Isolation of a raw starch-binding fragment from barley alpha-amylase

Barley -amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with -cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel -sheets in domain C and the ₇-and ₈-helices of the (/)₈ domain.     

Process optimization for the production of sugar for the bioethanol industry from sorghum,a non-conventional source of starch

A commercial preparation of -amylase, Biotempase, obtained from Biocon India Pvt. Ltd., and crude glucoamylase produced from Aspergillus sp. NA21 were used to hydrolyse sorghum powder, a non-conventional starchy substrate. Among various concentrations of starch (15–35%, dry weight/volume) tried for maximum liquefaction; slurry made with 25% substrate concentration proved optimal. An economical process of liquefaction was carried out using steam under pressure (0.2–0.3 bar, 104–105 °C) to liquefy a 25% slurry in just 45 min, contrary to a slower process carried out at 95 °C in a water bath. For liquefaction of starch a pH of 5.0 proved to be optimum. The dose of Biotempase as prescribed by the supplier could be reduced by 33% achieving the same degree of liquefaction, by addition of CaCl₂ to the starch slurry at the concentration of 200 mg/l. The conditions for the saccharification of liquefied starch were optimized to be 45 °C and pH 5.0, producing 90% saccharification in 24 h. Supplementation of divalent ions Ca²⁺, Mg²⁺ and Zn²⁺ in the process of saccharification showed no effect. Finally glucose was found to be the main hydrolysis product in the saccharification of sorghum starch.     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

The effects of interleukin-1 therapy on peripheral blood granulocyte function in humans

During a phase I trial of interleukin-1 (IL-1) in patients with ovarian carcinomas, the effects of this treatment on blood granulocyte respiratory burst and locomotive responses were examined. Differences in baseline granulocyte function in patients as well as dose-related effects of IL-1 treatment were observed. Patients enrolled early in the trial (low-dose patients) had significantly lower locomotive responses before treatment than their paired controls; these low responses normalized after 5 days of continuous-infusion IL-1 treatment. Patients enrolled later (high-dose patients) had normal locomotive responses before treatment and IL-1 treatment was associated with suppression of responses to selected stimuli at the end of treatment. Pretreatment respiratory burst responses in both low-and high-dose patient groups were essentially normal, but the rates of granulocyte H₂O₂ production following phorbol myristate acetate stimulation became significantly less than control values at the end of treatment. In vitro exposure of either patient or control cells to 150 U/ml IL-1 did not alter their locomotive or respiratory burst responses, suggesting the observed in vivo effects were not mediated directly by IL-1. Treatment with IL-1 is associated with changes in ex vivo granulocyte function that are related to the IL-1 dose. Treatment with low doses of IL-1 may provide a means of normalizing abnormal polymorphonuclear leukocyte function in some patients with ovarian malignancies.     

Biodegradation of a polyvinyl alcohol-starch blend plastic film

Attempts were made to elucidate the degradation mechanism of a polyvinyl alcohol (PVA)-starch blend plastic. A part of the starch fraction of this plastic was dissolved into an aqueous phase in a control test. Treatment with a PVA-degrading bacterium or enzyme gave a maximal weight loss of approximately 70% and film breakage occurred. Since this plastic contains 40% PVA, it is apparent that not only the PVA fraction but also a considerable portion of the starch fraction was lost from the film by treatment with the PVA-degrading enzyme. As the PVA-degrading bacterium and enzyme used here showed no starch-degrading activity, loss of the starch fraction seems to depend on its dissolution with degradation of the PVA fraction. These experimental results indicated that the degradation of the PVA fraction is an important requisite for complete degradation or decomposition of this plastic film.     

Parkinson’s disease: clinical aspects

Parkinsonism is a clinical syndrome characterized by akinesia, muscular rigidity, and resting tremor. The most frequent cause of parkinsonism is Parkinsons disease (PD). Progressive loss of substantia nigra neurons together with the occurrence of Lewy bodies are considered essential neuropathological features of PD. Recent neuropathological studies suggest that nigral degeneration is only part of a more extended brain degeneration that starts in the medulla oblongata and then spreads to the mesencephalon and cerebral cortex. Correspondingly, the clinical symptoms occurring in PD go far beyond parkinsonism. Depending on the disease stage, autonomic dysfunction, olfactory disturbances, depression, and dementia are frequently encountered in PD. These neuropathological and clinical observations have major implications for future research in PD. In particular, the analysis of the properties that the neuronal cell types involved in PD have in common and that might make them susceptible to degeneration is essential.     

The restrictive proteolysis of α-fodrin to a 120 kDa fragment is not catalyzed by calpains during thymic apoptosis

The -subunit (240 kDa) of fodrin was found to be digested selectively to a 120 kDa fragment during apoptosis of rat thymocytes in vivo and in vitro. This fragment was detected by an antibody (Ab) against full length -fodrin, but not by the anti-N-terminal sequence (GMMPR) of the -calpain-generated 150 kDa fragment Ab or the anti-PEST sequence of -fodrin Ab. On the other hand, basal levels of the 150 kDa fragment were constantly recognized by these three antibodies during apoptosis. The production of the 120 kDa fragment during apoptosis was not affected by the addition of calpain inhibitors such as Ac-LLLnal and E-64d, despite inhibition of the generation of the 150 kDa fragment. When x-irradiated thymocytes were incubated in the presence of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), both production of the 120 kDa fragment and apoptosis were suppressed. Purified - and m-calpain did not catalyze the formation of the 120 kDa fragment from purified -fodrin in vitro. These results suggest that a protease different from calpains is involved in the major process of -fodrin proteolysis to a 120 kDa fragment during thymic apoptosis.     

Histamine in immunotherapy of advanced melanoma: a pilot study

Sixteen patients with advanced metastatic malignant melanoma were treated with a high-dose infusion of interleukin-2 (IL-2; 18×10⁶ IU/m^–2 day^–1) together with daily subcutaneous (s.c.) injections of interferon (IFN; 3×10⁶ U/m^–2 day^–1) in 5-day cycles. Nine of these patients were given histamine (1 mg s.c.) twice daily during treatment with IL-2 and IFN. In the seven patients who did not receive histamine, one partial response (that is a reduction of more than 50% in the total tumour burden) was observed in a patient with skin and lymph node melanoma. In the eight histamine-treated patients evaluable for response, four partial responses were observed. Two other patients showed regression at one site of metastasis but tumours remained unchanged at other sites. Two histamine-treated patients showed complete resolution of extensive liver metastasis. Sites of response in histamine-treated patients also included the subcutis, lymph nodes, skeleton, spleen and muscle. Lung melanoma did not respond to histamine/IL-2/IFN. Three patients with lung tumours responded with significant (more than 50%) reduction of the volume of soft-tissue tumours, suggesting that the response to histamine may be organotropic. Survival was significantly prolonged in patients receiving histamine. Our data suggest that treatment with histamine may improve the antitumour efficacy of immunotherapy in metastatic melanoma.     

α-Amylase production by Bacillus subtilis and B. amyloliquefaciens in different PEG solutions

-Amylase production by Bacillus subtilis and Bacillus amyloliquefaciens was investigated in polyethyleneglycol (PEG)-containing growth medium. Five different molecular weight PEGs (600, 3000, 4000, 8000 and 20,000) were used. Enzyme production with B. subtilis increased 21% in medium containing 5% PEG 3000, but enzyme production with B. amyloliquefaciens increased 31% in medium containing 5% PEG 600 and 21% in medium containing 2% PEG 8000.     

Expression of receptor protein tyrosine phosphatase agr; mRNA in human prostate cancer cell lines

Receptor protein tyrosine phosphatase (RPTP) is transmembrane protein phosphatases, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTP mRNA in several normal human tissues. We further investigated the regulation of expression of RPTP mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5-dihydrotestosterone (DHT) down-regulates RPTP mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTP mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTP as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTP mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTP is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.     

Mutational analysis of human tumor necrosis factor-α

To understand the structure-function relationship of human tumor necrosis factor- (TNF-), mutational analysis was carried out on the lower regions (regions 1–6) of the molecule. The muteins were prepared as a soluble form by using a chaperonin co-expression system and the cytotoxic activities of the purified muteins were evaluated on TNF-sensitive murine fibrosarcoma L929 cells. Three regions (regions 1, 2 & 4) were found where mutations significantly influenced the bioactivity. In region 1 (residues 1–10), the number of deleted residues and the positioning of positive charges are important to achieve a maximum activity and in region 4 (residues 84–88), introduction of charged residues in one of the positions 86–88 significantly increased the cytotoxic activity. On the other hand, any mutation introduced in region 2 (residues 37–41) had a deleterious effect. The present study provides a structural basis for the design of highly potent TNF- as a therapeutic agent.Revisions requested 18 October 2004; Revisions received 22 November 2004     

Multiple Actions of the Human Immunodeficiency Virus Type-1 Tat Protein on Microglial Cell Functions

The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1-associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

Peptidylglycine α-amidating monooxygenase (PAM) immunoreactivity and messenger RNA in human pituitary and increased expression in pituitary tumours

Bioactivity of many peptides depends upon post-translational -amidation of inactive precursors by two enzyme activities known collectively as peptidylglycine -amidating monooxygenase (PAM). PAM enzymes are particularly abundant in the pituitary. The distribution of PAM immunoreactivity and messenger ribonucleic acid (mRNA) in the adult human pituitary and in pituitary tumours was investigated by use of immunocytochemistry and in situ hybridisation. Immunoreactivity was present in numerous cells of the anterior lobe: staining was intense in a proportion of gonadotrophs and folliculo-stellate cells, but weaker in the majority of somatotrophs and lactotrophs, a few corticotrophs and occasional thyrotrophs. PAM staining was also present in nerves, pituicytes and some endocrine cells within the posterior lobe (the human intermediate zone). Forty pituitary tumours of various types were immunoreactive for PAM; more intensely and uniformly stained than normal anterior lobe. In situ hybridisation with digoxigenin-labelled probes demonstrated intense labelling for PAM mRNA in numerous cells in normal anterior pituitary and in tumours. Many regulatory peptides that require amidation for activity, potential targets for PAM, are present in the pituitary. Many tumour growth factors also require amidation and PAM may regulate these mitogenic peptides in tumours.     

Effects of Schisandrin B and α-tocopherol on lipid peroxidation, in vitro and in vivo

Effects of Schisandrin B (Sch B) and -tocopherol (-TOC) on ferric chloride (Fe³⁺) induced oxidation of erythrocyte membrane lipids in vitro and carbon tetrachloride (CCl₄) induced lipid peroxidation in vivo were examined. While -TOC could produce prooxidant and antioxidant effect on Fe³⁺-induced lipid peroxidation, Sch B only inhibited the peroxidation reaction. Pretreatment with -TOC (3 mmol/kg/day × 3) did not protect against CCl₄-induced lipid peroxidation and hepatocellular damage in mice, whereas Sch B pretreatment (0.3 mmol/3.0 mmol/kg/day × 3) produced a dose-dependent protective effect on the CCl₄-induced hepatotoxicity. The ensemble of results suggests that the ability of Sch B to inhibit lipid peroxidation, while in the absence of pro-oxidant activity, may at least in part contribute to its hepatoprotective action.Abbreviations ALT alanine aminotransferase - CCl₄ carbon tetrachloride - Fe³⁺ ferric chloride - MDA malondialdehyde - Sch B Schisandrin B - TBA 2-thiobarbituric acid - TBARS thiobarbituric acid reactive substances - -TOC dl--tocopherol     

Structure of α-α-Hairpins with Short Connections in Globular Proteins

The analysis of conformations of more than 100 --hairpins with closely packed helical segments and connections up to four amino acid residues in length was carried out. Five types of the connections were revealed, and their and values on the Ramachandran map were found. Each type of --hairpins was shown to have a unique sequence pattern for hydrophobic and hydrophilic residues.     

Lipopolysaccharide Induction of MARCKS-Related Protein and Cytokine Secretion are Differentially Impaired in Microglia from LPS-nonresponsive (C3H/HeJ) Mice

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (100 ng/ml) than in normal (C3H/OuJ) microglia (10 ng/ml). By contrast, TNF production was not significantly increased in C3H/HeJ microglia even at 1 g LPS/ml. Microglia expressed PKC isoforms , , , and (but not and ); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lps^d mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.     

Application of solid-phase Ellman's reagent for preparation of disulfide-paired isomers of α-conotoxin SI

Disulfide-paired regioisomers of -conotoxin SIcan be accessed by orthogonal schemes using thecombination of S-9H-xanthen-9-yl (S-Xan) and S-acetamidomethyl (S-Acm)groups for cysteine protection. Following solid-phaseassemblies of the linear precursors, the peptides werecleaved from the solid support concurrent with removalof S-Xan protecting groups. The first disulfidebridges were formed in solution, using either thetraditional DMSO method or a recently introducedapproach featuring a solid-phase Ellman's reagent. The second disulfide bridges were oxidized by threedifferent methods: reactions mediated by thalliumtrifluoroacetate, iodine, or a sulfoxide/silylmixture. In general, yields depended primarily onwhich regioisomer was the target, rather than thespecific chemistry used for either disulfide-formingstep. However, the selectivities towards the desiredregio-isomers were reproducibly better using thesolid-phase Ellman's reagent, by comparison to theDMSO method. In the most favorable cases, completeselectivity was achieved, while even in cases wherethe net results using DMSO gave considerablescrambling, the corresponding experiments with thesolid-phase Ellman's reagent were more selective. Possible reasons why choice of oxidation method forthe first step affects the selectivity at the secondstep are discussed.