Moderate Labeling Research Articles

Gastrointestinal Leiomyosarcoma in an Egyptian Fruit Bat (Rousettus Aegyptiacus)

A 10-year-old female Egyptian fruit bat (Rousettus aegyptiacus) was evaluated for weakness and lethargy. Despite clinical improvement after supportive therapy, the bat was found dead the following day. Gross necropsy identified a mass associated with the duodenal wall and right renal cortex. Microscopically, the duodenal and gastric tunica muscularis and serosa and the right renal cortex were effaced by interlacing bundles of neoplastic spindle cells that were diffusely and strongly positive for vimentin and alpha-smooth muscle actin. The neoplastic cells also had mild to moderate cytoplasmic labeling for laminin and S100 and were negative for c-kit and desmin. On the basis of cell morphology and the immunophenotype, this tumor was diagnosed as a gastrointestinal leiomyosarcoma.

Distribution and expression of CD200 in the rat respiratory system under normal and endotoxin‐induced pathological conditions

In vivo and in vitro studies have clearly demonstrated that signaling mediated by the interaction of CD200 and its cognate receptor, CD200R, results in an attenuation of inflammatory or autoimmune responses through multiple mechanisms. The present results have shown a differential expression of CD200 in the respiratory tract of intact rats. Along the respiratory passage, CD200 was specifically distributed at the bronchiolar epithelia with intense CD200 immunoreactivity localized at the apical surface of some ciliated epithelial cells; only a limited expression was detected on the Clara cells extending into the alveolar duct. In the alveolar septum, double immunofluorescence showed intense CD200 immunolabeling on the capillary endothelia. A moderate CD200 labeling was observed on the alveolar type II epithelial cells. It was, however, absent in the alveolar type I epithelial cells and the alveolar macrophages. Immunoelectron microscopic study has revealed a specific distribution of CD200 on the luminal front of the thin portion of alveolar endothelia. During endotoxemia, the injured lungs showed a dose- and time-dependent decline of CD200 expression accompanied by a vigorous infiltration of immune cells, some of them expressing ionized calcium binding adapter protein 1 or CD200. Ultrastructural examination further showed that the marked reduction of CD200 expression was mainly attributable to the loss of alveolar endothelial CD200. It is therefore suggested that CD200 expressed by different lung cells may play diverse roles in immune homeostasis of normal lung, in particular, the molecules on alveolar endothelia that may control regular recruitment of immune cells via CD200-CD200R interaction. Additionally, it may contribute to intense infiltration of immune cells following the loss or inefficiency of CD200 under pathological conditions.

T-SNARE and v-SNARE in the GABAergic and glutamatergic synapses of the rat cerebellar cortex

Using light microscope immunohistochemistry, the distributions of the t-SNARE, SNAP-25 and syntaxin-1a, and v-SNARE, VAMP-2 (or synaptobrevin-2), have been analyzed in the rat cerebellar cortex and compared with those of GAD-65/67, marker of GABAergic neurons, and vGLUT-1 and vGLUT-2, markers of glutamatergic neurons. SNAP-25 immunoreactivity was detected throughout the cortex, highly diffused in the neuropil. In the molecular and Purkinje layers, the immunoreactivity surrounded dendrite trunks of the Purkinje neurons, and bodies of stellate, basket and Purkinje neurons, which appeared all immunonegative. In the granular layer, the immunoreactivity was less diffuse and formed small grains localized within the protoplasmic islands of Held (synaptic glomeruli). Immunoreactivity was also observed in the white matter. The distribution of syntaxin-1a immunoreactivity appeared superimposable to that of SNAP-25. VAMP-2 immunoreactivity was observed in small dots finely dispersed in the neuropil of the molecular/Purkinje layer; a moderate labelling of the Purkinje neuron bodies was also observed. In the granular layer, VAMP-2 immunoreactivity formed larger deposits, presumably corresponding to mossy fibre terminals. Experiments of double labelling immunohistochemistry revealed no co-localization between t-SNARE (SNAP-25) or v-SNARE (VAMP-2) and GAD-65/67. On the contrary, co-localization between SNAP-25 and VAMP-2, on one side, and v-GLUT-1 or vGLUT-2, on the other side, was frequently revealed. Briefly, in the molecular layer, v-GLUT-1 and vGLUT-2-positive terminals only partly displayed also SNAP-25 immunoreactivity; whereas in the granular layer, all v-GLUT-1 and vGLUT-2-positive terminals displayed this co-localization. The results of this study indicate that t-SNARE and v-SNARE largely characterize the glutamatergic synapses in the rat cerebellar cortex. The same proteins are lacking in the GABAergic synapses.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in breast cancer metastases

EpCAM (CD326) has diverse roles in cell adhesion and proliferation, and is known to be overexpressed in primary breast carcinomas (PBCs). While clinical and preclinical data suggest a role for EpCAM in metastases, the only prior study of EpCAM expression in breast cancer metastases suggested that EpCAM expression is decreased after first-line chemotherapy. This study evaluates EpCAM expression in metastatic breast carcinoma (MBC) versus matched PBC . Rapid autopsies were performed on 17 patients with widely metastatic breast cancer. Single patient tissue microarrays (TMAs) were constructed from archived PBC and post-mortem MBCs. In total, 169 spots from 17 PBCs and 895 spots from 195 MBCs were labeled for EpCAM by immunohistochemistry (IHC). Expression was scored as intensity (1-3) multiplied by percent membrane labeling (0-100%) and was subclassified as low (0-100), moderate (101-200), or high (201-300) labeling. PBCs exhibited exclusively low-moderate EpCAM labeling. EpCAM labeling was present in all metastases and was significantly increased in MBCs of 14 of 17 patients (P value range <0.05 to <0.0001, t test). In the remaining three patients, EpCAM labeling was nonsignificantly increased in 1 and unchanged in 2. High EpCAM labeling was verified using a different antibody for IHC, as well as in a separate series of surgically resected metastases compared to unmatched surgically resected primary breast cancers. In conclusion, EpCAM is highly expressed in MBCs compared to matched PBCs, verifying that it is a promising therapeutic target.

Cross‐basin comparison of phosphorus stress and nitrogen fixation in Trichodesmium

We investigated the phosphorus (P) status and N2 fixation rates of Trichodesmium populations from the North Pacific, western South Pacific, and western North Atlantic. Colonies of Trichodesmium were collected and analyzed for endogenous alkaline phosphatase (AP) activity using enzyme‐labeled fluorescence (ELF) and for nitrogenase activity using acetylene reduction. AP hydrolyzes dissolved inorganic phosphate (DIP) from dissolved organic phosphorus and is active in Trichodesmium colonies experiencing P stress. Across multiple stations in the subtropical North and South Pacific, there was low to moderate ELF labeling in Trichodesmium, although labeling was present in other taxa. In contrast, Trichodesmium ELF labeling in the North Atlantic ranged from low to high. Low ELF labeling corresponded with high DIP concentrations while high ELF labeling occurred only at North Atlantic stations with DIP concentrations #40 nmol L21, indicating that Trichodesmium was not experiencing dramatic P stress in the Pacific Ocean while P stress was evident in the western North Atlantic. However, nitrogenase activity was significantly higher in the P‐stressed western North Atlantic than in the Pacific Ocean (0.40‐1.30 compared to 0.01‐0.46 nmol C2H4 h−1 colony−1). These data underscore the differential basinlevel importance of P availability to Trichodesmium and suggest that factors other than P are constraining their N2 fixation rates in the Pacific.

Analysis of Cochlear Protein Profiles of Wistar, Sprague−Dawley, and Fischer 344 Rats with Normal Hearing Function

Differences in the expression of cochlear proteins are likely to affect the susceptibility of different animal models to specific types of auditory pathology. However, little is currently known about proteins that are abundantly expressed in inner ear. Identification of these proteins may facilitate the search for biomarkers of susceptibility and intervention targets. To begin to address this issue, we analyzed cochlear protein profiles of three strains of rats, Wistar, Sprague-Dawley, and Fischer 344, using a broad spectrum antibody microarray. Normal hearing function of the animals was ascertained using distortion product otoacoustic emissions (DPOAE). Of 725 proteins screened in whole cochlea, more than 80% were detected in all three strains. However, there were striking differences in the levels at which they occur. Among 213 proteins expressed at levels>or=2 fold of actin, only 7.5% were detected at these levels in all three strains. Myosin light chain kinase (MLCK) was immunolocalized in cuticular plate of outer hair cells (OHC) while mitogen activated protein (MAP) kinase-extracellular-signal regulated kinase1/2 (ERK1/2) was detected as foci in OHC, pillar cells, strial marginal cells, and fibroblasts of spiral ligament. A review of literature indicated that the expression of 7 (44%) of these 16 proteins were detected for the first time in the inner ear, although there were implications of the presence of some of these proteins. One of these abundant, but unstudied, proteins, MAP kinase activated protein kinase2 (MAPKAPK2), shows strong immunolabeling in pillar cells and inner hair cells (IHC). There was moderate MAPKAPK2 labeling in OHC, supporting cells, neurons, and marginal, intermediate, and basal cells. The current study provides the first, large cochlear protein profile of multiple rat strains. The diversity in expression of abundant proteins in these strains may contribute to differences in susceptibility of these strains to aging, noise, or ototoxic drugs.

Expression of KIT Receptor in Feline Cutaneous Mast Cell Tumors

Twenty-seven feline cutaneous mast cell tumors (MCTs) were selected for this retrospective study. Samples were routinely processed and stained with hematoxylin and eosin (HE) and toluidine blue, and tumors were classified as well-differentiated (19/27), atypical or poorly granulate (7/27), and pleomorphic (1/27). Immunohistochemistry to detect KIT protein was performed on all samples. The immunoreactivity was recorded by distribution within the tumor, cellular location, and intensity. Well-differentiated MCTs were predominantly characterized by diffuse cytoplasmic (8/19) and membranous stain (7/19); a diffuse distribution of KIT positive cells was displayed in most of these tumors as well (15/19). Atypical MCTs showed diffuse distribution of labeled cells (4/7), and diffuse cytoplasm immunostaining was seen most (5/7). The pleomorphic MCT showed diffuse cytoplasmic KIT stain, with moderate labeling intensity, typically displaying focal distribution in deeper areas of the neoplasm. According to the results, there was no correlation between the type of MCTs and KIT expression, although the use of feline KIT immunohistochemistry could be useful to assess the mast cell origin.

Gene-mediated Restoration of Normal Myofiber Elasticity in Dystrophic Muscles

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.

Localization of the transcription factor, sterol regulatory element binding protein-2 (SREBP-2) in the normal rat brain and changes after kainate-induced excitotoxic injury

The transcription factor sterol regulatory element binding protein-2 (SREBP-2) has a key role in regulating cholesterol biosynthesis. In view of ongoing cholesterol biosynthesis in the brain, the present study was carried out to examine the distribution of the SREBP-2 in the normal rat brain, and possible changes after kainate-induced excitotoxicity. Western immunoblot analysis of SREBP-2 showed a dense band at 70 kDa corresponding to the transcriptionally active form of SREBP-2 in homogenates from the rat hippocampus, cortex and striatum. SREBP-2 immunolabeled sections showed dense labeling of pyramidal neurons in field CA1 and CA3 of the hippocampus, moderately dense labeling of pyramidal and non-pyramidal neurons of the cerebral neocortex, and moderate labeling of putative medium spiny neurons in the caudate nucleus and putamen. Label was observed in the somatic cytoplasm, nucleus and apical dendrites of pyramidal neurons, and dendritic shafts and spines in the neuropil in the hippocampus. The expression of SREBP-2 was also elucidated after excitotoxic neuronal injury induced by intracerebroventricular injections of kainate. Real time RT-PCR analyses showed that mRNA level of SREBP-2 was significantly reduced at both 1 day and 2 weeks post-kainate injection. Immunohistochemical analyses showed significantly reduced SREBP-2 immunoreactivity in the kainate-induced lesions, in support of the real time RT-PCR results. Taken together, the above results show that there is high level of SREBP-2 expression in the normal hippocampus, and that neuronal injury results in a significant reduction of SREBP-2 expression in the damaged areas.

Localization of CB1 cannabinoid receptor mRNA in the brain of the chick ( Gallus domesticus)

The cannabinoid receptor one (CB1) is prevalent in the brains of many species. Receptor binding, in situ hybridization and immunohistochemical surveys have described the distribution of this receptor in a limited number of species. The current study used in situ hybridization to examine the expression of CB1 mRNA in the chick brain, a non-mammalian vertebrate. The results were compared to the observed patterns of expression for CB1 mRNA, protein, and agonist binding that have been reported for other avian species and mammals. Importantly, since CB1 receptors are typically located on neuronal terminals, comparison of the somatic mRNA expression with previously reported descriptions of the location of functional receptors, allows speculation about the circuits that make use of these receptors. The expression pattern for CB1 mRNA appears to be highly conserved across species in key areas such as the cerebellum and portions of the forebrain. For example, high levels of expression were observed in the avian amygdala and hippocampus, areas which express high levels of CB1 in mammals. The avian substantia nigra and ventral tegmental area, however, showed specific labeling. This finding is in stark contrast to the high levels of receptor binding or CB1 protein, but not CB1 mRNA in these areas of the mammalian brain. Moderate labeling was also seen throughout the hyperpallium and mesopallium. Throughout the brain, a number of regions that are known to be involved in visual processing displayed high levels of expression. For example, the tectum also had strong mRNA expression within layers 9–11 of the stratum griseum et fibrosum superficale and stratum album centrale.

A 72-kD B cell-associated surface glycoprotein expressed at high levels in hairy cell leukaemia and plasma cell neoplasms

The present paper describes two new MoAbs, GHI/75 and VMP55, which were raised against a glycoprotein enriched lysate of hairy cell leukaemia. These antibodies recognized a new antigen of 72 kD (unreduced) and 83 kD (reduced) molecular weight. GHI/75 and VMP55 gave very strong staining of plasma cells, moderate labelling of circulating B cells but only weak staining of monocytes, some tissue macrophages and lymphoid cells. Neither antibody reacted with neutrophils or any non-haematopoietic cells. Both antibodies, however, strongly labelled the tumour cells in hairy cell leukaemia, multiple myeloma, plasmacytoma and lymphoplasmacytic lymphomas. No staining was seen of the neoplastic cells in Hodgkin's disease, myeloid leukaemia or T cell lymphomas. The two antibodies, GHI/75 and VMP55, may be of value in the differential diagnosis of hairy cell leukaemias and plasma cell neoplasms. In addition, the ease with which their antigen can be purified provides the possibility for a detailed study of this molecule.

Espin actin‐cytoskeletal proteins are in rat type I spiral ganglion neurons and include splice‐isoforms with a functional nuclear localization signal

The espins are Ca(2+)-resistant actin-bundling proteins that are enriched in hair cell stereocilia and sensory cell microvilli. Here, we report a novel localization of espins to a large proportion of rat type I spiral ganglion neurons (SGNs) and their projections to the cochlear nucleus (CN). Moreover, we show that a fraction of these espins is in the nucleus of SGNs owing to the presence of splice-isoforms that contain a functional nuclear localization signal (NLS). Espin antibody labeled approximately 83% of type I SGNs, and the labeling intensity increased dramatically during early postnatal development. Type II SGNs and vestibular ganglion neurons were unlabeled. In the CN, espin-positive auditory nerve fibers showed a projection pattern typical of type I SGNs, with intense labeling in the nerve root region and posteroventral CN (PVCN). The anteroventral CN (AVCN) showed moderate labeling, whereas the dorsal CN showed weak labeling that was restricted to the deep layer. Espin-positive synaptic terminals were enriched around nerve root neurons and octopus cells in the PVCN and were also found on globular bushy cells and multipolar neurons in the PVCN and AVCN. SGNs expressed multiple espin transcripts and proteins, including splice-isoforms that contain a nonapeptide, which is rich in positively charged amino acids and creates a bipartite NLS. The nonapeptide was necessary to target espin isoforms to the nucleus and was sufficient to target an unrelated protein to the nucleus when joined with the upstream di-arginine-containing octapeptide. The presence of cytoplasmic and nuclear espins in SGNs suggests additional roles for espins in auditory neuroscience.

Differential projections from the vestibular nuclei to the flocculus and uvula‐nodulus in pigeons (Columba livia)

The pigeon vestibulocerebellum is divided into two regions based on the responses of Purkinje cells to optic flow stimuli: the uvula-nodulus responds best to self-translation, and the flocculus responds best to self-rotation. We used retrograde tracing to determine whether the flocculus and uvula-nodulus receive differential mossy fiber input from the vestibular and cerebellar nuclei. From retrograde injections into the both the flocculus and uvula-nodulus, numerous cells were found in the superior vestibular nucleus (VeS), the cerebellovestibular process (pcv), the descending vestibular nucleus (VeD), and the medial vestibular nucleus (VeM). Less labeling was found in the prepositus hypoglossi, the cerebellar nuclei, the dorsolateral vestibular nucleus, and the lateral vestibular nucleus, pars ventralis. In the VeS, the differential input to the flocculus and uvula-nodulus was distinct: cells were localized to the medial and lateral regions, respectively. The same pattern was observed in the VeD, although there was considerable overlap. In the VeM, the majority of cells labeled from the flocculus were in rostral margins on the ipsilateral side, whereas labeling from uvula-nodulus injections was distributed bilaterally throughout the VeM. Finally, from injections in the flocculus but not the uvula-nodulus, moderate labeling was observed in a paramedian area, adjacent to the medial longitudinal fasciculus. In summary, there were clear differences with respect to the projections from the vestibular nuclei to functionally distinct parts of the vestibulocerebellum. Generally speaking, the mossy fibers to the flocculus and uvula-nodulus arise from regions of the vestibular nuclei that receive input from the semicircular canals and otolith organs, respectively.

13C-labelling patterns of green leaf volatiles indicating different dynamics of precursors in Brassica leaves

To investigate the dynamics of precursor compounds of green leaf volatiles (GLV)s and other biogenic compounds released by mechanically damaged Brassica oleracea leaves , plants were exposed for two consecutive 16 h light phases to highly enriched 13CO 2. Analysis by GC-MS indicated (1) biogenic compounds released upon wounding, (2) a different labelling pattern between and (3) within compounds, and (4) evidence for spatial heterogeneity of the precursor pool extrapolated from points (1)–(3). First, GLVs comprised C 5 and C 6 molecules, with the GLV pentenyl acetate being reported here for the first time from higher plants. Second, the labelling pattern found in most GLVs indicates a low turnover of the precursor α-linolenic acid. Moderate labelling of dimethyldisulphide indicates a connection to an active plastidic methyl pool closely connected to CO 2 fixation, and very weak labelling of terpenes indicates a constitutive monoterpene pool. Third, not all GLVs exhibit similarly strong labelling patterns (hexenyl acetate vs. hexyl acetate), indicating different precursors. As the labelling patterns of alcohol and acetate moieties in the esters differ, with only the former being strongly labelled, the precursor of the acetate moiety, acetyl-CoA, is likely to derive from a different cellular pool to that used in chloroplastic fatty acid synthesis, or was rapidly synthesised after the end of labelling. Fourth, the exceptionally high relative abundance of labelled GLV and the low concentration of unlabelled molecules are likely to occur because recently synthesized α-linolenic acid is bound in lipids that are organised in distinct areas, or are chemically different from the older lipids. They must be preferentially used as precursors.

Expression of p53, bcl-2, and Ki-67 proteins in the inflammatory regenerative and dysplastic epithelial lesions of flat colonic mucosa.

The aim of the study was to define the distribution of p53, bcl-2 and Ki-67 proteins in the inflammatory-regenerative and dysplastic lesions of the colon mucosa. The relationship between the presentation of p53, bcl-2 and Ki-67 proteins and the intensity of the inflammatory-regenerative and dysplastic lesions in the colon flat mucosa was investigated as well. Biopsy specimens from 270 patients were examined: 74 were classified as inflammatory-regenerative and 196 as dysplastic lesions (108 mild, 58 moderate, and 30 severe dysplasia). The expression of all three proteins was assessed on the basis of location, quantity, and intensity of immunostaining, by counting antigen positive cells, in comparison with normal mucosa and adenocarcinoma. p53 protein appears only in sporadic cases (6.6%) of severe dysplasia. Bcl-2 expression appears significantly (p<0.005) more often in cases of mild dysplasia (61.1%) compared to inflammatory-regenerative mucosa (14.8%). In cases of mild dysplasia, bcl-2 positive cells were spreading from the lower third to the middle third of the crypts. Bcl-2 expression was maintained through the stadiums of moderate and severe dysplasia (75.8%), where antigen positive cells were found all along the crypts. A significant increase (p<0.005) in the expression of nuclear protein Ki-67 was noticed in the stadiums of moderate (labelling index =26.3) compared to mild dysplasia (labelling index=16.7), and severe (labelling index=36.7) compared to moderate dysplasia, where the zone of cellular proliferation was widen along the whole crypt length. In the process of the development of epithelial dysplasia in the flat mucosa of colon a degree of the gene p53 alteration is low and appears only in sporadic cases of severe dysplasia. Mutation of the bcl-2 gene is involved in the genesis of the lesion but not in its progression to carcinoma. Increased expression of Ki-67 protein speaks in favour of an increased cellular proliferation which, together with the above mentioned mechanisms, is involved in the process of occurrence and progression of epithelial dysplasia in the flat mucosa of colon.

Differential Effects of Estradiol on Encoding Properties of TMJ Units in Laminae I and V at the Spinomedullary Junction in Female Rats

To determine whether estrogen status modulated dorsal horn neural activity relevant to temporomandibular joint (TMJ) processing single units were recorded in superficial and deep laminae at the trigeminal subnucleus caudalis/upper cervical cord (Vc/C1-2) junction of ovariectomized (OvX) female rats under barbiturate anesthesia after 17beta-estradiol (E2) treatment for 2 days. E2 dose-dependently enhanced the response to intra-TMJ stimulation by adenosine triphosphate (ATP) of neurons classified as nociceptive specific (NS), but not wide dynamic range (WDR), in superficial laminae. ATP caused similar responses among NS and WDR neurons from deep laminae in all groups. By contrast, the cutaneous receptive field areas of WDR, but not NS, units in superficial and deep laminae were enlarged in high E2-treated (HE2) compared with low E2-treated (LE2) females. Units from untreated or vehicle-treated male rats displayed responses similar to those of LE2 females. TMJ units in superficial laminae from females were more likely to receive convergent cutaneous input and respond to jaw movement than males, independent of E2 treatment. Western blot analysis revealed similar levels of P2X2 and P2X3 receptor protein in Vc/C1-2 or trigeminal ganglion samples in all groups. Immunohistochemistry revealed dense terminal labeling for P2X3 receptors in superficial laminae and moderate labeling in deep laminae at the Vc/C1-2 junction. These data indicated a significant linkage between estrogen status and the magnitude of articular input evoked by ATP from TMJ neurons in the superficial laminae at the Vc/C1-2 junction, whereas estrogenic modulation of TMJ neurons in deep laminae affected only the convergent input from overlying facial skin.

Preparation of 99mTc-Carnosine and 99mTcO-(V)-DMSA Complexes, Biological Distribution, and Estimation of Their Gene Anti- Polymorphisms Induced by -Irradiation

Background: Two chelating agents (Carnosine and DMSA) were used to study their labeling conditions with technetium-99m followed by biological distribution investigation. Molecular studies were done via PCR/RFLP analysis of angiotensin II subtype II receptor gene for monitoring their antioxidant activity through free iron chelation leading to inhibition of Fenton reaction. Material and methods: Carnosine was labeled by mixing 4 mg with 30 mg glucose and 25 g SnCl2.2H2O, followed by pertechnetate and stand at room temperature for 60 minutes. Minor modification was done to prepare 99mTc(V)-DMSA tracer in one step, by adding pertechnetate solution to the lyophilized kit contains 1mg DMSA, 0.1 mg SnCl2.2H2O, and 30 mg glucose at pH 9. The biodistribution of the two tracers in normal and tumor-induced mice. The molecular investigation of the anti-oxidant activity of both carnosine and DMSA in 6 Gy - irradiated rats using the anti-inflammatory angiotensin II subtype II receptor gene (AT2RG) as indicator. Results: Carnosine and DMSA were labeled with Technetium-99m yielding 85% and 97%, respectively the ability of both tracers to localize in tumor sites but the priority to the 99mTc (V)-DMSA. Molecular studies showed strong antioxidant activity of carnosine but not enough to block radiation induced oxidative stress and Moderate antioxidant activity of DMSA was achieved by chelating free iron and iron released through oxidative stress. Maximum protection was achieved through the dual action of both DMSA and carnosine. Conclusion: moderate and high labeling yield were achieved for both 99mTc(V)DMSA and 99mTc-canosine respectively with higher selectivity of the former to tumor sites and maximum protection were achieved by the dual action of both chelating agents.

Preparation of 99mTc-Carnosine and 99mTcO-(V)-DMSA Complexes, Biological Distribution, and Estimation of Their Gene Anti- Polymorphisms Induced by -Irradiation

Background: Two chelating agents (Carnosine and DMSA) were used to study their labeling conditions with technetium-99m followed by biological distribution investigation. Molecular studies were done via PCR/RFLP analysis of angiotensin II subtype II receptor gene for monitoring their antioxidant activity through free iron chelation leading to inhibition of Fenton reaction. Material and methods: Carnosine was labeled by mixing 4 mg with 30 mg glucose and 25 g SnCl2.2H2O, followed by pertechnetate and stand at room temperature for 60 minutes. Minor modification was done to prepare 99mTc(V)-DMSA tracer in one step, by adding pertechnetate solution to the lyophilized kit contains 1mg DMSA, 0.1 mg SnCl2.2H2O, and 30 mg glucose at pH 9. The biodistribution of the two tracers in normal and tumor-induced mice. The molecular investigation of the anti-oxidant activity of both carnosine and DMSA in 6 Gy - irradiated rats using the anti-inflammatory angiotensin II subtype II receptor gene (AT2RG) as indicator. Results: Carnosine and DMSA were labeled with Technetium-99m yielding 85% and 97%, respectively the ability of both tracers to localize in tumor sites but the priority to the 99mTc (V)-DMSA. Molecular studies showed strong antioxidant activity of carnosine but not enough to block radiation induced oxidative stress and Moderate antioxidant activity of DMSA was achieved by chelating free iron and iron released through oxidative stress. Maximum protection was achieved through the dual action of both DMSA and carnosine. Conclusion: moderate and high labeling yield were achieved for both 99mTc(V)DMSA and 99mTc-canosine respectively with higher selectivity of the former to tumor sites and maximum protection were achieved by the dual action of both chelating agents.

Iodination of Proteins by IPy2BF4, a New Tool in Protein Chemistry

Iodination is a very useful method for protein characterization and labeling. However, derivatization chemistries used in most conventional iodination procedures may cause substantial alterations in protein structure and function. The IPy(2)BF(4) reagent [bis(pyridine)iodonium (I) tetrafluoroborate] has been shown to be an effective iodinating reagent for peptides. Herein we report the first application of IPy(2)BF(4) in protein iodination in an aqueous medium using three representative substrates: insulin, lysozyme, and the enzyme 1,3-1,4-beta-d-4-glucanohydrolase. Our results show that IPy(2)BF(4) has clear advantages over existing methods in that the reaction is quantitative, fast, and selective for the most accessible Tyr residues of a protein, and it preserves the functional integrity of the protein when moderate Tyr labeling levels are pursued.

Patterns of expression of the three cerebral cavernous malformation (CCM) genes during embryonic and postnatal brain development

Cerebral Cavernous Malformation (CCM) is a disease characterized by capillary-venous lesions mostly located in the central nervous system. It occurs both as a sporadic and hereditary autosomal dominant condition. Three CCM genes have been identified and shown to encode the KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3) proteins whose functions are so far unknown. In an attempt to get some insight into the role of the 3 CCM genes, we used in situ hybridization to conduct a comparative analysis of their expression pattern at several time points during murine embryonic, postnatal and adult stages particularly within the central nervous system. A strong expression of the 3 Ccm genes was detected in the various neuronal cell layers of the brain, cerebellum and spinal cord, from embryonic to adult life. By E14.5 a moderate labelling was observed in the heart, arterial and venous large vessels with all 3 Ccm probes. Ccm2 and Ccm3 mRNAs, but not Ccm1, were clearly detected within meningeal and parenchymal cortical vessels at P8. This expression was no more detected by P19 and in adult murine brain, strongly suggesting a role for these 2 proteins in the intensive angiogenesis process occuring within the central nervous system during this period.