Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.     

Nitrate regulation of anaerobic respiratory gene expression in narX deletion mutants of Escherichia coli K-12.

Previous studies have shown that narL+ is required for nitrate regulation of anaerobic respiratory enzyme synthesis, including formate dehydrogenase-N, nitrate reductase, and fumarate reductase. Insertions in the closely linked narX gene decrease, but do not abolish, nitrate regulation of anaerobic enzyme synthesis. Analysis of sequence similarities suggests that NarX and NarL comprise a two-component regulatory pair. We constructed lacZ operon and gene fusions to investigate the operon structure of narXL. We found evidence for a complex operon with at least two promoters; PXL-narX-PL-narL. We also investigated the role of NarX in nitrate regulation of anaerobic respiratory enzyme synthesis by constructing nonpolar loss of function narX alleles. These deletions were studied on narL+ lambda specialized transducing bacteriophage. The narX deletions had no effect on nitrate regulation in delta (narXL) strains. This finding suggest that the subtle effects of previously studied narX insertions are due to decreased expression of narL and that narX+ is not essential for normal nitrate regulation. The role of NarX in nitrate regulation remains to be determined.     

An analog of ACTH/MSH (4–9), ORG-2766, reduces cerebral uptake of morphine

The uptake of morphine was significantly reduced in most regions of the brains of conscious, unrestrained rats within 10 minutes after treatment with an analog of ACTH/MSH (4–9), ORG-2766. The effect was most obvious in regions with significant densities of enkephalin receptors, namely basal ganglia, hippocampus and cortex. The results explain, in part, how some fragments and analogs of ACTH/MSH may antagonize behavioral actions of morphine, even though some of these peptides lack significant opiate receptor binding properties. We believe that this effect of ORG-2766 is related to an action on the permeability characteristics of the brain microvasculature. The underlying mechanism is unknown.     

Selective-Differential Medium for Isolation and Differentiation of Pectinatus from Other Brewery Microorganisms

An agar medium, LL-agar (lactate-lead acetate) was designed to selectively differentiate members of the genus Pectinatus (S. Y. Lee, M. S. Mabee, and N. O. Jangaard, Int. J. Syst. Bacteriol. 28:582-594, 1978; S. Y. Lee, M. S. Mabee, N. O. Jangaard, and E. K. Horiuchi, J. Inst. Brew. 86:28-30, 1980) from other brewery microorganisms. Selectivity was achieved by the use of sodium lactate as the sole source of carbon and phenylethyl alcohol as an inhibitor for aerobic gram-negative bacteria and yeast. Differentiation was established by the introduction of lead acetate into the medium, which reacted with the H₂S liberated by Pectinatus and resulted in a blackening of the Pectinatus colonies while the other brewery organisms, when present, remained white. In combination with the Lee tube (J. E. Ogg, S. Y. Lee, and B. J. Ogg, Can. J. Microbiol. 25:987-990, 1979) and this medium, isolation of Pectinatus organisms from beer samples was accomplished with convenience and simplicity.     

1,25-Dihydroxyvitamin D3 increases serum levels of the vitamin K-dependent bone protein

1,25-dihydroxyvitamin D₃ increases serum levels of bone Gla protein (BGP). The maximal increase occurs 12 h after injection and is given by 350 ng 1,25(OH)₂D₃ per 180 g body weight. In both 2 and 11 month-old male rats, the maximal increase is about 3 times the normal level, while in 2 month old female rats, the maximal increase is 2 times the normal level. These effects of 1,25(OH)₂D₃ in rats parallel the previously described effects of the vitamin on BGP secretion by rat osteosarcoma cells in culture.BGP is the first bone-specific protein whose synthesis in animals is dramatically increased by 1,25(OH)₂D₃. The possible functions of BGP in the biological actions of 1,25(OH)₂D₃ on bone are discussed.     

Suppression of a thymus dependent humoral response in mice by concanavalin A in vivo

Mice treated with Concanavalin A prior to immunization with sheep erthyrocytes exhibit a markedly reduced plaque forming spleen cell response. This immunosuppressive effect could be reversed by using higher doses of antigen or priming the animals with nonimmunizing doses of antigen prior to Concanavalin A injection designed to either by-pass or enhance thymus derived lymphocyte functions. It was also demonstrated that Concanavalin A in vivo activated the thymus derived lymphocyte subpopulation in the spleen, and this activation was dose dependent and correlated with the immunosuppression observed. Animals injected with Concanavalin A at various times prior to whole body lethal irradiation would not support the plaque forming cell response of adoptively transferred normal syngeneic spleen cells. This effect was shown to be time and dose of Concanavalin A dependent. It was also shown that the route of injection of Concanavalin A prior to irradiation determined the results observed, in that the intravenous route resulted in the suppression of transferred cells, while the intraperitoneal route showed no effect. It is suggested that Concanavalin A induced immunosuppression of the humoral, thymus dependent immune response in mice results for the activation of a subpopulation of thymus derived suppressors cells, and that the effect is short lived, radiation resistant, and dose of Concanavalin A and antigen dependent.     

Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.     

HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days—Implications for HIV Remission

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to ‘purge’ the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different ‘reactivation founder’ viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.     

pHG165: A pBR322 copy number derivative of pUC8 for cloning and expression

During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.     

Susceptibility of dopamine D5 receptor targeted mice to cysteamine

BACKGROUND: Recently we demonstrated that gastric mucosa of rats can synthesize, store and release dopamine. Out of five different subtypes, mRNA of D5 (=D1b) dopamine receptor is very abundant in the gastric epithelium. D1 receptor selective dopamine agonists have been shown to protect against experimental gastro-duodenal lesions. AIMS: To test the hypothesis that protective effects of dopamine involve D5 receptors, mucosal lesions were induced in D5 receptor deficient (KO) and wild-type (WT) mice using cysteamine. Morphology and gastric acid secretion of D5 KO mice were also studied. METHODS: Single doses of 600 mg/kg, 300 mg/kg cysteamine or vehicle were administered subcutaneously to fasted animals. After 24 h, number and severity of gastro-duodenal lesions were analyzed. Basal and histamine-induced maximal gastric acid output were measured by a stomach-sac wash-through method. RESULTS: All the KOs in the 600 mg/kg cysteamine group died within 4 h showing symptoms of toxicity while three out of four WTs survived (P<0.05). Mortality after 300 mg/kg cysteamine was significantly higher in KOs versus the WTs: 6/14 versus 2/11, P<0.05. Gastric lesion-index was also significantly higher in KOs (median, middle quartile): four (3-9) versus 0 (0-0), P<0.05. Duodenal lesions did not develop from this single dose of cysteamine in either genotype. Basal and histamine-induced maximal gastric acid output were comparable in the two genotypes. CONCLUSIONS: This study demonstrates that loss of D5 receptor causes mucosal vulnerability and increased toxicity of cysteamine in genetically manipulated mice. Thus, D5 receptor subtype is indeed likely to be involved in protective effects of dopamine in the stomach.