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Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target., (2009 Wiley Periodicals, Inc.)

Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.

We report an approach of exploring the interaction between cationic surfactants and a type of structurally well-defined, spherical "Keplerate" polyoxometalate (POM) macroanionic molecular clusters, {Mo72V30}, in aqueous solution. The effectiveness of the interaction can be determined by monitoring the size change of the "blackberry" supramolecular structures formed by the self-assembly of {Mo72V30} macroions, which is determined by the effective charge density on the macroions. Long-chain surfactants (CTAB and CTAT) can interact with {Mo72V30} macroions stoichiometrically and lower their charge density. Consequently, the blackberry size decreases continuously with increasing surfactant concentration in solution. On the other hand, for short-chain surfactants (e.g., OTAB), a larger fraction of surfactants exist as discrete chains in solution and do not strongly interact with the macroions. This approach shows that a controllable amount of suitable surfactants can accurately tune the charge on large molecular clusters.

Background: Asthma is a chronic inflammatory disorder of the airways. The available treatment options have major limitations owing to low efficacy, associated adverse events and compliance issues. Therefore, the health burden of bronchial asthma is increasing globally at an alarming rate, providing a strong impetus for the development of new therapeutics. Myrica sapida is known traditionally in Ayurveda to possess anti-asthmatic activity. Hence, the present investigation was undertaken to evaluate the bronchodilator and anti-anaphylactic activity of the stem bark of Myrica sapida., Methods: Experimental models studied were acetylcholine induced bronchospasm in guinea pigs, egg albumin induced anaphylaxis in guinea pigs, in vitro studies on tracheal strip of egg albumin sensitized guinea pigs., Results: Treatment with ethanolic extract of M. sapida, 75 mg/kg, orally resulted in significant protection against acetylcholine aerosol induced bronchospasm and allergen induced anaphylaxis in guinea pigs. Ethanolic extract of M. sapida (75 mg/kg, p.o.) prevented the potentiation of responses and also produced a decrease in pD2 value of histamine and acetylcholine in guinea pig tracheal strip., Conclusion: These results suggest that M. sapida possesses bronchodilator activity, has potent inhibitory effect on immediate hyper-sensitivity reactions and decreases bronchial hyper-responsiveness.

The present investigation was undertaken to evaluate the bronchodilator and bronchial hyperreactivity of the stem bark of Myrica sapida. Experimental models studied were histamine induced bronchospasm in guinea pigs, bronchoalveolar lavage fluid (BALF) in egg albumin sensitized guinea pigs, histamine release from the lung tissues of sensitized guinea pigs and histopathological studies. Ethanolic extract of M. sapida (75 mg/kg, p.o., for 7 days) showed significant protection against histamine aerosol induced bronchospasm. Significant decrease in the total and differential leukocyte counts in BALF and prevention of egg albumin induced histamine release from chopped lung tissues of sensitized guinea pigs was observed on chronic administration of ethanolic extract of M. sapida (75 mg/kg, p.o., for 15 days). Histological examination of the section of lung from sensitized guinea pigs treated with ethanolic extract of M. sapida (75 mg/kg, p.o., for 15 days) was comparable to that of the control group. These results suggest that M. sapida possesses not only bronchodilator activity but also decreases bronchial hyperresponsiveness by decreasing the infiltration of inflammatory mediators like eosinophils, neutrophils in BALF and inhibiting histamine release from lungs of sensitized guinea pigs.

176 chromium-exposed and 30 control subjects were selected for this study. Blood samples (3 ml) were collected for the estimation of chromium. The data on chromium concentration indicated a significant higher level of chromium in the blood of the exposed workers as compared to the control. There was no significant correlation between the mean blood and environmental chromium level. This study suggests that exposure to chromium may have some effect on the health of workers, even though the dose response relationship could not be established between blood chromium and environmental chromium levels. This study suggests that exposure to chromium may have some effect on the health of workers, even though the dose response relationship could not be established between blood chromium and environmental chromium levels. Study also indicates that the environmental levels to Cr are well below the permissible levels at all the sites of the industry at the time of survey even though the blood Cr levels were observed high in 14.8% of workers and some of them were having Cr related morbidity. Therefore, preventive and engineering control measures are suggested to minimize the chromium exposure in the chromium based industry located in Gorwa industrial estate at Baroda, Gujarat. About three months period was taken to complete this study.

A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as approximately 500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.

Type I polyketide synthase (PKS) genes consist of modules approximately 3-6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.

To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of approximately 5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440-15445]. The major current impediment to the success of this tactic is the difficulty of building the approximately 5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp "synthons" with low error frequency by automated PCR-based gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon approximately 5-kb segments by using only three endonucleases and "ligation by selection." These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.

The authors examined 100 carbon disulphide (CS2) exposed male workers who had been employed ten years prior to study were selected for the study. They were virtually obliged to participate in the study by the Medical Labor Inspector and all of them participated voluntary. The aim was to assess the effects of occupational exposure to carbon disulphide concentrations below the threshold limit value (31 mg/m3) on the reproductive functions with special emphasis on miscarriages. Specially, workers history records were build up on number of children, miscarriages and general weakness, mental fatigue etc. It was found that the incidences of number of miscarriages against number of living children correlated well with environmental concentration of CS2. Where the average CS2 levels were 1.695 ppm, the incidences of miscarriages was 5.71% (group 1). Where as in group 2 environmental concentrations were 12.28 ppm and the incidences of miscarriages were 18.91%. It was also found that in the spinning department the exposure exceeds many times the Threshold Limit Values (TL V).

The chromosomes of several widely used laboratory derivatives of Streptomyces coelicolor A3(2) were found to have 1.06 Mb inverted repeat sequences at their termini (i.e. long-terminal inverted repeats; L-TIRs), which are 50 times the length of the 22 kb TIRs of the sequenced S. coelicolor strain M145. The L-TIRs include 1005 annotated genes and increase the overall chromosome size to 9.7 Mb. The 1.06 Mb L-TIRs are the longest reported thus far for an actinomycete, and are proposed to represent the chromosomal state of the original soil isolate of S. coelicolor A3(2). S. coelicolor A3(2), M600 and J1501 possess L-TIRs, whereas approximately half the examined early mutants of A3(2) generated by ultraviolet (UV) or X-ray mutagenesis have truncated their TIRs to the 22 kb length. UV radiation was found to stimulate L-TIR truncation. Two copies of a transposase gene (SCO0020) flank 1.04 Mb of DNA in the right L-TIR, and recombination between them appears to generate strains containing short TIRs. This TIR reduction mechanism may represent a general strategy by which transposable elements can modulate the structure of chromosome ends. The presence of L-TIRs in certain S. coelicolor strains represents a major chromosomal alteration in strains previously thought to be genetically similar.

Formaldehyde (HCHO) exposure in industry causes health impairment, especially immune profile. Male albino rats when exposed to HCHO at 5, 10, 15 mg/kg day for 30 days showed decrease in thyroid weight in 10, 15 mg/kg/day HCHO fed group with commensurate follicular regression (epithelial cells), decreased triiodothyronine (T3), thyroxin (T4) deficiency and enhanced thyroid stimulating hormone (TSH). Repeated chemical stimulation might have increased thyroid activity of the follicles and rapidly deteriorated the capacity for synthesis of thyroxin hormone and thus becomes atrophied. The chronic action of HCHO on thyroid may be due to a block at the source of thyroid hormone (s).

Directed neuronal, astroglial, and oligodendroglial cell migrations comprise a prominent feature of mammalian brain development. Because molecular motor proteins have been implicated in a wide spectrum of processes associated with cell motility, we initiated studies to define the pool of myosins in migrating cerebellar granule neurons and type-1 neocortical astrocytes. Our analyses identified two isoforms of a novel unconventional myosin, which we have cloned, sequenced, and designated myr 8a and 8b (eighth unconventional myosin from rat). Phylogenetic analysis indicates that myr 8 myosins comprise a new class of myosins, which we have designated class XVI. The head domain contains a large N-terminal extension composed of multiple ankyrin repeats, which are implicated in mediating an association with the protein phosphatase 1 (PP1) catalytic subunits 1alpha and 1gamma. The motor domain is followed by a single putative light-chain binding domain. The tail domain of myr 8a is comparatively short with a net positive charge, whereas the tail domain of myr 8b is extended, bears an overall neutral charge, and reveals several stretches of poly-proline residues. Neither the myr 8a nor the myr 8b sequence reveals alpha-helical coiled-coil motifs, suggesting that these myosins exist as monomers. Both immunoblot and Northern blot analyses indicate that myr 8b is the predominant isoform expressed in brain, principally at developmental time periods. The structural features and restricted expression patterns suggest that members of this novel class of unconventional myosins comprise a mechanism to target selectively the protein phosphatase 1 catalytic subunits 1alpha and/or 1gamma in developing brain.

1. Adult male albino rats (CF Strain) were administered i.p. CS2 dissolved in cotton seed oil at doses of 25, 50, 100 and 200 mg/kg b. wt. for a period of 60 days. Effect of CS2 on epididymis, adrenal weight, sperm count and sperm head shape abnormality was studied. 2. Epididymal weight remained unaltered in 25, 50 and 100 mg/kg CS2 treated groups, whereas in highest dose of CS2 treated (200 mg/kg) group a non-significant reduction in epididymis weight was observed. A slight increase in adrenal weight was observed in lower doses groups (25 and 50 mg/kg) while a considerable decrease in adrenal weight was noted in highest dose (200 mg/kg) of CS2 treated group in the present study. 3. An increase in sperm head shape abnormality and decrease in sperm count was observed in all the CS2 treated groups. However, the changes were statistically significant only after higher dose of CS2 treatment as compared to control. 4. This study suggests that CS2 may have the potential to induce adverse effects on male reproductive system of rats. Sperm head shape abnormality assay used in this study also elicits germ cell genotoxic potential of carbon disulphide.

Three groups of rats (n = 10) were subjected to intraperitoneal treatment of formaldehyde daily at doses of 5, 10 and 15 mg/kg body weight over a period of 30 days. Gradual diminution in body and testicular weight was observed in all treated groups. Leyding cell impairement was conspicuous in those given doses of 10 and 15 mg/kg. Inhibition of 3 beta-delta 5-hydroxy steroid dehydrogenase and accumulation of sudanophillic materials in testicular tissue of formaldehyde treated rats was recorded histochemically. Significant decline of serum testosterone was also observed in the same groups. Structural and functional impairement of Leydig cells after formaldehyde treatment caused steroidogenic inhibition.

Experiments were performed to investigate the effect of calcium on percent motility of cauda epididymal spermatozoa in vitro. Epididymal luminal contents collected by micropuncture were immediately diluted with different concentrations of calcium chloride in KRB buffer. Percent motility was recorded at different time intervals at room temperature. Results revealed that there was an immediate effect of calcium on sperm motility. The maximum motility was attained only on addition of 10(-5)M calcium chloride in the buffer, whereas a higher or lower concentration than the above in the medium was inhibitory. Higher calcium levels maintained the sperm motility for a longer duration with a slow, time-dependent decrease than the lower concentrations where a faster depression in motility occurred. It is concluded that calcium at an appropriate level in the medium is essential for optimal sperm motility.

Semen qualities were studied in workers with an average age of 30 years and occupationally exposed to lead in a printing press. Another sample with the same average age but not exposed to lead were taken as control subjects. The average lead content in blood and seminal plasma of the exposed group were 42.5 mcg/100 ml and 14.80 mcg/100 ml, respectively. Their sperm counts and percentage of motile sperm were significantly affected. Significantly higher percentages of abnormal spermatozoa were also observed in these semen samples. The levels of seminal acid phosphatase, succinic dehydrogenase, and fructose in them were also significantly found to be low compared with those from the unexposed subjects. Cytochemical study of sperm head DNA in the exposed groups showed a low surface reaction.