The significance of antibiotic production by Pseudomonas aureofaciens PA 147-2 for biological control of Phytophthora megasperma root rot of asparagus

Pseudomonas aureofaciens PA147-2 produces an antibiotic (Af⁺) which inhibits the growth of fungal phytopathogens on phosphate buffered potato dextrose agar (PBPDA). To determine the role of the antibiotic in disease suppression in vivo, PA147-2 and an antibiotic-deficient Tn5 mutant (Af^-) PA109, were tested for their ability to suppress root rot of Asparagus officinalis seedlings caused by Phytophthora megasperma var sojae, in the glasshouse. Seedlings coinoculated with the pathogen and the wildtype strain PA147-2, showed a significantly reduced level of infection and disease severity compared to seedlings inoculated with the pathogen alone. However, 100% of seedlings treated with Af^- mutant PA109 were diseased. Furthermore, a strain derived from mutant PA109, restored to Af⁺ through allele replacement of Tn5 by homologous recombination, gave similar levels of disease suppression as the wildtype. This suggests the antibiotic produced by PA147-2 is important for the control of P. megasperma in vitro as well as in planta. Treatment of seedlings with PA147-2 and derived strains including the Tn5 mutant (Af^-) strain improved plant weight by 40–100% in the presence and absence of the pathogen suggesting PA147-2 also has a direct growth stimulatory effect independent of antibiotic production.     

Entomophthora muscae (Entomophthorales: Entomophthoracae) mycosis in the onion fly,Delia antiqua (Diptera: Anthomyiidae)

Entomophthora muscae was identified as a common fungal pathogen of the onion fly, Delia antiqua, and the adult seed corn maggot, D. platura. Low infection levels also were found in populations of the cluster fly, Pollenia rudis (Diptera: Muscidae), and the tiger fly, Coenosia tigrina (Diptera: Muscidae). The disease cycle, as it affects D. antiqua in the onion agroecosystem, is described, including the etiology, symptomatology, and phenology. Natural infection levels approaching 100% were noted early in the spring and in late fall, impacting the 1st and 3rd generations of the D. antiqua population significantly. A lagged density-dependent disease response was noted at the gross population level, although more specific biological interactions may be involved in regulating the disease intensity.     

Endocrine and superovulatory responses in heifers pretreated with FSH or bovine follicular fluid

This study examined the effects of altered serum FSH concentration on subsequent ovarian response to superovulation. Synchronized heifers were assigned randomly on Day 1 of the cycle (estrus = Day 0) to three pretreatment groups that consisted of 6-d of saline (7ml, s.c., b.i.d.; Group I), FSH-P (0.5 mg, i.m., b.i.d.; Group II) or charcoal-extracted bovine follicular fluid (BFF; 7 ml, s.c., b.i.d.; Group III) injections. Superovulation was initiated on Day 7 and consisted of FSH-P in decreasing dosages over 4 d (4,3,2,1 mg; i.m., b.i.d.), with cloprostenol (500 mug) on the morning of the third day. A second replicate with 14 heifers was conducted using the same protocol but twice the pretreatment dosage of FSH-P (1 mg) and BFF (14 ml). Endogenous plasma FSH decreased during BFF and FSH-P pretreatments compared to controls (P < 0.02). Endogenous FSH concentrations in both primed groups (II and III) were similar to control values (Group I) 12 h after the start of superovulation. Basal LH concentrations were not different between pretreatment groups. The interval from cloprostenol treatment to the preovulatory LH surge in Group III was 21.3 and 23.9 h longer (P < 0.0001) than it was in Groups I and II. The postovulation progesterone rise was delayed in Group III. The number of corpora lutea (CL) was lowest in the BFF-primed group (4.2 +/- 0.8) compared with the FSH-primed (7.4 +/- 1.3) and the control (12.0 +/- 1.8; P < 0.003) groups. In the FSH-primed group (0.68 +/- 0.06 cm(3)), CL volumes were larger than in the control group (0.45 +/- 0.03 cm(3)), whereas in the BFF-primed group (0.27 +/- 0.02 cm(3)) CL volumes were smaller compared with the control group (P < 0.0001). Mean FSH concentrations for 48 h preceding superovulation and the number of CL per cow were positively correlated (r = 0.55; P < 0.004; n = 26). We concluded that both FSH-P and BFF pretreatments decreased the superovulatory response of heifers to FSH-P. The mechanism for this would appear to be associated with reduced endogenous FSH prior to the start of superovulation.     

Vagotonicity of Violence: Biochemical and Cardiac Responses to Violent Films and Television Programmes

In a search for a reproducible means of evoking different types of emotional stress it was found that in spite of increased adrenaline secretion slowing of the heart occurred when watching violent television programmes. Further evidence of increased vagal tone was provided by the “sinus arrhythmia” effect, a widening of the gap between the maximum and minimum heart rates during the respiratory cycle in parts of the humour, violence, and suspense sections of the television programme.Groups of people taken to see two particularly violent films showed similar evidence suggesting vagal overactivity, together with increases in plasma free fatty acids and decreases in triglycerides. As these changes occurred even with β-blockade it is suggested that they might be caused by non-sympathetically mediated changes in the levels of hormones, such as growth hormone, producing lipolysis.The ability to assess objectively an individual''s reaction to viewing violence might make it possible to judge the likely social impact of violent films and television programmes.     

Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.     

Cardiac Responses to Thermal,Physical, and Emotional Stress

We have studied the effect of a short period of exposure to the intense heat of a sauna bath on the electrocardiogram and plasma catecholamine, free fatty acid, and triglyceride concentrations in 17 subjects with apparently normal hearts and 18 persons with coronary heart disease. Similar observations were made on 11 of the 17 normal subjects and on 7 of the persons with coronary heart disease in response to exercise.Exposure to heat was associated with an increase in plasma adrenaline with no change in noradrenaline, free fatty acid, or triglyceride concentrations. Exercise was associated with the expected increase in both plasma noradrenaline and adrenaline concentrations. A heart rate up to 180 beats/min was observed in response to both heat and exercise. Apart from the ST-T changes inherent to sinus tachycardia, ST-T segment abnormalities were frequent in response to heat in both the subjects with normal and abnormal hearts, but little change occurred in the ST-T configuration when the subjects were exercised to produce comparable heart rates. Ectopic beats, sometimes numerous and multifocal, were observed in some subjects of both groups in response to heat, but not to exercise. It seems likely that the net unbalanced adrenaline component of the increased plasma catecholamine concentrations (which is also seen in certain emotional stress situations) is predominantly responsible for ischaemic-like manifestations of the electrocardiogram in susceptible subjects. The observations provide further validation for previously reported studies that it is the increased plasma noradrenaline in response to emotional stress that is associated with the release of free fatty acids and ultimate hypertriglyceridaemia, of probable importance in the aetiology of atheroma.     

A Comprehensive Panel of Near-Full-Length Clones and Reference Sequences for Non-Subtype B Isolates of Human Immunodeficiency Virus Type 1

Non-subtype B viruses cause the vast majority of new human immunodeficiency virus type 1 (HIV-1) infections worldwide and are thus the major focus of international vaccine efforts. Although their geographic dissemination is carefully monitored, their immunogenic and biological properties remain largely unknown, in part because well-characterized virological reference reagents are lacking. In particular, full-length clones and sequences are rare, since subtype classification is frequently based on small PCR-derived viral fragments. There are only five proviral clones available for viruses other than subtype B, and these represent only 3 of the 10 proposed (group M) sequence subtypes. This lack of reference sequences also confounds the identification and analysis of mosaic (recombinant) genomes, which appear to be arising with increasing frequency in areas where multiple sequence subtypes cocirculate. To generate a more representative panel of non-subtype B reference reagents, we have cloned (by long PCR or lambda phage techniques) and sequenced 10 near-full-length HIV-1 genomes (lacking less than 80 bp of long terminal repeat sequences) from primary isolates collected at major epicenters of the global AIDS pandemic. Detailed phylogenetic analyses identified six that represented nonrecombinant members of HIV-1 subtypes A (92UG037.1), C (92BR025.8), D (84ZR085.1 and 94UG114.1), F (93BR020.1), and H (90CF056.1), the last two comprising the first full-length examples of these subtypes. Four others were found to be complex mosaics of subtypes A and C (92RW009.6), A and G (92NG083.2 and 92NG003.1), and B and F (93BR029.4), again emphasizing the impact of intersubtype recombination on global HIV-1 diversification. Although a number of clones had frameshift mutations or translational stop codons in major open reading frames, all the genomes contained a complete set of genes and three had intact genomic organizations without inactivating mutations. Reconstruction of one of these (94UG114.1) yielded replication-competent virus that grew to high titers in normal donor peripheral blood mononuclear cell cultures. This panel of non-subtype B reference genomes should prove valuable for structure-function studies of genetically diverse viral gene products, the generation of subtype-specific immunological reagents, and the production of DNA- and protein-based subunit vaccines directed against a broader spectrum of viruses.One critical question facing current AIDS vaccine development efforts is to what extent human immunodeficiency virus type 1 (HIV-1) genetic variation has to be considered in the design of candidate vaccines (11, 21, 41, 72). Phylogenetic analyses of globally circulating viral strains have identified two distinct groups of HIV-1 (M and O) (33, 45, 61, 62), and 10 sequence subtypes (A to J) have been proposed within the major group (M) (29, 30, 45, 72). Sequence variation among viruses belonging to these different lineages is extensive, with envelope amino acid sequence variation ranging from 24% between different subtypes to 47% between the two different groups. Given this extent of diversity, the question has been raised whether immunogens based on a single virus strain can be expected to elicit immune responses effective against a broad spectrum of viruses or whether vaccine preparations should include mixtures of genetically divergent antigens and/or be tailored toward locally circulating strains (11, 21, 41, 72). This is of particular concern in developing countries, where multiple subtypes of HIV-1 are known to cocirculate and where subtype B viruses (which have been the source of most current candidate vaccine preparations [10, 21]) are rare or nonexistent (5, 24, 40, 72).Although the extent of global HIV-1 variation is well defined, little is known about the biological consequences of this genetic diversity and its impact on cellular and humoral immune responses in the infected host. In particular, it remains unknown whether subtype-specific differences in virus biology exist that have to be considered for vaccine design. Thus far, such differences have not been identified. For example, several studies have shown that there is no correlation between HIV-1 genetic subtypes and neutralization serotypes (38, 42, 46, 68). Some viruses are readily neutralized, while most are relatively neutralization resistant (42). Although the reasons for these different susceptibilities remain unknown, it is clear that neutralization is not a function of the viral genotype (38, 42, 46, 68). Similarly, recent studies have identified vigorous cross-clade cytotoxic T-lymphocyte (CTL) reactivities in individuals infected with viruses from several different clades (3, 6), as well as in recipients of a clade B vaccine (15). These results are very encouraging, since they suggest that CTL cross-recognition among HIV-1 clades is much more prevalent than previously anticipated and that immunogens based on a limited number of variants may be able to elicit a broad CTL response (6). Nevertheless, it would be premature to conclude that HIV-1 variation poses no problem for AIDS vaccine design. Only a comprehensive analysis of genetically defined representatives of the various groups and subtypes will allow us to judge whether certain variants differ in fundamental viral properties and whether such differences will have to be incorporated into vaccine strategies. Obviously, such studies require well-characterized reference reagents, in particular full-length and replication-competent molecular clones that can be used for functional and biological studies.Full-length reference sequences representing the various subtypes are also urgently needed for phylogenetic comparisons. Recent analyses of subgenomic (23, 52, 54, 58) as well as full-length (7, 18, 53, 60) HIV-1 sequences identified a surprising number of HIV-1 strains which clustered in different subtypes in different parts of their genome. All of these originated from geographic regions where multiple subtypes cocirculated and are the results of coinfections with highly divergent viruses (52, 60, 62). Detailed phylogenetic characterization revealed that most of them have a complex genome structure with multiple points of crossover (7, 18, 53, 60). Some recombinants, like the “subtype E” viruses, which are in fact A/E recombinants (7, 18), have a widespread geographic dissemination and are responsible for much of the Asian HIV-1 epidemic (69, 70). In other areas, recombinants appear to be generated with increasing frequencies since many randomly chosen isolates exhibit evidence of mosaicism (4, 8, 31, 66, 71). Since recombination provides the opportunity for evolutionary leaps with genetic consequences that are far greater than those of the steady accumulation of individual mutations, the impact of recombination on viral properties must be monitored. We therefore need full-length nonrecombinant reference sequences for all major HIV-1 groups and subtypes before we can map and characterize the extent of intersubtype recombination.The number of molecular reagents for non-subtype B viruses is very limited. There are currently only five full-length, nonrecombinant molecular clones available for viruses other than subtype B (45), and these represent only three of the proposed (group M) subtypes (A, C, and D). Moreover, only three clones (all derived from subtype D viruses) are replication competent and thus useful for studies requiring functional gene products (45, 48, 65). Given the unknown impact of genetic variation on correlates of immune protection, subtype-specific reagents are critically needed for phylogenetic, immunological, and biological studies. In this paper, we report the cloning (by long PCR and lambda techniques) of 10 near-full-length HIV-1 genomes from isolates previously classified as non-subtype B viruses. Detailed phylogenetic analysis showed that six comprise nonmosaic representatives of five major subtypes, including two for which full-length representatives have not been reported. Four others were identified as complex intersubtype recombinants, again emphasizing the prevalence of hybrid genomes among globally circulating HIV-1 strains. We also describe a strategy for the biological evaluation of long-PCR-derived genomes and report the generation of a replication-competent provirus by this approach. The effect of these reagents on vaccine development is discussed.