Evaluation of the Extract-Release Volume Phenomenon as a Rapid Test for Detecting Spoilage in Beef

Ground beef of several grades, obtained fresh and held refrigerated until spoiled, was presented to a test panel for scoring on color, odor, and tactile response (tackiness) as to degree of acceptance. Panel scores were correlated with total bacterial counts, ninhydrin-reactive substances, and ERV (extract-release volume) on the same meat. ERV correlated highest with bacterial counts the largest number of times; tackiness, odor, ninhydrin, and color followed in that order. Correlation between bacterial numbers and organoleptic qualities was best, with tackiness followed closely by odor, and then by color. Correlation between tackiness and odor was high. The degree of correlation between bacterial numbers, tackiness, and ERV was high enough to warrant the use of the ERV phenomenon as a rapid test of microbial quality of beef. An ERV value of 25 under the conditions of the test was supported as a divider between acceptable and unacceptable ground beef.     

N-acetyl-β-D-glucopyranosylamine: A potent T-state inhibitor of glycogen phosphorylase. A comparison with α-D-glucose

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to α-D-glucose (K_i = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of β-D-glucopyranosylamine, N-acetyl-β-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (K_i = 32 μM) and the α (K_i = 35 μM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase α and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4₃2₁2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 Å resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of α-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-α-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.     

Map4k4 suppresses Srebp-1 and adipocyte lipogenesis independent of JNK signaling

Adipose tissue lipogenesis is paradoxically impaired in human obesity, promoting ectopic triglyceride (TG) deposition, lipotoxicity, and insulin resistance. We previously identified mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a sterile 20 protein kinase reported to be upstream of c-Jun NH₂-terminal kinase (JNK) signaling, as a novel negative regulator of insulin-stimulated glucose transport in adipocytes. Using full-genome microarray analysis we uncovered a novel role for Map4k4 as a suppressor of lipid synthesis. We further report here the surprising finding that Map4k4 suppresses adipocyte lipogenesis independently of JNK. Thus, while Map4k4 silencing in adipocytes enhances the expression of lipogenic enzymes, concomitant with increased conversion of ¹⁴C-glucose and ¹⁴C-acetate into TGs and fatty acids, JNK1 and JNK2 depletion causes the opposite effects. Furthermore, high expression of Map4k4 fails to activate endogenous JNK, while Map4k4 depletion does not attenuate JNK activation by tumor necrosis factor α. Map4k4 silencing in cultured adipocytes elevates both the total protein expression and cleavage of sterol-regulated element binding protein-1 (Srebp-1) in a rapamycin-sensitive manner, consistent with Map4k4 signaling via mechanistic target of rapamycin complex 1 (mTORC1). We show Map4k4 depletion requires Srebp-1 upregulation to increase lipogenesis and further show that Map4k4 promotes AMP-protein kinase (AMPK) signaling and the phosphorylation of mTORC1 binding partner raptor (Ser792) to inhibit mTORC1. Our results indicate that Map4k4 inhibits adipose lipogenesis by suppression of Srebp-1 in an AMPK- and mTOR-dependent but JNK-independent mechanism.     

The cellular control enzyme polyADP ribosyl transferase is eliminated in cultured Fanconi anemia fibroblasts at confluency

Fanconi anemia (FA) is a hereditary disease of unknown pathogenic mechanisms, although mutations in seven different genes can be causative. Six of these genes have been cloned and sequenced. Only slight homology to the DNA of any other known gene has been found with the exception of FANCG which is identical to XRCC9. The function of these genes, including XRCC9, is presently unknown. Since pADP ribosyl transferase (pADPRT) plays a role in apoptosis, and apoptosis is affected in FA cells, we studied the correlation between pADPRT and FA cells. We reinvestigated the previously reported lack of pADPRT activity in fibroblasts from patients with Fanconi anemia. Here we describe the role of the lower redox potential of FA cells and demonstrate that this is an efficient strategy in the prevention of cell death due to the lack of energy under oxidative stress. This strategy is advantageous for the cells under the nonreplicative condition of confluency in which the risk of mutation is low and the prevention of apoptosis permits cell survival. pADPRT is not diminished to the same extent in all complementation groups of FA. It is prominent in FANCA, FANCG and FANCF cells, indicating that these genes control pADPRT diminution. Our experiments suggest that the pADPRT level is linked with the oxidoreduction reactions seen in FA.     

A modified static respiration assay and its relationship with an enzymatic test to assess compost stability and maturity

Despite the numerous compost stability and maturity tests, no universally accepted compost stability or maturity index exists. The fluorescein di-acetate (FDA) enzymatic assay, originating from soil studies, is examined here as a potential new compost stability test, and is compared to microbial respiration and phytotoxicity indices. Thirteen composts were used in the study from different source materials. Static microbial respiration activity indices calculated were the cumulative O₂ consumptions, O₂ consumption rates, total C-CO₂ production, the respiratory quotient and the bio C/N ratio. Compost phytotoxicity was quantified via a 7-day tomato seed germination assay. Results showed that the net fluorescein release rates correlated with all stability indices. The germination index marginally correlated with the fluorescein release rates, but not with any of the other stability indices. New limits to classify composts regarding their stability were proposed.     

Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice

Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp(b)) and native wild-type (wt) empty capsids (MVMp(e)), determined and refined to 3.25 and 3.75 A resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 A resolution in this study. A comparison of the MVMp(b) and MVMp(e) capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the "shoulder" of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.     

A role for dendritic cells in the dissemination of mycobacterial infection

The ability of mycobacteria to disseminate from the initial site of infection has an important role in immune priming and in the seeding of disease in multiple organs. To study this phenomenon, we used flow cytometry to analyse the distribution of green fluorescent protein-labelled BCG amongst different populations of antigen-presenting cells in the lungs of mice following intranasal infection, and monitored appearance of live bacteria in the draining mediastinal lymph nodes. BCG predominantly infected alveolar macrophages (CD11c(+)/CD11b(-)) and dendritic cells (CD11c(+)/CD11b(+)) in the lungs. The bacteria that disseminated to the lymph node were found in dendritic cells. The results are consistent with a model in which mycobacterial dissemination from the lung is initiated by the migration of infected dendritic cells to the draining lymph nodes.