enzyme that can use viral DNA as well as viral RNA as a template for DNA synthesis. Since both activities are essential for the production of new virus particles, blocking of this enzyme should reduce viral replication. In the present study the in vitro effect of zidovudine triphosphate on hepatitis B virus DNA polymerase activity and the in vivo effect of zidovudine on viral replication in chronic HBsAg-positive patients are investigated. Zidovudine triphosphate inhibited in vitro DNA polymerase activity by 50% at a concentration of 0.3 microM. Serum DNA polymerase activity was significantly reduced in 7 patients who received zidovudine (200 mg orally 4 times daily) for one week. A dose-response effect was suggested by the results found for 6 patients who received 100 mg, 200 mg and 300 mg orally 4 times daily for one week with 2 drug-free weeks between each course. We conclude that zidovudine may be of value for non-responders to alpha-interferon therapy or patients with high initial levels of viral replication prior to the start of interferon treatment.


Unexpected adverse reactions during a clinical trial in rural west Africa.

Ribavirin has been used widely in various clinical trials, without significant adverse effects beyond reversible, mild anemia. Since 1978 intravenous ribavirin has been used to treat Lassa fever in a remote area of Eastern Sierra Leone, West Africa. In March 1991, brief episodes of rigors in patients receiving ribavirin were reported. An immediate investigation found that 27/93 patients (29%) had records in 1990/91 of at least one episode, the strongest association being with survival of Lassa fever (P = 0.0001). The occurrence or number of rigors in an individual patient was unassociated with sex, age, weight, volume of loading dose, cumulative dose, administration of other drugs, use of intravenous lines or heparin traps. In a review of 12 years of ribavirin administration, 74/2117 injections sampled (3.5%) were associated with a record of rigors. Most occurred before 08.00 h (P less than 0.0001), between 0 and 30 min after injection, lasted 2-45 min, and clustered towards the end of the treatment course (P less than 0.0001). There was no association with drug lot or individual vials. Drug was being given as a bolus (less than 1 min). Since slowing the infusion rate, no further episodes have been reported. Epidemiologic techniques are important tools in rapid assessment of unexpected events particularly when conducting trials in remote locations.


New hypoxanthine nucleosides with RNA antiviral activity.

A series of novel C-2 functionalized hypoxanthine and purine ribonucleosides have been synthesized and evaluated against exotic RNA viruses of the family or genus alpha, arena, flavi, and rhabdo. Both specific and broad-spectrum antiviral activities were discovered but only with hypoxanthine nucleosides.


Animal models for anti-AIDS therapy.

Primate and non-primate species have been used to study the pathobiology of the simian immunodeficiency virus (SIV) and of the human immunodeficiency virus type 1 (HIV-1), respectively, and to develop new therapeutic regimes. Transgenic mice which express either the entire HIV-1 provirus or subgenomic fragments have been used to analyze viral gene products in vivo and may serve as models for the development of agents targeted to select viral functions. Chimeric mice which were created by transplanting human hematolymphoid cells into mice suffering from congenital severe combined immunodeficiency (scid/scid or so called SCID mice), can be infected with HIV-1 and allow one to study the entire HIV-1 replicative cycle. Type C murine leukemia virus models have been used to develop new prophylactic and therapeutic strategies but their use is restricted to the evaluation of select antiviral drug inhibition, targeted to retroviral genes common to both Lentivirinae and Oncovirinae. The role of various animal model systems in the development of anti-HIV-1 and anti-AIDS therapies is summarized.


In vivo anti-influenza virus activity of plant flavonoids possessing inhibitory activity for influenza virus sialidase.

Isoscutellarein (5,7,8,4'-tetrahydroxyflavone) from the leaf of Scutellaria baicalensis non-competitively inhibited (IC50, 20 microM) the hydrolysis of sodium p-nitrophenyl-N-acetyl-alpha-D-neuraminate by influenza virus sialidase with an apparent Ki value of 41 microM. Negligible inhibitory activity was observed for mouse liver sialidase at a concentration of 79 microM. Isoscutellarein also inhibited the replication of influenza virus A/WSN/33 in Madin-Darby bovine kidney cells with 50% virus inhibitory dose at 16 nmol/well and influenza virus A/PR/8/34 in the allantoic sac of embryonated egg with little toxic effects. The flavone showed significant anti-influenza virus activity in vitro similar to isoscutellarein-8-methylether (F36) (Nagai, T., Miyaichi, Y., Tomimori, T., Suzuki, Y. and Yamada H., 1990, Chem. Pharm. Bull. 38, 1329-1332), and more potent virucidal activity in ovo than F36. However, F36 completely prevented proliferation of mouse-adapted influenza virus A/PR/8/34 in mouse lung by the intranasal (0.5 mg/kg) and intraperitoneal (4 mg/kg) administrations, and it was more potent than the known anti-influenza virus substance, amantadine. Intranasal administration of F36 (0.5 mg/kg) also protected mice against a lethal influenza virus A/PR/8/34 infection. Isoscutellarein significantly inhibited lung virus proliferation when administered intranasally or orally to mice. F36 and isoscutellarein showed negligible toxic effect against mice. These results suggested that flavones, which have potent influenza virus sialidase inhibitory activity, have anti-influenza virus activity in vivo.


Murine retroviral disease-enhancing effects of a pyrimidinone immunomodulator.

(B10.A x A/WySn)F1 mice, infected with the Friend virus (FV) complex, were used as a predictive therapeutic model for AIDS. These infected mice exhibit many of the viral and immunologic manifestations of AIDS. Bropirimine (2-amino-5-bromo-6-phenyl-4[3H]pyrimidinone, ABPP) is an immunomodulating compound which has been shown to inhibit other viral infections. Oral (per os treatment) dosages of ABPP ranging from 50 to 400 mg/kg/day for 3 days resulted in increased numbers of infectious centers in the infected mice and increased splenomegaly and percentage of Ig+ (B cells) in spleens of infected and uninfected mice. Decreased percentages of total Thy-1.2+ (total T) cells and L3T4+ (T-helper) cells were seen in both uninfected and infected mice and a slightly decreased percentage of Ly-2+ (T-suppressor/cytotoxic) cells was observed in spleens of the infected mice. No effect on Ly2+ cells in spleens of uninfected mice was found. Intraperitoneal injection, single or multiple, of 20-200 mg/kg ABPP prior to FV injection resulted in increased spleen weights but had no effect on numbers of infectious centers in the spleens or on FV antibody titers in the plasma. Intraperitoneal treatment of uninfected mice with ABPP resulted in slight or no changes in percentages of Thy-1.2+, L3T4+ and Ly-2+ cells. Mice receiving multiple exposures of ABPP had an increase in percentage of splenic B cells and a depressed response to the T cell mitogen PHA. Treatment with ABPP induced the production of interferon (IFN); however, a state of hyporesponsive IFN production was seen following multiple administrations of ABPP. These data suggest that the immunomodulator ABPP may have an enhancing effect on this retroviral disease.


The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae.

The cell cycle of Saccharomyces cerevisiae contains a decision point in G1 called 'start', which is composed of two specific sites. Nutrient-starved cells arrest at the first site while pheromone-treated cells arrest at the second site. Functioning of the RAS-adenylate cyclase pathway is required for progression over the nutrient-starvation site while overactivation of the pathway renders the cells unable to arrest at this site. However, progression of cycling cells over the nutrient-starvation site does not appear to be triggered by the RAS-adenylate cyclase pathway in response to a specific stimulus, such as an exogenous nutrient. The essential function of the pathway appears to be limited to provision of a basal level of cAMP. cAMP-dependent protein kinase rather than cAMP might be the universal integrator of nutrient availability in yeast. On the other hand stimulation of the pathway in glucose-derepressed yeast cells by rapidly-fermented sugars, such as glucose, is well documented and might play a role in the control of the transition from gluconeogenic growth to fermentative growth. The initial trigger of this signalling pathway is proposed to reside in a 'glucose sensing complex' which has both a function in controlling the influx of glucose into the cell and in activating in addition to the RAS-adenylate cyclase pathway all other glucose-induced regulatory pathways in yeast. Two crucial problems remaining to be solved with respect to cell cycle control are the nature of the connection between the RAS-adenylate cyclase pathway and nitrogen-source induced progression over the nutrient-starvation site of 'start' and second the nature of the downstream processes linking the RAS-adenylate cyclase pathway to Cyclin/CDC28 controlled progression over the pheromone site of 'start'.


Genetic approaches to the study of mitochondrial biogenesis in yeast.

In contrast to most other organisms, the yeast Saccharomyces cerevisiae can survive without functional mitochondria. This ability has been exploited in genetic approaches to the study of mitochondrial biogenesis. In the last two decades, mitochondrial genetics have made major contributions to the identification of genes on the mitochondrial genome, the mapping of these genes and the establishment of structure-function relationships in the products they encode. In parallel, more than 200 complementation groups, corresponding to as many nuclear genes necessary for mitochondrial function or biogenesis have been described. Many of the latter are required for post-transcriptional events in mitochondrial gene expression, including the processing of mitochondrial pre-RNAs, the translation of mitochondrial mRNAs, or the assembly of mitochondrial translation products into the membrane. The aim of this review is to describe the genetic approaches used to unravel the intricacies of mitochondrial biogenesis and to summarize recent insights gained from their application.


Global regulators of chromosome function in yeast.

In the yeast Saccharomyces cerevisiae, several abundant, sequence-specific DNA binding proteins are involved in multiple aspects of chromosome function. In addition to functioning as transcriptional activators of a large number of yeast genes, they are also involved in transcriptional silencing, the initiation of DNA replication, centromere function and regulation of telomere length. This review will consider each of these proteins, focusing on what is known about the mechanisms of their multiple functions.


Nuclear transport and nuclear pores in yeast.

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.


The genetics of nuclear pre-mRNA splicing: a complex story.

The occurrence of introns in nuclear precursor RNAs (pre-mRNAs) is widespread in eukaryotes, and the splicing process that removes them is basically the same in yeasts as it is in higher eukaryotes. Splicing takes place in a very large, multi-component complex, the splicesome, and biochemical studies have been complicated by the large number of splicing factors involved. This review describes how genetic approaches used to study RNA splicing in Saccharomyces cerevisiae have complemented the biochemical studies and led to rapid advances in the field.


Molecular biology of translation in yeast.

The combination of genetic, molecular and biochemical approaches have made the yeast Saccharomyces cerevisiae a convenient organism to study translation. The sequence similarity of translation factors from yeast and other organisms suggests a high degree of conservation in the translational machineries. This view is also strengthened by a functional analogy of some proteins implicated in translation. Beautiful genetic experiments have confirmed existing models and added new insights in the mechanism of translation. This review summarizes recent experiments using yeast as a model system for the analysis of this complex process.