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The cDNA of a firefly luciferase from lantern mRNA of Lampyroidea maculata has been cloned, sequenced and functionally expressed. The cDNA has an open reading frame of 1647 bp and codes for a 548-residue-long polypeptide. Noteworthy, sequence comparison as well as homology modeling showed the highest degree of similarity with H. unmunsana and L. mingrelica luciferases, suggesting a close phylogenetic relationship despite the geographical distance separation. The deduced amino acid sequence of the luciferase gene of firefly L. maculata showed 93% identity to H. unmunsana. Superposition of the three-dimensional model of L. maculata luciferase (generated by homology modeling) and three dimensional structure of Photinus pyralis luciferase revealed that the spatial arrangements of Luciferin and ATP-binding residues are very similar. Putative signature of AMPbinding domain among the various firefly species and Lampyroidea maculata was compared and a striking similarity was found. Different motifs and sites have been identified in Lampyroidea maculata by sequence analysis. Expression and purification of luciferase from Lampyroidea maculata was carried out using Ni-NTA Sepharose. Bioluminescence emission spectrum was similar to Photinus pyralis luciferase.

Two Bacillus sp. strains, HR-08 and KR-8102, isolated from soil of the west and north parts of Iran were screened on gelatin agar medium for their ability to produce alkaline protease. The enzymes were active in a wide pH range (6.0-11.0) and stable in the alkaline range (7.0-12.0). The optimum temperatures for the protease from HR-08 and KR-8102 were 65 and 50 degrees C, respectively. The irreversible thermoinactivation of HR-08 and KR-8102 proteases showed that the stability of HR-08 enzyme was higher than that of KR-8102 and the half-lives of these enzymes were 95 and 32 min at 50 degrees C, respectively. In the presence of 10 mM Ca(2+), HR-08 retained 100, 90, and 20% of its initial activity after heating for 30 min at 50, 60, and 70 degrees C, respectively. Enzymes were inhibited by phenylmethylsulfonyl fluoride and iodoacetate. After inhibition by iodoacetate, both enzymes were reactivated by dithiothreitol. These data show that the enzymes seem to be thiol-dependent serine alkaline proteases. The enzymes especially from HR-08 were stable in the presence of H(2)O(2), surfactants, and local detergents; their activities were enhanced in the presence of 5 mM Fe(2+); and the presence of 5 mM metal ions such as Mg(2+), Cu(2+), and Mn(2+) produced almost no effect.

Hepatitis D (delta) virus (HDV) is a subviral pathogen agent and a satellite of Hepatitis B virus. Three distinct genotypes are described for HDV; genotype I is distributed worldwide but other genotypes appear to be more restricted geographically. In the present study, the entire nucleotide sequence of an HDV isolate from an Iranian patient (IR-1) was obtained using twelve pairs of primers to amplify six overlapping fragments covering the whole HDV genome by RT-nested PCR. Phylogenetic and pairwise alignments were done on this new isolate to determine IR-1 position among other isolates. Our results indicate that IR-1 contains 1676 nucleotides encoding 214 a.a. of the hepatitis delta antigen (HDAg). This new isolate belongs to genotype I with most sequence similarity to an Italian HDV isolate (92.6%). At amino acid level, predicted HDAg sequence of IR-1 revealed the most homology with those of Italian and Lebanese isolates. Data analysis confirmed genetic variability and heterogeneity of the HDV species isolated from different geographical areas.

Background: Rapid, sensitive and economical detection and identification of human herpesviruses as causative agents of central nervous system (CNS) infections are of clinical importance. The traditional methods for the detection of herpesviruses in CNS infections all suffer from limitations. PCR has a potential to overcome each of them., Objectives: The aims of this study were reducing the number of primers in multiplex PCR and increasing the sensitivity of the assay by nested PCR., Study Design: A multiplex nested consensus PCR (MNC-PCR) was developed for the simultaneous detection of major human herpesviruses. A pair of conserved primers was designed for detection of HSV-1, HSV-2, CMV and EBV and another pair of conserved primers for nested PCR. For VZV, a different pair of primers was designed and another pair of primers for nested PCR. A reduction in the number of designed primer pairs (from five pairs to two in both stages of PCR) is an advantage in this assay. One hundred forty-seven cerebral spinal fluid (CSF) samples from patients that showed clinical manifestation of CNS infections were tested. Results of MNC-PCR in CSF samples were compared with those of single PCR assay for each individual DNA virus. Sensitivity of the assay was determined with a plasmid containing VZV DNA binding protein gene and another plasmid for HSV-1 DNA polymerase gene. False negative results (due to the presence of inhibitor of DNA amplification in CSF samples) were avoided by the inclusion of beta2-microglobulin primers in the MNC-PCR assay as an internal control., Results: Positive results were obtained in 20 CSF samples (8 HSV-1, 2 HSV-2, 4 CMV, 3 VZV, 3 HSV-1/CMV, CMV/VZV and HSV-1/EBV coinfections). The comparison between single PCR and MNC-PCR showed a marked increase in sensitivity of MNC-PCR test, since six negative samples in single PCR proved positive in MNC-PCR (P<0.005). Sensitivity was determined 1-5 plasmid copies for VZV and 50-100 plasmid copies for HSV-1., Conclusions: The MNC-PCR assay presented in this study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of CSF samples.

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH1). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. Ka values of specific polyclonal antipeptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 x 10(10) M(-1) and 1.4 x 10(10) M(-1) respectively.