Translation of some maize small heat shock proteins is initiated from internal in-frame AUGs

Etiolated maize radicles (inbred Oh43) subjected to a brief heat shock synthesize a family of small heat shock proteins (approximately 18 kD) that is composed of at least 12 members. We previously described the cDNA-derived sequence of three maize shsp mRNAs (cMHSP18-1, cMHSP18-3, and cMHSP18-9). In this report, we demonstrate that the mRNA transcribed in vitro from one of these cDNAs (cMHSP18-9) is responsible for the synthesis of three members of the shsp family, and we suggest that cMHSP18-3 may be responsible for the synthesis of three additional members and cMHSP18-1 for the synthesis of two other members of this family. The fact that these genes do not contain introns, coupled with the observations reported herein, suggest that maize may have established another method of using a single gene to produce a number of different proteins.     

Inhibition of experimental autoimmune encephalomyelitis in Lewis rats by nasal administration of encephalitogenic MBP peptides: synergistic effects of MBP 68-86 and 87-99

Induction of mucosal tolerance by inhalation of soluble peptides with defined T cell epitopes is receiving much attention as a means of specifically down-regulating pathogenic T cell reactivities in autoimmune and allergic disorders. Experimental autoimmune encephalomyelitis (EAE) induced in the Lewis rat by immunization with myelin basic protein (MBP) and Freund's adjuvant (CFA) is mediated by CD4+ T cells specific for the MBP amino acid sequences 68-86 and 87-99. To further define the principles of nasal tolerance induction, we generated three different MBP peptides (MBP 68-86, 87-99 and the non-encephalitogenic peptide 110-128), and evaluated whether their nasal administration on day -11, -10, -9, -8 and -7 prior to immunization with guinea pig MBP (gp-MBP) + CFA confers protection to Lewis rat EAE. Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 at doses used conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage compared to that being used for individual peptides. Rats tolerized with MBP 68-86 + 87-99 nasally showed decreased T cell responses to MBP reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86 + 87-99 also had abrogated MBP-reactive IFN-gamma and tumor necrosis factor-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive transforming growth factor-beta and IL-4 mRNA expressing cells were observed in the two groups. Nasal administration of MBP 68-86 + 87-99 only slightly inhibited guinea pig spinal cord homogenate-induced EAE, and passive transfer of spleen mononuclear cells from MBP 68-86 + 87-99-tolerized rats did not protect na?ve rats from EAE. Finally, we show that nasal administration of MBP 68-86 + 87-99 can reverse ongoing EAE induced with gp-MBP, although higher doses are required compared to the dosage needed for prevention. In conclusion, nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.     

The discovery and structure-activity relationships of nonpeptide, low molecular weight antagonists selective for the endothelin ET(B) receptor

The systematic modification of the ETA selective N-(5-isoxazolyl)benzene-sulfonamide endothelin antagonists to give ETB selective antagonists is reported. The reversal in selectivity was brought about by substitution of the 4-position with aryl and substituted aryl groups. Of all the aromatic substituents studied, the para-tolyl group gave rise to the most active and selective ETB antagonist. Larger substituents caused a decrease in both ETB activity and selectivity. A similar trend was observed by substitution at the 5-position of the N-(5-isoxazolyl)-2-thiophenesulfonamide ETA receptor antagonists. The para-tolyl group was again found to be optimal for the ETB activity and selectivity. The structural features that were found to be favorable for binding to the ETB receptor, that is, the presence of a linear, conjugated pi-system of definite shape and size, have been successfully incorporated into the design of ETB selective polycyclic aromatic sulfonamides antagonists.     

Thoracic duct-cutaneous fistula in a patient with cirrhosis of the liver: successful treatment with a transjugular intrahepatic portosystemic shunt

Patients with cirrhosis of the liver have increased hepatic and gastrointestinal lymph flow that may contribute to the formation of ascites and pleural effusions. Increased lymph flow, which is due to postsinusoidal portal hypertension, causes a high rate of flow through the thoracic duct. Because of the high flow rates, disrupted lymphatic vessels in patients with cirrhosis of the liver may fail to close, a situation that results in chylous ascites, pleural effusions, or chylous fistulas. Chylous fistulas deplete proteins, fluid, and lymphocytes and thus lead to volume depletion and coagulopathy. Herein we describe an unusual case in which a high-output traumatic thoracic duct-cutaneous fistula developed in a patient with cirrhosis and led to volume depletion and coagulopathy. Correction of the portal hypertension with placement of a transjugular intrahepatic portosystemic shunt led to closure of the fistula and normalization of accompanying metabolic abnormalities.     

Risk factors for the development of oesophageal cancer as a second primary tumour

Exposure to irradiation or chemotherapy as well as prolonged exposure to risk factors, such as alcohol and tobacco, may induce a second primary carcinoma of the oesophagus. To estimate the potential risk of previous treatment regimens, we performed a case-control study. In the Tumour Registry of The Netherlands Cancer Institute, from 1955, 27 cases of squamous cell carcinoma of the oesophagus were identified following treatment for malignant lymphoma (n = 11), breast cancer (n = 8) and lung cancer (n = 8). The median interval was 6.6 years (range 1-16). Preferably 3 controls from the same tumour registry were matched to each case on the basis of sex, age, primary tumour, location of primary treatment (academic or general hospital), calendar year at diagnosis of primary tumour and duration of follow-up. Clinical data and details of treatment were obtained from the medical records. In patients who had smoked for more than 5 years, there was a 3.2-fold increased risk of oesophageal carcinoma (P = 0.04); for those with a regular alcohol intake the relative risk was 3.3 (P = 0.01). There was no significant relationship between irradiation of the mediastinum and subsequent risk for oesophageal cancer. The number of chemotherapy-treated patients was too small to calculate the relative risk associated with cytostatic drugs. In conclusion, oesophageal cancer as second primary cancer is extremely rare. Risk factors include the well known abuse of alcohol and tobacco. No significant relationship with previous mediastinal irradiation could be demonstrated.     

The biosynthesis of mycolic acids in Mycobacterium tuberculosis. Enzymatic methyl(ene) transfer to acyl carrier protein bound meromycolic acid in vitro

A closely related family of enzymes from Mycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived from S-adenosyl-L-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of [methyl-3H]SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S-adenosyl-L-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M. smegmatis or M. tuberculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates. These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate. Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids. These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain.     

3,5-dihydroxyphenylglycine is a highly selective agonist for phosphoinositide-linked metabotropic glutamate receptors in the rat hippocampus

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein-coupled glutamate receptors that are linked to multiple second messenger systems in the CNS. In this study the selectivity of mGluR agonists for different mGluR second messenger effects was characterized in slices of the rat hippocampus. The mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid and (2S,3S,4S)alpha-(carboxycyclopropyl)glycine produced multiple effects on second messengers that included enhanced phosphoinositide hydrolysis in both adult and neonatal rat hippocampus, inhibition of forskolin-stimulated cyclic AMP (cAMP) formation in adult tissue, and increases in basal cAMP formation in the neonatal hippocampus. In contrast, 3,5-dihydroxyphenylglycine was potent and effective in increasing phosphoinositide hydrolysis in both adult and neonatal hippocampus but unlike the other mGluR agonists did not inhibit forskolin-stimulated cAMP formation (in the adult) or substantially enhance basal cAMP formation (in the neonate). Thus, in the rat hippocampus mGluR agonist-mediated increases or decreases in cAMP formation are not secondary to mGluR-mediated changes in phosphoinositide hydrolysis. Furthermore, 3,5-dihydroxyphenylglycine can be used to activate subpopulations of mGluRs coupled to phosphoinositide hydrolysis with minimal effects on cAMP-mGluR second messenger systems.     

Synthesis and in vitro antitumor activity of some amino-deoxy 7-hexofuranosylpyrrolo[2,3-d]pyrimidines

7-(6-amino-6-deoxy-beta-D-glucofuranosyl)-5-cyanopyrrolo[2,3 -d]pyrimidine (22) and 7-(3-amino-methyl-3-deoxy-beta-D-allofuranosyl)-5- cyanopyrrolo[2,3-d]pyrimidine (28) were synthesized by sequentially coupling silylated 4-amino-6-bromo-5- cyanopyrrolo[2,3-d]pyrimidine with the corresponding protected sugars 9 and 17, followed by deblocking and catalytic hydrogenation. Conversion of the 5-nitrile in 22 and 28 into a carboxamide gave the corresponding sangivamycin derivatives 23 and 29. Whereas 5'-aminomethyl nucleosides 22 and 23 inhibited the growth of four different human tumor cell lines at microM concentrations, the 3'-aminomethyl analogs 28 and 29 were much less active against these cells.