Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Survival of F-specific RNA coliphage,feline calicivirus,and Escherichia coli in water: a comparative study

The relationship between the survival of enteric viral pathogens and their indicators (coliform bacteria and coliphages) is not well understood. We compared the survival rates of feline calicivirus (FCV), Escherichia coli, and a male-specific RNA coliphage MS2 at 4, 25, and 37 degrees C for up to 28 days in dechlorinated water. The survival rates of E. coli and FCV, a surrogate of noroviruses (NV), had a high degree of correlation at 4 and 25 degrees C, while MS2 phage survived significantly longer (P < 0.05) at these two temperatures. At 37 degrees C, the survival rates for all three organisms were highly correlated. Decimal reduction values indicating the number of days needed for 90% reduction in titer (D values) decreased for all three organisms as storage temperatures increased. FCV had the shortest D value among all three organisms at all temperatures investigated. These findings indicate that F-specific RNA phages may be useful indicators of NV in the environment.     

Effect of chromium picolinate on histopathological alterations in STZ and neonatal STZ diabetic rats

Earlier studies from our laboratory have indicated insulin sensitizing action of chromium picolinate as the mechanism of its anti-diabetic activity in experimental models of type I and type II diabetes. In the present investigation, we have evaluated the effects of chronic administration of chromium picolinate on the functional and histological alterations of streptozotocin (STZ)-induced diabetes in rats. Type I diabetes was induced by intravenous injection of STZ (40 mg/kg) in adult rats, whereas, type II diabetes was induced by intraperitoneal injection of STZ (90 mg/kg) in 2-day old rat pups which in adulthood develop abnormalities resembling type II diabetes. Chromium picolinate was administered at 8 μg/ml in drinking water for 6 weeks and was found to improve glucose tolerance and increase insulin sensitivity of STZ-diabetic rats. This treatment decrease elevated serum creatinine and urea levels as well as elevated serum levels of hepatic enzymes of both groups of diabetic rats. Histopathological studies of kidney and liver show decrease in the intensity and incidence of vacuolations, cellular infiltration and hypertrophy of STZ and nSTZ (neonatal STZ) diabetic rats. Chronic treatment with chromium picolinate however, did not alter the normal function or morphology of control rats. Chronic chromium picolinate at the therapeutic doses that improved glucose tolerance, was observed to have no hepatotoxic or nephrotoxic potential. It was rather found to improve renal and hepatic function and to reduce abnormalities associated with STZ-diabetes. Chromium picolinate could play an important role in the long term management of diabetes mellitus.     

Effect of chronic treatment with losartan on streptozotocin-induced renal dysfunction

The present investigation was undertaken to study the effect of chronic treatment with angiotensin (AT₁) receptor antagonist losartan (2 mg/kg, p.o., 6 weeks) on streptozotocin (STZ) induced (45 mg/kg, i.v., single dose) renal dysfunctions in diabetic rats. Injection of streptozotocin produced not only the cardinal symptoms of diabetes mellitus like loss of body weight, hyperglycemia, and hypoinsulinemia but also the renal dysfunctions. Losartan treatment significantly prevented all these changes except STZ-induced hypoinsulinemia. There was a significant elevation of blood pressure in diabetic rats and treatment with losartan significantly brought it back to normal. Renal dysfunction in diabetic rats was characterized by a significant decrease in creatinine clearance, elevated levels of electrolytes and renal hypertrophy. Treatment with losartan prevented these changes. A good correlation was found between biochemical parameters and histopathological abnormalities. Our data suggests that, losartan may be considered as the drug of choice when there is a co-existence of diabetes mellitus and hypertension with compromised kidney function.     

Effect of sarpogrelate on altered STZ-diabetes induced cardiovascular responses to 5-hydroxytryptamine in rats

Sarpogrelate, a specific 5-HT_2A receptor antagonist is reported to produce a number of beneficial cardiovascular effects in diabetes mellitus. In the present investigation we have studied the effects of sarpogrelate on 5-HT receptors in heart and platelets in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg) and sarpogrelate (1 mg/kg, i.p.) was administered daily for 6 weeks. Injection of STZ produced significant loss of body weight, polyphagia, polydypsia, hyperglycemia, hypoinsulinemia, hypertension and bradycardia. Treatment with sarpogrelate significantly lowered fasting glucose levels with corresponding increase in insulin levels. It also significantly prevented STZ-induced polydypsia, hyperphagia, hypertension, and bradycardia but not the loss of body weight. 5-HT produced dose-dependent positive inotropic effect that was found to be decreased significantly in STZ-diabetic rats. Hearts obtained from sarpogrelate treated diabetic rats did not show any decrease in responsiveness to 5-HT. Relative platelet aggregation per se was found to be higher in STZ-diabetic rats as compared to control and this was significantly prevented by sarpogrelate treatment. 5-HT produced a dose-dependent increase in platelet aggregation in non-diabetic and sarpogrelate treated diabetic rats. However, 5-HT failed to produce any increase in platelet aggregation in untreated diabetic rats. Our data suggest that STZ-induced diabetes may produce down-regulation of cardiac 5-HT_2A receptors and increased platelet aggregation. Treatment with sarpogrelate seems to prevent STZ-induced down-regulation of 5-HT receptors and increase in platelet activity in diabetic rats.     

Phosphorus magnetic resonance spectroscopy: its utility in examining the membrane hypothesis of schizophrenia

A novel approach to understanding the pathophysiology of schizophrenia has been the investigation of membrane composition and functional perturbations, referred to as the "Membrane Hypothesis of Schizophrenia." The evidence in support of this hypothesis has been accumulating in findings in patients with schizophrenia of reductions in phospholipids and essential fatty acids various peripheral tissues. Postmortem studies indicate similar reductions in essential fatty acids in the brain. However, the use of magnetic resonance spectroscopy (MRS) has provided an opportunity to examine aspects of membrane biochemistry in vivo in the living brain. MRS is a powerful, albeit complex, noninvasive quantitative imaging tool that offers several advantages over other methods of in vivo biochemical investigations. It has been used extensively in investigating brain biochemistry in schizophrenia. Phosphorus MRS (31P MRS) can provide important information about neuronal membranes, such as levels of phosphomonoesters that reflect the building blocks of neuronal membranes and phosphodiesters that reflect breakdown products. 31P MRS can also provide information about bioenergetics. Studies in patients with chronic schizophrenia as well as at first episode prior to treatment show a variety of alterations in neuronal membrane biochemistry, supportive of the membrane hypothesis of schizophrenia. Below, we will briefly review the principles underlying 31P MRS and findings to date. Magnetic resonance spectroscopy (MRS) is a powerful, albeit complex, imaging tool that permits investigation of brain biochemistry in vivo. It utilizes the magnetic resonance imaging hardware. It offers several advantages over other methods of in vivo biochemical investigations. MRS is noninvasive, there is no radiation exposure, does not require the use of tracer ligands or contrast media. Because of it is relatively benign, repeated measures are possible. It has been used extensively in investigating brain biochemistry in schizophrenia.     

Carbon concentration mechanisms in photosynthetic microorganisms

Unicellular green algae and cyanobacteria have mechanism to actively concentrate dissolved inorganic carbon into the cells, only if they are grown with air levels of CO2. The carbon concentration mechanisms are commonly known as "CCM" or "DIC-pumps". The DIC-pumps are environmental adaptation that function to actively transport and accumulate inorganic carbon (HCO3- and CO2; Ci) within the cell and then uses this Ci pool to actively increase the concentration of CO2 at the site of ribulose bisphosphate carboxylase-oxygenase (Rubisco), the primary CO2-fixing enzyme. The current working model for dissolved inorganic carbon concentration mechanism in unicellular green algae includes several isoforms of carbonic anhydrase (CA), and ATPase driven active transporters at the plasmalemma and at the inner chloroplast envelopes. In the past fifteen years, significant progress has been made in isolating and characterizing the various isoforms of carbonic anhydrase at the biochemical and molecular level. However, we have an inadequate understanding of active transporters that are located on the plasmalemma and at the chloroplast envelopes. In this mini-review we focus on certain aspects of the induction, function and significance of the dissolved inorganic carbon concentration mechanisms in aquatic photosynthetic microorganisms.     

Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase

We have reported a simple method of determination of plasma oxalate using a Cl(-) and NO(3)(-) insensitive oxalate oxidase purified from grain sorghum leaf and commercially available peroxidase from horseradish [Pundir et al., Ind. J. Biochem. Biophys., 35 (1998) 120-122]. The present report describes the immobilization of both the enzymes onto alkylamine glass, their kinetic properties and application for discrete analysis of plasma oxalate. In the analytic method, H(2)O(2) generated from plasma oxalate by immobilized oxalate oxidase is measured colorimetrically at 520 nm by oxidative coupling with 4-aminophenazone, and phenol catalyzed by immobilized peroxidase. The minimum detection limit of the method is 2.5 micromol/l. Analytic recovery of added oxalate in plasma was 89. 5+/-4.1% (mean+/-S.D.). The within and between day CV for plasma oxalate measurement were <9.37 and <11.0%, respectively. The normal range of plasma oxalate as measured by the present method was 3.6 to 5.7 micromol/l. The method is not only free from interference by plasma Cl(-) and NO(3)(-) but also provides the reuse of glass beads and thus reduces the cost of analysis for routine.     

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.     

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