PhLPs and PhLOPs in the phosducin family of G beta gamma binding proteins

In this study, we identify new isoforms of the retinal phosducin and investigate the expression of the phosducin family, showing that an isoform, PhLP1, has sequence homology with Phd and Gbeta gamma binding capability, whereas two isoforms (phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gbeta gamma. Original identification of PhLP1 and the PhLOPs was from a human retina cDNA library, using a PCR product for library hybridization screening that contained a predicted functional epitope domain. The screen identified Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina or forskolin-stimulated Y79 retinoblastoma cells; however, the new isoforms are below the level of detection on Northern blot analysis. The predicted amino acid translation of each homologue revealed major differences, arising from either splice variants or gene duplication of Phd. To test the functional interaction of all phosducin isoforms with Gbeta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gbeta gamma was verified for GST-Phd and demonstrated for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound Gbeta gamma. Comparable results were observed when the GST-phosducin fusion proteins selectively sequestered Gbeta gammas from retinal extracts or when functional Gbeta gamma interactions were assessed using surface plasmon resonance technology. Phosducin and its isoforms are widely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an 11-amino acid conserved epitope domain (TGPKGVINDWR) that controls the high-affinity binding of Gbeta gamma; these isoforms are implicated in the G-protein signaling pathway. The phosducin-like orphan proteins (PhLOPs) fail to bind Gbeta gamma, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways.     

Individual significance and growing complexity: contributions of Sigmund Freud and René Spitz to developmental psychology]

Contributions of Sigmund Freud and René Spitz to developmental psychology are presented in terms of today's research. The work of each of these pioneers draws our attention to the need for increasing our knowledge about the meaning of individual experience and increasing complexity in the course of development. Freud's contribution to today's developmental thinking is reviewed in terms of his observations of play, his schematic perspectives on developmental processes, and his innovative theoretical approaches involving nonconscious mental activity in the context of constructivism. Spitz's contribution to today's thinking is reviewed according to a similar array of topics. These include his observational assessments of infants, his schematic perspectives on developmental processes, and his innovative theoretical approaches involving affective communications in the context of caregiving.     

O-GlcNAcylation of key nuclear and cytoskeletal proteins: reciprocity with O-phosphorylation and putative roles in protein multimerization

BACKGROUND: Registered mortality from cryptogenic fibrosing alveolitis (CFA) in England and Wales has increased substantially since the specific International Classification of Diseases code for CFA was introduced in 1979. However, since a significant proportion of deaths from CFA are misclassified as post inflammatory fibrosis (PIF), it is possible that the observed rise in CFA mortality is due to diagnostic transfer from this code. To investigate this, and to assess mortality trends in other countries, annual CFA and PIF mortality data from England and Wales, USA, Australia, Scotland, Canada, New Zealand, and Germany were analysed. METHODS: Crude annual mortality rates were calculated and rates standardised by Poisson regression to allow assessment of changes over time and comparison between countries, sexes, and age groups. The relative trends in mortality from CFA and PIF were assessed by calculating the annual ratio of CFA to PIF deaths. RESULTS: Men were more likely than women to die from both CFA and PIF in all countries. The highest standardised CFA mortality rate occurred in England and Wales, and the lowest in Germany. Since 1979 mortality from CFA has increased in England and Wales, Australia, Scotland and Canada, but there was no trend in CFA mortality in New Zealand or Germany. In the USA mortality from CFA was low and has fallen. Mortality from PIF increased in all countries except New Zealand and Germany, and the highest PIF mortality, together with the greatest increase over time, was seen in the USA. Changes over time in the annual ratio of CFA to PIF deaths in all countries were small, implying that diagnostic transfer is not a major cause of the increasing CFA mortality. CONCLUSIONS: Mortality from CFA continues to increase in England and Wales and in many other countries. Diagnostic transfer from PIF does not appear to be a major cause of this.     

Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis

We describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-cultured Mycobacterium tuberculosis. The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT. Dead mycobacteria were unable to reduce MTT. Rifampin-sensitive M. bovis (BCG) and M. tuberculosis exposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected. The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU. By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains. The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results. The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria. The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistant M. tuberculosis that is affordable even by low-income countries where tuberculosis is a major public health problem.     

Quiescence versus apoptosis: Myc abundance determines pathway of exit from the cell cycle

When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states.     

Molecular genetic studies of sporadic pituitary tumors

Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype.     

Synergistic effects of relaxation and susceptibility in differentiation between compartmentalized and noncompartmentalized tissues

RATIONALE AND OBJECTIVES: This study illustrates the synergistic effects of relaxation- and susceptibility-based contrast enhancement. Using a combination of gadolinium (Gd) and dysprosium (Dy) complexes and a sequence capable of taking advantage of the particular relaxation behavior of this combination, the difference between compartmentalized and noncompartmentalized regions was significantly enhanced. METHODS: Magnetic resonance imaging of the rat kidney was performed before and immediately after the administration of a combination of Gd and Dy chelates (Gd-DTPA-BMA and Dy-DTPA-BMA). RESULTS: The signal intensity (SI) of the renal parenchyma was reduced by 85%, whereas the collecting tubes had a 100% increase of their SI as demonstrated by a short repetition time (600 msec), long echo time (50 msec), and spin-echo sequence. CONCLUSIONS: The high R2* effect, specific to the compartmentalized tissues, associated with the moderately high R1 and R2 developed in the remaining areas, results in an important improvement in tissue differentiation, which potentially is useful for the evaluation of pathological changes as in tubular necrosis.     

Reduced COX-2 protein in colorectal cancer with defective mismatch repair

Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.