Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein

Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5' regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl-D-aspartate receptors.     

Brain cytokines and chemokines: roles in ischemic injury and pain

Cytokines and chemokines were originally identified as essential mediators for inflammatory and immune responses. Enhanced production and release of cytokines/chemokines are observed also in the central nervous system (CNS) under diverse pathological conditions. There is growing evidence showing that brain cytokines/chemokines play crucial roles in the neuro-glio-vascular interaction underlying the pathology of various brain disorders and therefore are potential targets for development of novel and effective therapeutics for CNS diseases. Here the evidence of the involvement of cytokines/chemokines in ischemic brain injury and pain is reviewed.     

Altered fractionation

For improving radiotherapy treatment results, altered fractionation (AF) is one of the most important biological factors to modify the conventional fractionation schedule. AF is classified into two categories. One is decreasing in dose-per-fraction and increasing in total dose, so-called hyperfractionation (HF), which expands the difference in radio-sensitivity between tumor and normal tissue. On the other hand, shortening of overall treatment time, so-called accelerated hyperfractionation (AHF), prevents accelerated repopulation of tumor cells during radiotherapy. AF is rarely recognized as a standard therapy despite many reports about its efficacy against various cancers, totally. This is often used for head and neck cancer. However, the problem is that they usually improve local-control, but do not always improve survival. Although AHF is recognized as one of a standard treatment for small cell lung cancer, it is still objectionable and disputable. Besides these, efficacy of AF against non-small cell lung cancer, bladder cancer and malignant glioma, has been reported. However, AF is not considered as a standard treatment. Accompanied with spread out of stereotactic irradiation, dose-fractionation-time relationship becomes to be more important subject, especially hypofractionation, to clarify the new aspect of AF.     

Genetic polymorphism of cholesterol 7alpha-hydroxylase (CYP7A1) and colorectal adenomas: Self Defense Forces Health Study

Bile acids have long been implicated in colorectal carcinogenesis, but epidemiological evidence is limited. Cholesterol 7alpha-hydroxylase (CYP7A1) is the rate-limiting enzyme producing bile acids from cholesterol. A recent case-control study showed a decreased risk of proximal colon cancer associated with the CC genotype of the CYP7A1 A-203C polymorphism. The present study examined the relationship between the CYP7A1 A-203C polymorphism and colorectal adenoma, which is a well-established precursor lesion of colorectal cancer. The study subjects comprised 446 cases of colorectal adenomas and 914 controls of normal total colonoscopy among men receiving a preretirement health examination at two hospitals of the Self Defense Forces (SDF). The CYP7A1 genotype was determined by the polymerase chain reaction-restriction fragment length polymorphism method. Statistical adjustment was made for age, hospital, rank in the SDF, smoking, alcohol use, body mass index, physical activity and parental history of colorectal cancer. The CYP7A1 polymorphism was not measurably related to the overall risk of colorectal adenomas. However, the CC genotype was associated with a decreased risk of proximal colon adenomas, but not of distal colon and rectal adenomas. Adjusted odds ratios of proximal colon adenomas (95% confidence intervals) for the AC and CC genotype versus AA genotype were 0.82 (0.54-1.24) and 0.56 (0.34-0.95), respectively. The findings add to evidence for the role of bile acids in colorectal carcinogenesis. The CC genotype of the CYP7A1 A-203C polymorphism probably renders lower activity of the enzyme synthesizing bile acids.     

Development of MPS IVA mouse (Galnstm(hC79S.mC76S)slu) tolerant to human N-acetylgalactosamine-6-sulfate sulfatase

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disease caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. In recent studies of enzyme replacement therapy for animal models with lysosomal storage diseases, cellular and humoral immune responses to the injected enzymes have been recognized as major impediments to effective treatment. To study the long-term effectiveness and side effects of therapies in the absence of immune responses, we have developed an MPS IVA mouse model, which has many similarities to human MPS IVA and is tolerant to human GALNS protein. We used a construct containing both a transgene (cDNA) expressing inactive human GALNS in intron 1 and an active site mutation (C76S) in adjacent exon 2 and thereby introduced both the inactive cDNA and the C76S mutation into the murine Galns by targeted mutagenesis. Affected homozygous mice have no detectable GALNS enzyme activity and accumulate glycosaminoglycans in multiple tissues including visceral organs, brain, cornea, bone, ligament and bone marrow. At 3 months, lysosomal storage is marked within hepatocytes, reticuloendothelial Kupffer cells, and cells of the sinusoidal lining of the spleen, neurons and meningeal cells. The bone storage is also obvious, with lysosomal distention in osteoblasts and osteocytes lining the cortical bone, in chondrocytes and in the sinus lining cells in bone marrow. Ubiquitous expression of the inactive human GALNS was also confirmed by western blot using the anti-GALNS monoclonal antibodies newly produced, which resulted in tolerance to immune challenge with human enzyme. The newly generated MPS IVA mouse model should provide a good model to evaluate long-term administration of enzyme replacement.