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Green versus sol-gel synthesis of zinc oxide nanoparticles (ZnO NPs) were carried out and antimicrobial activity was evaluated against a wide panel of bacterial and fungal pathogens. Five plants seed extracts were used for green synthesis from Brassicaceae family, whereas the sol-gel method was adopted in the case of chemical synthesis. The ZnO NPs were characterized using Ultraviolet-Visible (UV-Vis), Energy Dispersive X-ray (EDX), X-ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) techniques. The antimicrobial potential of the synthesized NPs was assessed against bacterial and fungal strains by disc diffusion method. The size of the ZnO NPs was in the range of 100–150 nm. The ZnO NPs showed promising antimicrobial activity and zones of inhibition were in the range of 10–20 mm, whereas Streptomycin (standard drug) showed the zone of inhibition of 25 mm. The synthesized ZnO NPs furnished promising antibacterial and antifungal potential. The green route of synthesis is rather safe and eco-friendly when compared to chemical route and could be used for the fabrication of ZnO NPs as an antimicrobial agent.

Antifibrotic prescribing for Idiopathic Pulmonary Fibrosis (IPF) is limited by the National Institute of Health and Clinical Effectiveness (NICE) to patients with a forced vital capacity (FVC) of 50–80%. 38% of IPF patients on the British Thoracic Society registry have an FVC above 80%. Methods This is a retrospective single centre cohort study of IPF patients with baseline FVC above 80%, between January 2007 and September 2018. We assessed electronic records to collect data on patient demographics, treatment and lung function changes over time. Data and statistics are described as in Table1. A linear mixed model was performed to assess the change in FVC and DLCO over time. Results 161 patients with baseline FVC above 80% were included, 74.5% (n=120) were male. The mean age was 72.7 ±7.4. 128 patients initially were treated with antifibrotics through compassionate use programmes (CUP) (42 (26.1%) on pirfenidone and 104 (64.6%) on nintedanib) compared to 33 patients who had no treatment, as the CUP had closed. Patient demographics, duration of treatment, adverse events and reasons for discontinuation are presented in Table 1. Patients without antifibrotic therapy had a statistically higher baseline FVC compared to other groups (3.55 (100%) vs. 2.88 (89%) pirfenidone vs 2.96 (92%) nintedanib) (p Conclusion Untreated patients had higher FVC and DLCO compared to treated cohorts, which makes comparison of lung function decline difficult. Despite this, one in five untreated patients with an average FVC of 100% still die within a median of 2.5 years while antifibrotic therapy was associated with a median survival to 3–3.5 years in a cohort with lower baseline lung function. Lung function decline in treated cohorts is similar to that seen in clinical trials (-2.64%nintedanib and -3.24%pirfenidone).

An experiment was conducted to assess the effect of applying different rates of urea treated with nBTPT (N-(n-butyl) thiophosphoric triamide) alone, or a combination of both nBTPT and DCD (Dicyandiamide) and DMPP (3, 4-Dimethylpyrazol-phosphate), on spinach productivity at experimental area of Faculty of Agriculture, Gomal University, Dera Ismail Khan during 2012-13. The experiment was arranged in a Complete Randomized Design (CRD) with nine treatments and four replications. Composite soil samples were collected before the experiment for various physico-chemical analysis viz soil texture, soil pH, organic matter, bulk density, NH4-N, NO3-N, extractable P and exchangeable K. The results showed that treatment (T6) with dose of Urea@150 kg/ha + GA3 @60 gram/ha was more effective than agrotain alone i.e tallest plant height (11.54 cm), maximum number of leaves (69), highest fresh weight (22.37g), highest dry weight (4.64 g), maximum biological yield (227 g pot-1) and highest net return (13.42 million). Such findings may be due to availability of nitrogen for longer period and reduced loss on nitrogen by volatilization. The results further suggested that applying urea treated with Gibberellic acid (GA3) or Agrotain significantly increased the spinach growth. However, combine application of Urea, Urease, Nitrification Inhibitors and Plant Growth Regulators on Spinach productivity was better than urease inhibitor alone (agrotain).

The effects of a urease inhibitor [N-(n-butyl) thiophosphoric triamide (nBTPT– trade name Agrotain®)] alone and a combination of nitrification inhibitor dicyandiamide (DCD) and nBTPT, defined as a double inhibitor (DI), on urea fertilizer use efficiency in pastoral systems were investigated. The treatments comprised urea alone, urea with nBTPT, urea with DI at a low rate (LDI), and urea with DI at a high rate (HDI). Each treatment had three replicates and was applied at two rates – 30 and 60 kg nitrogen (N) ha−1 to three trial sites (North Island-1, North Island-2 and South Island) in New Zealand during the 2005–2006 seasons. The trials at North Island-1 and South Island received six applications of fertilizers, giving total N application rates of 180 and 360 kg N ha−1 over an18-month period. The North Island-2 trial received these treatments five times during a one-year period, giving total N application rates of 150 and 300 kg N ha−1. Soil samples collected after the first fertilizer applicatio...

The essential oil from the leaves of Curcuma longa L. Kasur variety grown in Pakistan was extracted by hydro-distillation. Chemical constituents of the essential oil were identified by gas chromatography/mass spectrometry. The chromatographic analysis of oil showed 25 constituents, out of which nine chemical constituents were identified. The eucalyptol (10.27%) was the major component of the essential oil. α-pinene (1.50%), β-phellandrene (2.49%), β-pinene (3.57%), limonene (2.73%), 1,3,8-p-menthatriene (1.76%), ascaridole epoxide (1.452%), 2-methylisoborneol (2.92%), 5-isopropyl-6-methyl-hepta-3, dien-2-ol (2.07%) were also present in considerable quantity. The antimicrobial properties of leaves of Curcuma longa were tested by disc diffusion method against various human pathogens, including eight fungal and five bacterial strains. Essential oil showed maximum resistance against Fusarium miniformes MAY 3629 followed by Bacillus subtilis ATCC 6633 whereas; it exhibited least resistance against Fusarium oxysporium ATCC 48122. The results of the antimicrobial assay revealed that essential oil showed significant inhibitory activity against the tested organisms.

In this study, we investigated the role of lysozyme on the viability of Bacillus cereus , Bacillus subtilis , Bacillus pumilus and Bacillus anthracis (Sterne) in egg white (EW), ground beef and milk. At 35 °C in EW, growth rates (GR) for B. cereus , B. subtilis , B. pumilus and B. anthracis were 0.005, −0.018, −0.028 and −0.029 OD 600 /h, respectively. Heat-treating EW at 55 and 60 °C reduced the inactivating effect of EW by 3.1 and 10.5-fold, respectively. Addition of lysozyme (2 mg/ml) to 60 °C-treated EW increased the inactivation rate 5.76-fold, indicating involvement of lysozyme in B. anthracis inactivation. B. anthracis inactivation was influenced by pH, as shown by a progressive increase in inactivation rate from 0.25 to −4.42 logs CFU/h over a pH range of 6.0–8.5. Adding 2 mg/ml lysozyme to milk and ground beef also suppressed the growth of B. anthracis 3.3 and 6.5-fold, respectively. These data indicate that lysozyme, as a natural component of EW or potential additive in other foods, could reduce biothreat risks presented by bioterror agents.

We have developed multiplex real-time PCR assays that utilize DNA-intercalating dyes, SYBR Green I (SG) and EvaGreen (EG), with two primer sets (set 1=qacEδ1, tetA and aacA-aphD; set 2=tetA, marA, and floR) to simultaneously amplify the qacEδ1, tetA, aacA-aphD, marA, and floR genes. Validity of the multiplex PCR assays was confirmed by testing 83 bacterial isolates, including Staphylococcus aureus (28 isolates), Enterococcus spp. (17 isolates), Salmonella enterica serovar Typhimurium (8 isolates), Citrobacter spp. (9 isolates), Escherichia coli (14 isolates) and Aeromonas veronii (7 isolates), and performing sequence analysis of representative PCR products. Agarose gel analysis revealed the presence of correct size PCR products, and the differences in their thermal melting (T(m)) curves were used to distinguish various PCR products. Although T(m) peaks of different amplicons after EG-based singleplex and multiplex PCR assays were resolved nicely, only one or two peaks were seen for SG-bound amplicons. EG-based multiplex real-time PCR assays provided better peak resolution. There was a good correlation with a better linear relationship between the C(t) and log input DNA concentration for the set 1 and set 2 genes in EG-based assays (R(EG)(2)=0.9813and0.9803) than in SG-based assays (R(SG)(2)=0.5276and0.6255). The sensitivities of detection were 2.5-25fg and 25-250fg of template DNA in EG and SG-based singleplex and multiplex PCR assays, respectively. The assays, which could be completed in less than 45min, offer sensitive and rapid detection of qacEδ1, aacA-aphD, marA, floR, and tetA genes from a diverse group of multiple antibiotic-resistant bacterial strains.

A procedure based on QuEChERS extraction and a simultaneous liquid-liquid partition clean-up was developed. The procedure involved extraction of hydrated soil samples using acetonitrile and clean-up by liquid-liquid partition into n-hexane. The hexane extracts produced were clean and suitable for determination using gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated by analysis of soil samples, spiked at five levels between 1 and 200 microg kg(-1). The recovery values were generally between 70 and 100% and the relative standard deviation values (%RSDs) were at or below 20%. The procedure was validated for determination of 19 organochlorine (OC) pesticides. These were hexachlorobenzene (HCB), alpha-HCH, beta-HCH, gamma-HCH, heptachlor, heptachlor epoxide (trans), aldrin, dieldrin, chlordane (trans), chlordane (cis), oxychlordane, alpha-endosulfan, beta-endosulfan, endosulfan sulfate, endrin, p,p'-DDT, o,p'-DDT, p,p'-DDD and p,p'-DDE. The method achieved low limits of detection (LOD; typically 0.3 microg kg(-1)) and low limits of quantification (LOQ; typically 1.0 microg kg(-1)). The method performance was also assessed using five fortified soil samples with different physico-chemical properties and the method performance was consistent for the different types of soil samples. The proposed method was compared with an established procedure based on Soxtec extraction. This comparison was carried out using six soil samples collected from regions of Pakistan with a history of intensive pesticide use. The results of this comparison showed that the two procedures produced results with good agreement. The proposed method produced cleaner extracts and therefore led to lower limits of quantification. The proposed method was less time consuming and safer to use. The six samples tested during this comparison showed that soils from cotton growing regions contained a number of persistent OC residues at relatively low levels (

Guillain–Barre Syndrome (GBS) is a neuromuscular disorder and campylobacteriosis is known to trigger the onset of the disorder. A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 497-bp region of the UDP-galactose 4-epimerase ( gal E) gene sequence in campylobacters responsible for triggering the onset of GBS. The identity of the PCR product was confirmed by Hind III endonuclease restriction digestion, which produced the predicted 430 and 67-bp DNA fragments. The assay could detect the presence of the gene in Campylobacter suspensions containing as few as 5 cells ml −1 . The assay detected the presence of the gene in 17 of the 20 campylobacters isolated from chicken, 9 of the 13 campylobacters isolated from turkey and 7 of the 7 campylobacters isolated from human stools. All Campylobacter strains isolated from chicken, turkey and clinical samples were resistant to multiple antibiotics. The assay failed to detect the presence of the gene in five different microaerophilic strains of Helicobacter spp., E. coli and Salmonella spp. The entire diagnostic assay, including template preparation, amplification and electrophoresis, can be completed within 6 h.

A one year field experiment was conducted to assess the efficiency of urea fertilizer applied to pasture near Lincoln University, New Zealand. Urea with or without () molybdenum (Mo) were applied to field plot, with a urease inhibitor [N-(n-butyl) thiophosphoric triamide (nBTPT), nBTPT + elemental sulphur (S), and nitrification inhibitor dicyandiamide (DCD) + nBTPT defined as a double inhibitor (DI) in spring 2005. Mo was sprayed at the rate of 50 g Mo/ha/yr once. Urea various inhibitor and S treatments were broadcast at a rate of 30 kg N/ha 5 times over one year. The Mo alone treatment increased pasture dry matter (PDM) yield by 8.9%. Molybdenum, when applied together with urea+nBTPT+S, urea+nBTPT and urea alone, caused an initial depression in PDM yield by 14.2, 13 and 5.6% respectively. However, these depressions in yield disappeared from the second pasture cut. Over a one year period, Mo applied with urea+nBTPT, urea+DI and urea+nBTPT+S produced 18179, 16716 and 18253 kg DM ha-1 respectively, compared to 16171 kg ha-1 for urea+Mo. Pastures which received no Mo, but were treated with urea+nBTPT+S, or urea+nBTPT produced 18982 and 18276 kg DM ha-1 respectively, compared to 16363 kg ha-1 for urea alone, giving an increase of 16% and 12% over urea alone. Pastures receiving Mo, together with urea+nBTPT or urea+nBTPT+S, also showed improvement in N uptake and N recovery, compared to urea alone. Applying urea with nBTPT and S have the most potential to improve urea efficiency.

The structural intermediates in the capsid assembly and DNA packaging pathway of Vibrio vulnificus bacteriophage 71A-6, a rod-shaped double-stranded DNA podovirus, were isolated by ultracentrifugation and studied by electron microscopy, SDS-PAGE and pulsed-field gel electroIphoretic analysis. Bacteriophage 71A-6 synthesized rod-shaped capsids (mean length=200±8 nm; mean width=47±3 nm n =50) during its development. Several host proteins that probably help in the assembly and maturation of the capsids were attached to these capsids as spherical structures. A capsid-DNA or DNA packaging complex that consisted of the mature capsids, DNA and a 42·5-kDa protein was also isolated. The size of the capsids increased in length and decreased in width (mean length=220±8 nm; mean width=45±3 nm n =50) either during or after the DNA packaging. The capsid fractions contained about 12 phage structural proteins and eight host proteins. At least three proteins were tentatively identified: a 38·5-kDa major capsid protein, a 35·2-kDa tail protein and 42·5-kDa packaging initiator or terminator protein. The size of the bacteriophage 71A-6 genome was determined to be 143·0-kb by pulsed-field gel electrophoresis. The total mass of all the mature phage proteins corresponded to only 14·0% of the coding capacity of phage genome.

Salmonella typhimurium definitive type 104 (DT104) is a virulent pathogen for humans and animals with many strains having multiple drug resistance characteristics. The organism typically carries resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant). A multiplex PCR method was developed to simultaneously amplify four genes, florfenicol (flo(st)), virulence (spvC), invasion (invA), and integron (int) from S. typhimurium DT104 (ACSSuT-type). Twenty-two ACSSuT-resistant DT104 isolates in our collection gave 100% positive reactions to this PCR assay by amplifying 584-, 392-, 321- and 265-bp PCR products, using primers specific to the respective target genes. One Salmonella strain DT23, ACSSuT-resistant, phage type 711 failed to amplify the 584-bp fragment, indicating that this method is specific for DT104-type ACSSuT-resistant S. typhimurium strains. One clinical and one bovine ASSuT-resistant strains that were sensitive to chloramphenicol and florfenicol did not yield a 584-bp fragment, indicating the absence of the flo(st) gene. This method will be useful for rapid identification of ACSSuT-type DT104 strains from clinical, food and environmental samples.

A comparative analysis of the two most dominant erythromycin-resistance determinant genes in Staphylococcus sppnamely, the ermA and ermC genes, was carried out. Sixty erythromycin-resistant strains of Staphylococcus spp. were tested, of which 24 were avian and 36 were clinical isolates. Our results indicated the prevalence of ermA over the ermC gene as opposed to the widely held opinion of the ermC gene being the most dominant resistance determinant gene. A multiplex-PCR assay was developed to detect the presence of ermA and ermC genes. Two pairs of primers, specific for the detection of ermA and ermC genes, were used in a multiplex-polymerase chain reaction (PCR) assay to yield amplified DNA products of 610 and 520 bp, respectively. Their digestion with restriction enzyme FokI that yielded a 477 bp and a 132 bp digestion product for ermA and a 333 bp and a 187 bp digestion product for ermC confirmed the authenticity of PCR products. The method could be used to amplify the ermA and ermC genes with as little as 5 pg of template DNA. The use of excess primers or the template DNA resulted in gene-specific amplification and no non-specific amplification was observed by changing the primer to primer or template to primer ratios. Furthermore, no amplification from erythromycin-sensitive S. aureus strain was observed. Using this assay, the poultry strains were found to contain either ermA alone (50%) or a combination of ermA (100%) and ermC (50%) both. The clinical strains contained either ermA (94.5%) or ermC (5.5%) but never both. The gene-specific internal probes were also used to verify the above findings and a high degree of correlation between the multiplex PCR and Southern hybridization data was observed.

An improved, simple, cost-effective and non-radioactive procedure for in-gel hybridization is described for the detection of signal in dried agarose gels. Large and small digoxigenin-labelled DNA and oligonucleotide probes hybridized efficiently and specifically with the complementary DNA sequences in the gel. The signal-to-noise ratios for the gels dried at 55 degrees C at 1 atmospheric pressure were 3-3.5-fold higher than the gels dried at 25 degrees C under vacuum. The method shows an increased sensitivity over currently available non-radioactive methods for in-gel hybridization. A single copy of a gene insert could be detected by the use of this procedure.

Aeromonas trota is recognized as an important enteropathogen, and its haemolysin (aerolysin) is purported to be one of the virulence factors. Rapid detection and identification of A. trota is important for early and specific diagnosis of the infectious diseases that it causes. Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a species-specific sequence of the aerA gene, which encodes the aerolysin of A. trota. A DNA fragment of 622 bp was amplified from both lysed cells and isolated DNA from A. trota. The identity of the amplified 622 bp fragment was confirmed by digestion with BamH I restriction endonuclease, which produced the predicted 557 and 65 bp fragments. The lower limit for detection of the aerA gene by PCR amplification was 10 pg of total DNA or 10-15 cells ml-1. Primer specificity for A. trota was determined by the PCR assay with cells of 55 strains of Aeromonas sppincluding all of the 14 currently recognized DNA hybridization groups. A strain of Aeromonas enteropelogenes that had been reclassified as A. trota was also PCR positive. The method described here can be used to detect aerolysin-producing A. trota (hybridization group 13) strains from environmental and clinical samples without the use of selective media or additional biochemical tests.

Methods used previously for the determination of aflatoxins B1, B2, G1 and G2, ochratoxins A and B, cyclopiazonic acid, zearalenone, sterigmatocystin, and moniliformin in maize were applied successfully to rice bran. However, recovery of deoxynivalenol and other trichothecene mycotoxins spiked into samples was lower than expected and no citrinin could be recovered. Forty samples of rice bran used in the animal feed industry were examined for the presence of 20 mycotoxins. The level of contamination of rice bran by mycotoxins was low. Aflatoxin B1 was present in 29 (72.5%) samples, usually together with related aflatoxins, up to a total of 28 micrograms/kg after allowing for recovery losses. Ochratoxin A at a level of 12 and 3 micrograms/kg was confirmed in two samples, while most samples also appeared to contain small concentrations of ochratoxin A. A chromatographic peak corresponding to cyclopiazonic acid occurred in several samples and one sample appeared to contain a low level of moniliformin. Positive confirmation of these last two mycotoxins was difficult and these findings must be regarded with caution. No other toxins were detected.

The influences of concentration of acrylamide, pH, temperature, duration of storage of encapsulated cells and presence of different metals and chelators on the ability of immobilized cells of a Rhodococcus sp. to degrade acrylamide were evaluated. Immobilized cells (3 g) rapidly degraded 64 and 128 mM acrylamide in 3 and 5 h, espectively, whereas free cells took more than 24 h to degrade 64 mM acrylamide. An acrylamide concentration of 128 mM inhibited the growth of the free cells. Immobilized bacteria were slow to degrade acrylamide at 10 °C. Less than 60% of acrylamide was degraded in 4 h. However, 100% of the compound was degraded in less than 3 h at 28 °C and 45 °C. The optimum pH for the degradation of acrylamide by encapsulated cells was pH 7.0. Less than 10% of acrylamide was degraded at pH 6.0, while ca. 60% of acrylamide was degraded at pH 8.0 and 8.5. Copper and nickel inhibited the degradation, suggesting the presence of sulfhydryl (-SH) groups in the active sites of the acrylamide degrading amidase. Iron enhanced the rates of degradation and chelators (EDTA and 1,10 phenanthroline) reduced the rates of degradation suggesting the involvement of iron in its active site(s) of the acrylamide-degrading-amidase. Immobilized cells could be stored up to 10 days without any detectable loss of acrylamide-degrading activity.

Analytical methods have been developed for the reliable detection and estimation of 22 mycotoxins in maize gluten and other maize products used in the animal feed industry. The mycotoxins are aflatoxins B1, B2, G1 and G2, ochratoxins A and B, citrinin, cyclopiazonic acid, zearalenone, sterigmatocystin, deoxynivalenol, nivalenol together with seven related trichothecene mycotoxins, fumonisins B1 and B2 and moniliformin. For most of the mycotoxins, recoveries obtained were 60% or greater and reproducibility data were better than +/- 40%. In general, the analysis of maize gluten proved more difficult than for other maize products. In total 40 samples of maize gluten, and 27 samples of other maize products were examined. Aflatoxins were not found above the reporting limit (1-5 micrograms/kg) in any sample while ochratoxin A was detected in only two samples of maize gluten at 2 micrograms/kg. No sterigmatocystin or cyclopiazonic acid were detected although the limits of detection for these toxins were poor. Twenty percent of maize gluten samples contained zearalenone at up to 500 micrograms/kg while all other maize products also contained this mycotoxin. Highest levels occurred in screenings and meal while the lowest amounts were in flaked maize and germ. Many samples contained a multi-toxin mixture of trichothecenes, fumonisins and moniliformin. The most highly contaminated samples of maize screenings and maize meal contained a mixture of zearalenone, deoxynivalenol, nivalenol, fumonisin B1 and B2, moniliformin, each in mg/kg amounts and lower amounts of other trichothecenes such as 15-acetoxy deoxynivalenol, HT-2 toxin and T-2 toxin. Fumonisins occurred in all except two gluten samples and occurred up to a level of 32 mg/kg in maize screenings, 13 mg/kg in maize meal and 8 mg/kg in maize germ.

This field study evaluated the effects of applying a combination of urease (UI) and nitrification inhibitors (NI) on ammonia (NH3) and nitrous oxide (N2O) emission from urine patches, using zeolite, single superphosphate (SSP) and urea fertilizer as a carrier. The trial was conducted on a Typic Haplustepts silt loam soil, near Lincoln, Canterbury, New Zealand during 2009-11. The treatments in 2009 included: a control (no urine or inhibitor), urine alone at 600 kg N ha(-1), and urine with either double inhibitor (DI) a mixture (1:7 ratio w/w) of UI (N-(n-butyl) thiophosphoric triamide (nBTPT - trade name Agrotain®) and NI, dicyandiamide (DCD) or DCD alone at 10 kg ha(-1) using zeolite and SSP as carriers. In 2010 trials, both zeolite and urea were used as carriers for DI and DCD. DI-zeolite and DI-urea were equally effective and reduced the average NH3 losses from applied urine over two years by 34.6% in autumn and 40% in spring respectively. The nBTPT in DI-SSP was decomposed by the free acid produced during its dissolution and therefore increased NH3 emission as does DCD alone. DCD consistently increased NH3 emissions by 39% and 15.6% in autumn and spring respectively. Spring application resulted in NH3-N losses of 16.9% as a percentage of the total N applied compared to 8.4% in autumn. Over the two years, the DI reduced N2O emissions by 53% in autumn and 46% in spring over urine alone treatment; the equivalent reductions for DCD were 42% and 39% for autumn and spring, respectively. These results suggest that applying DI in autumn and spring using zeolite or urea carrier five days prior to grazing has the most potential to reduce NH3 and N2O losses from specific urination event than using DCD alone; and therefore warrants further research to improve its longevity.

A constitutively expressed aliphatic amidase from a Rhodococcus sp. catalyzing acrylamide deamination was purified to electrophoretic homogeneity. The molecular weight of the native enzyme was estimated to be 360,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500. The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0. The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively. Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme. No labile sulfide was detected. The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+). However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents. The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp. strain R312.

A strain of Pseudomonas sp. capable of utilizing acrylamide as the sole source of carbon and nitrogen was tested for its ability to degrade an aliphatic amide mixture containing acrylamide, propionamide, butyramide, and methacrylamide. Batch cultures of Pseudomonas sp. degraded butyramide and propionamide at the rate of 115 μmol/h and at the rate of 127 μmol/h to butyric and propionic acids and ammonia in 48 h. Acrylamide and methacrylamide were degraded to acrylic acid and methacrylic acid in 72 h at the rate of 97 and 78 μmol/h, respectively. Faster rates of degradation could be obtained when the bacteria was immobilized

To determine whether there was an association between the size of the pancreas and the type of diabetes, ultrasonography of the pancreas was performed on 57 diabetic patients: 14 with Type 1 (insulin-dependent) diabetes, 10 insulin-treated and 33 tablet-treated patients with Type 2 (non-insulin-dependent) diabetes, and 19 non-diabetic subjects. The pancreas of patients with Type 1 diabetes was markedly smaller (p < 0.0001) than the pancreas in non-diabetic subjects. The pancreas of patients with Type 2 diabetes was more moderate in size: larger (p < 0.001) than that of Type 1 diabetic patients but smaller (p < 0.5) than the pancreas of the control group. Pancreatic size of patients with Type 2 diabetes was also related to basal insulin secretion with insulin-deficient patients (low or undetectable C-peptide) having smaller (p < 0.05) pancreases than those with normal insulin secretion. There was no difference in the size of the pancreas in the different treatment groups of Type 2 diabetic patients. Pancreatic size did not correlate with age, body mass index or the duration of diabetes. We conclude that the pancreas is a smaller organ in patients with diabetes mellitus and that the decrement in size is maximal in insulin-dependent/insulin-deficient subjects. Ultrasonography, therefore, can potentially serve to discriminate between the different types of diabetes.

Two bacterial isolates capable of utilizing acrylamide as the sole source of carbon and nitrogen were isolated from herbicide-contaminated soil samples and identified as Pseudomonas sp. and Xanthomonas maltophilia. Batch cultures of Pseudomonas sp. and X. maltophilia completely degraded 62.8 mM acrylamide to acrylic acid and ammonia in 24 and 48 h, respectively. Pseudomonas sp. And X. maltophilia, when immobilized in calcium alginate, markedly increased the rate of degradation of acrylamide over batch cultures. Cells of the isolates immobilized in calcium alginate degraded acrylamide to acrylic acid and ammonia in less than 6 h. Initial metabolism of acrylamide by immobilized cells of Pseudomonas sp. followed by inoculation with nonimmobilized cells after 6 h totally removed acrylamide and its metabolites in 72 h. A similar procedure with X. maltophilia resulted in the total metabolism of acrylamide in 96 h. An inducible, intracellular amidase was responsible for the hydrolysis of acrylamide to acrylic acid and ammonia. The specific activity of Pseudomonas sp. amidase was higher than the specific activity of X. maltophilia amidase.Key words: acrylamide, biodegradation, immobilization.

A mixed microbial culture was isolated from an environment contaminated with organic cyanides and polychlorinated biphenyls (PCBs). This mixed culture could utilize acetonitrile as the sole source of carbon and nitrogen, and biphenyl as the sole source of carbon. This mixed culture also utilized nitriles, their respective amides, and several PCBs as growth substrates. Studies involving the radiolabeled compounds indicated that nearly 70% of [14C]acetonitrile and 51% of [14C]biphenyl were recovered as 14CO2. The end products resulted during the degradation of acetonitrile and biphenyl were identified as ammonia and benzoate, respectively. It was also observed that the mixed culture that can degrade biphenyl loses its capability when transferred repeatedly into a medium supplemented with acetonitrile as growth substrate. The present study suggests that the application of mixed rather than pure microbial culture is more effective in bioremediation of toxic chemicals.

The efficiency of an analytical method for the determination of aflatoxins in groundnut meal has been statistically examined. The procedure consists of a phenyl bonded phase (PH) clean-up of an acetone: water (85∶15) extract followed by HPLC quantification. Average recoveries from spiked groundnut meal extracts were calculated to be 101.3% and 101.8%, with limits of detection of 7.4 and 2.62 μg/kg for aflatoxins-B1 and-B2 respectively. Higher recoveries of aflatoxin-B1 from naturally contaminated samples were recorded using the proposed procedure than those recorded using the official AOAC (CB) method although the precisions of the two methods were not found to differ at the 5% significance level. Similar recoveries of aflatoxin-B2 were recorded for both methods but the proposed procedure was found to be more precise. The proposed PH-HPLC method was far less time consuming and more economical on solvents than the CB procedure.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.

A bacterium capable of utilizing either acetonitrile as the sole source of carbon and nitrogen or biphenyl as the sole source of carbon was isolated from soil and identified as Pseudomonas aeruginosa. The bacterium also utilized other nitriles, amides, and polychlorinated biphenyls (PCBs) as growth substrates. Acetonitrile- or biphenyl-grown cells oxidized these substrates without a lag. In studies with [14C]acetonitrile, nearly 74% of the carbon was recovered as 14CO2 and 8% was associated with the biomass. In studies with [14C]biphenyl, nearly 68% of the carbon was recovered as 14CO2 and nearly 6% was associated with the biomass. Although higher concentrations of acetonitrile as the sole sources of nitrogen inhibited the rates of [14C]biphenyl mineralization, lower concentrations (0.05%, w/v) gave a 77% stimulation in 14CO2 recovery. Pseudomonas aeruginosa metabolized acetonitrile to ammonia and acetic acid and biphenyl to benzoic acid. The bacterium also simultaneously utilized biphenyl as the sole carbon source and acetonitrile as the sole nitrogen source. However, biphenyl utilization increased only after the depletion of acetronitrile. Metabolites of the mixed substrate were ammonia and benzoic acid, which completely disappeared in the later stages of incubation. Nitrile hydratase and amidase were resposible for the transformation of acetonitrile to acetic acid and ammonia. Key words: biodegradation, acetonitrile, biphenyl, Pseudomonas aeruginosa.

Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified as Chromobacterium sp. and Pseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation and P. aeruginosa grew slightly faster than Chromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (14C)acetonitrile indicated 57% of acetonitrile was degraded by Pseudomonas aeruginosa as compared to 43% by Chromobacterium. The isolates utilized different nitrile compounds as carbon substrates.

In-gel hybridization with digoxigenin (DIG)-labeled probes has been shown to detect complementary DNA sequences in dried agarose gels. Gels dried at room temperature or at 55 degrees C in an oven do not show detectable changes in the sensitivity of detection. However, gels dried under vacuum seem to lose the sensitivity by approx 8- to 10-fold. In-gel hybridization after blotting high molecular weight T7 DNA (40 kbp) onto nylon membranes has been demonstrated to transfer the DNA to the membrane inefficiently. In-gel hybridization of DIG-labeled probes with the complementary DNA sequences has been determined to detect as little as 0.05 ng of 40-kbp T7 DNA and single copies of the erythromycin resistance marker gene ermA. Nonradioactive in-gel hybridization provides better quantitation of nucleic acids than filter hybridization in Southern and Northern blot analysis.

A gram-negative, rod-shaped bacterium capable of utilizing L-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed L-asparaginase was detected and it deaminated L-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM L-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on L-fructose, D-galactose, saccharose, or maltose, and in cells cultured on L-asparagine as the sole nitrogen source. The pH and temperature optimum of L-asparaginase was 8.5 and 37-42 degrees C, respectively. The half-life of the enzyme at 30 degrees C and 37 degrees C was 10 and 8 h, respectively.

A technique referred to as 'linked-column immunoassay clean-up', was developed for the simultaneous determination of aflatoxins and ochratoxin A in a range of dry cereal-based pet foods and wild bird food. In addition a method also based on clean-up using immunoaffinity columns was used for the determination of fumonisin mycotoxins in samples known or suspected to contain maize. One hundred samples of pet foods consisting of 35 samples of domestic bird seeds and 15 samples of wild bird food were examined for aflatoxins B1, B2, G1 and G2 and ochratoxin A. Twenty samples of these samples were also examined for fumonisins B1 and B2. Limits of detection were about 0.5 micrograms/kg for each aflatoxin and ochratoxin A and 3 and 8 micrograms/kg for fumonisins B1 and B2 respectively. Eighty-four percent of the samples contained no measurable concentrations of mycotoxins. A low level of aflatoxin B1 was found in a sample of cat food and a concentration of 370 micrograms/kg aflatoxin B1 in one sample of peanuts marketed for wild birds. Ochratoxin A was detected in 10% of samples but in low concentrations, the highest of 7 micrograms/kg occurring in a sample of bird food. Fumonisins were found in 30% of the 20 samples tested with a maximum of 750 micrograms/kg total fumonisins being found in a sample of cat food. Five samples each of dog and cat foods were examined for mould count and fungal species as received and after storage under controlled simulated damp conditions. Mould counts in all 10 samples examined were very low when received. The samples in which moisture content had been increased contained visible mould after storage. Penicillium, Eurotium and Aspergillus were the predominant species. However, no ochratoxin A or aflatoxins were detected in these spoiled samples.

An amidase capable of degrading acrylamide and aliphatic amides was purified to apparent homogeneity from Klebsiella pneumoniae NCTR 1. The enzyme is a monomer with an apparent molecular weight of 62,000. The pH and temperature optima of the enzyme were 7.0 and 65 degrees C, respectively. The purified amidase contained 11 5,5-dithiobis(2-nitrobenzoate) (DTNB)-titratable sulfhydryl (SH) groups. In the native enzyme 1.0 SH group readily reacted with DTNB with no detectable loss of activity. Titration of the next 3.0 SH groups with DTNB resulted in a loss of activity of more than 70%. The remaining seven inaccessible SH groups could be titrated only in the presence of 8 M guanidine hydrochloride. Titration of SH groups was strongly inhibited by carboxymethylation and KMnO4, suggesting the presence of SH groups at the active site(s). Inductively coupled plasma-atomic emission spectrometry analysis indicated that the native amidase contains 0.33 mol of cobalt and 0.33 mol of iron per mol of the native enzyme. Polyclonal antiserum against K. pneumoniae amidase was raised in rabbits, and immunochemical comparisons were made with amidases from Rhodococcus sp., Mycobacterium smegmatis, Pseudomonas chlororaphis B23, and Methylophilus methylotrophus. The antiserum immunoprecipitated and immunoreacted with the amidases of K. pneumoniae and P. chlororaphis B23. The antiserum failed to immunoreact or immunoprecipitate with other amidases.

The reaction of methanol to olefins was studied over the following zeolite type materials: ZSM-5, SAPO-34 and chabazite (mineral). A strongly shape-selective catalyst (8-ring channels) is needed to suppress the formation of aromatics, and thereby get the highest olefin selectivity. Olefin yields slightly above 80 mol% at close to 100% conversion could be obtained with the H-SAPO-34 catalyst. Fast deactivation is the main limitation in the MTO reaction. The deactivation rate, which was much smaller in the case of H-ZSM-5 catalysts, appears partly structure dependent, but a large difference in deactivation rate between the isostructural catalysts chabazite and SAPO-34 was observed. Temperature programmed desorption of ammonia showed the chabazite and ZSM-5 to contain acid sites with higher acidity than is found in SAPO-34. The acid site content varies as follows: H-chabazite > SAPO-34 > H-ZSM-5.

A bacterium capable of utilizing acetonitrile (methyl cyanide) as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas aeruginosa. This bacterium could also utilize and oxidize numerous lower-mol-wt nitrile compounds and their corresponding amides as growth substrates. A metabolite of acetonitrile in the culture medium was determined to be ammonia. The accumulation of ammonia in the culture medium was proportional to the concentration of the substrate and the inoculum. Cell extracts of the bacterium contained activities corresponding to nitrile aminohydrolase (E C 3.5.5.1) and amidase (E C 3.5.1.4), which regulate the degradation of acetonitrile. Both enzymes were inducible and hydrolyzed a wide range of substrates, and it was determined that the specific activity of amidase was far greater than the activity of nitrile aminohydrolase.

A gram-negative rod-shaped bacterium capable of utilizing acrylonitrile as the sole source of nitrogen was isolated from industrial sewage and identified as Klebsiella pneumoniae. The isolate was capable of utilizing aliphatic nitriles containing 1 to 5 carbon atoms or benzonitrile as the sole source of nitrogen and either acetamide or propionamide as the sole source of both carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae was capable of hydrolyzing 6.15 mmol of acrylonitrile to 5.15 mmol of acrylamide within 24 h. The acrylamide was hydrolyzed to 1.0 mmol of acrylic acid within 72 h. Another metabolite of acrylonitrile metabolism was ammonia, which reached a maximum concentration of 3.69 mM within 48 h. Nitrile hydratase and amidase, the two hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds, were induced by acrylonitrile. The optimum temperature for nitrile hydratase activity was 55 degrees C and that for amidase was 40 degrees C; both enzymes had pH optima of 8.0.

A bacterium capable of utilizing high concentrations of acetonitrile as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas putida. This bacterium could also utilize butyronitrile, glutaronitrile, isobutyronitrile, methacrylonitrile, propionitrile, succinonitrile, valeronitrile, and some of their corresponding amides, such as acetamide, butyramide, isobutyramide, methacrylamide, propionamide, and succinamide as growth substrates. Acetonitrile-grown cells oxidized acetonitrile with a K m of 40.61 mM. Mass balance studies with [ 14 C]acetonitrile indicated that nearly 66% of carbon of acetonitrile was released as 14 CO 2 and 14% was associated with the biomass. Metabolites of acetonitrile in the culture medium were acetic acid and ammonia. The acetate formed in the early stages of growth completely disappeared in the later stages. Cell extracts of acetonitrile-grown cells contained activities corresponding to nitrile hydratase and amidase, which mediate the breakdown of actonitrile into acetic acid and ammonia. Both enzymes were intracellular and inducible and hydrolyzed a wide range of substrates. The specific activity of amidase was at least 150-fold higher than the activity of the enzyme nitrile hydratase.