Effects of oral calcium bolus supplementation on intracellular polymorphonuclear leukocyte calcium levels and functionality in primiparous and multiparous dairy cows.

The objectives of this study were (1) to characterize Ca levels and polymorphonuclear leukocyte (PMN) function in primiparous and multiparous animals following oral Ca bolus supplementation, and (2) to determine differential responses of boluses containing a lower dose of Ca than traditionally used in primiparous animals on Ca levels and PMN function. Jersey × Holstein crossbred animals (n = 104) were enrolled within 24 h of parturition. All animals were blocked by time relative to calving and randomly assigned to treatment. The Ca boluses were composed of a mixture of Ca chloride, Ca sulfate, and Ca propionate. For objective 1, animals were assigned to control (CON; no Ca supplementation), or a series of 2 Ca boluses given 24 h apart for a total of 50 g of Ca. Objective 2 treatments included control (CON; no Ca supplementation), a series of 2 Ca boluses given 24 h apart containing 50 g of Ca, or a series of 2 Ca boluses given 24 h apart containing 25 g of Ca. Blood samples were collected on d 1 (<24 h), 2, 3, 5, and 7 relative to parturition. Total serum Ca, serum haptoglobin, PMN intracellular Ca, PMN intracellular Ca after stimulation with an environmental Escherichia coli, PMN L-selectin surface expression, and PMN phagocytic and oxidative burst activities were analyzed. For objective 1 a tendency was detected for a treatment difference on basal intracellular PMN Ca and a treatment difference on E. coli-stimulated intracellular PMN Ca. We detected a parity × DIM effect for PMN oxidative burst intensity. However, no other interactions or parity effects on other functional PMN variables were detectable. In primiparous animals, we found a treatment difference for E. coli-stimulated intracellular PMN Ca among animals given 50 g of Ca but no treatment difference on basal intracellular PMN Ca. The 50 g of Ca treatment increased both PMN phagocytosis and oxidative burst intensities. Supplementing animals with 50 g of oral Ca increased intracellular PMN Ca and influenced PMN function.

Pre-weaning plane of nutrition and Mannheimia haemolytica dose influence inflammatory responses to a bovine herpesvirus-1 and Mannheimia haemolytica challenge in post-weaning Holstein calves.

The objectives of this study were to determine whether plane of nutrition (PON) of milk replacer previously provided to calves, and dosage level of Mannheimia haemolytica (MH), influenced inflammatory responses to a combined viral-bacterial respiratory challenge. Holstein calves (1 d of age; n = 30) were assigned to treatments in a 2 × 3 factorial with pre-weaning PON and MH dose as main effects (n = 5 per treatment). Calves were fed either a low (LPN; n = 15) or a high PON (HPN; n = 15) from birth through weaning. Calves fed LPN were fed 436 g of dry matter (DM) per day of milk replacer until weaning, and HPN calves were fed 797 g of DM per day of milk replacer from d 1 to 10 and 1,080 g of DM per day from d 11 until weaning. Calf starter and water were offered ad libitum. Calves were step-down weaned beginning at d 54 and moved into an enclosed barn at d 70. Indwelling rectal temperature (RT) recording devices and jugular catheters were inserted at d 80. Calves were challenged with 1.5 × 108 plaque-forming units (pfu) per mL of bovine herpesvirus-1 (BHV-1) in each nostril at d 81 and with either 106, 107, or 108 cfu of MH at d 84. Blood samples were collected at varying intervals relative to BHV-1 and MH challenges. Four LPN calves either died or were euthanized soon after the 144-h observation period, whereas all HPN calves survived the entire observation period. As dosage of MH administered increased, acute and systemic inflammatory responses increased. Higher doses of MH resulted in increased leukocyte, neutrophil, and haptoglobin concentrations in infected calves. Data from the current study suggest that the highest dose, 108 cfu, triggered weaned calves' acute disease response, whereas the lower doses, 106 and 107 cfu, caused more moderate inflammation and disease. The effects of PON on inflammation responses to the disease challenge indicated that calves previously fed the LPN diet had more severe pathophysiological responses. Calves fed LPN showed higher peripheral neutrophil and leukocyte counts and serum haptoglobin concentrations following the BHV-1 challenge. Additionally, following the MH challenge, LPN calves had higher peripheral neutrophil counts, neutrophil-to-lymphocyte ratios, and serum tumor necrosis factor-α concentrations. These data demonstrate that higher doses of MH increase the acute inflammatory response and prolong inflammation, and that calves previously fed LPN responded more severely to the combined viral-bacterial respiratory challenge.

A combined phase contrast imaging and heterodyne interferometer system for multiscale fluctuation measurements (invited).

In this work, a novel combined diagnostic capable of measuring multiscale density fluctuations that extend from magnetohydrodynamic (MHD) to the lower range of electron temperature gradient turbulence has been designed, installed, and operated at DIII-D. The combined diagnostic was constructed by adding a heterodyne interferometer to the pre-existing phase contrast imaging (PCI) system, both of which measure line-integrated electron density fluctuations. The port-space footprint is minimized via use of a single 10.6 µm probe beam. With temporal bandwidths in excess of 1 MHz, the PCI measures high-k (1.5 cm-1 < |k R | ≤ 25 cm-1) fluctuations with sensitivity 3 × 1 0 13 m - 2 / kHz , while the interferometer simultaneously measures low-k (|k R | < 5 cm-1) fluctuations with sensitivity 3 × 1 0 14 m - 2 / kHz . The intentional mid-k overlap has been empirically verified with sound-wave calibrations and allows quantitative investigation of multiscale effects that are predicted to be significant in the reactor-relevant T e ∼ T i regime. Furthermore, via correlation with the primary DIII-D interferometer, the toroidal mode numbers of core-localized MHD can be measured.

A phase contrast imaging-interferometer system for detection of multiscale electron density fluctuations on DIII-D.

Heterodyne interferometry and phase contrast imaging (PCI) are robust, mature techniques for measuring low-k and high-k electron density fluctuations, respectively. This work describes the first-ever implementation of a combined PCI-interferometer. The combined system uses a single 10.6 µm probe beam, two interference schemes, and two detectors to measure electron density fluctuations at large spatiotemporal bandwidth (10 kHz

Incretin-based medications for type 2 diabetes: an overview of reviews.

AIMS: To summarize evidence from and assess the quality of published systematic reviews evaluating the safety, efficacy and effectiveness of incretin-based medications used in the treatment of type 2 diabetes. METHODS: We identified systematic reviews of randomized controlled trials or observational studies published in any language that evaluated the safety and/or effectiveness of glucagon-like peptide-1 (GLP-1) receptor agonists or dipeptidyl-peptidase-4 (DPP-4) inhibitors. Data sources used include the Cochrane Library, PubMed, EMBASE, Web of Science, International Pharmaceutical Abstracts, table of contents of diabetes journals, and hand-searching of reference lists and clinical practice guidelines. The methodological quality of systematic reviews was independently assessed by two reviewers using the Assessment of Multiple Systematic Reviews (AMSTAR) checklist. Our study protocol was registered with PROSPERO (2013:CRD42013005149). The primary outcomes were pooled treatment effect estimates for glycaemic control, macrovascular and microvascular complications, and hypoglycaemic events. RESULTS: We identified 467 unique citations of which 84 systematic reviews met our inclusion criteria. There were 51 reviews that evaluated GLP-1 receptor agonists and 64 reviews that evaluated DPP-4 inhibitors. The median (interquartile range) AMSTAR score was 6 (3) out of 11 for quantitative and 1 (1) for non-quantitative reviews. Among the 66 quantitative systematic reviews, there were a total of 718 pooled treatment effect estimates reported for our primary outcomes and 1012 reported pooled treatment effect estimates for secondary outcomes. CONCLUSIONS: Clinicians and policy makers, when using the results of systematic reviews to inform decision-making with regard to round clinical care or healthcare policies for incretin-based medications, should consider the variability in quality of reviews.

Associations between early alcohol and tobacco use and prolonged time to puberty in boys.

BACKGROUND: Previous research has demonstrated a relationship between prepubertal alcohol and tobacco use and delayed pubertal characteristics in girls. Although, laboratory research indicates that alcohol and tobacco use inhibits sexual maturation in male rats, human research in this area is lacking. To address this question among boys, we conducted a study to explore the association between early use of alcohol and tobacco and time to development of secondary sexual characteristics. METHODS: The study population included 3199 boys interviewed between the ages of 11 and 21. Participants reported the ages at which they first experienced body hair growth, deepening of the voice and facial hair growth. Early alcohol and tobacco use were defined as first use preceding the age of pubertal development among those reporting regular consumption patterns. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using Cox proportional hazard models. RESULTS: Early alcohol use was associated with longer time to body hair growth (HR 0.77; 95% CI 0.69-0.87), voice changes (HR 0.72; 95% CI 0.64-0.82) and facial hair growth (HR 0.77; 95% CI 0.68-0.86), after adjusting for tobacco use and age at interview. Tobacco use was not independently associated with the puberty indicators after controlling for alcohol use and age at interview. CONCLUSIONS: Our findings are consistent with the hypothesis that alcohol may inhibit puberty onset in boys, an association that has been previously observed among young girls. Thus, alcohol may be an exposure deserving more scrutiny as a disruptor to normal pubertal development.

Direct observation of weight counselling in primary care: alignment with clinical guidelines.

Primary care physicians provide care to a disproportionate number of overweight and obese patients and are uniquely positioned to help patients manage their weight in the context of a continuity relationship. The US National Heart, Lung and Blood Institute (NHLBI) developed evidence-based guidelines for the effective and efficient care of overweight/obese patients, but little is known about the use of these guidelines in practice. To determine the content of weight discussions and assess the elements of the NHLBI guidelines that were accomplished, office visits of 544 adult, overweight/obese patients to 28 primary care physicians were observed and audio recorded. Associations between type of weight management discussion and patient, physician and visit characteristics were examined. Fifty per cent (n = 270) of visits included weight discussions; 47% and 38% included use of at least one NHLBI assessment or treatment element during discussions about weight, respectively. Only 35% (n = 193) of discussions included an assessment and treatment strategy; none included all NHLBI-recommended elements. Overall, adherence to guidelines was poor, particularly with regard to reporting body mass index to the patient, measuring waist circumference and setting realistic weight loss goals. Weight discussions did not clearly vary by the patient, physician or visit characteristics examined. These findings suggest opportunities to develop and further tailor resources for improved physician training in patient weight management communication and treatment techniques that are both consistent with current standards for effective, evidence-based care and efficient enough for routine use during busy primary care visits.

Effect of diagnostic ultrasound during the fetal period on learning and memory in mice.

BACKGROUND: An experiment was conducted to find out whether in utero exposure to diagnostic ultrasound leads to changes in postnatal behavior in adult mice. METHODS: A total of 15 pregnant Swiss albino mice were exposed to diagnostic levels of ultrasound (3.5 MHz, 65 mW/cm(2), intensity((spatial peak-temporal peak)) (I(SPTP))=1 mW/cm(2), intensity((spatial average-temporal average)) (I(SATA))=240 mW/cm(2)) for 30 min on day 14 or 16 of gestation. All exposed as well as control animals were left to complete gestation and parturition. Their offspring were used in our further studies. They were monitored during early postnatal life for standard developmental markers (such as pinna detachment, eye opening and fur development) and postnatal mortality was recorded up to 6 weeks of age. The litters were subjected to behavioral tests for learning and memory at 4 months of age. Representative animals from each group were sacrificed and the hippocampal region of the brain was assayed for biogenic amines, noradrenaline, dopamine, serotonin (5-HT) and 5-HT's metabolite, 5-hydroxy indoleacetic acid (5-HIAA), in order to determine whether ultrasound exposure produced any biochemical changes in the hippocampal region of the brain. Coronal sections from the dorsal hippocampus from the representative animals from each group were processed for staining and the number of neurons was counted. RESULTS: Neither the standard developmental markers (such as pinna detachment, eye opening and fur development) nor the postnatal mortality was affected by ultrasound exposure. However, there was a significant impairment in learning (hole board test) and memory functions (shuttle box test) in both the exposure groups. Significant reductions in the biogenic amines and the decrease in the neuronal density were found only in day 14th pc ultrasound-exposed group compared with the control animals. The 16th day exposure group is relatively resistant to ultrasound-induced impairment of brain functions. CONCLUSIONS: The results suggest that the early fetal brain is highly susceptible to induction of neurobehavioral changes by ultrasound exposure.

The evidence based wound healing activity of Lawsonia inermis Linn.

The ethanol extract of Lawsonia inermis (200 mg/kg/day) was used to evaluate the wound healing activity on rats using excision, incision and dead space wound models. The animals were divided into three groups of six each in the excision model and two groups of six each in the incision model and dead space models. The topical application was made in the case of excision wound model, whereas, oral treatment was done with incision and dead space wound models. The following differences were noted in the group of experimental animals which were treated with an extract of L. inermis when compared with the control and reference standard animals: a high rate of wound contraction (p < 0.001), a decrease in the period of epithelialization (p < 0.001), high skin breaking strength (p < 0.001), a significant increase in the granulation tissue weight (p < 0.001) and hydroxyproline content (p < 0.05). The extract-treated animals showed 71% reduction in the wound area when compared with controls which was 58%. Histological studies of the tissue obtained on day 10 from the extract-treated group showed increased well organized bands of collagen, more fibroblasts and few inflammatory cells when compared with the controls which showed inflammatory cells, scanty collagen fibres and fibroblasts. Enhanced wound contraction, increased skin breaking strength, hydroxyproline and histological findings suggest the use of L. inermis in the management of wound healing.

Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins.

Cancer cells display an altered distribution of DNA methylation relative to normal cells. Certain tumor suppressor gene promoters are hypermethylated and transcriptionally inactivated, whereas repetitive DNA is hypomethylated and transcriptionally active. Little is understood about how the abnormal DNA methylation patterns of cancer cells are established and maintained. Here, we identify over 20 DNMT3B transcripts from many cancer cell lines and primary acute leukemia cells that contain aberrant splicing at the 5' end of the gene, encoding truncated proteins lacking the C-terminal catalytic domain. Many of these aberrant transcripts retain intron sequences. Although the aberrant transcripts represent a minority of the DNMT3B transcripts present, Western blot analysis demonstrates truncated DNMT3B isoforms in the nuclear protein extracts of cancer cells. To test if expression of a truncated DNMT3B protein could alter the DNA methylation patterns within cells, we expressed DNMT3B7, the most frequently expressed aberrant transcript, in 293 cells. DNMT3B7-expressing 293 cells have altered gene expression as identified by microarray analysis. Some of these changes in gene expression correlate with altered DNA methylation of corresponding CpG islands. These results suggest that truncated DNMT3B proteins could play a role in the abnormal distribution of DNA methylation found in cancer cells.

Evaluation of wound healing activity of Vanda roxburghii R.Br(Orchidacea): a preclinical study in a rat model.

The extract of Vanda roxburghii was administered topically to rats at a dose of 150mgkg(-1) day(- 1) for 10 days and was studied for its effect on wound healing, using the excision wound model. A 60% reduction in wound diameter was observed in the test group rats receiving the extract compared to controls (48%). Significant increases in wet and dry granulation tissue weights (P < .001), hydroxyproline (P < .001), and hexosamine (P < .003) contents were detected. An increase in protein content was also detected in the test group (P > .05, ns). These findings are consistent with wound healing at cellular levels. The pro-healing action may be attributed either to increased collagen deposition or to better alignment and maturation or both. The test wounds (extract-treated wounds) were, on average, fully healed by the 13th day, whereas the control group healed, on average, by the 20th day. These data suggest that the extract of Vanda roxburghii administered topically has wound-healing potential in rats.

Disease fingerprinting with cDNA microarrays reveals distinct gene expression profiles in lethal type 1 and type 2 cytokine-mediated inflammatory reactions.

Development of polarized immune responses controls resistance and susceptibility to many microorganisms. However, studies of several infectious, allergic, and autoimmune diseases have shown that chronic type-1 and type-2 cytokine responses can also cause significant morbidity and mortality if left unchecked. We used mouse cDNA microarrays to molecularly phenotype the gene expression patterns that characterize two disparate but equally lethal forms of liver pathology that develop in Schistosoma mansoni infected mice polarized for type-1 and type-2 cytokine responses. Hierarchical clustering analysis identified at least three groups of genes associated with a polarized type-2 response and two linked with an extreme type-1 cytokine phenotype. Predictions about liver fibrosis, apoptosis, and granulocyte recruitment and activation generated by the microarray studies were confirmed later by traditional biological assays. The data show that cDNA microarrays are useful not only for determining coordinated gene expression profiles but are also highly effective for molecularly "fingerprinting" diseased tissues. Moreover, they illustrate the potential of genome-wide approaches for generating comprehensive views on the molecular and biochemical mechanisms regulating infectious disease pathogenesis.

Localization of the chromosome 22 breakpoints in two cases of acute megakaryoblastic leukemia with t(1;22)(p13;q13).

Acute megakaryoblastic leukemia with t(1;22)(p13;q13) is a rare malignancy occurring in infants and young children. The genes involved in t(1;22)(p13;q13) are unknown. In this study, dual-color fluorescence in situ hybridization (FISH) experiments with 15 probes were performed on the metaphase cells obtained from one patient to systematically narrow the region of the breakpoint on chromosome 22 and localize it to RP5-1042K10. A 22.3-kb FISH probe derived from RP5-1042K10 was used to further refine the locus of the breakpoint in this case. Southern blot analysis covering of genomic DNA from a second patient detected DNA rearrangement at a site close to the breakpoint observed with the 22.3-kb probe in the first case. A partially characterized gene, KIAA 1438, is in the vicinity of the breakpoints determined by FISH and Southern blot experiments, suggesting that this gene plays a role in this malignancy.

Chromosomal instability in chromosome band 12p13: multiple breaks leading to complex rearrangements including cytogenetically undetectable sub-clones.

During fluorescence in situ hybridization (FISH) analysis of metaphase cells from 70 patients with lymphoid and myeloid hematologic malignancies and chromosomal rearrangements involving band 12p13, we identified nine patients (four with lymphoid malignancies, four with myeloid malignancies and one with biphenotypic leukemia) who showed more complicated rearrangements than we had expected from conventional cytogenetic study. In six patients, multiple breaks occurred in small segments of 12p with subsequent translocations and insertions of these segments into other chromosomes, sometimes to unexpected regions. In three patients additional chromosome breaks resulted in a sub-clone which was cytogenetically indistinguishable from the main clone in each patient based on the cytogenetic analysis. These subtle molecular events were detected exclusively in a region covering TEL/ETV6 and KIP1/CDKN1B. Seven of nine had a previous history of chemo/radiotherapy; all the patients showed complex karyotypes, even though they were newly diagnosed with leukemia. Survival data were available in five patients, and all survived less than 6 months. These findings suggest that the 12p13 region, especially the above-mentioned region, is genetically unstable and fragile. It is likely that multiple chromosome breaks were induced through mutagens used in chemo/ radiotherapy, and are associated with a sub-group of patients with an extremely bad prognosis.

Mechanism of monoterpene cyclization: stereochemical aspects of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase.

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimately cyclizes to the various monoterpene skeletons. Previous experimental approaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and racemic methanogeranyl diphosphate, in attempts to dissect the cryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthases and by the inability to resolve chiral monoterpene products at the microscale. These approaches were revisited using three recombinant monoterpene synthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene olefin synthases, (-)-limonene synthase from spearmint and (-)-pinene synthase from grand fir, yielded essentially only achiral, olefin products (corresponding to the respective analogs and homologs of myrcene, trans-ocimene and cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogeranyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipated analog and homolog of the tertiary intermediate linalyl diphosphate (i.e., 6,7-dihydrolinalyl diphosphate and homolinalyl diphosphate, respectively). In the case of recombinant (+)-bornyl diphosphate synthase from common sage, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinalool and homolinalool also comprised significant components of the respective product mixtures, indicating greater access of water to the active site of this enzyme compared to the olefin synthases; again, no direct evidence for the production of 6,7-dihydrolinalyl diphosphate or homolinalyl diphosphate was obtained. Resolution of the terpenol products of (+)-bornyl diphosphate synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homolinalool from (2R,3S)-methanogeranyl diphosphate. The opposite stereochemistries of these products indicates water trapping from opposite faces of the corresponding tertiary carbocationic intermediates of the respective reactions, a phenomenon that appears to result from the binding conformations of these substrate analogs. Although these experiments failed to provide direct evidence for the tertiary intermediate of the tightly coupled isomerization-cyclization sequence, they did reveal a mechanistic difference between the olefin synthases and bornyl diphosphate synthase involving access of water as a participant in the reaction.

The guanine protein coupled receptor rhodopsin is developmentally regulated in the free-living stages of Schistosoma mansoni.

Schistosoma mansoni parasites inhabit three distinct environments including water, intermediate molluscan hosts, and definitive vertebrate hosts. Determining how schistosomes interact with these environments may be one mechanism by which suitable vaccines or novel chemotherapeutic targets will be identified. Towards this end, we describe the identification of a 36-kDa S. mansoni protein that shares extensive sequence similarity to light absorbing rhodopsin guanine protein coupled receptors (GPCRs). This protein, S. mansoni rhodopsin (SmRHO), is the first molecularly characterized GPCR described in schistosomes. Sequence analysis reveals that SmRHO shares extensive phylogenetic conservation among rhodopsins/opsins expressed in water-dwelling invertebrates, possibly indicative of orthology. We demonstrate here that SmRHO is expressed in the free-living, light responsive miracidia and cercaria stages and is down-regulated in the adult, vertebrate residing forms. Moreover, we show that SmRHO is localized to sub-tegumental structures found towards the anterior end of cercariae. As SmRHO may be implicated in schistosome photoreception processes, we have begun a search for additional parasite encoded GPCR super-family members, which may be associated with chemoreception, chemotaxis, and olfaction. Identifying and characterizing new GPCRs may uncover hidden aspects of parasite biology useful towards the development of novel intervention strategies.

Refinement of the smallest commonly deleted segment of chromosome 20 in malignant myeloid diseases and development of a PAC-based physical and transcription map.

A deletion of the long arm of chromosome 20, del(20q), is a recurring abnormality in malignant myeloid diseases. In previous studies, we delineated a commonly deleted segment (CDS) of 5 Mb within band 20q12 flanked by D20S206 (proximal) and D20S481 (distal). We have generated a detailed physical map of P1 artificial chromosome (PAC) clones of this interval as well as a transcriptional map. The contig consists of 81 clones to which 152 markers (27 genes, 45 unique expressed sequence tags (ESTs) or UniGenes, 24 polymorphisms, and 56 sequence-tagged sites) have been mapped. Using PAC clones for fluorescence in situ hybridization analysis of myeloid leukemia cells with reciprocal translocations of 20q, or unbalanced rearrangements leading to loss of 20q, we have narrowed the CDS to an approximately 250-kb interval encompassing two overlapping PACs, P201E16 and P29M7 (between EST AA368224 and D20S481). This interval is gene-rich and contains 5 characterized genes, 4 UniGenes, and 9 single ESTs. The development of a transcriptional map and the identification of the smallest CDS will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 20q.

Anergic T lymphocytes selectively express an integrin regulatory protein of the cytohesin family.

It has been proposed that the maintenance of T cell anergy depends on the induction of negative regulatory factors. Differential display of reverse transcribed RNA was used to identify novel genes that might mediate this function in anergic Th1 clones. We report that anergic Th1 clones do indeed express a genetic program different from that of responsive T cells. Moreover, one gene, the general receptor of phosphoinositides 1 (GRP1), was selectively induced in anergic T cells. The GRP1, located in the plasma membrane, regulated integrin-mediated adhesion and was invariably associated with unresponsiveness in multiple models of anergy. T cells expressing retrovirally transduced GRP1 exhibited normal proliferation and cytokine production. However, GRP1-transduced T cells were not stable and rapidly lost GRP1 expression. Thus, although GRP1 may not directly mediate T cell anergy, it regulates cell expansion and survival, perhaps through its integrin-associated activities.

Molecular characterization of a human DNA kinase.

Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5'-hydroxyl termini and dephosphorylate 3'-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5'-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.

Identification of Nck family genes, chromosomal localization, expression, and signaling specificity.

Already a dozen molecules share binding to the Src homology (SH) 3 domains of human Nck, an SH3-SH3-SH3-SH2 adapter protein. We reason that there may be multiple gene members of Nck to accommodate the large binding repertoires. Here we report identification of novel human and mouse Nck genes and rename them as the Nckalpha and Nckbeta genes (including the human Nckalpha, human Nckbeta, mouse Nckalpha, and mouse Nckbeta genes). Nckalpha and Nckbeta share 68% amino acid identity, whereas the two Nckalpha and two Nckbeta across the species show 96% identity to each other. The human Nckbeta gene is mapped to 2q12, whereas the human Nckalpha gene has previously been mapped at 3q21. Antibodies specifically against Nckalpha and Nckbeta detect Nckalpha and Nckbeta with an identical molecular mass in the same cells of various origins. Ectopically expressed Nckbeta, but not its SH2 domain mutant, strongly inhibits epidermal growth factor- and platelet-derived growth factor-stimulated DNA synthesis. Consistently, epidermal growth factor receptor and platelet-derived growth factor receptor preferentially interact with Nckbeta over Nckalpha in vitro. This study indicates that Nck is a multiple gene family and that each gene may have its own signaling specificity. Because previous anti-Nck (human Nckalpha) antibodies cross-react with Nckbeta, reassessment of those studies with specific Nck genes would be necessary.