Electron microscope studies on the development of the external zone of the mouse median eminence

Summary The development of the external zone of the median eminence of the mouse was studied in the electron microscope. The examination follows the development of the embryo from the 15th day of the gestation period and the juvenile growth until 24 days of age.Single terminals of the tubero-infundibular neurons of the external zone were found to extend to the outer basement membrane of the perivascular space of the portal primary capillary plexus in the 16 day-old embryo. In the 18 day-old embryo a narrow external zone has developed. Organization of the external zone into the adult pattern is accomplished at the age of three to four weeks. Small agranular as well as large granular vesicles are present in the tubero-infundibular nerve terminals even in the 16 day-old embryo.Changes in the organization of the nerve endings along the outer perivascular basement membrane in relation to the ependymal vascular feet were considered.     

Attenuation of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice achieved by lowering dietary lipid

We examined the development of cardiac hypertrophy in juvenile visceral steatosis (JVS) mice, a model of systemic carnitine deficiency, by varying the amount of lipid in the diet. Cardiac hypertrophy was markedly attenuated by decreasing soy bean oil (SBO) from 5% (w/w) to 1%. Triglyceride contents of the ventricles of JVS mice fed 1% SBO were significantly lower than in JVS mice fed 5% SBO. The addition of medium-chain triglycerides metabolically utilized by JVS mice did not affect the development of cardiac hypertrophy. On the other hand, the mRNA levels of atrial natriuretic peptide and skeletal alpha-actin, which are related to cardiac hypertrophy, were also attenuated by decreasing lipid in the diet. Adenylate energy charge and creatine phosphate in the heart of JVS mice at the early stage of hypertrophy were not significantly different from control mice given the same laboratory chow (4.6% of lipid). Although urinary prostaglandin F(2alpha) levels were found to be increased in JVS mice at 15 days of age when they developed cardiac hypertrophy, administration of aspirin was not efficacious. We, therefore, propose that the proportion of lipid in the diet is important in the development of cardiac hypertrophy in carnitine-deficient JVS mice, and that this is not related to prostaglandin formation.     

Temporal and differential proteomic profile of molecular mediators associated with chronic and acute wound healing

The underlying pathophysiology of nonhealing chronic wounds is poorly understood due to the changes occurring at the gene level and the complexity arising in their proteomic profile. Here, we elucidated the temporal and differential profile of the normal and diabetic wound-healing mediators along with their interactions and associated pathways. Skin tissues corresponding to normal and diabetic wounds were isolated at Days 0, 3, 6, and 9 representing different healing phases. Temporal gene expression was analyzed by quantitative real-time PCR. Concurrently, differential protein patterns in the wound tissues were identified by Nano LC-ESI-TOF mass spectrometry and later confirmed by Western blot analysis. Gene ontology annotation, protein-protein interaction, and protein pathway analysis were performed using DAVID, PANTHER, and STRING bioinformatics resources. Uniquely identified proteins (complement C3, amyloid beta precursor protein, and cytoplasmic linker associated protein 2) in the diabetic wound tissue implied that these proteins are involved in the pathogenesis of diabetic wound. They exhibit enhanced catalytic activity, trigger pathways linked with inflammation, and negatively regulate wound healing. However, in the normal wound tissue, axin 1, chondroitin sulfate proteoglycan 4, and sphingosine-1-phosphate receptor were identified, which are involved in proliferation, angiogenesis, and remodeling. Our findings demonstrate the correlation between elevated gene expression of tumor necrosis factor-α, interleukin (IL)-1β, and identified mediators: aryl hydrocarbon receptor nuclear translocator, 5′-aminolevulinate synthase 2, and CXC-family, that inflicted an inflammatory response by activating downstream MAPK, JAK-STAT, and NF-κB pathways. Similarly, in normal wound tissue, the upregulated IL-4 and hepatocyte growth factor levels in conjunction with the identified proteins, serine/threonine-protein kinase mTOR and peroxisome proliferator-activated receptor gamma, played a significant role in the cellular response to platelet-derived growth factor stimulus, dermal epithelialization, and cell proliferation, processes associated with the repair mechanism. Furthermore, Western blot analysis indicated elevated levels of inflammatory markers and reduced levels of proliferative and angiogenic factors in the diabetic wound.     

Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System

Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli’s natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or “coliroids,” rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.     

Attenuation of DNA damage and mRNA gene expression in hypoxic rats using natural antioxidants

This study aimed to explore the efficiency of carnosine (Cs) and/or l ‐arginine (Agn) in the downregulation of apoptotic and inflammatory molecule expression and DNA damage caused hepatic injury in response to sodium nitrite (Sd)‐induced hypoxia in rats. Rats were injected with Sd; Agn or/and Cs were administrated prior to Sd intoxication. Sd significantly decreased hemoglobin concentration and Bcl‐2 mRNA expression, while increased expressions of apoptotic markers (Bax and caspase), tumor necrosis factor‐α, nuclear factor kappa B, and C‐reactive protein and the oxidative DNA damage in hepatic tissue. Moreover, administration of Agn or/and Cs exhibited a modulation of the previous parameters. However, concurrent treatment with the forementioned antioxidants modulated these levels. It was concluded that the treatment with the combination of Agn and Cs was the most effective regimen in ameliorating Sd toxicity accompanied by hypoxic stress.     

<Emphasis Type="Italic">Cyclospora cayetanensis</Emphasis> and other intestinal parasites associated with diarrhea in a rural area of Jordan

Cryptosporidium spp. and Cyclospora cayetanensis have emerged as important causes of epidemic and endemic diarrhea in immunocompetent and immunocompromised hosts. The exact modes of transmission in certain rural areas are still unclear. Reports of water-borne and food-borne outbreaks suggest that fecally contaminated water or food acts as a vehicle of transmission. Two hundred stool samples of patients with gastroenteritis from four health centers in a rural area of Jordan were examined using formalin-ethyl acetate concentration, wet preparation, and modified acid-fast staining methods. Oocysts of C. cayetanensis and Cryptosporidium spp. were found in 6% and 8% of the samples respectively, mainly those of children. Parasites such as Entamoeba histolytica, Giardia lamblia, and other enteropathogens were also observed. The results reflect the seasonality of natural cyclosporiasis and cryptosporidiosis, being higher in the spring. The risk factors that were found by the Fisher test to be significant and might be associated with illness are the source of drinking water, contact with animals, and eating unwashed vegetables (p<0.028, p<0.0005, p<0.00005 respectively).     

Complete genome sequence of Burkholderia rhizoxinica, an Endosymbiont of Rhizopus microsporus

Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic fungus Rhizopus microsporus. The vertically transmitted endosymbiont not only delivers the antimitotic macrolide rhizoxin to its host but is also essential for vegetative spore formation of the fungus. To shed light on the genetic equipment of this model organism, we sequenced the whole genome of B. rhizoxinica HKI 0454, thus providing the first genomic insight into an intracellular mutualist of a fungal species. The 3.75-Mb genome consists of a chromosome and two strain-specific plasmids. The primary metabolism appears to be specialized for the uptake of fungal metabolites. Besides the rhizoxin biosynthesis gene cluster, there are 14 loci coding for nonribosomal peptide synthetase (NRPS) assembly lines, which represent novel targets for genomic mining of cryptic natural products. Furthermore, the endosymbionts are equipped with a repertoire of virulence-related factors, which can now be studied to elucidate molecular mechanisms underlying bacterial-fungal interaction.