T-cell receptor alpha-chain gene is split in a human T-cell leukemia cell line with a t(11;14)(p15;q11).

Chromosomal rearrangements in malignant T-cell disease frequently involve the chromosome bands containing the T-cell receptor genes. The RPMI 8402 cell line, which was established from the leukemia cells of a patient with T-cell acute lymphoblastic leukemia, is characterized by a translocation involving chromosome 14 (band q11) and chromosome 11 (band p15) [t(11;14)(p15;q11)]. By using in situ chromosomal hybridization and Southern blot analysis to examine RPMI 8402 cells, we determined that the break at 14q11 occurs within the variable region sequences of the T-cell receptor alpha-chain gene (TCRA); the break at 11p15 occurs between the HRAS1 gene and the genes for insulin and the insulin-like growth factor 2. These results suggest that the TCRA sequences activate a cellular gene located at 11p15 in malignant T-cell disorders.     

Identification of cDNA clones encoding a precursor of rat liver cathepsin B.

Recent studies have suggested that many lysosomal enzymes, including cathepsin B (EC 3.4.22.1), may be synthesized as larger precursors and proteolytically processed to their mature forms. To determine the structure of the primary translation product of cathepsin B, we have screened a phage cDNA library for clones encoding rat liver cathepsin B. We synthesized two extended DNA oligonucleotides to use as hybridization probes: a 50-mer corresponding to the coding segment for residues 215-231 of mature cathepsin B and a 54-mer corresponding to residues 117-134. After screening 600,000 plaques, five clones were obtained that hybridized to the 32P-labeled 50-mer; of these, two (lambda rCB3 and lambda rCB5) also reacted with the 54-mer. DNA sequence analysis confirmed that lambda rCB3 and lambda rCB5 both encoded rat liver cathepsin B, and the translated sequence is in agreement with the sequence determined [Takio, K., Towatari, T., Katunuma, N., Teller, D. C. & Titani, K. (1983) Proc. Natl. Acad. Sci. USA 80, 3666-3670], except for a tryptophan for glycine substitution at residue 78 and the presence of two amino acids at the junction site of the light and heavy chains. Moreover, the DNA sequence reveals an open reading frame extending beyond the 5' (NH2 terminus), and the predicted COOH terminus of the coding sequence for the mature protein is extended by six amino acids. These results confirm that the biosynthesis of cathepsin B involves a larger precursor form and demonstrate the effectiveness of long oligonucleotide probes for screening to detect rare cloned mRNAs.     

Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fcgamma receptors I and II/III

The SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) adapter protein is expressed in T cells and myeloid cells, whereas its homologue BLNK (B cell linker protein) is expressed in B cells. SLP-76 and BLNK link immunoreceptor tyrosine-based activation motif-containing receptors to signaling molecules that include phospholipase C-gamma, mitogen-activated protein kinases, and the GTPases Ras and Rho. SLP-76 plays a critical role in T cell receptor, FcvarepsilonRI and gpVI collagen receptor signaling, and participates in signaling via FcgammaR and killer cell inhibitory receptors. BLNK plays a critical role in B cell receptor signaling. We show that murine bone marrow-derived macrophages express both SLP-76 and BLNK. Selective ligation of FcgammaRI and FcgammaRII/III resulted in tyrosine phosphorylation of both SLP-76 and BLNK. SLP-76(-/-) bone marrow-derived macrophages display FcgammaR-mediated tyrosine phosphorylation of Syk, phospholipase C-gamma2, and extracellular signal regulated kinases 1 and 2, and normal FcgammaR-dependent phagocytosis. These data suggest that both SLP-76 and BLNK are coupled to FcgammaR signaling in murine macrophages.     

A tagging-via-substrate technology for detection and proteomics of farnesylated proteins

A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.     

Construction and selection of recombinant plasmids containing full-length complementary DNAs corresponding to rat insulins I and II.

We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)-RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli chi 1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.     

Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia

Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M(4) subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M(4) receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M(4) receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D(432)) in the third extracellular loop of the human M(4) receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.     

Composition of common bile duct stones in Chinese patients during and after endoscopic sphincterotomy

AIM: Endoscopic sphincterotomy (ES) is a well-established therapeutic modality for the removal of common bile duct (CBD) stones. After ES there are still around 10% of patients that experience recurrent CBD stones. The aim of this study is to investigate the composition of CBD stones before and after ES and its clinical significance in Chinese patients. METHODS: From January 1996 to December 2003, 735 patients with CBD stones received ES at Kaohsiung Veterans General Hospital and stone specimens from 266 patients were sent for analysis. Seventy-five patients had recurrent CBD stones and stone specimens from 44 patients were sent for analysis. The composition of the stones was analyzed by infrared (IR) spectrometry and they were classified as cholesterol or bilirubinate stones according to the predominant composition. Clinical data were analyzed. RESULTS: In the initial 266 stone samples, 217 (82%) were bilirubinate stones, 42 (16%) were cholesterol stones, 3 were calcium carbonate stones, 4 were mixed cholesterol and bilirubinate stones. Patients with bilirubinate stones were significantly older than patients with cholesterol stones (66±13 years vs56±17 years, P= 0.001). In the 44 recurrent stone samples, 38 (86%) were bilirubinate stones, 3 (7%) were cholesterol stones, and 3 were mixed cholesterol and bilirubinate stones. In 27 patients, both initial and recurrent stone specimens can be obtained, 23 patients had bilirubinate stones initially and 2 became cholesterol stones in the recurrent attack. In the four patients with initial cholesterol stones, three patients had bilirubinate stones and one patient had a cholesterol stone in the recurrent attack. CONCLUSION: Bilirubinate stone is the predominant composition of initial or recurrent CBD stone in Chinese patients. The composition of CBD stones may be different from initial stones after ES.     

Initial experience on rifampin and pyrazinamide vs isoniazid in the treatment of latent tuberculosis infection among patients with silicosis in Hong Kong

OBJECTIVE: To compare the adverse effects and treatment adherence between 2 months of rifampin plus pyrazinamide (2RZ) and 6 months of isoniazid (6H). BACKGROUND: Patients with silicosis in Hong Kong are at high risk of acquiring tuberculosis. A previous study showed that treatment with 6H reduced the risk of silico-tuberculosis by one half. METHOD: Patients with silicosis and a Mantoux skin test reaction > or =10 mm were randomized to receive either 2RZ or 6H daily. Liver function testing was done monthly during the initial 2 months. The adverse effects and treatment adherence were compared between the two regimens. RESULTS: Forty patients (mean age, 61.6 +/- 9.1 years) and 36 patients (mean age, 57.6 +/- 9.7 years) were randomized to the 2RZ and 6H arms, respectively (p > 0.05) [+/- SD]. Baseline characteristics were comparable. Nineteen patients in the 2RZ arm had peak alanine transaminase (ALT) levels > 1.5 times the upper limit of normal (ULN) in comparison with only five study subjects of the 6H arm (47.5% vs 13.9%, p < 0.01). Fourteen patients (35%) in the 2RZ arm and 1 patient (2.8%) in the 6H arm had peak ALT levels more than five times the ULN (p < 0.001). Only seven patients had symptoms suggestive of hepatitis; none of the patients had jaundice. All recovered after withholding treatment. In the 2RZ study arm, none of the baseline characteristics predicted hepatotoxicity. Other adverse effects were generally mild and comparable between both study arms. Treatment was stopped prematurely in 45% and 36.1% of patients in the 2RZ and 6H arms, respectively (p = 0.43). The main reasons were hepatotoxicity for the 2RZ arm and voluntary withdrawal after experiencing other minor adverse effects for the 6H arm. CONCLUSION: A higher incidence of hepatotoxicity was associated with rifampin plus pyrazinamide than isoniazid in the treatment of latent tuberculosis infection among patients with silicosis in Hong Kong.     

Evolution of the insulin superfamily: cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus.

Although insulin and the insulin-like growth factors (IGFs) share marked similarities in amino acid sequence and biological activity, their evolutionary origins have not been resolved. To investigate this issue, we recently cloned a cDNA encoding an insulin-like peptide (ILP) from a primitive chordate species, amphioxus (Branchiostoma californiensis). The deduced sequence of amphioxus preproILP indicates that it is a hybrid molecule containing features characteristic of both insulin and IGF. Like proinsulin, amphioxus proILP contains a C-peptide, which is flanked by paired basic residues and is probably removed by proteolysis. However, proILP also contains an extended carboxyl-terminal peptide region that can be divided into D and E domains similar to those of proIGF. Sequence comparisons show that the amphioxus ILP A and B domains are equally homologous to those of human insulin and IGF-I and -II. Based on these results and the exon-intron organization of the amphioxus ILP gene, we propose that IGF emerged at a very early stage in vertebrate evolution from an ancestral insulin-type gene.     

Occurrence of matrix metalloproteinases and tissue inhibitors of metalloproteinases in tuberculous pleuritis

OBJECTIVE: Matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) have been found in high concentrations in pleural effusions. Because MMP and TIMP may play a part in the causation of the fibrosis seen in tuberculous (TB) pleuritis their occurrence was examined. DESIGN: Pleural effusion fluid and plasma concentrations of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, TIMP-1 and TIMP-2 were determined by ELISA in 21 patients with TB pleuritis. To adjust for the total protein content, respective ratios were calculated. Activities of MMP-2 and MMP-9 were measured by gelatine zymography and the MMP-9/MMP-2 ratios calculated. Pleural effusions and plasma of 15 patients with congestive heat failure (CHF) and plasma of 15 healthy persons (CON) served as controls. RESULTS: Immunoreactive pleural fluid concentrations of MMP-1, MMP-2, MMP-8, and MMP-9 were higher in TB compared to CHF, but plasma concentrations were not different between the groups. TB pleural fluid concentrations of MMP-1, MMP-2, TIMP-1, and TIMP-2 were higher compared to TB plasma. MMP-3 was found in trace amounts only. The MMP-9/total protein ratios in pleural fluid were higher in TB compared to CHF (0.4492+/-0.1633 vs 0.0364+/-0.0145, P<0.005) but the TIMP-1 ratios were lower (139.0+/-28.7 vs 517.8+/-183.7, P<0.0005). In TB pleural fluid vs TB plasma, the respective MMP-1, MMP-2, TIMP-1, and TIMP-2 ratios were increased (0.46+/-0.10 vs 0.17+/-0.02; 25.2+/-2.8 vs 4.2+/-0.9; 139.0+/-28.7 vs 27.8+/-8.2; 0.67+/-0.13 vs 0.18+/-0.04, P<0.0005 each). Gelatine zymography demonstrated MMP-2 and MMP-9 bands of different brightness in TB effusions but in CHF effusions the MMP-9 band was barely visible. The MMP-9/MMP-2 effusion ratios were therefore higher in TB compared to CHF (0.46+/-0.15 vs 0.05+/-0.04, P<0.0005). CONCLUSION: Compartmentalized MMP-1, MMP-2, TIMP-1, and TIMP-2 and, compared to CHF, a surplus of MMP-1, MMP-2, MMP-8, and MMP-9 in the pleural space obviously contribute to the fibrotic reactions in TB pleuritis.