Import of a putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase into mitochondria

A putative precursor of rat liver mitochondrial glutamic oxaloacetic transaminase which was about 2,000 daltons larger than the subunits of the mature enzyme synthesized in vitro was sensitive to proteases (trypsin and chymotrypsin). When this precursor was incubated with isolated mitochondria in the absence of protein synthesis, it was processed to the mature form; the mature form co-sedimented with mitochondria and was resistant to externally added proteases. Mature enzyme did not compete with this transport.     

Quick-freeze,deep-etch visualization of the ‘cytoskeletal spring’ of cochlear outer hair cells

Summary The lateral membrane system of the cochlear outer hair cell, consisting of the lateral plasma membrane, pillars, filamentous lattice and subsurface cisternae, is considered to be involved in the contractile movement of the isolated cochlear outer hair cell. The filamentous lattice, called the cytoskeletal spring, has been identified in the demembranated cochlear outer hair cell treated with the detergent Triton X-100. In this study, the quick-freeze, deep-etch method was applied to demonstrate the three-dimensional organization of both the filamentous and membranous structures of the lateral membrane system of cochlear outer hair cells. Treatment with saponin revealed that the inner leaflet of the lateral plasma membrane of the cochlear outer hair cell possesses more membrane particles than the outer leaflets, and that the pillars are closely associated with membrane particles in the inner leaflet of the lateral membrane. The presence of filamentous bridges between the filamentous lattice and the subsurface cisternae was also detected. We propose that the lateral membrane system in the cochlear outer hair cell may play an important role in the tuning mechanisms within the cochlea in normal hearing.     

Characterization of three mouse monoclonal antibodies that react with high-molecular-mass glycopeptides isolated from F9 mouse teratocarcinoma cells

Three IgM mouse monoclonal antibodies, NL-9, Thy-22, and HL-5, which were produced primarily against human hematopoietic cells, were tested for their reactivity with various mouse cell lines and were found to react predominantly with mouse embryonal carcinoma cells. Thy-22 reacted with 2-cell-stage mouse embryos, whereas the other two antibodies were not reactive at this stage. All three antibodies, however, reacted with 8-cell-stage embryos. At the blastocyst stage, Thy-22 reacted with the entire surface of the trophectoderm cells, whereas the reactivity of NL-9 and HL-5 was weaker and was polarized on the mural trophectoderm. Immunohistological examination of 6th-day mouse embryos using anti-complement immunofluorescence demonstrated that the embryonic ectoderm was positive for all three antibodies: the reaction of NL-9 and Thy-22 was uniformly distributed over these cells, whereas HL-5 predominantly stained the luminal aspects of the cells lining the proamniotic cavity. Visceral-endoderm cells and trophoblastic cells were positive with all three monoclonal antibodies, whereas the parietal endoderm, extraembryonic ectoderm, and ectoplacental cone were negative. In 19th-day fetuses and adult tissues, certain epithelial cells were stained by these three antibodies. The biochemical nature of the antigens detected was also investigated. Farr's assay showed that both NL-9 and Thy-22 precipitated approximately 10% of the high-molecular-mass glycopeptides isolated from F9 cells, while HL-5 reacted with about 5% of these glycopeptides. The reactivity of the three antibodies against the glycopeptides was completely inhibited by the presence of X-hapten-conjugated silica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum Procalcitonin Level and SOFA Score at Discharge from the Intensive Care Unit Predict Post-Intensive Care Unit Mortality: A Prospective Study

Purpose

The final decision for discharge from the intensive care unit (ICU) is uncertain because it is made according to various patient parameters; however, it should be made on an objective evaluation. Previous reports have been inconsistent and unreliable in predicting post-ICU mortality. To identify predictive factors associated with post-ICU mortality, we analyzed physiological and laboratory data at ICU discharge.

Methods

Patients admitted to our ICU between September 2012 and August 2013 and staying for critical care>2 days were included. Sequential Organ Failure Assessment (SOFA) score; systemic inflammatory response syndrome score; white blood cell count; and serum C reactive protein, procalcitonin (PCT), interleukin-6 (IL-6), lactate, albumin, and hemoglobin levels were recorded. The primary end point was 90-day mortality after ICU discharge. Two hundred eighteen patients were enrolled (195 survivors, 23 non-survivors).

Results

Non-survivors presented a higher SOFA score and serum PCT, and IL-6 levels, as well as lower serum albumin and hemoglobin levels. Serum PCT, albumin, and SOFA score were associated with 90-day mortality in multiple logistic regression analysis. Hosmer-Lemeshow test showed chi-square value of 6.96, and P value of 0.54. The area under the curve (95% confidence interval) was 0.830 (0.771–0.890) for PCT, 0.688 (0.566–0.810) for albumin, 0.861 (0.796–0.927) for SOFA score, and increased to 0.913 (0.858–0.969) when these were combined. Serum PCT level at 0.57 ng/mL, serum albumin at 2.5 g/dL and SOFA score at 5.5 predict 90-day mortality, and high PCT, low albumin and high SOFA groups had significantly higher mortality. Serum PCT and SOFA score were significantly associated with survival days after ICU discharge in Cox regression analysis.

Conclusions

Serum PCT level and SOFA score at ICU discharge predict post-ICU mortality and survival days after ICU discharge. The combination of these two and albumin level might enable accurate prediction.     

Active integration: new strategies for transgenesis

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed “Active Transgenesis” to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac’s specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.     

A complement-dependent cytotoxicity-enhancing anti-CD20 antibody mediating potent antitumor activity in the humanized NOD/Shi-scid, IL-2Rγnull mouse lymphoma model

Engineering the Fc region of monoclonal antibodies (mAb) in order to enhance effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC) is likely to a be promising approach for next-generation mAb therapy. Here, we report on such an antibody, 113F, a novel CDC-enhancing variant of rituximab, and determine the tumor-associated factors influencing susceptibility to 113F-induced CDC. The latter included the quantity of complement inhibitors present, such as CD55 and CD59. We report that compared to rituximab, 113F mediated highly enhanced CDC against primary CD20-expressing lymphoma cells in vitro. Currently, a major problem in the field of immunotherapy research is the lack of suitable small animal models to evaluate human CDC in vivo. Therefore, we established a novel human tumor-bearing NOD/Shi-scid, IL-2Rγ^null mouse model, in which human complement functions as the CDC mediator. We demonstrated that rituximab exerted significant antitumor effects via human CDC in this humanized mouse. The finding of specific localization of human C1q on CD20-expressing tumor cell membranes was consistent with the observation that human CDC indeed contributed to the antitumor effect in this model. Moreover, 113F exerted significantly more potent antitumor effects than rituximab in this in vivo model. The detection of more abundant dense signals from C1q using 113F compared to rituximab was consistent with the concept that this reagent represented a CDC-enhancing mAb. In the near future, the efficacy of this type of CDC-enhancing antibody will be determined in clinical trials in humans.     

Absence of Diabetes and Pancreatic Exocrine Dysfunction in a Transgenic Model of Carboxyl-Ester Lipase-MODY (Maturity-Onset Diabetes of the Young)

Background

CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas.

Methods

We established a monotransgenic floxed (flanking LOX sequences) mouse line carrying the human CEL mutation c.1686delT and crossed it with an elastase-Cre mouse to derive a bitransgenic mouse line with pancreas-specific over-expression of CEL carrying this disease-associated mutation (TgCEL). Following confirmation of murine pancreatic expression of the human transgene by real-time quantitative PCR, we phenotyped the mouse model fed a normal chow and compared it with mice fed a 60% high fat diet (HFD) as well as the effects of short-term and long-term cerulein exposure.

Results

Pancreatic exocrine function was normal in TgCEL mice on normal chow as assessed by serum lipid and lipid-soluble vitamin levels, fecal elastase and fecal fat absorption, and the normoglycemic mice exhibited normal pancreatic morphology. On 60% HFD, the mice gained weight to the same extent as controls, had normal pancreatic exocrine function and comparable glucose tolerance even after resuming normal diet and follow up up to 22 months of age. The cerulein-exposed TgCEL mice gained weight and remained glucose tolerant, and there were no detectable mutation-specific differences in serum amylase, islet hormones or the extent of pancreatic tissue inflammation.

Conclusions

In this murine model of human CEL-MODY diabetes, we did not detect mutation-specific endocrine or exocrine pancreatic phenotypes, in response to altered diets or exposure to cerulein.     

Interaction of Yeast Phosphoglyceromutase with Nucleotides

Moderate cell growth occurred after a long lag phase of about 100 hr when oxygen-sensitive hydrogen bacterium N34 was cultivated chemoautotrophically under 40% O₂. A decrease in cell growth or viable count was not observed during the lag phase. These cells grown under 40 % O₂ were oxygen-resistant because when used as inocula for fresh 40 % O₂-culture, the growth lag period was less than 10 hr. Nine oxygen-sensitive colonies developed from a single oxygen-sensitive cell respectively. When these colonies were inoculated into 40% O₂-culture, they showed an almost equal lag period and growth rate. These results suggest that cell growth in 40% O₂-culture inoculated with oxygen-sensitive strain N34 occurred not by selection of oxygen-resistant variants which might preexist but by adaptation of very oxygen-sensitive cells to high oxygen tension. Oxygen-resistance thus developed was maintained after successive subcultures under 10% O₂ for more than one year.     

Quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid enhance the Fenton reaction in phosphate buffer.

Quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid enhanced the Fenton reaction in phosphate buffer, respectively. The enhancement by quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid of the Fenton reaction may be partly related to their respective actions in the biological systems such as a neurotoxic effect (quinolinic acid), a marked growth-inhibitory action on rice seeding (alpha-picolinic acid and fusaric acid), and an antiseptic (2,6-pyridinedicarboxylic acid). The ultraviolet-visible absorption spectrum of the mixture of alpha-picolinic acid with ferrous ion showed a characteristic visible absorbance band with a lambda(max) at 443 nm, suggesting that alpha-picolinic acid chelate of Fe2+ ion forms in the solution. Similar characteristic visible absorbance band was also observed for the mixture of Fe2+ ion with quinolinic acid (or fusaric acid, or 2,6-pyridinedicarboxylic acid). The chelation seems to be related to the enhancement by quinolinic acid, alpha-picolinic acid, fusaric acid, and 2,6-pyridinedicarboxylic acid of the Fenton reaction. alpha-Picolinic acid was reported to be a toxic substance isolated from the culture liquids of blast mould (Piricularia oryzae CAVARA). On the other hand, it has also been known that chlorogenic acid protects rice plants from the blast disease. The chlorogenic acid inhibited the formation of the hydroxyl radical in the reaction mixture of alpha-picolinic acid, FeSO4(NH4)2SO4, and H2O2. Thus the inhibition may be a possible mechanism of the protective action of the chlorogenic acid against the blast disease.