Enzymatic removal of zeins from the surface of maize starch granules

Alcohol-extractable, hydrophobic zein proteins contaminate starch granule surfaces and can be removed by enzymatic digestion with thermolysin. The goal of this research was to find practical alternatives to thermolysin that might be used during the corn wet-milling process. All of the commercial thermostable alkaline proteases studied (SP 709, Neutrase, and Spezyme FAN) removed the zein proteins from various types of cornstarch, as demonstrated by the lack of protein bands below 30 kDa under the reducing conditions of SDS-PAGE gel. Each enzyme removed the zein proteins as effectively as thermolysin removed them. However, the removal of the zein protein did not reduce the quantity of free fatty acids associated with the starch. Journal of Industrial Microbiology & Biotechnology (2000) 24, 71–74. Received 27 May 1999/ Accepted in revised form 01 October 1999     

Chronic hypoxia attenuates resting and exercise-induced VEGF, flt-1, and flk-1 mRNA levels in skeletal muscle.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic mitogen. However, chronic hypoxia is generally not found to increase mammalian skeletal muscle capillarity. We sought to determine the effect of chronic hypoxia (8 wk, inspired O2 fraction = 0.12) on skeletal muscle gene expression of VEGF, its receptors (flt-1 and flk-1), basic fibroblast growth factor, and transforming growth factor-beta1. Wistar rats were exposed to chronic hypoxia (n = 12) or room air (n = 12). After the exposure period, six animals from each group were subjected to a single 1-h treadmill exercise bout (18 m/min on a 10 degrees incline) in room air while the remaining six animals served as rest controls. Morphological analysis revealed that chronic hypoxia did not increase skeletal muscle capillarity. Northern blot analyses showed that chronic hypoxia decreased resting VEGF, flt-1, and flk-1 mRNA by 23, 68, and 42%, respectively (P < 0.05). The VEGF mRNA response to exercise was also decreased (4.1- and 2.7-fold increase in room air and chronic hypoxia, respectively, P < 0.05). In contrast, neither transforming growth factor-beta1 nor basic fibroblast growth factor mRNA was significantly altered by chronic hypoxia. In conclusion, prolonged exposure to hypoxia attenuated gene expression of VEGF and its receptors flt-1 and flk-1 in rat gastrocnemius muscle. These findings may provide an explanation for the lack of mammalian skeletal muscle angiogenesis that is observed after chronic hypoxia.     

Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.     

In vivo neutralization of TNF-alpha promotes humoral autoimmunity by preventing the induction of CTL.

Neutralization of TNF-alpha in humans with rheumatoid arthritis or Crohn's disease has been associated with the development of humoral autoimmunity. To determine the effect of TNF-alpha neutralization on cell-mediated and humoral-mediated responses, we administered anti-TNF-alpha mAb to mice undergoing acute graft-vs-host disease (GVHD) using the parent-into-F(1) model. In vivo neutralization of TNF-alpha blocked the lymphocytopenic features characteristic of acute GVHD and induced a lupus-like chronic GVHD phenotype (lymphoproliferation and autoantibody production). These effects resulted from complete inhibition of detectable antihost CTL activity and required the presence of anti-TNF-alpha mAb for the first 4 days after parental cell transfer, indicating that TNF-alpha plays a critical role in the induction of CTL. Moreover, an in vivo blockade of TNF-alpha preferentially inhibited the production of IFN-gamma and blocked IFN-gamma-dependent up-regulation of Fas; however, cytokines such as IL-10, IL-6, or IL-4 were not inhibited. These results suggest that a therapeutic TNF-alpha blockade may promote humoral autoimmunity by selectively inhibiting the induction of a CTL response that would normally suppress autoreactive B cells.     

Primary structure of bovine matrix Gla protein, a new vitamin K-dependent bone protein

The complete amino acid sequence of bovine bone matrix Gla protein (MGP) was determined by automatic sequence analysis of the intact protein and of peptides isolated from tryptic and BNPS-skatole digests. This 79-residue, vitamin K-dependent protein contains a single disulfide bond and 4.8 gamma-carboxyglutamate (Gla) residues, one each at positions 37, 41, 48, and 52, and 0.8 Gla and 0.2 Glu at position 2. There is sufficient sequence homology between MGP and bone Gla protein (BGP) to indicate that these two bovine bone proteins arose by gene duplication and subsequent divergent evolution. Although MGP has a very low solubility in water compared to BGP, there is no hydrophobic domain in MGP which could account for its insolubility, and the overall fraction of hydrophobic residues is 32% for MGP compared to 43% for BGP. MGP is the first vitamin K-dependent protein to be discovered which has several non-gamma-carboxylated residues to the NH2-terminal side of its Gla residues. The presence of NH2-terminal Glu residues between the putative targeting domain for the gamma-carboxylase in the MGP leader sequence and the mid-molecule Gla residues suggests that the gamma-carboxylase may have additional, as yet unrecognized, specificity requirements which determine the susceptibility of Glu residues for gamma-carboxylation.     

Prolonged delirium without anticholinergic signs following amitriptyline overdose.

In a case of acute intoxication with a tricyclic antidepressant (amitriptyline) delirium was prolonged without there being prominent peripheral anticholinergic or electrocardiographic signs. Administration of physostigmine, repeated when necessary, reversed the delirium.     

The cDNA cloning and immunological characterization of hamster p53.

We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75% homologous to human and mouse p53, respectively. Immunoprecipitations of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.     

Exposure to nuclear magnetic resonance imaging procedure attenuates morphine-induced analgesia in mice

Adult male mice exposed to a Nuclear Magnetic Resonance Imaging (NMRI) procedure during the mid-dark period and injected with morphine (10 mg/kg) failed to exhibit the normal nocturnally enhanced morphine analgesia response to a thermal stimulus that was displayed by mice exposed to a sham imaging procedure and treated with morphine (p less than .01). When tested during the mid-light period, animals exposed to the NMRI procedure and given morphine displayed attenuated analgesia levels relative to sham exposed mice (p less than .01) treated with morphine. However, the morphine induced analgesia was not totally abolished since the imaged mice still exhibited analgesia relative to saline treated mice (p less than .01). These results suggest that the magnetic and/or radio-frequency fields associated with the NMRI procedure alter both day- and night-time responses to morphine. These results may reflect magnetic field induced alterations in neuronal calcium binding and/or alterations in nocturnal pineal gland activity.     

A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin

The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)