Truncated, inactive N-acetylglucosaminyltransferase III (GlcNAc-TIII) induces neurological and other traits absent in mice that lack GlcNAc-TIII

N-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complex N-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3(Delta)) or for a targeted mutation in the Mgat3 gene (previously called Mgat3(neo), but herein renamed Mgat3(T37) because the allele generates inactive GlcNAc-TIII of approximately 37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutral N-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3(T37/T37) homozygotes in a mixed or 129(SvJ) background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned by Mgat3(T37/T37) mothers were rescued by wild-type foster mothers. None of these Mgat3(T37/T37) traits were exhibited by Mgat3(Delta/Delta) mice or by heterozygous mice carrying the Mgat3(T37) mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3(T37) and Mgat3(Delta) mutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed in Mgat3(T37/T37) brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse.     

Reversibility of oxygen induced inactivation of nitrogenase in some enterobacteria

Aerobic and microaerobic diazotrophs possess numerous oxygen restriction strategies to protect nitrogenase from inactivation by oxygen without interfering with energy generation through oxidative phosphorylation. Protection by conformational change in nitrogenase was first detected and described in Azotobacter. This strategy once considerd unique for Azotobacter has been shown in this study to occur in Citrobacterfreundii (Braak) Werkman and Gillen and Klebsiella pneumoniae subspecies rhinoscleromatis (Trevisan) Migula also. However, in these enteric bacteria the entire enzyme is not protected probably due to the absence of any respiratory protection similar to that found in the aerobe, Azotobacter.     

Role of root-mediated interactions in phytotoxic interference of Ageratum conyzoides with rice (Oryza sativa)

Ageratum conyzoides L. (billy goat weed; Asteraceae) is an annual invasive weed native of tropical America and has now naturalized worldwide, particularly in Southeast Asia. The present study investigated the nature and potential of root-mediated allelopathic interference of A. conyzoides against rice (Oryza sativa). Root and shoot length and biomass accumulation of rice were significantly reduced (by 18–30%) when grown in the rhizosphere soil of the weed indicating the release of putative allelochemicals from the weed into the soil. The growth of rice was also progressively reduced in the soil amended with increasing amounts of root residues (5, 10 and 20 g kg⁻¹ soil) of A. conyzoides. The addition of activated charcoal, an inert material with high affinity for organic biomolecules, partly ameliorated the negative effects of root residues amended in the soil. Further, there was no negative effect on the availability of soil nutrients in the root-amended soils. These were rather nutrient rich with greater electrical conductivity, and higher amount of organic matter, thus indicating no role in observed growth reduction. The reduction in allelopathic effects of root residue upon charcoal addition further indicated that putative phytotoxins released from the weed roots are water-soluble phenolic compounds. A significant amount of water-soluble phenolics were present in rhizosphere (∼6-times higher) and root-amended soils (∼5–10-fold higher) and their content was reduced (to ∼3.6–7.0-fold higher) when charcoal was added. The observed growth reduction in Ageratum rhizospheric or root-amended soils was concomitant with the amount of phenolic compounds. Upon HPLC analyses, these were identified as p-coumaric acid, gallic acid, ferulic acid, p-hydroxybenzoic acid and anisic acid. Under laboratory conditions, these phenolic acids reduced the root length and seedling weight of rice individually as well as in equimolar mixture, though no synergistic effect was noticed. The study concludes that root exudates and residues of A. conyzoides suppress the growth of rice by releasing phenolic allelochemicals into the soil rhizosphere and not through alteration of soil nutrients, and allelopathy plays a significant role in root-mediated negative interference of A. conyzoides.     

Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla _NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla _NDM-1 Klebsiella pneumonia and bla _NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla _NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.     

Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery

The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance (¹H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98 ± 0.089 and 10.84 ± 0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by ¹H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35 ± 56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.KEY WORDS: back-of-the-eye, drug delivery, formulation, mixed nanomicelles, posterior, rabbits, rapamycin/sirolimus, retina/choroid, sclera, topical eye drops     

The Effect of Life History on Retroviral Genome Invasions

Endogenous retroviruses (ERV), or the remnants of past retroviral infections that are no longer active, are found in the genomes of most vertebrates, typically constituting approximately 10% of the genome. In some vertebrates, particularly in shorter-lived species like rodents, it is not unusual to find active endogenous retroviruses. In longer-lived species, including humans where substantial effort has been invested in searching for active ERVs, it is unusual to find them; to date none have been found in humans. Presumably the chance of detecting an active ERV infection is a function of the length of an ERV epidemic. Intuitively, given that ERVs or signatures of past ERV infections are passed from parents to offspring, we might expect to detect more active ERVs in species with longer generation times, as it should take more years for an infection to run its course in longer than in shorter lived species. This means the observation of more active ERV infections in shorter compared to longer-lived species is paradoxical. We explore this paradox using a modeling approach to investigate factors that influence ERV epidemic length. Our simple epidemiological model may explain why we find evidence of active ERV infections in shorter rather than longer-lived species.     

Absolute Leukocyte Telomere Length in HIV-Infected and Uninfected Individuals: Evidence of Accelerated Cell Senescence in HIV-Associated Chronic Obstructive Pulmonary Disease

Combination antiretroviral therapy (cART) has extended the longevity of human immunodeficiency virus (HIV)-infected individuals. However, this has resulted in greater awareness of age-associated diseases such as chronic obstructive pulmonary disease (COPD). Accelerated cellular senescence may be responsible, but its magnitude as measured by leukocyte telomere length is unknown and its relationship to HIV-associated COPD has not yet been established. We measured absolute telomere length (aTL) in peripheral leukocytes from 231 HIV-infected adults. Comparisons were made to 691 HIV-uninfected individuals from a population-based sample. Subject quartiles of aTL were assessed for relationships with measures of HIV disease severity, airflow obstruction, and emphysema severity on computed tomographic (CT) imaging. Multivariable regression models identified factors associated with shortened aTL. Compared to HIV-uninfected subjects, the mean aTL in HIV-infected patients was markedly shorter by 27 kbp/genome (p<0.001); however, the slopes of aTL vs. age were not different (p=0.469). Patients with longer known durations of HIV infection (p=0.019) and lower nadir CD4 cell counts (p=0.023) had shorter aTL. Shorter aTL were also associated with older age (p=0.026), smoking (p=0.005), reduced forced expiratory volume in one second (p=0.030), and worse CT emphysema severity score (p=0.049). HIV-infected subjects demonstrate advanced cellular aging, yet in a cART-treated cohort, the relationship between aTL and age appears no different from that of HIV-uninfected subjects.