A procedure for easy removal of acrylamide gel rods from the casting glass tubes

A new method specific for the determination of subpicomole quantities of tryptophan has been developed by elaboration of the Pictet-Spengler reaction. It permitted reproducible quantitation of tryptophan in less than 1 μl of plasma ultrafiltrate or 1 mg of brain tissue. Samples deproteinized by trichloroacetic acid were boiled for 15 min with formaldehyde and potassium ferricyanide at controlled acidity, where tryptophan was converted to a single new product identified as 9-hydroxymethyl-β-carboline. It was quantitated by either direct spectrofluorometry or a reversed-phase HPLC system developed for β-carbolines. Under our conditions, peptides containing N-terminal tryptophan such as Trp-Leu and delta sleep-inducing peptide gave N-(9-hydroxymethyl-β-carboline-3-carbonyl) peptides which retained all amino acid residues except tryptophan.     

Correlation of the expression of double-stranded RNA-dependent protein kinase (p68) with differentiation in head and neck squamous cell carcinoma

p68 is an inducible protein kinase which is believed to be an important factor in the regulation of both viral and cellular protein synthesis. We have produced a monoclonal antibody (TJ4C4) which specifically detects p68, and which can be used to detect this antigen in formalin-fixed, paraffin-embedded tissues. Because p68 plays an important role in cellular protein synthesis, we hypothesized that it may correlate with normal and neoplastic cellular differentiation. One hundred and seventy-seven head and neck squamous cell carcinoma specimens, representing 82 patients, were studied. The relative amount, frequency, and distribution of p68 expression were determined by microscopic evaluation of ABC immunoperoxidase-stained specimens. A spectrum of immunoreactivity was detected in 156 of 177 tumors, as well as within the normal squamous epithelium. Normal, actively proliferating cells, such as the basal layer of squamous epithelium, expressed comparatively little p68. Increased p68 expression was noted to parallel the morphologic features of cellular differentiation. In neoplastic tissue, p68 expression also increased with the degree of cellular differentiation. These data demonstrate that the expression of p68 parallels the degree of cellular differentiation in squamous cell carcinoma of the head and neck region, as well as within normal squamous mucosa. Therefore, p68 may provide an objective biologic measure of cellular differentiation which does not depend on morphologic features.     

A versatile gel casting cum electrophoresis apparatus

A simple apparatus for vertical.,in situ, polyacrylamide or agarose gel casting as well as for the subsequent electrophoresis is described. The apparatus is completely leakproof and does not require any special device like clamps, O-rings, gaskets, grease etc. for sealing. Slab gels of various thickness (0.04 to 1.0 cm) can be made and the apparatus can be used for analytical or preparative purposes. Gel rods can also be cast and run in the device. Forward as well as reverse polarity electrophoresis of a sample can be run simultaneously in the apparatus. NCL Communication No.: 3077.     

Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.     

Connective Tissue Growth Factor Overexpression in Cardiomyocytes Promotes Cardiac Hypertrophy and Protection against Pressure Overload

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca²⁺ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF''s profibrotic function in the heart.     

Process optimization for biodiesel production from mahua (Madhuca indica) oil using response surface methodology

A central composite rotatable design was used to study the effect of methanol quantity, acid concentration and reaction time on the reduction of free fatty acids content of mahua oil during its pretreatment for making biodiesel. All the three variables significantly affected the acid value of the product, methanol being the most effective followed by reaction time and acid catalyst concentration. Using response surface methodology, a quadratic polynomial equation was obtained for acid value by multiple regression analysis. Verification experiments confirmed the validity of the predicted model. The optimum combinations for reducing the acid level of mahua oil to less than 1% after pretreatment was 0.32 v/v methanol-to-oil ratio, 1.24% v/v H2SO4 catalyst and 1.26 h reaction time at 60 degrees C. After the pretreatment of mahua oil, transesterification reaction was carried out with 0.25 v/v methanol-to-oil ratio (6:1 molar ratio) and 0.7% w/v KOH as an alkaline catalyst to produce biodiesel. The fuel properties of mahua biodiesel so obtained complied the requirements of both the American and European standards for biodiesel.     

Theiler's Murine Encephalomyelitis Virus L* Amino Acid Position 93 Is Important for Virus Persistence and Virus-Induced Demyelination

The DA strain and other members of the TO subgroup of Theiler''s murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection associated with an inflammatory white matter demyelinating disease. TO subgroup strains synthesize an 18-kDa protein, L*, out of frame with the polyprotein from an initiation codon 13 nucleotides downstream from the polyprotein''s AUG codon. We previously generated a mutant virus from our infectious DA full-length clone that has a change of the L* AUG codon to ACG (with no change in the polyprotein''s amino acid sequence). Studies of this mutant virus showed that L* was key to the TO subgroup phenotype because the mutant had a decreased ability to persist and demyelinate. This work was initially called into question because a similar mutant derived from a different full-length DA infectious clone persisted and demyelinated similarly to wild-type DA virus (O. van Eyll and T. Michiels, J. Virol. 74:9071-9077, 2000). We now report that (i) the sequence of the L* coding region differs in the two infectious clones, resulting in a Ser or Leu as the predicted amino acid at position 93 of L* (with no change in the polyprotein''s amino acid sequence), (ii) the difference in this amino acid is key to the phenotypic differences between the two mutants, and (iii) the change in amino acid 93 may affect L* phosphorylation. It is of interest that this amino acid only appears critical in determining the virus phenotype when L* is present in a significantly reduced amount (i.e., following translation from an ACG initiating codon).The DA strain and other members of the TO subgroup of Theiler''s murine encephalomyelitis virus (TMEV) induce an acute subclinical gray matter central nervous system (CNS) infection in SJL mice that is followed by a chronic inflammatory white matter demyelinating disease; virus persists in the CNS for the life of the mouse (for a review, see reference 16). The DA virus demyelinating disease serves as a model of multiple sclerosis since the two are similar in white matter pathology and both disease processes appear to be immune mediated. In contrast, the GDVII strain and other members of the GDVII subgroup of TMEV cause an acute fatal neuronal infection and fail to persist.The study of the TMEV model system is an especially attractive one for molecular pathogenesis studies because TMEV is a relatively simple virus with only four structural proteins in the virion; the nucleotide sequence and deduced amino acid sequence are known for several TMEV strains; the three-dimensional crystal structure of two TMEV strains has been solved; infectious TMEV clones are available, easing the preparation of recombinant or mutated viruses; TMEV-neutralizing monoclonal antibody sites and T-cell epitopes are known; and the experimental and natural host for TMEV, the mouse, is easily manipulated and is well studied immunologically and genetically. An understanding of the determinants of the TMEV disease phenotypes (neurovirulence, virus persistence, restricted infection, demyelination) may be valuable in clarifying the pathogenesis of picornaviral diseases, as well as and non-virus-induced CNS diseases such as amyotrophic lateral sclerosis and multiple sclerosis.The genome of all picornaviruses contains a long open reading frame that is translated into a polyprotein. One remarkable feature of the TO subgroup strains is that an 18-kDa protein, L*, is synthesized out of frame with the polyprotein from an initiation codon that is 13 nucleotides (nt) downstream from the polyprotein''s AUG codon (8) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.The DA virus genome (A) and parental wild-type and L* mutant viruses (B). (A) The DA virus genome with a 5′ untranslated region (5′ UTR), polyprotein (LP1P2P3), and 3′ UTR, as well as L*, which is out of frame with the polyprotein''s reading frame; the regions of the genome are not drawn to size. The polyprotein''s initiating AUG codon is at nt 1066, while the L* AUG codon is at nt 1079. (B) Parts of the nucleotide sequences of the L* proteins of two wild-type parental DA viruses (DA, TMDA) and the mutant L* proteins derived from one or the two parental wild-type infectious clones, with a replacement of the initiating AUG codon with an ACG codon, as well as a C or U at nt 1359 coding for a Ser or Leu at amino acid 93.We previously engineered a mutant virus, called DAL*-1, which had a change of the L* AUG codon to ACG (but with no change in the amino acid sequence of the polyprotein) (4) from our infectious clone pDAFL3 (17) (Fig. ​(Fig.1B).1B). The DAL*-1 virus had a decreased ability to persist and demyelinate, suggesting that L* is key to the distinctive TO subgroup phenotype. Subsequent studies showed that L* interfered with virus clearance by CD4⁺ T cells, allowing the virus to persist (10). This work was initially called into question by Michiels and colleagues (21) because a mutant DA virus, OV48, with the same mutation as DAL*-1 (as well as an additional replacement of an AUG codon, in the fifth codon of the L* reading frame, to ACG) had no effect on the ability of the DA virus to persist or demyelinate; however, this mutant was engineered from a different full-length DA clone, pTMDA1 (11). In a subsequent study, they further investigated the importance of L* in virus persistence and demyelination by demonstrating that an L* mutant that contained a stop codon in the L* sequence (with no change in the polyprotein''s amino acid sequence) had a significant decrease in virus persistence and demyelination (22). We suspected that the contrasting disease phenotypes induced by the DAL*-1 and OV48 viruses might have resulted from a sequence difference(s) in L*. In the present study, we demonstrate that a change in L* of nucleotide (nt) 1356/amino acid 93 led to the change in phenotype of DAL*-1 versus OV48.     

Bacillinaphthin A: A New Naphthohydroquinone from the Endophyte Bacillus subtilis NPROOT3

The present study explores the endophyte associated with the halophyte Salicornia brachiata for uncovering new biologically important compounds. Thus, HPLC-PDA guided chemical investigation of the ethyl acetate extract of the Bacillus subtilis NPROOT3 led to the isolation of a new compound named bacillinaphthin A ( 1 ) along with previously known rubinaphthin A ( 2 ). The structure of 2 was determined by a comparison of HR-ESI-MS, ¹H and ¹³C nuclear magnetic resonances (NMR) with those of reported data, whereas the structure of new compound 1 was elucidated by interpretation of 1D- and 2D-NMR and MS data. Bacillinaphthin ( 1 ) and rubinaphthin ( 2 ) feature 1,4-dihydroxy-2-naphthoic acid derivatives which have been isolated herein for the first time from the genus Bacillus. Bacillinaphthin ( 1 ) is a new congener of 2 with an additional succinic acid side chain attached to the sugar moiety. Production of succinoglycan compounds was reported to regulate symbiosis, hence the isolation of 1 exhibits an example of chemical ecology between the halophyte and its endophyte. In silico tools were used to assess the bioactive potential of both isolated molecules.