Expression in and purification of Hepatitis B surface antigen (S-protein) from methylotrophic yeast Pichia pastoris

The study describes expression and purification of recombinant hepatitis B small surface antigen (rHBsAg hereafter) in methylotrophic yeast Pichia pastoris strain GS115. For expression of the rHBsAg, a single copy of 678 bp cDNA was inserted at the unique EcoRI site downstream of the alcohol oxidase (AOX 1) promoter of the 8.2 kb pHIL-D2 vector. The cDNA-pHIL-D2 construct was used to transform the strain GS115, resulting in a Mut(S) (Methanol Utilizing Slow) phenotype in which the 226 amino acids containing active and full-length rHBsAg protein could be expressed intra-cellularly during slow growth and induction with methanol. The recombinant protein from the Mut(S) expressor was harvested by cell disruption, and purified first by adsorption-desorption on aerosil followed by two-step chromatographic separation i.e. anion exchange on DEAE resin followed by gel permeation on Superdex 75. Reversed passive hem-agglutination assay (RPHA) was used to test the antigenicity while SDS-PAGE was performed to check the purity of the 27 kDa rHBsAg and its aggregates. The results showed that disruption at 12 Kpsi (three cycles), or 30 Kpsi (1 cycle), desorption with 10mM carbonate buffer (pH 9-10), and storage at 4 degrees C without detergent did not adversely affect the antigenicity of the rHbsAg. However, the presence of detergents such as TritonX100 and deoxycholate in the disruption and desorption buffers, respectively resulted in reduced antigenicity during storage both at 4 and -20 degrees C in spite of higher initial yields.     

Preparation and Evaluation of Gelatin/Sodium Carboxymethyl Cellulose Polyelectrolyte Complex Microparticles for Controlled Delivery of Isoniazid

The ratio of gelatin to sodium carboxymethyl cellulose (SCMC) at which maximum yield was obtained was optimized. This optimized ratio of gelatin to SCMC along with other parameters was used to prepare microparticles of different sizes. Vegetable oil was used as emulsion medium. Effect of various factors like amount of surfactant, concentration of polymer on the formation, and size of the microparticles was investigated. These microparticles were used as carrier for isoniazid. Among different cross-linkers, glutaraldehyde was found to be the most effective cross-linker at the temperature and pH at which the reaction was carried out. The loading efficiency and release behavior of loaded microparticles were found to be dependent on the amount of cross-linker used, concentration of drug, and time of immersion. Maximum drug loading efficiency was observed at higher immersion time. The release rate of isoniazid was more at higher pH compared to that of at lower pH. The sizes of the microparticles were investigated by scanning electron microscope. In all the cases, the microparticles formed were found spherical in shape except to those at low stirring speed where they were agglomerated. Fourier transform infrared study indicated the successful incorporation of isoniazid into the microparticles. Differential scanning calorimetry study showed a molecular level dispersion of isoniazid in the microparticles. X-ray diffraction study revealed the development of some crystallinity due to the encapsulation of isoniazid.     

Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder characterized by smooth-muscle tumors of the skin and uterus and/or renal cancer. Although the identification of germline mutations in the fumarate hydratase (FH) gene in European families supports it as the susceptibility gene for HLRCC, its role in families in North America has not been studied. We screened for germline mutations in FH in 35 families with cutaneous leiomyomas. Sequence analysis revealed mutations in FH in 31 families (89%). Twenty different mutations in FH were identified, of which 18 were novel. Of these 20 mutations, 2 were insertions, 5 were small deletions that caused frameshifts leading to premature truncation of the protein, and 13 were missense mutations. Eleven unrelated families shared a common mutation: R190H. Eighty-one individuals (47 women and 34 men) had cutaneous leiomyomas. Ninety-eight percent (46/47) of women with cutaneous leiomyomas also had uterine leiomyomas. Eighty-nine percent (41/46) of women with cutaneous and uterine leiomyomas had a total hysterectomy, 44% at age < or =30 years. We identified 13 individuals in 5 families with unilateral and solitary renal tumors. Seven individuals from four families had papillary type II renal cell carcinoma, and another individual from one of these families had collecting duct carcinoma of the kidney. The present study shows that mutations in FH are associated with HLRCC in North America. HLRCC is associated with clinically significant uterine fibroids and aggressive renal tumors. The present study also expands the histologic spectrum of renal tumors and FH mutations associated with HLRCC.     

Unusual structural features of hydantoin lesions translate into efficient recognition by Escherichia coli Fpg

Oxidation of guanine (G) and 8-oxoguanine (OG) with a wide variety of oxidants yields the hydantoin lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). These two lesions have garnered much recent attention due to their unusual structures and high mutagenic potential. We have previously shown that duplexes containing Gh and Sp are substrates for the base excision repair glycosylase Escherichia coli Fpg (EcFpg). To evaluate the recognition features of these unusual lesions, binding and footprinting experiments were performed using a glycosylase inactive variant, E3Q EcFpg, and 30 bp duplexes containing the embedded lesions. Surprisingly, E3Q EcFpg was found to bind significantly more tightly ( approximately 1000-fold) to duplexes containing Gh or Sp over the corresponding duplexes containing OG. This may be a consequence of the helix-destabilizing nature of the hydantoin lesions that facilitates their recognition within duplex DNA. Though DNA binding affinities of E3Q EcFpg with Gh- and Sp-containing duplexes were found to be similar to each other, hydroxyl radical footprinting using methidium-propyl-EDTA (MPE)-Fe(II) revealed subtle differences between binding of E3Q EcFpg to the two lesions. Most notably, in the presence of E3Q EcFpg, the Sp nucleotide (nt) is hyperreactive toward cleavage by MPE-Fe(II)-generated hydroxyl radicals, suggestive of the formation of an intercalation site for the MPE-Fe(II) reagent at the Sp nt. Interestingly, increasing the duplex length from 18 to 30 bp enhanced the excision efficiency of Gh and Sp paired with C, G, or T by EcFpg such that these substrates are processed as efficiently as the signature substrate lesion, OG. Moreover, the base removal activity with these two lesions was more efficient than removal of OG when in a base pairing context opposite A. The high affinity and efficient activity of EcFpg toward the hydantoin lesions suggest that EcFpg mediates repair of the lesions in vivo. Notably, the facile activity of EcFpg toward Gh and Sp in base pairing contexts with G and A, which are likely to be present after DNA replication, would be detrimental and enhance mutagenesis.     

Behaviour of a ‘sticky-desynaptic’ mutant in pearl millet

In the progeny after selfing of a normally open pollinated variety (L.S. 326-3) of pearl millet (Pennisetum americanum (L.) Leeke, n=7) one plant exhibited desynapsis, chromosome stickiness and high sterility. Meiosis was normal until diplotene. Thereafter, it was characterized by dissociation of bivalents into univalents and formation of nonspecific congregations of chromosomes at diakinesis, shrinkage of cytoplasm and occurrence of unoriented sticky chromatin masses at metaphase I, relaxation of stickiness, unbalanced chromosome numbers at the poles and laggards at anaphase I, and presence of other irregularities in subsequent stages. Meiosis was completed. Male and female sterility was high. This meiotic mutant thus has multiple effects and is inherited as a monogenic recessive and designated as st.     

Methotrexate binds in a non-productive orientation to human dihydrofolate reductase in solution, based on NMR spectroscopy

Dihydrofolate reductase (DHFR) is an intracellular target enzyme for folate antagonist drugs, including methotrexate. In order to compare the binding of methotrexate to human DHFR in solution with that observed in the crystalline state, NMR spectroscopy has been used to determine the conformation of the drug bound to human DHFR in solution. In agreement with what has been observed in the crystalline state, NOE's identified protein and methotrexate protons indicate that methotrexate binds in a non-productive orientation. In contrast to what has been reported for E. coli DHFR in solution, only one bound conformation of methotrexate is observed.     

Characterization of Sr9h,a wheat stem rust resistance allele effective to Ug99

Key message

Wheat stem rust resistance gene SrWeb is an allele at the Sr9 locus that confers resistance to Ug99.

Abstract

Race TTKSK (Ug99) of Puccinia graminis f. sp. tritici, the causal fungus of stem rust, threatens global wheat production because of its broad virulence to current wheat cultivars. A recently identified Ug99 resistance gene from cultivar Webster, temporarily designated as SrWeb, mapped near the stem rust resistance gene locus Sr9. We determined that SrWeb is also present in Ug99 resistant cultivar Gabo 56 by comparative mapping and an allelism test. Analysis of resistance in a population segregating for both Sr9e and SrWeb demonstrated that SrWeb is an allele at the Sr9 locus, which subsequently was designated as Sr9h. Webster and Gabo 56 were susceptible to the Ug99-related race TTKSF+ from South Africa. Race TTKSF+ possesses unique virulence to uncharacterized Ug99 resistance in cultivar Matlabas. This result validated that resistance to Ug99 in Webster and Gabo 56 is conferred by the same gene: Sr9h. The emergence of pathogen virulence to several resistance genes that are effective to the original Ug99 race TTKSK, including Sr9h, suggests that resistance genes should be used in combinations in order to increase resistance durability.