Direct inactivation of viruses by MCP-1 and MCP-2, natural peptide antibiotics from rabbit leukocytes.

Six homologous peptides were purified to homogeneity from rabbit granulocytes or alveolar macrophages and tested for their ability to inactivate herpes simplex virus type 1 (HSV-1). Two of the peptides, MCP-1 and MCP-2, showed considerable in vitro neutralizing activity, whereas four structurally homologous peptides (NP-3a, NP-3b, NP-4, and NP-5) were relatively ineffective. Inactivation of HSV-1 by MCP-1 or MCP-2 depended on peptide concentration and on the time, temperature, and pH of the incubation. HSV-2, vesicular stomatitis virus, and influenza virus A/WSN were also susceptible to direct neutralization by MCP-1 or MCP-2, whereas cytomegalovirus, echovirus type 11, and reovirus type 3 were not. We speculate that MCP-1 and MCP-2, peptides that are abundant in rabbit granulocytes and lung macrophages, may contribute to antiviral defenses by mediating the direct inactivation of HSV-1 and selected other viruses.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Methylation: a regulator of HIV-1 replication?

Pakistan, the second most populous Muslim nation in the world, has started to finally experience and confront the HIV/AIDS epidemic. The country had been relatively safe from any indigenous HIV cases for around two decades, with most of the infections being attributable to deported HIV positive migrants from the Gulf States. However, the virus finally seems to have found a home-base, as evidenced by the recent HIV outbreaks among the injection drug user community. Extremely high-risk behavior has also been documented among Hijras (sex workers) and long-distance truck drivers. The weak government response coupled with the extremely distressing social demographics of this South-Asian republic also helps to compound the problem. The time is ripe now to prepare in advance, to take the appropriate measures to curtail further spread of the disease. If this opportunity is not utilized right now, little if at all could be done later.     

Avian predation on a parasitic fly of cervids during winter: can host‐related cues increase the predation risk?

The deer ked (Lipoptena cervi) is an ectoparasitic fly on cervids that has expanded its distribution rapidly in Northern Europe. However, the regulating biotic factors such as predation remain unknown. The host‐independent pupal stage of the fly lasts for several months. Blackish pupae are visible against snow, especially on the bedding sites of hosts, and are thus exposed to predators. To evaluate the role of predation on the invasion dynamics and evolution of L. cervi, we monitored pupal predation on artificial bedding sites in three geographical areas in Finland during winter. We explored: (1) possible predators; (2) magnitude of predation; and (3) whether predation risk is affected by host‐derived cues. We demonstrate that pupae are predated by a number of tit species. Any reddish brown snow discoloration on bedding sites, indicating heavy infestation of the host, serves as an exploitable cue for avian predators, thereby increasing the risk of pupal predation. The ability of tits to use this host‐derived cue seems to be dependent on the prevalence of L. cervi and the period of invasion history, which suggests that it may be a learned behavioural response. Predation by tits may potentially affect the L. cervi population dynamics locally. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 275–286.     

Biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid in the isolated perfused rat liver

The isolated perfused rat liver was used to examine the hepatic extraction, biliary secretion and effect on bile flow of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid. The naturally occurring taurine and glycine conjugates of these bile acids were used for comparisons. The 2-fluoro-beta-alanine conjugates were extracted by the liver to a similar extent as the taurine and glycine conjugates. The biliary secretion rate and increase in bile flow were similar for all the cholic acid conjugates. On the other hand, the maximal biliary secretion rate of the 2-fluoro-beta-alanine conjugate of chenodeoxycholate was similar to that of the glycochenodeoxycholate, but 47% lower than that of taurochenodeoxycholate. In addition, the 2-fluoro-beta-alanine conjugate of chenodeoxycholate produced a decrease in bile flow that was comparable to that observed with the glycochenodeoxycholate (54% vs. 74%), but which was greater than that produced by the taurochenodeoxycholate (12%). In summary, these data demonstrate that the biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid are not markedly different from those of the naturally occurring taurine and glycine conjugates. These data also suggest that the amino acid moiety can influence the biliary secretion and cholestatic properties of chenodeoxycholic acid conjugates.     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

The physiology and ecological implications of efficient growth

The natural habitats of microbes are typically spatially structured with limited resources, so opportunities for unconstrained, balanced growth are rare. In these habitats, selection should favor microbes that are able to use resources most efficiently, that is, microbes that produce the most progeny per unit of resource consumed. On the basis of this assertion, we propose that selection for efficiency is a primary driver of the composition of microbial communities. In this article, we review how the quality and quantity of resources influence the efficiency of heterotrophic growth. A conceptual model proposing innate differences in growth efficiency between oligotrophic and copiotrophic microbes is also provided. We conclude that elucidation of the mechanisms underlying efficient growth will enhance our understanding of the selective pressures shaping microbes and will improve our capacity to manage microbial communities effectively.     

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.