Ribozymes: A modern tool in medicine

Since the discovery of ribozymes and self-splicing introns, it has been estimated that this biological property of RNA combined with other recombinant DNA technologies would become a tool to combat viral diseases and control oncogenes. These goals seem like a distinct possibility now. However, there is still a lot to be learned about the mobility of RNA inside the cells and the cellular factors that can impede ribozyme action in order to capitalize fully on the targeted RNA inactivation property of ribozymes. The most effective approach to maximize ribozyme function in a complex intracellular environment is to understand as much as possible about the intracellular fate of the RNA that is being targeted. As new techniques in cell biology become available, such understanding will be less problematic. Fundamental studies of ribozyme structure and mechanism of catalysis are flourishing both at the academic and industrial level and it can be expected that many new developments will continue to take place in these areas in the near future. Here, we review the design, stability and therapeutic application of these technologies illustrating relevant gene targets and applications in molecular medicine. Relevant problems in implementation of the technology, group I and II introns and the differences in applications, ribozyme structure and the application of this technology to virus attack and oncogene downregulation are discussed. Also some of the latest RNA-based technologies such as siRNA, RNA/DNA duplexes and RNA decoys have been introduced.     

Microtubule-dependent oligomerization of tau. Implications for physiological tau function and tauopathies

The accumulation of abnormal tau filaments is a pathological hallmark of many neurodegenerative diseases. In 1998, genetic analyses revealed a direct linkage between structural and regulatory mutations in the tau gene and the neurodegenerative disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Importantly, the FTDP-17 phenotype is transmitted in a dominant rather than a recessive manner. However, the underlying molecular mechanisms causing disease remain uncertain. The most common molecular mechanism generating dominant phenotypes is the loss of function of a multimeric complex containing both mutant and wild-type subunits. Therefore, we sought to determine whether tau might normally function as a multimer. We co-incubated 35S-radiolabeled tau and biotinylated tau with taxol stabilized microtubules, at very low molar ratios of tau to tubulin. Subsequent covalent cross-linking followed by affinity-precipitation of the biotinylated tau revealed the formation of microtubule-dependent tau oligomers. We next used atomic force microscopy to independently assess this conclusion. Our results are consistent with the hypothesis that tau forms oligomers upon binding to microtubules. In addition to providing insights into normal tau action, our findings lead us to propose that one mechanism by which mutations in tau may cause cell death is through the formation of tau complexes containing mutant tau molecules in association with wild-type tau. These wild-type-mutant tau complexes may possess altered biological and/or biophysical properties that promote onset of the FTDP-17 phenotype, including neuronal cell death by either altering normal tau-mediated regulation of microtubule-dependent cellular functions and/or promoting the formation of pathological tau aggregates.     

Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) for purification of xylanase

It is shown that eudragit S-100, a copolymer of methylacrylic acid and methylmethacrylate, undergoes three-phase partitioning. It was found that 95% eudragit S-100 could be recovered as the interfacial precipitate by using 30% (w/v) ammonium sulfate, 1:1 ratio of t-butanol to polymer solution at 40 degrees C. Three-phase partitioning of proteins uses simultaneous addition of ammonium sulfate and t-butanol to precipitate proteins in an interfacial layer separating the aqueous phase and organic solvent. Exploiting the affinity of xylanases towards eudragit S-100, it was possible to purify xylanase from Aspergillus niger; 60% recovery of activity with 95-fold purification could be obtained by this process. The purified enzyme showed A single band on SDS-PAGE. The technique shows promise to develop into a general method that could be termed "macroaffinity ligand-facilitated three-phase partitioning (MLFTPP).     

CYP1A1*2 and CYP1A1*3 polymorphisms cosegregate in the Indian population

Several ethnic groups have been genotyped for polymorphisms at the CYP1A1 gene locus that encodes the enzyme that catalyzes the initial step in the metabolism of polycyclic aromatic hydrocarbons. Two of the CYP1A1 polymorphisms, namely, CYP1A1*2 and CYP1A1*3 are reported to cosegregate among the Japanese and to a lesser extent in Caucasians, but not in people of African descent. In the absence of such information in the Indian population, the frequency of the CYP1A1*2 polymorphism was determined in this study, using DNA samples from 649 ethnic Indians who had been earlier genotyped for the CYP1A1*3 polymorphism. Analysis of the combined genotype data revealed that the two polymorphisms cosegregate in the Indian population.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Three phase partitioning for extraction of oil from soybean

Three phase partitioning, a method generally used for protein separation, has been evaluated for extraction of oil from soybean. 82% oil was extracted within 1 h using this process which required simultaneous addition of t-butanol (1:1, v/v) and 30% ammonium sulphate to the soybean slurry.     

Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens

The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts. and measure 4.4-5.4 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) with a length to width ratio 1.0-1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice. Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.     

Imaging real-time aggregation of amyloid beta protein (1-42) by atomic force microscopy

Amyloid beta protein (AbetaP) is the major fibrillar constituent of senile plaques. However, no causative role for AbetaP-fibers in Alzheimer's disease (AD) pathology is established. Globular AbetaPs are continuously released during normal cellular metabolism at pico- to nano-molar concentration. We used atomic force microscopy (AFM) to examine aggregation of freshly prepared AbetaP(1-42) and to examine the role of AbetaP concentration, imaging medium (air, water, or PBS) and agonists/antagonists on AbetaP-fibrillogenesis. At even very high and non-physiological AbetaP concentrations, 24-48 h of real-time AFM imaging (a) in water show only multiple layers of globular aggregates and no fibrils and (b) in PBS show mainly the globular structures and some short fibrils. On-line addition of Zn, an agonist for AbetaP-fibrillogenesis, induced a slow but non-fibrillar aggregation of globular AbetaPs. EDTA, a chelator of Zn and calcium (a modulator of AbetaP-mediated toxicity) induced a reversible change in the Zn-mediated aggregation. These results strongly suggest that no AbetaP-fibers are formed for the physiologically relevant concentration and thus the plaque-associated fibers may not account for the AD pathophysiology.