Evidence of a Hardening in the Cosmic Ray Proton Spectrum at around 166 TeV Observed by the GRAPES-3 Experiment.

We present the measurement of the cosmic ray proton spectrum from 50 TeV to 1.3 PeV using 7.81×10^{6} extensive air shower events recorded by the ground-based GRAPES-3 experiment between 1 January 2014 and 26 October 2015 with a live time of 460 day. Our measurements provide an overlap with direct observations by satellite and balloon-based experiments. The electromagnetic and muon components in the shower were measured by a dense array of plastic scintillator detectors and a tracking muon telescope, respectively. The relative composition of the proton primary from the air shower data containing all primary particles was extracted using the multiplicity distribution of muons which is a sensitive observable for mass composition. The observed proton spectrum suggests a spectral hardening at ∼166 TeV and disfavors a single power law description of the spectrum up to the Knee energy (∼3 PeV).

Measurement of the Electrical Properties of a Thundercloud Through Muon Imaging by the GRAPES-3 Experiment.

The GRAPES-3 muon telescope located in Ooty, India records rapid (∼10 min) variations in the muon intensity during major thunderstorms. Out of a total of 184 thunderstorms recorded during the interval of April 2011-December 2014, the one on December 1, 2014 produced a massive potential of 1.3 GV. The electric field measured by four well-separated (up to 6 km) monitors on the ground was used to help estimate some of the properties of this thundercloud, including its altitude and area that were found to be 11.4 km above mean sea level and ≥380 km^{2}, respectively. A charging time of 6 min to reach 1.3 GV implied the delivery of a power of ≥2 GW by this thundercloud that was moving at a speed of ∼60 km h^{-1}. This work possibly provides the first direct evidence for the generation of gigavolt potentials in thunderclouds that could also possibly explain the production of highest-energy (100 MeV) gamma rays in the terrestrial gamma-ray flashes.

Analysis of volatile aroma constituents of wine produced from Indian mango (Mangifera indica L.) by GC-MS.

Volatile aroma compounds are synthesized by wine yeast during wine fermentation. In this study the volatile aroma composition of two varieties of mango wine were determined to differentiate and characterize the wines. The wine was produced from the fruits of two varieties of mango cultivars namely Banginapalli and Alphonso. The volatile compounds formed in mango wine were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Thirty-two volatile compounds in wines were determined of which four were new and unidentified present in lower concentration. Apart from the ethanol (8.5 ± 0.28 and 7.2 ± 0.28% v/v), 1-propanol (54.11 ± 0.33 and 42.32 ± 0.57 mg/l), isobutyl alcohol (102 ± 1.57 and 115.14 ± 2.88 mg/l) and isoamyl alcohol (123 ± 2.88 and 108.40 ± 0.23 mg/1) were found to be the major flavouring higher alcohols in the mango wines produced from the fruits of Banginapalli and Alphonso respectively. Ethyl acetate (35 ± 0.57 and 30.42 ±1.15 mg/l) was the major ester component in both wines produced. Besides, other esters like ethyl octonoate, ethyl hexanoate and ethyl decanoate were also present in the wines. Cyclohexane methanol (1.45 ± 0.11 mg/l) was present only in wine made from Banginapalli and ß-phenylethyl butanoate (0.62 ± 0.01 mg/1) was found only in Alphonso wine. The results demonstrate that the wine prepared from Banginapalli variety had better aroma composition and good taste than that from the Alphonso variety.

Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches.

The proteolytic enzymes are the most important group of commercially produced enzymes. The production of alkaline protease was optimized using a newly isolated Bacillus sp. RKY3. The fermentation variables were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodological approach. Four significant variables (corn starch, yeast extract, corn steep liquor, and inoculum size) were selected for the optimization studies. The statistical model was constructed via central composite design (CCD) using three screened variables (corn starch, corn steep liquor, and inoculum size). An overall 2.3-fold increase in protease production was achieved in the optimized medium as compared with the unoptimized basal medium. Enzyme activity increased significantly with optimized medium (939 u ml(-1)) when compared with unoptimized medium (417 u ml(-1)).

Improvement of ethanol production in very high gravity fermentation by horse gram (Dolichos biflorus) flour supplementation.

AIMS: To determine the effect of osmotic stress on yeast and to investigate the protective role of horse gram flour during very high gravity (VHG) ethanol fermentation. METHODS AND RESULTS: Saccharomyces cerevisiae was inoculated into high sugar (30-40%, w/v) containing medium with and without supplementation of horse gram flour. The fermentation experiments were carried out in batch mode. The effect of 4 or 6% of horse gram flour to the medium on the metabolic behaviour and viability of yeast was studied. Significant increase in ethanol yield up to 50% and dramatic decrease in glycerol production up to 100% was observed in the presence of horse gram flour. The fermentation rate was increased from 3 to 5 days with increased viable cell count. The physical and chemical factors of horse gram flour may aid in reducing the osmotic stress of high gravity fermentation of ethanol as well as enhancing ethanol yield. CONCLUSIONS: It was found that horse gram flour not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of high ethanol concentration by using VHG sugar fermentation eliminates the expensive steps in the conventional process and saves time.

Schwann cells express active agrin and enhance aggregation of acetylcholine receptors on muscle fibers.

To explore novel roles of glial cells in synaptic function and formation, we examined the expression of agrin in frog Schwann cells and tested their role in the aggregation of acetylcholine receptors (AChRs). Using reverse transcription-PCR, we found that Schwann cells along nerve fibers in tadpoles expressed only the inactive agrin isoform B0 but began to also express active agrin isoforms B11 and B19 at approximately metamorphosis. During nerve regeneration in the adult, the expression of these active agrin isoforms in Schwann cells was upregulated, including the appearance of the most potent isoform, B8. This upregulation was induced by regenerating axons but not by nerve injury per se. In muscle cultures, the presence of adult Schwann cells enhanced the number and the total area of AChR aggregates 2.2- and 4.5-fold, respectively, and this enhancement was eliminated by heparin treatment. Furthermore, adult Schwann cells in culture expressed active agrin isoforms and produced agrin protein. Using a novel technique to selectively ablate perisynaptic Schwann cells (PSCs) at the neuromuscular junction, we found that PSCs also expressed active agrin isoforms B11 and B19, and these active isoforms were upregulated, including the appearance of B8, during reinnervation. Observation in vivo showed that extrajunctional AChR aggregates were associated with PSC sprouts after nerve injury and subsequent reinnervation. These results suggest that, contrary to the prevailing view that only neurons express active agrin, glial cells also express active agrin and play a role in the aggregation of AChRs both in vitro and in vivo.

Sequential chemiluminescent detection of target DNAs without stripping and reprobing.

We present a simple method for sequential chemiluminescent detections of two different DNA loci on a single Southern blot. First, an enzyme-linked DNA probe for a unique sequence is detected with a horse-radish peroxidase (HRP) substrate followed by the detection of another enzyme-linked DNA probe for a different unique sequence with an alkaline phosphatase (AP) substrate that simultaneously inhibits the chemiluminescence generated by HRP. Such sequential detection steps eliminate the need to strip and reprobe blots and can be performed with no intervening steps.

Growth and gene expression in diploid epithelial cell lines derived from normal human parotid gland.

Secretions of salivary glands are essential for the maintenance of oral health. Due to the lack of suitable in vitro models, studies to examine biochemical and molecular mechanisms of the cellular secretions have been difficult. Furthermore, adequate quantities of human epithelial cells could not be obtained, because normal diploid cells are believed to exhibit a limited lifespan of two to three passages (40-50 population doublings). This report describes for the first time the development of two diploid epithelial acinar cell lines, HPAM1 and HPAF1, derived from the normal human parotid gland. The cell lines are propagated in serum-free medium comprised of keratinocyte basal medium supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. The HPAM1 cell line has been passaged more than 50 times (> 189 population doublings) and HPAF1 more than 40 times (> 185 population doublings). Both cell lines exhibit normal diploid karyotypes, lack transformed phenotypes and are non-tumorigenic in nude mice. Both cell lines produce tissue-specific proteins, i.e. alpha-amylase 1, basic proline-rich protein, and cystatins; and express the corresponding genes as determined by RT-PCR analyses. These results demonstrate that normal diploid human cells do not inherently exhibit limited life-span in vitro and can, under optimum conditions, be propagated indefinitely.

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.

Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level.

The insulin-dependent tyrosine kinase activity (TKA) of the insulin receptor (IR) plays an essential role in insulin signaling. Thus, dysregulation of IR-TKA might be an important element in the states of insulin resistance. A phosphorylated rat hepatic glycoprotein (pp63) acting as an inhibitor of IR-TK has been described. In search of the human homolog of pp63, we isolated a cDNA clone from a human liver lambda gt11 cDNA library. DNA sequence analysis reveals identity with the mRNA product of a human gene AHSG encoding a serum protein, alpha 2-Heremans Scmid-glycoprotein (alpha 2HSG), with heretofore unknown physiological function. Northern blot analysis demonstrates a 1.8-kilobase mRNA in human liver and HepG2 hepatoma cells. alpha 2HSG, purified from human serum, specifically inhibits insulin-stimulated IR autophosphorylation in vitro and in vivo as well as exogenous substrate tyrosine phosphorylation. alpha 2HSG also inhibits both insulin-induced tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the p85 subunit of phosphatidylinositol-3 kinase in H-35 hepatoma cells. alpha 2HSG inhibits insulin-dependent mitogenesis, but does not affect insulin-stimulated induction of the metabolic enzyme tyrosine aminotransferase. alpha 2HSG does not compete with insulin for binding to IR. Finally, the action of alpha 2HSG is specific toward the IR-TK; its effect does not extend to insulin-like growth factor-I-stimulated TKA. Our results allow us to assign a biochemical function for human alpha 2HSG, namely regulation of insulin action at the IR-TK level.

Isolation and characterization of a recombinant heat shock protein of Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus, a common environmental fungus, is responsible for a number of lung disorders, including allergy and infection, in human beings. For immunodiagnosis of these diseases, standardized, pure, and relevant antigens are not currently available. METHODS: A complementary DNA library of A. fumigatus was constructed with messenger RNA isolated from 96-hour-old culture of the organism. Fusion proteins expressed with the cDNA were characterized and evaluated. RESULTS: One of the clones, which reacted with both rabbit anti-A. fumigatus serum and a pool of sera from patients with allergic bronchopulmonary aspergillosis, expressed a 65 kd protein of A. fumigatus. The recombinant protein reacted with immunoglobulin E and immunoglobulin G antibodies in the sera from patients with allergic bronchopulmonary aspergillosis. The deduced amino acid sequence of the partially sequenced complementary DNA of the clone is homologous with the hsp 90 family of heat shock proteins in human beings and other organisms. CONCLUSION: The immunodominant nature and the homology to human heat shock proteins suggest a possible role for this protein in protective immunity and autoimmunity.

Specific amplification of Aspergillus fumigatus DNA by polymerase chain reaction.

We have developed a specific and sensitive method to detect the human pathogen Aspergillus fumigatus by polymerase chain reaction (PCR) with an objective of detecting the organism in peripheral blood and urine which can be obtained by non-invasive procedures. A pair of oligonucleotide primers for PCR were designed based on the published partial protein sequences of an 18 KD IgE-binding protein of A. fumigatus Asp f1 and the ribotoxins mitogillin and restrictocin of A. restrictus, and alpha-sarcin of A. giganteus. The primers were specific in amplifying an expected 315 bp region of the homologous genes in A. fumigatus and A. restrictus but not in A. giganteus. Also, there was no amplification of human DNA or DNA of A. flavus, A. niger, A. fischeri, Penicillium sp., Candida albicans and Pneumocystis carinii. The sensitivity of the PCR detection of A. fumigatus DNA is about 20 pg on an ethidium bromide gel and 0.6 pg by Southern analysis using a 32P-labelled internal oligonucleotide. In preliminary analysis of 13 urine specimens of patients suspected of invasive aspergillosis (IA), two were PCR positive, one of whom died of IA with brain lesion. Further analyses of both urine and blood specimens of IA are in progress to determine the comparative utility of PCR over the conventional antigen tests.

Detection of Pneumocystis carinii in a rat model of infection by polymerase chain reaction.

The polymerase chain reaction (PCR) was employed to detect Pneumocystis carinii in organs of infected rats. Using a pair of oligonucleotides designed to the dihydrofolate reductase (DHFR) gene of rat P. carinii, specific amplification of an expected 415 bp region of P. carinii DHFR DNA of this organism was achieved, while no amplification occurred with the human, Candida albicans, and Mycobacterium avium and tuberculosis DNAs. Using rat P. carinii isolated from in vitro cultures and infected lung homogenates, the minimum detection level by PCR on an ethidium bromide gel was about 200 organisms and by Southern analysis with radiolabelled DHFR probe the detection level improved to 20 organisms. This level of sensitivity is sufficient to detect P. carinii specific band on the gel in infected rat lung and other organs. This PCR technique is potentially useful for detecting P. carinii in bronchoalveolar lavage (BAL) fluids of AIDS patients and for quantifying the organisms in tissues and in in vitro cultures where a high background with conventional stains makes it harder to determine the number of organisms.

Proliferation patterns of latent Pneumocystis carinii in rat organs during progressive stages of immunosuppression.

Pneumocystis carinii is known to proliferate mainly in the lung of an immunocompromised host. In AIDS and other immune disorders sporadic extrapulmonary presence of this organism has been documented. Occasionally, P. carinii does not appear to infect the lung. These observations have been based on the detection of P. carinii by conventional staining techniques. We have sought to determine the extent of these infections by the polymerase chain reaction (PCR) in a rat model. Harlan Sprague-Dawley rats weighing between 110 and 130 g were immunosuppressed with dexamethasone (1.2 mg/l) in drinking water. During progressive stages of immunosuppression 2 rats were sacrificed at 2, 3, 4 and 5 wk, and the lung, liver, kidney, spleen and bone marrow were taken. Sonicated crude extracts of the tissues were used as template DNA for the amplification of the dihydrofolate reductase (DHFR) gene of P. carinii. All the PCR products were analyzed by Southern hybridization with radiolabelled DHFR DNA. These analyses revealed a general trend of P. carinii proliferation first in bone marrow at 2 wk, followed by liver at 3 wk, and lung at 5 wk on immunosuppression. Kidney and spleen infections were infrequent. Although P. carinii appears to proliferate in the lung at later stages of immunosuppression, the degree of proliferation is several-fold greater than in extrapulmonary organs. The extrapulmonary proliferation of P. carinii, however small, may possibly suppress hematopoietic stem cell differentiation in bone marrow, and may also contribute to the pathology present in various organs.

Sequences of three VSG mRNAs expressed in a mixed population of Trypanosoma brucei rhodesiense.

A cDNA library was constructed from a mixed population of bloodstream Trypanosoma brucei rhodesiense expressing at least three different VSGs, one of which is immunologically similar to a VSG present during the metacyclic stage. Complete sequence determinations of three full length VSG cDNAs showed that the 5' spliced leader sequence is located 20-26 nucleotides upstream of the start codons of each of the three VSG mRNAs. Two of the VSGs (472 and 487 aa) are members of one C-terminal isotype group while the other, metacyclic-like, VSG (517 aa) is a member of the other C-terminal group. Since VSGs of different sequences have similar three-dimensional structures, comparison of many different VSGs sequences should lead to a more detailed understanding of the relationship between primary and tertiary structures of proteins in general. GenBank accession numbers: M33823, M33824, and M33825.

Age-related changes in muscle ammonia detoxification potential in exhausted rats.

The changes in the pattern of production and detoxification of ammonia have been studied in the skeletal muscles and blood of rats of different age groups (1, 3, 6, 12 and 24 months), subjected to exhaustive exercise. The protein profiles at exhaustion showed a sharp drop in all muscles and the decrement was more in the senile rats. In general, the muscle and blood ammonia content increased with age with a corresponding increase in AMP deaminase activity implicating the possibility of elevated purine nucleotide deamination during senescence. However, glutamate oxidation was decreased and urea and glutamine formation was increased consequent to ammonia production during senescence under intensive physical stress. The possible alterations in protein levels and ammonia production and its disposal in different skeletal muscle types of senile exhausted rats have been discussed in relation to detoxication capacity of the fibre types.