Activity-Dependent Modulation of Odorant Receptor Gene Expression in the Mouse Olfactory Epithelium

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.     

Low-energy X-radiation for prevention of restenosis results in localized inhibition of V79 fibroblast cell proliferation

Local delivery of high-energy ionizing radiation by using 3 or 3 emitters to injured vessels demonstrated inhibition of cell proliferation (CP) and neointima formation. Low-energy ('soft') X-radiation (LEXR) offers logistic and safety advantages over the use of disposable radioisotopes. This study evaluated the efficacy of LEXR in penetration and inhibition of CP at doses similar to those prescribed for the use of radioisotopes for prevention of restenosis. Serial measurements in an ion chamber detected the attenuation of LEXR using potentials of 17 and 40 kV at a distance of 17 cm of air through 0-10 mm depths of serum-containing tissue culture medium. The effect of inhibition on CP was determined by exposing V79 fibroblasts to a potential of 17 kV in order to deliver a prescribed dose of 13 Gy at a dose rate of 2.17 Gy/min to the surface of the cells. Complete inhibition of CP at a height of 0.00 mm occurred with 13 Gy; however, a 50% attenuation of the dose was measured at a medium depth of 1.22 mm and was associated with a reduction of 60% of the CP. LEXR demonstrated an ability to inhibit CP at doses equivalent to those used in techniques involving 3 and 3 irradiation. Under such conditions, the dose gradient is too high, especially for large vessels. However, a catheter-based LEXR that could be inserted into the artery with the capability of varying effective energy would be ideal for intravascular applications.     

Linkage of <Emphasis Type="Italic">Patr-AL</Emphasis> to <Emphasis Type="Italic">Patr-A</Emphasis> and-<Emphasis Type="Italic">B</Emphasis> in the major histocompatibility complex of the common chimpanzee (<Emphasis Type="Italic">Pan troglodytes</Emphasis>)

Patr-AL is a recently described gene found only in the common chimpanzee, but closely related in structure to the highly polymorphic Patr-A and HLA-A genes of the chimpanzee and human MHCs, respectively. Unlike Patr-A and HLA-A, the Patr-AL gene has little polymorphism and is not fixed in the chimpanzee genome. To determine whether Patr-AL is located in the MHC or elsewhere, we compared segregation of the Patr-AL gene with segregation of Patr-A and - B alleles in chimpanzee families. The results demonstrate that Patr-AL is an MHC class I gene present on different MHC haplotypes as defined by their combination of Patr-A and B alleles.     

Rapid Extraction of DNA From Escherichia coli and Cryptosporidium parvum for Use in PCR

The Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were evaluated for their ability to rapidly extract PCR-quality DNAs from Escherichia coli O157:H7 and Cryptosporidium parvum. All methods provided satisfactory DNA from E. coli, and the Xtra Amp and Instagene reagents provided satisfactory DNA from C. parvum.     

Development of Allogeneic NK Cell Adoptive Transfer Therapy in Metastatic Melanoma Patients: In Vitro Preclinical Optimization Studies

Natural killer (NK) cells have long been considered as potential agents for adoptive cell therapy for solid cancer patients. Until today most studies utilized autologous NK cells and yielded disappointing results. Here we analyze various modular strategies to employ allogeneic NK cells for adoptive cell transfer, including donor-recipient HLA-C mismatching, selective activation and induction of melanoma-recognizing lysis receptors, and co-administration of antibodies to elicit antibody-dependent cell cytotoxicity (ADCC). We show that NK cell activation and induction of the relevant lysis receptors, as well as co-administration of antibodies yield substantial anti-cancer effects, which are functionally superior to HLA-C mismatching. Combination of the various strategies yielded improved effects. In addition, we developed various clinically-compatible ex vivo expansion protocols that were optimized according to fold expansion, purity and expression of lysis receptors. The main advantages of employing allogeneic NK cells are accessibility, the ability to use a single donor for many patients, combination with various strategies associated with the mechanism of action, e.g. antibodies and specific activation, as well as donor selection according to HLA or CD16 genotypes. This study rationalizes a clinical trial that combines adoptive transfer of highly potent allogeneic NK cells and antibody therapy.     

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.     

The association of APOE genotype with cognitive function in persons aged 35 years or older

APOE genotype is associated with the risk of Alzheimer's disease. In the present study, we investigated whether APOE genotype was associated with cognitive function in predominantly middle-aged persons. In a population-based cohort of 4,135 persons aged 35 to 82 years (mean age (SD), 55 (12) years), cognitive function was measured with the Ruff Figural Fluency Test (RFFT; worst score, 0 points; best score, 175 points). APOE genotype (rs429358 and rs7412) was determined by polymerase chain reaction. The mean RFFT score (SD) of the total cohort was 69 (26) points. Unadjusted, the mean RFFT score in homozygous APOE ε4 carriers was 4.66 points lower than in noncarriers (95% confidence interval, -9.84 to 0.51; p?=?0.08). After adjustment for age and other risk factors, the mean RFFT score in homozygous APOE ε4 carriers was 5.24 points lower than in noncarriers (95% confidence interval, -9.41 to -1.07; p?=?0.01). The difference in RFFT score was not dependent on age. There was no difference in RFFT score between heterozygous APOE ε4 carriers and noncarriers. The results indicated that homozygous APOE ε4 carriers aged 35 years or older had worse cognitive function than heterozygous carriers and noncarriers.     

Rapid B cell receptor-induced unfolded protein response in nonsecretory B cells correlates with pro- versus antiapoptotic cell fate

The adaptive unfolded protein response (UPR) is essential for the development of antibody-secreting plasma cells. B cells induced by lipopolysaccharide (LPS) to differentiate into plasma cells exhibit a nonclassical UPR reported to anticipate endoplasmic reticulum stress prior to immunoglobulin production. Here we demonstrate that activation of a physiologic UPR is not limited to cells undergoing secretory cell differentiation. We identify B cell receptor (BCR) signaling as an unexpected physiologic UPR trigger and demonstrate that in mature B cells, BCR stimulation induces a short lived UPR similar to the LPS-triggered nonclassical UPR. However, unlike LPS, BCR stimulation does not induce plasma cell differentiation. Furthermore, the BCR-induced UPR is not limited to cells in which BCR induces activation, since a UPR is also induced in transitional immature B cells that respond to BCR stimulation with a rapid apoptotic fate. This response involves sustained up-regulation of Chop mRNA indicative of a terminal UPR. Whereas sustained Chop expression correlates with the ultimate fate of the BCR-triggered B cell and not its developmental stage, Chop-/- B cells undergo apoptosis, indicating that CHOP is not required for this process. These studies establish a system whereby a terminal or adaptive UPR can be alternatively triggered by physiologic stimuli.     

Molecular machines

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Highlights? Elastic models of proteins capture large-scale motions of motors. ? Function of motors is embedded in native contacts and evolutionary patterns. ? Reaction path models motor motions by a tube of minimum free energy path. ? Milestoning correlates structure and function of atomic models of motors.