Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.     

Proteins associated with human parainfluenza virus type 3.

The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins were present in detergent- and salt-resistant viral cores. Of these, three proteins designated NP0, NP1, and NP2 of 68,000, 58,000, and 52,000 daltons, respectively, were stably associated with 50S RNA in CsCl gradient-purified nucleocapsids. The amounts of NP1 and NP2 were variable, and these proteins were shown to be structurally related to the major nucleocapsid protein (NP0) by partial Staphylococcus aureus V8 protease mapping. The other core proteins included a 240K protein designated L (candidate for the viral polymerase) and an 84K protein designated as the phosphoprotein (P) on the basis of a predominant incorporation of Pi. The viral envelope had four prominent proteins (72, 53, 40, and 12K) under reducing conditions of electrophoresis. The 72 and 53K proteins were specifically labeled with [3H]glucosamine and [3H]mannose. When sulfhydryl reagents were removed, a new 62K protein was visualized in place of the 72, 53, and 12K proteins. The 53 and 12K proteins were interpreted to be the two subunits (F1 and F2) of the fusion protein, and the 72K protein was designated as the HN (hemagglutinin-neuraminidase) glycoprotein. The unglycosylated 40K protein represented the viral matrix protein (M). Immunoprecipitation of infected cell lysates with rabbit hyperimmune antiserum against purified virus confirmed the viral origin of these polypeptides.     

Apolipoprotein A-I metabolism in subjects with a PstI restriction fragment length polymorphism of the apoA-I gene and familial hypoalphalipoproteinemia

Familial hypoalphalipoproteinemia (hypoalpha), characterized by a decreased high density lipoprotein level, is associated with an increased incidence of premature cardiovascular disease. Restriction fragment length polymorphism analysis of genomic DNA has detected a polymorphism for the PstI restriction endonuclease near the apoA-I gene, with either a 2.2 or a 3.3 kb fragment. The latter has been previously found to occur with significantly higher frequency in probands of families with familial hypoalpha. ApoA-I was isolated from three unrelated subjects with familial hypoalpha and the 3.3 kb PstI polymorphism of the apoA-I gene, and from normal control subjects. The apoA-I from the hypoalpha subjects was structurally normal as determined by amino acid analysis and by two-dimensional gel electrophoresis. When normal apoA-I and hypoalpha apoA-I were simultaneously injected into either normal controls or hypoalpha subjects, both forms of apoA-I were catabolized at the same rate in the same subject, indicating that the hypoalpha apoA-I is also metabolically normal. Analysis of the kinetics of metabolism of apoA-I in the hypoalpha subjects, compared to the normal controls, revealed that the reduced plasma levels of apoA-I were due to an increased apoA-I fractional catabolic rate, and that the synthetic rate was normal. Based on these results, we conclude that the apoA-I gene in these hypoalpha subjects is normal, and the PstI polymorphism near the apoA-I gene, which is associated with familial hypoalpha, is likely to be a marker for a mutant gene closely linked to, but not in, the apoA-I gene.     

Use of tumour marker immunoreactivity to identify primary site of metastatic cancer.

OBJECTIVES--To determine whether variations in the expression of tumour related antigens can predict the origin of tumours. DESIGN--Immunohistological study of tumour marker expression in primary adenocarcinomas and respective metastatic deposits. Antibodies to the following tumour markers were used: polymorphic epithelial mucin (NCRC-11 and SM3), carcinoembryonic antigen, carcinoembryonic antigen with non-specific antigen co-specificity, CA125, CA19.9, prostate specific antigens, and thyroglobulin. SETTING--Histopathology department of teaching hospital. SUBJECTS--100 pathology sections of metastatic adenocarcinoma and their related primary tumours. MAIN OUTCOME MEASURES--Concordance of reactivity between primary and metastatic tumours. Reactivity profiles of tumour sites. RESULTS--The correct primary site of origin was predicted in 70% (33/47) of tumours in men and 54% (27/43) tumours in women with antibodies SM3, 288, CA19.9, CA125, and PSA (men only). Specificities ranged from 68% for breast tumour to 98% for prostate tumour. CONCLUSION--Use of tumour markers in patients presenting with metastatic adenocarcinoma of unknown origin can help localise the probable primary sites and reduce the need for extensive and expensive imaging techniques.     

Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor).

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

FSHIP2 regulates epithelial cell polarity through its lipid product,which binds to Dlg1, a pathway subverted by hepatitis C virus core protein

The main targets of hepatitis C virus (HCV) are hepatocytes, the highly polarized cells of the liver, and all the steps of its life cycle are tightly dependent on host lipid metabolism. The interplay between polarity and lipid metabolism in HCV infection has been poorly investigated. Signaling lipids, such as phosphoinositides (PIs), play a vital role in polarity, which depends on the distribution and expression of PI kinases and PI phosphatases. In this study, we report that HCV core protein, expressed in Huh7 and Madin–Darby canine kidney (MDCK) cells, disrupts apicobasal polarity. This is associated with decreased expression of the polarity protein Dlg1 and the PI phosphatase SHIP2, which converts phosphatidylinositol 3,4,5-trisphosphate into phosphatidylinositol 4,5-bisphosphate (PtdIns(3,4)P2). SHIP2 is mainly localized at the basolateral membrane of polarized MDCK cells. In addition, PtdIns(3,4)P2 is able to bind to Dlg1. SHIP2 small interfering RNA or its catalytically dead mutant disrupts apicobasal polarity, similar to HCV core. In core-expressing cells, RhoA activity is inhibited, whereas Rac1 is activated. Of interest, SHIP2 expression rescues polarity, RhoA activation, and restricted core level in MDCK cells. We conclude that SHIP2 is an important regulator of polarity, which is subverted by HCV in epithelial cells. It is suggested that SHIP2 could be a promising target for anti-HCV treatment.     

Magnetic core-shell nanoparticles for drug delivery by nebulization

Background

Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe₃O₄ magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated.

Results

Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting.

Conclusion

We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.     

Understanding the Persistence of Plague Foci in Madagascar

Plague, a zoonosis caused by Yersinia pestis, is still found in Africa, Asia, and the Americas. Madagascar reports almost one third of the cases worldwide. Y. pestis can be encountered in three very different types of foci: urban, rural, and sylvatic. Flea vector and wild rodent host population dynamics are tightly correlated with modulation of climatic conditions, an association that could be crucial for both the maintenance of foci and human plague epidemics. The black rat Rattus rattus, the main host of Y. pestis in Madagascar, is found to exhibit high resistance to plague in endemic areas, opposing the concept of high mortality rates among rats exposed to the infection. Also, endemic fleas could play an essential role in maintenance of the foci. This review discusses recent advances in the understanding of the role of these factors as well as human behavior in the persistence of plague in Madagascar.     

Neutral Genomic Microevolution of a Recently Emerged Pathogen,Salmonella enterica Serovar Agona

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks.