Neuroprotective impact of prothymosin alpha-derived hexapeptide against retinal ischemia-reperfusion.

Prothymosin alpha (ProTα) has robustness roles against brain and retinal ischemia or serum-starvation stress. In the ProTα sequence, the active core 30-amino acid peptide/P30 (a.a.49-78) is necessary for the original neuroprotective actions against ischemia. Moreover, the 9-amino acid peptide sequence/P9 (a.a.52-60) in P30 still shows neuroprotective activity against brain and retinal ischemia, though P9 is less potent than P30. As the previous structure-activity relationship study for ProTα may not be enough, the possibility still exists that any sequence smaller than P9 retains potent neuroprotective activity. When different P9- and P30-related peptides were intravitreally injected 24h after retinal ischemia in mice, the 6-amino acid peptide/P6 (NEVDEE, a.a.51-56) showed potent protective effects against ischemia-induced retinal functional deficits, which are equipotent to the level of P30 peptide in electroretinography (ERG) and histological damage in Hematoxylin and Eosin (HE) staining. Further studies using ERG and HE staining suggested that intravitreal or intravenous (i.v.) injection with modified P6 peptide/P6Q (NEVDQE) potently inhibited retinal ischemia-induced functional and histological damage. In an immunohistochemical analysis, the ischemia-induced loss of retinal ganglion, bipolar, amacrine and photoreceptor cells were inhibited by a systemic administration with P6Q peptide 24h after the ischemic stress. In addition, systemic post-treatment with P6Q peptide significantly inhibited retinal ischemia-induced microglia and astrocyte activation in terms of increased ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) intensity, respectively, as well as their morphological changes, increased number and migration. Thus, this study demonstrates the therapeutic significance of modified P6 peptide P6Q (NEVDQE) derived from 6-amino acid peptide (P6) in ProTα against ischemic damage.

Epidemiological Aspect and common Bacterial and Fungal isolates from Suppurative Corneal Ulcer in Mymensingh Region.

This prospective study was done to find out the epidemiological factors of suppurative corneal ulcer and the common causative bacterial and fungal isolates from the, patients with suppurative corneal ulcer in secondary and tertiary level hospital at Mymensingh region. A total 100 samples of corneal scrapings were collected purposively from clinically diagnosed suppurative corneal ulcer patients from March 18, 2012 to March 17, 2013. Out of the total 100 samples, bacterial species were 29(29%) cases and the fungal spacies were 71(71%) identified by the culture in blood agar, chocolate agar and sabouraud's agar media and also by microscopic examination. The bacterial species were streptococcus pneumonae 12 cases (12%), Staphylococcus aureus 9 cases (9%), pseudomonas in 6 cases (6%), and Streptococcus pyoganes 2 cases (2%). Fungal species were aspergillus fumigatus 61 cases (61%), aspergillus niger 10 cases (10%). Out of the study populations, most of the populations were from the age group of 41 to 60 years (39 %), followed 21 to 40 years (34%) age group. Considering the sex, male were 67%, female were 33%. The majority of patients came from the rural area of Mymensingh region; occupationally they were farmers (44%). Ocular trauma due to agricultural materials was the most common associated factor (71%). The etiological and epidemiological pattern of suppurative corneal ulcer varies significantly with geographical region, patient population and health of the cornea. The present study was carried out to explore the epidemiological pattern, causative bacterial and fungal specie by laboratory procedure from corneal scraping and to invent a prospective guide line for the management of corneal ulcer in the community.

Bulk growth of ninhydrin single crystals by solvent evaporation method and its characterization for SHG and THG applications.

Ninhydrin is a well-known compound generally used in amino acid synthesis and also for detecting the latent fingerprints on porous surfaces. Single crystals can be grown by dissolving the compound in double distilled water at ambient temperature, and can be used as a potential material for second and third harmonic generation applications. The grown specimen was subjected to different characterization techniques in order to find out its suitability for device fabrication. Its lattice dimensions have been confirmed by X-ray powder diffraction and its crystalline quality has been assessed by high resolution X-ray diffraction and X-ray topography methods. The presence of functional groups was identified from HETCOR analysis and confirmed the absence of impurities during crystallization. Its optical properties have been examined by photoluminescence and birefringence analyses. Its thermal parameters such as thermal diffusivity, thermal conductivity and specific capacity have been carried out by following photopyroelectric method. Third order nonlinear optical measurements have been carried out using Z-scan technique and its nonlinear optical absorption coefficient has been determined.

Involvement of LPA1 receptor signaling in cerebral ischemia-induced neuropathic pain.

We demonstrated previously that the lysophosphatidic acid-1 (LPA1) receptor plays a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. The present study revealed that mild cerebral ischemia by left transient middle cerebral artery occlusion (tMCAO) for 15min causes the hypersensitive responses (paw withdrawal) to the nociception by electrical stimuli to the paw by the use of Neurometer Current Perception Threshold/C (CPT/C). The hypersensitivity or neuropathic pain was only observed by the stimulation with 250 and 2000, but not 5Hz, which are the characterized sine-wave frequencies of Aδ-, Aß- or C-fibers, respectively. The significant neuropathic pain was observed from day 2 through week 2 on the right paw after tMCAO, while there was slight but significant pain sensitivity on the left paw at day 7. The neuropathic pain on the contralateral side at week 2 after tMCAO was completely abolished in LPA1(-/-) mice. These results suggest that LPA1 receptor signaling plays key roles in the development of central neuropathic pain following cerebral ischemia as well as the peripheral neuropathic pain following partial sciatic nerve injury.

Epidemiology and outcome of non viral keratitis.

This cross sectional study was done to evaluate the epidemiological pattern of fungal keratitis. It was done among 120 patients, selected randomly, at the cornea clinic, BNSB Eye Hospital, Mymensingh during the period of April 2008 to July 2008. In this study Chi-square test was done and males were predominant. Rural patients were mostly affected (93%). Study showed bacterial infection (41.66%) and fungal infection (39.16%). Among the fungus infected patient, 49% had history of trauma and 51% had no history of trauma (p>0.05), about 30% had vegetative injury and 19% had injury other than vegetative. Fungus was identified under microscope, 95% of which was confirmed by culture. Fifty five percent (55%) patients of bacterial infection and 31% patients of fungal infection attended with hypopyon (p<0.05). For fungal keratitis, trauma considered as the important predisposing factor. Immuno-compromised condition, ocular surface disease and climatic effects should also be kept in consideration. Microscopic investigation at the beginning of the treatment might help to achieve better outcome.

Smad7 induces hepatic metastasis in colorectal cancer.

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-beta type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-beta/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

Restoration of TGF-beta signalling reduces tumorigenicity in human lung cancer cells.

Members of the transforming growth factor-beta (TGF-beta) family regulate a wide range of biological processes including cell proliferation, migration, differentiation, apoptosis, and extracellular matrix deposition. Resistance to TGF-beta-mediated tumour suppressor function in human lung cancer may occur through the loss of type II receptor (TbetaRII) expression. In this study, we investigated the expression pattern of TbetaRII in human lung cancer tissues by RT-PCR and Western blot analyses. We observed downregulation of TbetaRII in 30 out of 46 NSCLC samples (65%) by semiquantitative RT-PCR. Western blot analyses with tumour lysates showed reduced expression of TbetaRII in 77% cases. We also determined the effect of TbetaRII expression in lung adenocarcinoma cell line (VMRC-LCD) that is not responsive to TGF-beta due to lack of TbetaRII expression. Stable expression of TbetaRII in these cells restored TGF-beta-mediated effects including Smad2/3 and Smad4 complex formation, TGF-beta-responsive reporter gene activation, inhibition of cell proliferation and increased apoptosis. Clones expressing TbetaRII showed reduced colony formation in soft-agarose assay and significantly reduced tumorigenicity in athymic nude mice. Therefore, these results suggest that reestablishment of TGF-beta signalling in TbetaRII null cells by stable expression of TbetaRII can reverse malignant behaviour of cells and loss of TbetaRII expression may be involved in lung tumour progression.

Characterization of a proximal element in the rat preadipocyte factor-1 (Pref-1) gene promoter.

Preadipocyte factor-1 (Pref-1) was shown to negatively regulate adipocyte differentiation. We recently reported that ZOG, a rat homolog of Pref-1, was specifically expressed in the adrenal zona glomerulosa. Results of the investigation of Pref-1 expression in preadipocyte and in undifferentiated adrenal cortex suggested that down-regulation of Pref-1 gene was closely correlated with the differentiation process. In this study we demonstrate that an upstream region (from -76 to -47) of the rat Pref-1 gene was essential for its expression in adrenocortical carcinoma-derived H295R cells. A nucleotide sequence found in this region, GCGTGGGCGTGGGCGGGGG (Egr/GC-box), seemed to contain three elements, two early growth response (Egr) elements and one GC-box, overlapping each other. Mutations of four or five nucleotides in a 7-nucleotides-stretch in the midst of the Egr/GC-box eliminated the binding of Sp1/3, abolished the activation by Egr-factor(s) and diminished the Pref-1 promoter activity. When mutations were introduced into the outside of the middle portion, the binding of Sp1/3 to the Egr/GC-box was abolished similarly. However, the decrease in the promoter activity was less than that found with the construct mutated at the middle. These results indicated that an element present at the 7-nucleotides-stretch in the midst of the Egr/GC-box might be important for the Pref-1 promoter activity, and this proximal element was possibly activated by a still-unidentified nuclear factor(s). This element would function as the promoter of the Pref-1 gene in H295R cells, but not in HeLa cells.

An outbreak of gangrenous syndrome among buffaloes and cattle in West Bengal: clinicopathological studies.

Gangrenous syndrome/Degnala disease was recorded in a large number of buffaloes and cattle in Murshidabad district of West Bengal, India. Fusarium spp. had been isolated from the mouldy paddy straw which were fed to the animals. There was a reduction in the incidence of the disease following withdrawal of the mouldy paddy straw. Histopathological examination showed necrosis and loss of architectural details in the skin.

Cloning of a novel kinase (SIK) of the SNF1/AMPK family from high salt diet-treated rat adrenal.

PCR-coupled cDNA subtraction hybridization was adapted to identify the genes expressed in the adrenocortical tissues from high salt diet-treated rat. A novel cDNA clone, termed salt-inducible kinase (SIK), encoding a polypeptide (776 amino acids) with significant similarity to protein serine/ threonine kinases in the SNF1/AMPK family was isolated. An in vitro kinase assay demonstrated that SIK protein had autophosphorylation activity. Northern blot revealed that SIK mRNA levels were markedly augmented by ACTH treatment both in rat adrenal glands and in Y1 cells. SIK may play an important role in the regulation of adrenocortical functions in response to high plasma salt and ACTH stimulation.

Cloning of a membrane-spanning protein with epidermal growth factor-like repeat motifs from adrenal glomerulosa cells.

The three zones of adrenal cortex are thought to arise from a single multipotential stem cell, but the mechanisms underlying the zonal differentiation during embryonic development of adrenal cortex are poorly understood. Employing subtraction cloning strategy, we isolated three distinct clones that were specifically expressed in the rat glomerulosa zone. One clone, named zona glomerulosa specific clone, encoded a membrane-spanning protein with a signal peptide at the N-terminus, six epidermal growth factor-like repeat motifs, and a transmembrane domain near the C-terminus. It was identified as a rat homolog of preadipocyte factor-1 (Pref-1), a factor involved in maintaining the undifferentiated status of preadipocyte. Immunohistochemical studies confirmed the presence of Pref-1 protein in the glomerulosa zone. Detailed examination revealed that the zone is divided into two layers; the first is a few-cells-thick layer present underneath the capsule (expressing both Pref-1 protein and aldosterone synthase cytochrome P450), and the second layer is beneath the first (containing Pref-1 protein but not aldosterone synthase). Moreover, another cell layer was found beneath the second layer and above the fasciculata zone, whose cells contained no Pref-1 protein, aldosterone synthase, or 11beta-hydroxylase. These findings suggest that a recently reported aldosterone synthase- and 11beta-hydroxylase-less cell layer between the two zones is composed of two kinds of cell: Pref-1 protein-positive and -negative cells. The level of Pref-1 message in the adrenal glands of animals having various pituitary-adrenal axis activities, as well as various plasma salt concentrations, correlated with the total number of glomerulosa cells. However, the specific content of Pref-1 message in a cell was fairly constant. When the adrenal gland was surgically enucleated and the remaining capsule regenerated, the level of Pref-1 transcript was significantly suppressed at the early phase. At this phase, only a minor population of the cortical cells expressed Pref-1 protein, most of these cells already expressing a fasciculata/reticularis-specific marker, inner zone antigen. These findings suggest that the capsular cells, mostly composed of the glomerulosa cells, may have potential for differentiating into other zones' cells, and the down-regulation of Pref-1 expression may be an important step in the adrenal zonal differentiation.

Implication of ZOG protein (zona glomerulosa-specific protein) in zone development of the adrenal cortex.

The three zones of adrenal cortex are thought to arise from a single multipotential stem cell. Immunohistochemical studies of fetal and adult adrenals using an antibody against a previously-cloned ZOG protein, a rat homolog of Pref-1, were conducted to explore its roles in the differentiation of cortical tissues. At the early embryonic stage, ZOG was already expressed in adrenogonadal primordial cells. The ZOG-positive cells gradually formed the adrenal primordium by E14.5. By E17.5 the expression was repressed in the inner part of the aggregate and these cells began to express CYP11B1. The ZOG-positive cells at this stage existed at the periphery of the aggregate but they did not express CYP11B2 yet. Not until E20.5 did the aldosteronogenic cells appear among the ZOG-positive cells at the outermost part of the gland. Based on these and the other findings the zonal development of the adrenal cortex is discussed.

Zona glomerulosa-specific factor: cloning and function.

The rat adrenal cortex is composed of three zones: the zona glomerulosa, the zona fasciculata, and the zona reticularis. Several investigators have claimed the presence of a zona intermedia between the zonae glomerulosa and fasciculata. The cells of zona glomerulosa, a few layers of cells just beneath the adrenal capsule, synthesize and secrete aldosterone, whereas those of zonae fasciculata and reticularis secrete glucocorticoids and androgens, respectively. The function of the cells in zona intermedia is unclear, because they express neither aldosterone synthase nor 11 beta-hydroxylase. To investigate the mechanism underlying the zonal differentiation of adrenocortical steroidogenesis, attempts have been made to isolate and characterize zone-specifically expressed proteins such as steroidogenic enzymes and putative regulatory factors. Having subtracted the mRNAs present in the decapsulated adrenal gland from those in the adrenal capsule, we successfully isolated three distinct clones, each specifically expressed in the zona glomerulosa. One clone encoded a protein named zona glomerulosa-specific factor (ZOG), which had a putative signal peptide at the N-terminus, six tandem epidermal growth factor (EGF)-like repeats, and a transmembrane domain in the central portion and a short cytosolic stretch at the C-terminus. Immunohistochemical studies using the antibody raised against ZOG confirmed the presence of the protein in all layers of cells in the zona glomerulosa. In contrast, cells possessing aldosterone synthase were present only in the periphery of zona glomerulosa, just beneath the capsule. These findings suggest that there are at least two kinds of zona glomerulosa cells in the rat adrenal cortex, one expressing aldosterone synthase as well as ZOG, and another expressing only ZOG. The cells in the zona intermedia did not express ZOG, aldosterone synthase, or 11 beta-hydroxylase, but did express Ad4BP. ZOG was not detected in zonae fasciculata and reticularis where 11 beta-hydroxylase was present.

Polyadenylation-mediated translational regulation of maternal P450(11beta) mRNA in frog oocytes.

Northern blot analysis of bullfrog tissues using a cDNA probe of cytochrome P450(11beta) showed that a large amount of message was present in the ovary as well as in the adrenal tissue. Two kinds of mRNA of different sizes were found in the ovary. Sequence determination of the two cDNAs and analysis by reverse-transcription polymerase chain reaction indicated that the protein encoded by the larger mRNA was identical to the adrenal enzyme, while the protein encoded by the smaller had a truncated sequence lacking an extension peptide necessary for the protein transport to the mitochondria. The mRNAs were present in the oocytes but not in the follicular cells, and their content in an oocyte varied little during its maturation. Immunoblot analyses of the mitochondrial fraction of oocytes failed to demonstrate the presence of P450(11beta) protein. In contrast the eggs were found to contain a large amount of enzymatically active protein. Interestingly the mRNA has a cis-element called cytoplasmic polyadenylation element at its 3' untranslated region. When poly(A) tails of the message prepared from eggs and oocytes were examined by RNase H digestion or reverse-transcription polymerase chain reaction, those of eggs were about 150 nucleotides longer than those of oocytes. These results suggest that translation of the message is stimulated during the oocyte maturation as a result of enhanced polyadenylation at its 3'-end. Finally a finding is presented that progesterone was converted to 11beta-hydroxyprogesterone by the frog P450(11beta), implying that the enzyme expressed in eggs may control a level of progesterone which is needed to initiate the oocyte maturation.

Cytochrome P450(11 beta): ectopic expression in oocyte and its physiological implication.

Cytochrome P450(11 beta) (steroid 11 beta-hydroxylase) catalyzes an essential step for biosynthesis of glucocorticoid as well as mineralocorticoid, and therefore is believed to exist specifically in the adrenocortical mitochondria. We previously isolated a complementary DNA clone encoding this enzyme from bullfrog (Rana catesbeiana) adrenal tissues and characterized the enzyme expressed in COS cells. To explore the tissue distribution of the enzyme we performed Northern blot analysis. Unexpectedly we found that a large amount of the mRNA exists in the ovary as well as the adrenal gland. It was present in oocytes, but not in follicular cells. The P450 protein was not found during early stages of oocyte development, and at the final stage of maturation the message appeared to be translated. Evidence was obtained that the stage-dependent translation of the mRNA was due to the regulated polyadenylation at its 3'-end. In oocytes of various organisms several maternal mRNAs are known to add poly(A) tails in the cytoplasm during the progesterone-induced maturation.

Cytochrome P450(11 beta): structure-function relationship of the enzyme and its involvement in blood pressure regulation.

Cytochrome P450(11 beta) is deeply involved in the final steps of biosynthesis of mineralocorticoids. This paper deals with following issues about this enzyme. (1) The structure and function of the enzymes of various animal species are discussed. By making alignment of amino acid sequences of the enzymes, we identified peptide domains essential for the enzyme actions such as a putative steroid binding domain and a heme binding region. Estimates of molecular similarity among the P450(11 beta) family enzymes suggested that the enzymes having both 11 beta-hydroxylation activity and aldosterone (ALDO) synthetic activity of certain animals such as frog, cattle and pig are more similar to the ALDO synthases of the other animals, such as rat, mouse and human, than the 11 beta-hydroxylases of these animals. (2) The molecular nature of the P450(11 beta) family enzymes of genetically hypertensive rats as well as adrenal regeneration hypertension (ARH) rats is examined. (i) Mutation was found in the P450(11 beta) gene of Dahl's salt-resistant normotensive rat. Steroidogenic activity expressed by the mutated gene accounted well for abnormal plasma levels of steroid hormones in this rat. (ii) 11 beta-, 18- and 19-Hydroxylation activities of adrenal mitochondrial prepared from spontaneously hypertensive rat (SHR), Wistar-Kyoto rat (WKY), and stroke-prone (SP)-SHR were not significantly different from each other. Levels of mRNA of ALDO synthase in adrenal glands of 50-week-old SHR was significantly lower than those of 10-week-old SHR, WKY and SHR-SP. (iii) No significant difference in 19-hydroxylation activity was found between adrenal mitochondria prepared from ARH rat and those from control rat. The level of message of ALDO synthase was lower in adrenal glands of ARH rat.

Frog cytochrome P-450 (11 beta,aldo), a single enzyme involved in the final steps of glucocorticoid and mineralocorticoid biosynthesis.

A cDNA for cytochrome P-450(11 beta,aldo) was cloned from a library of bullfrog interrenal tissue (tissue corresponding to the mammalian adrenal gland). The 1919-bp cDNA encoded a protein of 517 amino acids. Its amino acid sequence was highly similar to the sequences of bovine P-450(11 beta) and rat P-450(11 beta,aldo) when P-450(11 beta) family enzymes reported to date were examined. The enzyme expressed in COS7 cells had the 11 beta-hydroxylation, 18-hydroxylation activities and aldosterone synthetic activity. Northern-blot and immunoblot analyses suggested that a single P-450(11 beta) enzyme was expressed in bullfrog interrenal tissue. These results suggest that a single enzyme catalyzes the final steps of glucocorticoid and mineralocorticoid biosynthesis in bullfrog interrenal tissue as in bovine adrenal gland. A phylogenetic tree of CYP11B genes suggests that the frog enzyme diverged at an earlier evolutionary time from other vertebrate enzymes. Immunohistochemical and in situ hybridization studies indicated that steroidogenic cells existed in the outer region of interrenal tissue more densely than in the inner region, whereas some medullary cells made clusters like islets. Most of the cells were diffusely distributed in the tissue.