Myristicyclins A and B: antimalarial procyanidins from Horsfieldia spicata from Papua New Guinea.

An antimalarial screen for plants collected from Papua New Guinea identified an extract of Horsfieldia spicata as having activity. Isolation of the active constituents led to the identification of two new compounds: myristicyclins A (1) and B (2). Both compounds are procyanidin-like congeners of myristinins lacking a pendant aromatic ring. Myristicyclin A was found to inhibit the ring, trophozoite, and schizont stages of Plasmodium falciparum at similar concentrations in the mid-µM range.

Asexual blood stages of Plasmodium falciparum exhibit signs of secondary necrosis, but not classical apoptosis after exposure to febrile temperature (40 C).

It has been shown by others that after cultures of Plasmodium falciparum were exposed to a febrile temperature of 40 C, parasitemia was reduced in the subsequent generation, suggesting a temperature-induced inhibition of trophozoites and schizonts. In the current study, influences unique to cultivation were ruled out, demonstrating that 40 C impacted the parasites directly. Metabolic profiling of DNA synthesis, protein synthesis, and glucose utilization clearly indicated that febrile temperatures had a direct effect on parasite development, beginning 20-24 hr after erythrocyte invasion. The mechanism of parasite death was investigated for evidence of temperature-induced apoptosis. Lack of typical physiological hallmarks, namely, caspase activation, characteristic mitochondrial membrane potential changes, and DNA degradation as indicated by DNA laddering, eliminated 'classical' apoptosis as a mechanism of parasite death. Parasites dying under the influence of heat, staurosporine, and chloroquine initially appeared pyknotic by light and electron microscopy (as in apoptosis), but eventual swelling and lysis of the food vacuole membrane led to secondary necrosis. Chloroquine did induce DNA laddering, but it was later attributed to occult white blood cell contaminants. While not apoptosis, the results do not rule out other forms of temperature-induced programmed cell death.

Cell death in bovine parvovirus-infected embryonic bovine tracheal cells is mediated by necrosis rather than apoptosis.

The helper-independent bovine parvovirus (BPV) was studied to determine its effect on host embryonic bovine tracheal (EBTr) cells: whether the ultimate outcome of infection results in apoptotic cell death or cell death by necrosis. Infected cells were observed for changes marking apoptosis. Observations of alterations in nuclear morphology, membrane changes, apoptotic body formation, membrane phosphatidylserine inversions, caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. On the other hand, at the end of the virus replication cycle, infected cells released viral haemagglutinin and infectious virus particles, as would be expected from cell membrane failure. Moreover, the infected cells released lactate dehydrogenase (LDH), release of which is a marker of necrosis. LDH release into the cell medium correlated directly with viral m.o.i. and time post-infection. Furthermore, assessment of mitochondrial dehydrogenase activity was consistent with cell death by necrosis. Taken together, these findings indicate that cell death in BPV-infected EBTr cells is due to necrosis, as defined by infected-cell membrane failure and release of the cell contents into the extracellular environment.

Geographical and temporal conservation of antibody recognition of Plasmodium falciparum variant surface antigens.

The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunity, but their diversity makes them questionable vaccine candidates. We determined levels of VSA-specific immunoglobulin G (IgG) in human plasma collected at four geographically distant and epidemiologically distinct localities with specificity for VSA expressed by P. falciparum isolates from three African countries. Plasma levels of VSA-specific IgG recognizing individual parasite isolates depended on the transmission intensity at the site of plasma collection but were largely independent of the geographical origin of the parasites. The total repertoire of immunologically distinct VSA thus appears to be finite and geographically conserved, most likely due to functional constraints. Furthermore, plasma samples frequently had high IgG reactivity to VSA expressed by parasites isolated more than 10 years later, showing that the repertoire is also temporally stable. Parasites from patients with severe malaria expressed VSA (VSASM) that were better recognized by plasma IgG than VSA expressed by other parasites, but importantly, VSASM-type antigens also appeared to show substantial antigenic homogeneity. Our finding that the repertoire of immunologically distinct VSA in general, and in particular that of VSASM, is geographically and temporally conserved raises hopes for the feasibility of developing VSA-based vaccines specifically designed to accelerate naturally acquired immunity, thereby enhancing protection against severe and life-threatening P. falciparum malaria.

Coronamycins, peptide antibiotics produced by a verticillate Streptomyces sp. (MSU-2110) endophytic on Monstera sp.

Coronamycin is a complex of novel peptide antibiotics with activity against pythiaceous fungi and the human fungal pathogen Cryptococcus neoformans. It is also active against the malarial parasite, Plasmodium falciparum, with an IC(50) of 9.0 ng ml(-1). Coronamycin is produced by a verticillate Streptomyces sp. isolated as an endophyte from an epiphytic vine, Monstera sp., found in the Manu region of the upper Amazon of Peru. Bioassay-guided fractionation of the fermentation broths of this endophyte on silica gel and HPLC chromatography yielded two principal, inseparable, peptides with masses of 1217.9 and 1203.8 Da. Three other minor, but related components, are also present in the preparation. Amino acid analysis of coronamycin revealed residues of component 1, component 2, methionine, tyrosine and leucine at a ratio of 2:2:1:1:3. Other compounds with antifungal activities are also produced by this endophytic streptomycete.

Kakadumycins, novel antibiotics from Streptomyces sp NRRL 30566, an endophyte of Grevillea pteridifolia.

An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics - the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2-0.3 microg x ml(-1) in contrast to echinomycin at 1.0-1.2 microg x ml(-1). Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD(50)s in the range of 7-10 ng x ml(-1). In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.

Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans.

Munumbicins A, B, C and D are newly described antibiotics with a wide spectrum of activity against many human as well as plant pathogenic fungi and bacteria, and a Plasmodium sp. These compounds were obtained from Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by bioassay-guided HPLC. The major components were four functionalized peptides with masses of 1269.6, 1298.5, 1312.5 and 1326.5 Da. Numerous other related compounds possessing bioactivity, with differing masses, were also present in the culture broth extract in lower quantities. With few exceptions, the peptide portion of each component contained only the common amino acids threonine, aspartic acid (or asparagine), glutamic acid (or glutamine), valine and proline, in varying ratios. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had an MIC of 2.5 microg x ml(-1) against a methicillin-resistant strain of Staphylococcus aureus, whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis. However, the most impressive biological activity of any of the munumbicins was that of munumbicin D against the malarial parasite Plasmodium falciparum, having an IC(50) of 4.5+/-0.07 ng x ml(-1). This report also describes the potential of the munumbicins in medicine and agriculture.