Strain improvement for tannase production from co-culture of Aspergillus foetidus and Rhizopus oryzae

Spores from the co-culture of Aspergillus foetidus and Rhizopus oryzae were subjected to UV, heat and NTG (3-nitro,5-methylguanidine) mutagenesis. A few colonies were screened from the selected media for tannase study. Amongst all, the best mutant isolated from the heat treatment (60 degrees C for 60 min) was SCPR 337. The maximum yield of gallic acid and tannase in case of mutant strain was 95.2% and 53.6 U/ml with an incubation period of 30 h as compared to wild strain where the incubation period was 48 h with an enzyme activity of 44.2 U/ml and gallic acid yield of 94%, respectively. The mutant was sensitive to tetracycline and was also an over-producer of protease and amylase.     

Energetics of gating at the apo–acetylcholine receptor transmitter binding site

Acetylcholine receptor channels switch between conformations that have a low versus high affinity for the transmitter and conductance for ions (R↔R*; gating). The forward isomerization, which begins at the transmitter binding sites and propagates ∼50 Å to the narrow region of the pore, occurs by approximately the same sequence of molecular events with or without agonists present at the binding sites. To pinpoint the forces that govern the R versus R* agonist affinity ratio, we measured single-channel activation parameters for apo-receptors having combinations of mutations of 10 transmitter binding site residues in the α (Y93, G147, W149, G153, Y190, C192, and Y198), ε (W55 and P121), or δ (W57) subunit. Gating energy changes were largest for the tryptophan residues. The αW149 energy changes were coupled with those of the other aromatic amino acids. Mutating the aromatic residues to Phe reduces the R/R* equilibrium dissociation constant ratio, with αY190 and αW149 being the most sensitive positions. Most of the mutations eliminated long-lived spontaneous openings. The results provide a foundation for understanding how ligands trigger protein conformational change.     

Effect of growth temperature,induction, and molecular chaperones on the solubilization of over-expressed cellobiose phosphorylase from <Emphasis Type="Italic">Cellvibrio Gilvus</Emphasis> under <Emphasis Type="Italic">in vivo</Emphasis> conditions

In vivo folding of many proteins can be facilitated by growth temperature, extent of induction, and molecular chaperones, which prevent over-expressed protein from being trapped into insoluble inclusion bodies. In the present report, we describe the role of molecular chaperones and growth temperature on the solubilization of overexpressed Cellobiose Phosphorylase (CBP) in Escherichia coli. The growth of host at low temperature enhanced enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yielded higher enzyme in soluble fraction. The synergistic effect of low temperature and induction on the prevention of inclusion bodies was also evident from our results. In addition, co-expression of the target gene with two types of molecular chaperones (GroESL and KODHsp) was also attempted. However, none of these chaperones enhanced the solubilization under in vivo conditions. Nevertheless, effective role of low growth temperature coupled with low level of induction appeared to be an attractive feature for producing recombinant protein.     

Thermostability of acid phosphatase inSelinum vaginatum Clarke andAcer caesium Wall. grown at low and high altitudes

Acid phosphatase isolated from low altitude grown plants of two high altitude plant species,Selinum vaginatum Clarke andAcer caesium Wall, displayed higher thermostability than that from plants of the same species grown at high altitude. The isozyme composition, however, remained unchanged inSelinum vaginatum. InA. caesium, one of four isozymes, was thermolabile in the samples from high altitude and was lost after 10 min heating of the extracts at 60 °c. In the samples from low altitude, this isozyme was not detected and a band with slightly lower Rf value was present which was thermostable. The described changes in the thermal properties of acid phosphatase reflect an adaptive step towards high temperature acclimation at low altitude.     

Intraoperative cytology (squash smear) in neurosurgical practice – pitfalls in diagnosis experience based on 3057 samples from a single institution

OBJECTIVES: The smear technique is challenging for a neuropathologist where rapid and accurate diagnosis is to be given on small biopsies. The present study, a large retrospective analysis of squash smears in neurosurgical practice, was conducted to assess the usefulness, accuracy and the diagnostic pitfalls of smear diagnosis. METHODS: The authors analysed 3057 central nervous system (CNS) lesions sent for intraoperative cytology (IC) during the years 1988-2005. The stain used was 1% alcoholic toluidine blue. The smear diagnosis was compared with the histological diagnosis to evaluate the diagnostic accuracy. RESULTS: Diagnostic accuracy irrespective of lesion and site ranged from 83.0% to 86.0% per year (mean=85%). The highest rate of correlation among common brain tumours was noted in schwannoma (96.6%) and pituitary adenoma (92.2%), followed by meningiomas (88.9%), astrocytomas (88.4%), chordomas (86.4%) and neurocytomas (86.9%). Infections as a whole contributed 380 cases. The most common infection was tuberculosis. CONCLUSION: This is the largest series reported from India to the best of our knowledge. Squash smear technique is a very reliable and rapid method of intraoperative diagnosis. Knowledge of clinical and neuroimaging details helps the experienced neuropathologist to improve the diagnostic accuracy.     

2-difluoromethyloestrone 3-O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).     

Ultrasonographic evaluation of uterine involution and postpartum follicular dynamics in French jennies (Equus asinus)

Uterine involution and follicular dynamics during postpartum period were studied ultrasonographically in French jennies. For the study of uterine involution in postpartum jennies (n = 6, Group S), sonographic measurements of different parts of the uterus and endometrium were made at three-day interval, starting from the day of foaling and continued up to 33 days postpartum. Uterine dimensions were also recorded in non-pregnant jennies (n = 3, Group C) throughout a cycle and compared with the dimensions of Group S jennies observed on the day of complete involution. Follicular dynamics of first and second postpartum ovulatory cycles were studied and compared with that of the single estrous cycle of Group C jennies. Jugular venous blood samples of Group S jennies were collected at weekly intervals for 49 days, commencing at the appearance of first preovulatory follicle, to support the sonographic findings. The average involution period was 22.5 +/- 1.7 days. However, it was significantly delayed (P < 0.05) in jennies which came into first postpartum ovulatory heat within Day 9 than those who came later (25.0 +/- 1.0 versus 20.0 +/- 1.0). The endometrial layer was not discernible beyond Day 15 postpartum and thus was found to be unreliable index of uterine involution. The follicular growth rate (mm per day) and diameter (mm) of preovulatory follicle in postpartum jennies were similar to that in normal cycling jennies (P > 0.05). The first and second ovulations occurred at 14.6 +/- 0.8 and 39.0 +/- 0.8 days postpartum in Group S jennies. All the corpora lutea, either echogenic or centrally non-echogenic were functionally similar and had similar life span (P > 0.05). In conclusion, the postpartum reproductive events related to uterine involution and ovarian cyclicity apparently resemble that of mares.     

Phosphine-containing HYNIC derivatives as potential bifunctional chelators for (99m)Tc-labeling of small biomolecules

Two prototype phosphine-containing HYNIC chelators, HYNIC-Kp-DPPB and HYNIC-Ko-DPPB (HYNIC = 6-hydrazinonicotinamide; K = lysine; and DPPB = diphenylphosphine-benzoic acid), have been synthesized and characterized by NMR ((1)H, (13)C, and (31)P) and LC-MS. Macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], were prepared by reacting the phosphine-containing HYNIC chelator with (99m)TcO(4)(-) in the presence of excess tricine and stannous chloride. Results from this study clearly demonstrated that both HYNIC-Kp-DPPB and HYNIC-Ko-DPPB are able to form highly stable macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], when tricine is used as the coligand. Radio-HPLC data suggest that the complex [(99m)Tc(HYNIC-Kp-DPPB)(tricine)] exists as only one detectable isomer in solution while the complex [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] has three isomers. It was also found that three isomers of [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] interconvert at elevated temperatures, suggesting that the presence of these isomers might be due conformational changes in the macrocyclic Tc chelate. The LC-MS data for both macrocyclic (99m)Tc complexes are completely consistent with the proposed composition. The phosphine-containing HYNIC chelators described in this study may have the potential as bifunctional chelators for (99m)Tc labeling of small biomolecules.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.