A Morphological Study of Myocardial Bridges in the Fetal Heart.

Introduction:Myocardial bridges (MB) are congenital anomalies of hearts observed as muscle fibers covering epicardial branches of the coronary artery. The left anterior descending artery (LAD) was found to be commonly showing myocardial bridges (MBs). Clinically, MBs were claimed to cause varied symptomatology. The data on the morphology and prevalence of MBs in fetuses was limited, despite the commonly accepted congenital origin. Material and methods:Fetal hearts obtained from 37 fetuses from the donation program were used. The hearts were dissected out from the thorax by standard dissection procedure. The pericardium and epicardium were dissected. The coronary arteries were delineated, and MBs were observed and noted. The coronary artery segment having MBs, its distance from the ostium as well as the direction and length of the MBs were studied. Results:The MBs were observed in 20 out of 37 fetal hearts studied over the left anterior descending, right coronary, posterior interventricular and circumflex arteries. The mid or distal part of the coronary arteries frequently exhibited MBs. The mean length of the MB was 4.2 mm, with MBs being situated about 1.5 cm away from the coronary ostium. The oblique pattern of MB was more frequently noted. Conclusion:The morphology and prevalence of fetal MBs showed common occurrence in the LAD artery, with a predominant oblique morphological pattern.

Dyke-Davidoff-Masson syndrome.

Dyke-Davidoff-Masson syndrome (DDMS) refers to atrophy or hypoplasia of one cerebral hemisphere, due to an insult to the developing brain in fetal or early childhood period. Age of presentation depends on the time of neurologic insult, and characteristic changes may be seen only in adolescence. Male gender and left hemisphere are more frequently involved. A 17-year-old female adolescent with a history of recurrent refractory seizures, hemiplegia and mental retardation reported to Department of Radiology for computed tomography (CT) assessment of brain. On examination, she had facial asymmetry, delayed milestones, and spastic hemiplegia. The CT brain showed right cortical atrophy with ventricular dilatation, prominent sulci, and shifting of falx to the right side. Bone window image showed asymmetry in skull vault thickness, the width of diploic space, the size of paranasal air sinuses and inclination of the petrous ridge between the affected and normal sides. As the above case deviates from the usual presentation of male left sided DDMS, hence the report.

Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Identification of a Mutant Kinase/ATP Analog Pair.

INTRODUCTIONThis protocol describes a method for studying protein kinases and their substrates by generation of mutant kinase enzymes that can incorporate ATP analogs into the substrate. This method was used to identify substrates of extracellular signal-regulated kinase 2 (ERK2). Once mutations (called "pocket mutations") have been generated in the kinase, the next step is to screen ATP analogs for their compatibility with the kinase mutant. This screening is best performed in a two-step process. The first step involves assaying the ability of an ATP analog to inhibit the incorporation of radioactive phosphate from normal [γ-32P]ATP into a known substrate in an in vitro kinase reaction. Because certain analogs might be able to interact with the ATP-binding site of the kinase but be unable to be used by the kinase as an ATP source, it is also necessary to directly test the ability of the mutant kinase to phosphorylate a substrate with analog ATP. Doing this requires either radiolabeled ATP analogs or, preferably, a phospho-specific antibody to the phosphorylation site on the known substrate. (A phospho-specific antibody allows a large number of ATP analogs to be screened without the need for radioactivity.) The kinase/ATP analog pair that provides the best phosphorylation of the substrate is then used for future experiments. Only those analogs that cannot be used by the wild-type kinase and other cellular kinases are chosen.

Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Generation of [{gamma}-32P]ATP Analog from ADP Analog.

INTRODUCTIONThe generation of a high-specific-activity [γ-(32)P]ATP analog is essential for detecting direct substrates of a kinase. Synthesis of radiolabeled ATP analog from unlabeled ADP analog involves a succession of phosphotransfer reactions that are performed in two stages. The first stage involves transfer of phosphate from [γ-(32)P]ATP to nucleotide diphosphate kinase (NDPK) to make [(32)P]NDPK. The second stage involves transfer of labeled phosphate from [(32)P]NDPK to ADP analog to generate [γ-(32)P]ATP analog. In this protocol, radiolabeled [γ-(32)P]cpATP is prepared from cyclopentyl ADP (cpADP) and [γ-(32)P]ATP by a phosphate transfer reaction using NDPK.

Using genetically engineered kinases to screen for novel protein kinase substrates: phosphorylation of kinase-associated substrates.

INTRODUCTIONThis protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [γ-(32)P]ATP analog. This technique has been used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates; however, the methodology can be applied to other protein kinases as well.

Using genetically engineered kinases to screen for novel protein kinase substrates: phosphorylation of substrates in cell lysates with exogenous kinase.

INTRODUCTIONThis protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves the addition of recombinant mutant kinase and [γ-(32)P]ATP analog to cell lysates. This technique has been successfully used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates in cell lysates from SKOV-3 ovarian cells; however, the methodology can be applied to other protein kinases as well.

Identifying specific kinase substrates through engineered kinases and ATP analogs.

Intracellular signaling by protein kinases controls many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effect on cellular functions. One of the recent developments in this area takes advantage of the similarity in the ATP binding domains of protein kinases, where a few conserved amino acids containing large side chains come in close contact with the N-6 position of bound ATP. Mutation of one or more of these residues generates a "pocket" in the ATP binding site that allows the mutant kinase, but not other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N-6 position. The use of such a mutated kinase and radiolabeled ATP analogs allows for the specific labeling of direct substrates of the kinase within a mixture of cellular proteins. We have recently reported the generation of "pocket" mutants of extracellular regulated kinase 2 (ERK2) and their use in the identification of two novel substrates of ERK2. In this report, we discuss the generation and characterization of ERK2 mutants that utilize analogs of ATP and describe the methodology used to identify ERK2-associated substrates. We also describe the direct labeling of ERK2 substrates in cell lysates. These methodologies can be adapted for use with other protein kinases to increase the understanding of intracellular signal transduction.

Identification of novel ERK2 substrates through use of an engineered kinase and ATP analogs.

The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified by differential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rsk1. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade.

Characterization of the initiator tRNA gene locus and identification of a strong promoter from Mycobacterium tuberculosis.

An initiator tRNA gene, metA, and a closely linked fragment of a second initiator-tRNA-like sequence, metB, from Mycobacterium tuberculosis H37Ra have been cloned and characterized. The promoter region of metA shows the presence of conserved sequence elements, TAGCCT and TTGGCG, with resemblance to -10 and -35 promoter regions. The deduced sequence of the mature tRNA contains the three unique features of the eubacterial initiator tRNAs represented by (i) a C:U mismatch at position 1:72, (ii) three consecutive base pairs, 29-31G:C39-41 in the anticodon stem, and (iii) a purine:pyrimidine (A:U) base pair at position 11:24 in the dihydrouridine stem. A putative hairpin structure consisting of an 11 bp stem and a three-base loop found in the 3' flanking region is followed by a stretch of T residues and may serve as a transcription terminator. Analysis of the expression of metA and of its promoter using chloramphenicol acetyltransferase fusion constructs in Mycobacterium smegmatis shows that metA is a functional gene driven by a strong promoter. Furthermore, the overexpressed transcripts are fully processed and formylated in vivo. The metB clone shows the presence of sequences corresponding to those downstream of position 30 of the tRNA. However, the CCA sequence at the 3' end has been mutated to CCG. Interestingly, the 3' flanking sequences of both the genes are rich in GCT repeats. The metB locus also harbours a repeat element, IS6110. A method to prepare total RNA from mycobacteria (under acidic conditions) to analyse in vivo status of tRNAs is described.