Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis

The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromosomal anomalies in stage D1 prostate adenocarcinoma primary tumors and lymph node metastases detected by fluorescence in situ hybridization

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N2-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 mumol/mol creatinine. The present results indicate that the peak found with elution from the C18 column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.     

The emerging role of glutamate as a new pharmacologic target.

Neurotransmitters serve as functional substrates for receptor signaling, as well as dynamic mediators of psychotropic drug activity. Identifying dysregulations in specific neurotransmitter systems has greatly contributed to increased understanding of the pathophysiology of various disorders. Several primary neurotransmitters have received the greatest attention as they relate to neuropsychiatric disorders--namely, acetylcholine, and the biogenic monoamines (norepinephrine, dopamine, and serotonin). In addition to these 'classic' neurotransmitters, various amino acids, such as gamma-aminobutyric acid (GABA), glutamate, aspartate, and glycine, have also been identified as having neurotransmitter properties within the central nervous system (CNS). The excitatory amino acid glutamate, the most prevalent neurotransmitter in the CNS and a primary mediator of excitatory synaptic transmission, has begun receiving attention. Clinicians will benefit from this overview of glutamate's neurobiology, pharmacological activity, and suspected involvement in the pathophysiology of various disease states. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Localization of corticotropin-releasing factor in primary and secondary lymphoid organs of the rat

Cells of the immune system produce a variety of neuropeptides or peptide hormones, either constitutively or upon induction, and possess specific neuropeptide receptors that display ligand-receptor interactions similar to those described in the central nervous system (CNS). These findings suggest that specific subsets of lymphoid cells can produce and respond to peptides previously thought to be principally neural mediators. Recently, corticotropin releasing factor (CRF) mRNA was detected in the rat thymus and spleen, although the cells that synthesize CRF were not identified. We examined the localization of CRF and its mRNA in the rat spleen, thymus, and mesenteric lymph nodes using immunocytochemistry (ICC) and in situ hybridization (ISH), respectively. Immunoreactive CRF was present in cells in the marginal zone and red pulp of the spleen, in connective tissue septa and the subcapsular region of the thymus, and in the medullary cords and sinuses of the mesenteric lymph nodes. Dual ICC/ISH for CRF and its mRNA, respectively, demonstrated CRF mRNA over CRF-immunoreactive cells, suggesting CRF synthesis. Double-label ICC for CRF and markers for specific immunocyte subsets suggest that CRF+ cells in the spleen and thymus are macrophages. CRF+ cells in primary and secondary lymphoid organs reside in compartments that are innervated by sympathetic nerves, and some cells appears to be contacted by noradrenergic sympathetic nerve fibers, suggesting that CRF release may be influenced by the sympathetic nervous system, as it is in the hypothalamo-pituitary-adrenal axis. The presence of CRF in organs of the immune system suggests that this neuropeptide may modulate immune functions after paracrine release.     

Analysis of physician attitudes concerning requests for autopsy

To quantify the role of failure to request consent as a determinant of the autopsy rate, questionnaires asking whether an autopsy had been requested and the reasons for that decision were distributed to primary physicians after each death in a consecutive series of 75 patient deaths. Autopsies were requested in only 56% of cases. Common reasons to request an autopsy included unanswered medical questions (37%), medical education (22%), research protocol participation (16%), or routine policy (14%). When autopsies were not requested, the most common reason was the belief that there were no outstanding medical questions (64%). Follow-up interviews with 14 oncologists and hematologists revealed that 8 generally request autopsies (usually to contribute to medical education or to discover unexpected findings) and 6 generally do not (usually because no unexpected findings are anticipated). Attempts to increase the current low autopsy rate should address the question of when and why physicians are willing to request this procedure.