Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs.     

A Retroviral Chimeric Capsid Protein Reveals the Role of the N-Terminal β-Hairpin in Mature Core Assembly

The human immunodeficiency virus (HIV) is an enveloped virus constituted by two monomeric RNA molecules that encode for 15 proteins. Among these are the structural proteins that are translated as the gag polyprotein. In order to become infectious, HIV must undergo a maturation process mediated by the proteolytic cleavage of gag to give rise to the isolated structural protein matrix, capsid (CA), nucleocapsid as well as p6 and spacer peptides 1 and 2. Upon maturation, the 13 N-terminal residues from CA fold into a β-hairpin, which is stabilized mainly by a salt bridge between Pro1 and Asp51. Previous reports have shown that non-formation of the salt bridge, which potentially disrupts proper β-hairpin arrangement, generates noninfectious virus or aberrant cores. To date, however, there is no consensus on the role of the β-hairpin. In order to shed light in this subject, we have generated mutations in the hairpin region to examine what features would be crucial for the β-hairpin's role in retroviral mature core formation. These features include the importance of the proline at the N-terminus, the amino acid sequence, and the physical structure of the β-hairpin itself. The presented experiments provide biochemical evidence that β-hairpin formation plays an important role in regard to CA protein conformation required to support proper mature core arrangement. Hydrogen/deuterium exchange and in vitro assembly reactions illustrated the importance of the β-hairpin structure, its dynamics, and its influence on the orientation of helix 1 for the assembly of the mature CA lattice.     

T-817MA, a neuroprotective agent, attenuates the motor and cognitive impairments associated with neuronal degeneration in P301L tau transgenic mice

Tau pathology is implicated in mechanisms of neurodegenerative tauopathies, including Alzheimer’s disease (AD) and hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). It has been reported that transgenic mice expressing FTDP-17 mutation P301L of human tau (P301L mice) display extensive tau pathology and exhibit behavioral deficits with aging. In this study, we investigated the effects of T-817MA, a neuroprotective agent, on the motor and cognitive impairments associated with neuronal degeneration in P301L mice. T-817MA prevented the progression of motor deficit and the loss of spinal cord motor neurons in P301L mice. Furthermore, T-817MA significantly attenuated the spatial memory impairment and the reduction in synaptic terminal density in the hippocampal dentate gyrus of P301L mice. These results indicate that T-817MA improved the motor and cognitive impairments as a result of inhibiting neuronal degeneration derived from tau pathology in the P301L mice. Therefore, it is expected that T-817MA has a therapeutic potential for tau-related neurodegenerative diseases such as AD.     

Mutations that mimic phosphorylation of the HIV-1 matrix protein do not perturb the myristyl switch

Recent studies indicate that the matrix domain (MA) of the HIV-1 Gag polyprotein directs Gag to the plasma membrane for virus assembly via a phosphatidylinositol-4,5-bisphosphate (PIP(2))-dependent myristyl switch mechanism. MA also has been reported to direct nuclear trafficking via nuclear import and export functions, and some studies suggest that nuclear targeting may be regulated by MA phosphorylation (although this proposal remains controversial). We have prepared and studied a series of HIV-1 MA mutants containing Ser-to-Asp substitutions designed to mimic phosphorylation, including substitutions in regions of the protein involved in protein-protein interactions and known to influence the myristyl switch (S6D, S9D, S67D, S72D, S6D/S9D, and S67D/S72D). We were particularly interested in substitutions at residue 6, since conservative mutations adjacent to this site strongly perturb the myristyl switch equilibrium, and this site had not been genetically tested due to its involvement in post-translational myristylation. Our studies reveal that none of these mutations, including S6D, influences the PIP(2)- or concentration-dependent myristyl switch equilibrium. In addition, all of the mutants bind liposomes with affinities that are only slightly reduced in comparison with the native protein. In contrast, the myristylated mutants bind liposomes with substantially greater affinity than that of the native, unmyristylated protein. These findings support the hypothesis that phosphorylation is unlikely to significantly influence membrane-mediated intracellular trafficking.     

Effects of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> MA2 isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol diet

The objective of this study was to evaluate the effects of Lactobacillus plantarum MA2, an isolate from Chinese traditional Tibet kefir, on cholesterol-lowering and microflora of rat in vivo. Rats were fed on cholesterol-enriched experimental diet, supplemented with lyophilized L. plantarum MA2 powder, with a dose of 10¹¹ cells/day per mice. The results showed that L. plantarum MA2 feeding significantly lowered serum total cholesterol, low-density lipoprotein cholesterol, and triglycerides level, while there was no change in high-density lipoprotein cholesterol. In addition, liver total cholesterol and triglycerides was also decreased. However, fecal cholesterol and triglycerides was increased significantly (P < 0.05) in comparison with the control. Also, L. plantarum MA2 increased the population of lactic acid bacteria and bifidobacteria in the fecal, but it did not change the number of Escherichia coli as compared to control. Moreover, pH, moisture, and organic acids in the fecal were also measured. The present results indicate the probiotic potential of the L. plantarum MA2 strain in hypocholesterolemic effect and also increasing the probiotic count in the intestine.     

Varied truncation and clustering characterize some short repeats identified in micronucleus-specific DNA of Tetrahymena thermophila

There are over 6000 internally eliminated DNA sequences (IESs) in the Tetrahymena genome that are deleted in a programmed fashion during the development of a polyploid, somatic macronucleus from a diploid germline micronucleus. Recently, based on several results, a homology and small RNA-based mechanism has been proposed for the efficient elimination of IES elements. Since the RNAi machinery is proposed to be intimately involved in silencing potentially harmful repeats such as transposons and viruses, characterization of repeats and the conditions for their developmental elimination from the somatic genome is warranted. Three short (500–600 bp) repeat families, members of which had been experimentally identified in IESs, that is, in micronucleus-specific DNA, are examined here using the Tetrahymena genome database. Members of all three families display varied degrees of truncation and are represented in macronuclear sequences. A 200 bp segment of one of the families can appear in the genome on its own, or as part of a 600 bp repeat detected experimentally, or in association with an unrelated 1 kb sequence to form a 1.2 kb repeat that is also frequently truncated. The 1 kb sequence contains a 300 bp section similar to a repeat associated with a non-long terminal repeat-like element and is often found accompanied by several more copies of this shorter repeat. These observations indicate that transposition may have had a role in the evolution of the short repeat families.     

Natively unfolded nucleic acid binding P8 domain of SeMV polyprotein 2a affects the novel ATPase activity of the preceding P10 domain

Open reading frame (ORF) 2a of Sesbania mosaic virus (SeMV) codes for polyprotein 2a (Membrane anchor-protease-VPg-P10-P8). The C-terminal domain of SeMV polyprotein 2a was cloned, expressed and purified in order to functionally characterize it. The protein of size 8 kDa (P8) domain, like viral protein genome linked (VPg), was found to be natively unfolded and could bind to nucleic acids. Interestingly, P10-P8 but not P8 showed a novel Mg²⁺ dependent ATPase activity that was inhibited in the presence of poly A. In the absence of P8, the ATPase activity of the protein of size 10 kDa (P10) domain was reduced suggesting that the natively unfolded P8 domain influenced the P10 ATPase.     

GnRH secreting cultured fetal rat hypothalamic cells do not degrade GnRH

The possible degradation of GnRH in the hypothalamus was investigated. Rat fetal hypothalamic cells were kept in culture for two weeks and basal and stimulated GnRH release was measured by highly sensitive RIA. These intact hypothalamic cells did not degrade GnRH during 4 hours of incubation, but a 50% degradation occurred after 24 hours incubation followed by HPLC using specifically tritium labeled GnRH or RIA. The rate of degradation or the kinetic of degradation did not change by increasing GnRH concentration in the medium or by mechanical instead of enzymatic dispersion of the cells. For comparison GnRH degradation in homogenized hypothalamic tissue and in synaptosomal preparation was measured and rapid degradation was found. Our results suggest that intact hypothalamic cells under physiological circumstances do not degrade extracellular GnRH.