Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.     

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.     

Induction of blindness by formoguanamine hydrochloride in adult male roseringed parakeets (Psittacula krameri)

Formoguanamine (2,4-diamino-s-triazine) was known to be an effective chemical agent in inducing blindness in poultry chicks, but not in adult birds. The present study was undertaken to demonstrate the influences, if any, of this chemical on the visual performance and retinal histology in an adult sub-tropical wild bird, the roseringed parakeet (Psittacula krameri). Formoguanamine (FG) hydrochloride was subcutaneously injected into adult parakeets at a dosage of 25 mg (dissolved in 0.75 ml physiological saline)/100 g body weight/day, for two consecutive days while the control birds were injected only with a placebo. The effects were studied after 10, 20 and 30 days of the last treatment of FG. Within 24 h of the treatment of FG, about 90% of the total birds exhibited lack of visual responses to any light stimulus and even absence of pupillary light reactions. The remaining birds became totally blind on the day following the last injection of FG and remained so till the end of investigation. At the microscopic level, conspicuous degenerative changes were noted in the outer pigmented epithelium and the photoreceptive layer of rods and cones in the retinas of FG treated birds. A significant reduction in the thickness of the outer nuclear layer was also found in the retinas of FG treated parakeets, compared to that in the control birds. However, the inner cell layers of the retina in the control and FG administered parakeets were almost identical. It deserves special mention that the effects of FG, noted after 30 days of last treatment, were not very different from those noted just after 10 days of treatment. Collectively, the results of the present investigation demonstrate that FG can be used as a potent pharmacological agent for inducing irreversible blindness through selective damage in retinal tissue even in the adult wild bird, thereby making FG treatment an alternative euthanasic device to a cumbersome, stressful, surgical method of enucleation of the ocular system for laboratory studies.     

Marker assisted detection of gene (1Dx5) and translocation (1B/1R) in wheat genotypes

Detection of 1Dx5 gene and presence of 1B/1R wheat rye translocation were studied in nineteen elite Indian wheat genotypes using AS-PCR and STS markers, respectively. Fifteen genotypes had 1B/1R translocation whereas ten showed presence of 1Dx5 gene. More than 50 per cent of the genotypes tested were found positive for both 1Dx5 and 1B/1R translocation. The results are in conformity with HMW glutenin SDS-PAGE profile for 1Dx5 and cytological observations for 1B/1R translocation.     

Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro

Elevation of cAMP can overcome myelin inhibitors to encourage regeneration of the CNS. We show that a consequence of elevated cAMP is the synthesis of polyamines, resulting from an up-regulation of Arginase I, a key enzyme in their synthesis. Inhibiting polyamine synthesis blocks the cAMP effect on regeneration. Either over-expression of Arginase I or exogenous polyamines can overcome inhibition by MAG and by myelin in general. While MAG/myelin support the growth of young DRG neurons, they become inhibitory as DRGs mature. Endogenous Arginase I levels are high in young DRGs but drop spontaneously at an age that coincides with the switch from promotion to inhibition by MAG/myelin. Over-expressing Arginase I in maturing DRGs blocks that switch. Arginase I and polyamines are more specific targets than cAMP for intervention to encourage regeneration after CNS injury.     

Bone grafts and bone morphogenetic proteins in spine fusion

Spinal fusions are being performed for various pathologies of the spine. Stabilizing vertebral segments by eliminating motion across those segments becomes critical in dealing with pathologies of the spine that lead to instability. The use of autograft has been the gold standard for spine fusion. However, due to complications such as donor site morbidity, increased operating time, and limited supply, the use of allograft as a graft extender has become an acceptable practice especially in fusions spanning multiple segments. The discovery and isolation of novel proteins (i.e., bone morphogenetic proteins, BMPs), which initiate the molecular cascade of bone formation, have experimentally been shown in numerous animal studies to be as effective as autografts. Although the use of BMPs has exciting applications in spine surgery, long-term clinical studies must be evaluated for its efficacy in various applications in humans. The use of biomimetic materials such as hydroxyapatite (HA), or tricalcium phosphate (TCP) has also been examined in several animal models as bone graft substitutes or carriers. Although these materials have shown some promise in specific site applications, more work remains in elucidating an efficacious combination of these materials and BMPs that can be as effective as autografts. This review will present the status of bone grafts, bone morphogenetic proteins, gene therapy, and work that has been done to facilitate spinal fusion and simultaneously eliminate the need for bone graft. This revised version was published online in July 2006 with corrections to the Cover Date.     

First synthesis of phosphonobile acids and preliminary studies on their aggregation properties

The synthesis of three novel phosphonobile acids from natural bile acids is reported. The CMC of phosphonodeoxycholic acid (PDCA) at pH 8.2 was found to be lower than that of the parent deoxycholic acid (DCA). PDCA micelles were also found to have higher microviscosity compared to DCA micelles, suggesting higher hydrophobicity and tighter packing in the interior of PDCA micelles. PDCA aggregated further to form an aqueous gel at pH 4.     

Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.