Ultrasonographic measurement of caudal vena cava to aorta ratio during fluid resuscitation of dogs with spontaneous circulatory shock.

OBJECTIVES: To describe the change in the caudal vena cava to aorta ratio (CVC:Ao) ratio during fluid resuscitation of circulatory shock in dogs and compare these results with those of the physical examination and blood lactate. MATERIALS AND METHODS: Perfusion parameters and blood lactate were recorded at admission. An abdominal point-of-care ultrasound protocol was performed, during which the caudal vena cava to aorta ratio was measured on the spleno-renal view. Measurements were performed within 5 minutes before and after a 10 mL/kg crystalloid fluid bolus. Investigators were not blinded to therapeutic interventions. RESULTS: Twenty-nine dogs with physical signs of circulatory shock were enrolled. Caudal vena cava to aorta ratios were below reference interval in 28 of 29 dogs. After bolus administration, median caudal vena cava diameter increased by 0.14 cm (0.69 to 0.83 cm) and median aorta diameter increased by 0.03 cm (0.87 to 0.90 cm) and caudal vena cava to aorta ratio returned to within reference range in 65% of dogs (13/29). Bolus administration was associated with an increase in median caudal vena cava to aorta ratio of 0.10 (95% CI:0.05 to 0.16, P=0.0005). Blood lactate did not change significantly. Heart rate and capillary refill time decreased significantly after fluid bolus (heart rate: estimate=-19 bpm, 95% CI:-30 to -8, P=0.002; capillary refill time: estimate=-1.0 s, 95% CI:-1.3 to -0.7, P < 0.0001). CLINICAL SIGNIFICANCE: In this population of dogs with circulatory shock, the caudal vena cava to aorta ratio significantly increased after a fluid bolus. Future studies that implement blinding of the outcome assessors are warranted to confirm these findings.

Long-term experience and Visual Acuity outcomes in Peters Anomaly cases.

PURPOSE: To report the results in a series of Peters Anomaly cases, and propose management and treatment approaches according to the alterations associated with each case. MATERIAL AND METHODS: A retrospective analysis was performed on the records of 27 patients (32 eyes) clinically diagnosed with Peters Anomaly. Each patient was subjected to different treatment modalities according to the type of Peters Anomaly, anywhere from medical follow-up clinics to a Penetrating Keratoplasty procedure (PKP). RESULTS: Of the 27 patients (32 eyes), 74% were male and 26% female, with 18.5% (5) being bilateral and 81.5% (22) unilateral. The mean number of years of follow-up was 10.2 years (Range: 3.5 to 18 years). The results of long-term VA correlate directly with the type of Peters Anomaly. For the total number of patients, the VA results were LogMAR 1.71 ±â€¯1.04. The results by groups were: Type I with only medical monitoring LogMAR 0.3 ±â€¯0, Type I with only Optical Iridectomy (OI) LogMAR 0.97 ±â€¯0.78, Type I with PKP LogMAR 1.22 ±â€¯0.97, Type II without a compromised posterior pole with PKP LogMAR 2.41 ±â€¯0.80, and Type II with a compromised posterior pole with PKP LogMAR 2.56 ±â€¯0.48. CONCLUSIONS: The result of VA and long-term corneal failure is directly related to the type of Peters Anomaly. Patients with Type I who only required medical follow-ups had the most favourable prognosis. Patients who underwent Peripheral Iridectomy followed and patients in which PKP was performed had an inferior prognosis.

Impact of flow and temperature on non-dyspnoeic dogs' tolerance undergoing high-flow oxygen therapy.

OBJECTIVES: To prospectively describe the impact of gas flow rate and temperature on dog's tolerance of high-flow nasal oxygen therapy during recovery from anaesthesia, hypothesizing that higher flow rates and temperatures will decrease tolerance. MATERIALS AND METHODS: Twelve non-dyspnoeic client-owned dogs recovering from general anaesthesia were included in this study. After extubation, a nasal cannula was positioned and high-flow nasal oxygen therapy was initiated. Two flow rates (two or four time the theoretical minute ventilation: HF2 and HF4), each of them combined with two temperatures (31 and 37°C: T31 and T37), were randomly applied (four conditions per dog). For each condition, cardiovascular and respiratory parameters (heart rate, respiratory rate, systolic arterial blood pressure and pulse oximeter oxygen saturation), sedation score and tolerance score were recorded at initiation (T0 ) and after 10 minutes of accommodation (T10 ). RESULTS: Sedation scores were not significantly different between the four conditions. Cardiovascular and respiratory parameters were not significantly different between any condition at both T0 and T10 . Tolerance scores were good and not significantly different between any flow rate or temperature (HF2-T31: 4 (2-4), HF4-T31: 4 (2-4), HF2-T37: 4 (2-4), HF4-T37: 4 (1-4)). CLINICAL SIGNIFICANCE: The gas flow rates and temperatures studied have no impact on tolerance during the recovery period of non-dyspnoeic dogs, and high-flow nasal cannula is well tolerated. Further studies are required to confirm these results in dyspnoeic dogs.

Apolipoprotein A-I and platelet factor 4 are biomarkers for infliximab response in rheumatoid arthritis.

OBJECTIVES: The use of biologicals such as infliximab has dramatically improved the treatment of rheumatoid arthritis (RA). However, factors predictive of therapeutic response need to be identified. A proteomic study was performed prior to infliximab therapy to identify a panel of candidate protein biomarkers of RA predictive of treatment response. METHODS: Plasma profiles of 60 patients with RA (28 non-responders (as defined by the American College of Rheumatology 20% improvement criteria (ACR20)) negative and 32 responders (ACR70 positive) to infliximab) were studied by surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) technology on two types of arrays, an anion exchange array (SAX2) and a nickel affinity array (IMAC3-Ni). Biomarker characterisation was carried out using classical biochemical methods (purification by ammonium sulfate precipitation or metal affinity chromatography) and identification by matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS analysis. RESULTS: Two distinct protein profiles were observed on both arrays and several proteins were differentially expressed in both patient populations. Five proteins at 3.86, 7.77, 7.97, 8.14 and 74.07 kDa were overexpressed in the non-responder group, whereas one at 28 kDa was increased in the responder population (sensitivity>56%, specificity>77.5%). Moreover, combination of several biomarkers improved the sensitivity and specificity of the detection of patient response to over 97%. The 28 kDa protein was characterised as apolipoprotein A-I and the 7.77 kDa biomarker was identified as platelet factor 4. CONCLUSIONS: Six plasma biomarkers are characterised, enabling the detection of patient response to infliximab with high sensitivity and specificity. Apolipoprotein A-1 was predictive of a good response to infliximab, whereas platelet factor 4 was associated with non-responders.

Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73.

Exosomes are membrane vesicles secreted by hematopoietic cells upon fusion of late multivesicular endosomes with the plasma membrane. Dendritic cell (DC)-derived exosomes induce potent antitumor immune responses in mice, resulting in the regression of established tumors (Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G. Raposo, and S. Amigorena. 1998. Nat. Med. 4:594-600). To unravel the molecular basis of exosome-induced immune stimulation, we now analyze the regulation of their production during DC maturation and characterize extensively their protein composition by peptide mass mapping. Exosomes contain several cytosolic proteins (including annexin II, heat shock cognate protein hsc73, and heteromeric G protein Gi2alpha), as well as different integral or peripherally associated membrane proteins (major histocompatibility complex class II, Mac-1 integrin, CD9, milk fat globule-EGF-factor VIII [MFG-E8]). MFG-E8, the major exosomal component, binds integrins expressed by DCs and macrophages, suggesting that it may be involved in exosome targeting to these professional antigen-presenting cells. Another exosome component is hsc73, a cytosolic heat shock protein (hsp) also present in DC endocytic compartments. hsc73 was shown to induce antitumor immune responses in vivo, and therefore could be involved in the exosome's potent antitumor effects. Finally, exosome production is downregulated upon DC maturation, indicating that in vivo, exosomes are produced by immature DCs in peripheral tissues. Thus, DC-derived exosomes accumulate a defined subset of cellular proteins reflecting their endosomal biogenesis and accounting for their biological function.

The phagosome proteome: insight into phagosome functions.

Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead-containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.

Biological autoimmunity screening in hepatitis C patients by anti-HepG2 lysate and anti-heat shock protein 70.1 autoantibodies.

Viruses require viral and cellular chaperones during their life cycle and interactions of these molecules with the immune system are probable during the infection. Thus, an anti-chaperone antibody response has been firstly investigated in hepatitis C patients in this paper. A HepG2-lysate antigen (90, 79, 72, 70, 62, 54 and 48 kDa) was assayed in sera from 59 (19F/40M) chronic hepatitis C patients without cirrhosis before therapy. Forty of them were positive for anti-HepG2 lysate antigen antibodies and this test may evaluate biological autoimmunity. Hsp70.1, Hsp90 and calreticulin levels were significantly higher in this antigen than in a control HepG2 antigen. Secondly, Hsp70.1 was identified as Hsp 70 kDa protein-1 by proteomic analysis and studied as a possible antibody target. Fourteen out of 59 patients were positive for anti-Hsp70.1 antibodies that were inversely correlated with alanine aminotransferase levels, the Metavir activity index and viraemia. Finally, for comparative purposes, 50 sera from systemic lupus erythematosus (SLE) patients have been tested: eight and 41 of them were positive for anti-Hsp70.1 and anti-HepG2 lysate antigen antibodies, respectively. Therefore, anti-Hsp70.1 autoantibodies may be produced and can partially lead to biological autoimmunity in chronic hepatitis C patients.

[Preanalytical guidelines for clinical proteomics investigation of biological fluids]. / Recommandations préanalytiques pour les analyses de protéomique clinique des fluides biologiques.

Research of new diagnosis or prognosis biomarkers is a major challenge for the management of patients with complex pathologies like cancer. Clinical proteomics is one of the recent approaches to identify these biomarkers in biological fluids. Over the last five years, many problems related to the variability and the quality control of these analyses have been observed. This was notably related to the different preanalytical status of each sample. A strong need for standardization of the critical preanalytical phases (collection, transport, processing, storage...) has been therefore recognized. With this goal in mind, working groups of the "Institut national du cancer" (INCa) and the "Société française de biologie clinique" (SFBC) proposed here preanalytical proteomics guidelines for the most common biological fluids: plasma, serum, urine and cerebrospinal fluid. To goal is to provide the basis for the harmonization of the procedures in clinical laboratories and biobanks to allow an optimal use of biological collections.

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Molecular cloning and characterization of a radial spoke head protein of sea urchin sperm axonemes: involvement of the protein in the regulation of sperm motility.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Hemorrhagic, Hemostatic, and Thromboelastometric Disorders in 35 Dogs with a Clinical Diagnosis of Leptospirosis: A Prospective Study.

BACKGROUND: Leptospirosis in dogs is occasionally associated with a hemorrhagic syndrome, the pathophysiology of which is not fully understood. HYPOTHESIS/OBJECTIVES: To characterize hematologic, hemostatic, and thromboelastometric abnormalities in dogs with leptospirosis and to study their association with hemorrhagic diatheses and outcomes. ANIMALS: Thirty-five client-owned dogs. METHODS: A prospective observational single cohort study was conducted. Results from the CBC, coagulation tests (prothrombin, activated partial thromboplastin and thrombin times, fibrinogen, fibrin(ogen) degradation products, and D-dimer concentrations), rotational thromboelastometry (TEM), signalment, hemorrhagic diatheses, occurrence of disseminated intravascular coagulation (DIC) at admission, and survival to discharge were recorded. RESULTS: The most common hematologic and hemostatic abnormalities were anemia (30/35), thrombocytopenia (21/35), and hyperfibrinogenemia (15/35). Eight dogs were diagnosed with DIC. A normal TEM profile was found in 14 dogs, a hypercoagulable profile in 14 dogs, and a hypocoagulable profile in 7 dogs. The 8 dogs with hemorrhagic diatheses at admission had significantly decreased platelet counts (P = .037) and increased D-dimer concentrations (P = .015) compared with other dogs. Dogs with a hypocoagulable profile exhibited more hemorrhagic diatheses compared with the dogs that had normal and hypercoagulable profiles (P = .049). The mortality rate was lower in dogs with a hypercoagulable profile than in those with a hypocoagulable profile (21% vs 57%; P = .043). Disseminated intravascular coagulation was not a significant prognostic factor. CONCLUSIONS AND CLINICAL IMPORTANCE: Thromboelastometric parameters were altered in dogs with both hypercoagulable and hypocoagulable profiles. A hypocoagulable profile was significantly correlated with hemorrhagic diathesis and higher mortality rate.

Metabolomic, proteomic and biophysical analyses of Arabidopsis thaliana cells exposed to a caesium stress. Influence of potassium supply.

The incorporation and localisation of 133Cs in a plant cellular model and the metabolic response induced were analysed as a function of external K concentration using a multidisciplinary approach. Sucrose-fed photosynthetic Arabidopsis thaliana suspension cells, grown in a K-containing or K-depleted medium, were submitted to a 1 mM Cs stress. Cell growth, strongly diminished in absence of K, was not influenced by Cs. In contrast, the chlorophyll content, affected by a Cs stress superposed to K depletion, did not vary under the sole K depletion. The uptake of Cs was monitored in vivo using 133Cs NMR spectroscopy while the final K and Cs concentrations were determined using atomic absorption spectrometry. Cs absorption rate and final concentration increased in a K-depleted external medium; in vivo NMR revealed that intracellular Cs was distributed in two kinds of compartment. Synchrotron X-ray fluorescence microscopy indicated that one could be the chloroplasts. In parallel, the cellular response to the Cs stress was analysed using proteomic and metabolic profiling. Proteins up- and down-regulated in response to Cs, in presence of K+ or not, were analysed by 2D gel electrophoresis and identified by mass spectrometry. No salient feature was detected excepting the overexpression of antioxidant enzymes, a common response of Arabidopsis cells stressed whether by Cs or by K-depletion. 13C and 31P NMR analysis of acid extracts showed that the metabolome impact of the Cs stress was also a function of the K nutrition. These analyses suggested that sugar metabolism and glycolytic fluxes were affected in a way depending upon the medium content in K+. Metabolic flux measurements using 13C labelling would be an elegant way to pursue on this line. Using our experimental system, a progressively stronger Cs stress might point out other specific responses elicited by Cs.

[Development of tissue microarray technology (TMA) for immunohistochemical study of molecular expression profiling in prostate cancer (part 1)]. / Desarrollo de un microarray tisular (TMA) para el estudio inmunohistoquímico del patrón de expresión molecular en el cáncer de próstata (parte 1).

Tissue microarray technology (TMA) is nowadays considered as a powerful tool for the high-throughput analysis of molecular expression pattern of cancer. In this manuscript we show the experience of both groups in the design and building of a TMA for the study of protein expression pattern of prostatecancer as well as a summary of the technical points to analyze the results obtained with this technology. Today, different data generated by the immunostained tissues are studied to achieve a molecular profile in different clinical scenarios.

Mapping of the pyrophosphate binding sites of beef heart mitochondrial F1-ATPase by photolabelling with azidonitrophenyl [alpha-32P]pyrophosphate.

4-Azido-2-nitrophenyl [alpha-32P]pyrophosphate (azido-[alpha-32P]PPi) mimics ADP and PPi by some of its binding properties when assayed in the absence of photoirradiation with mitochondrial F1-ATPase. Upon photoirradiation, both alpha- and beta-subunits of F1-ATPase were covalently labelled. Following chemical and enzymatic cleavages of each of the two photolabelled subunits, peptides containing the covalently bound radioactivity were separated by HPLC and identified by amino acid sequencing. Bound azido-[alpha-32P]PPi was found to be concentrated in two distant sequences of the alpha-subunit, namely Asp194-Thr221 and Lys386-Met437, and in a single sequence of the beta-subunit Glu294-Met358 with most of the photoprobe bound to beta-Tyr-311 and beta-Tyr-345. These results are discussed in terms of a model in which the pyrophosphate binding sites of F1 are located in regions of the alpha- and beta-subunits exposed at the interface between the two subunits and correspond to non-catalytic and catalytic adenine nucleotide binding sites, respectively.

Technical Advance: Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins.

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane alpha-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.

TIMP-1/MMP-9 imbalance in an EBV-immortalized B lymphocyte cellular model: evidence for TIMP-1 multifunctional properties.

Tissue inhibitors of metalloproteinases (TIMPs) were initially described as agents controlling metalloproteinase activity. The purpose of this study was to investigate the expression and the roles of TIMP-1 secreted by Epstein-Barr-virus (EBV)-immortalized B lymphocytes. TIMP-1 was isolated from conditioned medium of interleukin (IL)-1beta stimulated EBV-B lymphocytes; purified TIMP-1 was identified by mass spectrometry and immunochemistry. TIMP-1-free MMP-9 was quantified after purification by zymography and enzyme-linked immunosorbent assay. EBV-B lymphocyte-secreted TIMP-1 inhibited MMP-9 gelatinolytic activity resulting in decreased B-cell transmigration as measured in vitro. The release of huge amounts of TIMP-1 in proportion to MMP-9 from B lymphocytes after EBV transformation was shown to be correlated with secretion of IL-10 and dependent on culture time. In contrast, there was little TIMP-1 and almost no IL-10 released from native B cells, suggesting a possible IL-10 mediated autocrine regulation mechanism of TIMP-1 synthesis. The MMP-9/TIMP-1 imbalance observed in the culture medium of EBV-B lymphocytes (TIMP-1>MMP-9) and of native B cells (MMP-9>TIMP-1) is suggestive of a new function for TIMP-1. We propose that TIMP-1 acts as a survival factor controlling B-cell growth and apoptosis through an autocrine regulation process involving IL-10 secreted by EBV-B lymphocytes.

Cloning and characterization of cDNAs expressed during chick development and encoding different isoforms of a putative zinc finger transcriptional regulator.

Development proceeds through successive activation of different sets of genes by specific transcription factors as a consequence of cell interactions and signaling. It is thus of primary interest to identify new putative transcriptional regulators. We report here the isolation of chicken clones bearing sequences coding for a chicken zinc finger protein (chZFp) which contains four pairs of zinc fingers of mixed type C2-H-C/C2-H2. At least five chZFp isoforms are produced through differential splicing of four small exons. The amino acid domains encoded by these four exons are highly conserved across species. Northern blot analysis and RNase-protection assays showed that chZFp transcripts are present in brain, heart, skin and liver during chick development. Reverse transcription mediated polymerase chain reaction (RT-PCR) experiments suggested that the relative amount of some chZFp isoforms increases at critical stages of development and skin morphogenesis. Finally, the main chZFp isoforms are able to directly interact in vitro with the scaffold attachment factor-A (SAF-A, also known as heterogenous nuclear ribonucleoprotein U) through both their aminoterminal and carboxyterminal domains.

Differences between coagulation and cytokine profiles in dogs of different ages.

In human medicine, age is a risk factor for thromboembolic diseases associated with hypercoagulable and antifibrinolytic states, but information in veterinary medicine is limited. This study compared the thromboelastometric (TEM) profiles of two groups of dogs of distinct ages. Ten healthy old (>10 years) Beagles and 10 healthy young (<3 years) Beagles were recruited. White blood cell counts and haematocrit were significantly lower in the old group compared to the young group, and fibrinogen, total proteins, globulins and monocyte chemoattractant protein-1 plasma concentrations were significantly higher in the old group. Comparisons of the TEM profiles indicated a hypercoagulable profile and a decrease in fibrinolytic activity in all old Beagles. The findings support the need to consider age as a possible risk factor for thrombosis in dogs.

Characterization of MEC 14.7, a new monoclonal antibody recognizing mouse CD34: a useful reage for identifying and characterizing blood vessels and hematopoietic precursors.

Endothelial cell-specific molecules are potential targets for new therapeutic strategies in the control of inflammatory reactions, immune responses and neoangiogenesis. We describe the production and characterization of MEC 14.7, a monoclonal antibody directed to murine endothelial cells recognizing a glycosylated protein with an apparent molecular mass of about 100 kDa in cultured endothelioma cell lysate and about 80 kDa in lung lysate. MEC 14.7 antigen was selectively expressed by the endothelium in vivo, particularly in small vessels and neoformed capillaries and by developing vascular structures in embryonal bodies. Deglycosylation of the molecule with neuraminidase, O- and N-glycanase showed that the MEC 14.7 epitope is neuraminidase-sensitive. MEC 14.7 antigen was purified from lung lysates by chromatographic techniques, and sequenced internal peptides indicated it was identical with murine CD34. Thus the apparent molecular mass of CD34 is heterogeneous, depending on the glycosylation state in the different cell types. Immunomagnetic isolation and culture of MEC 14.7-positive bone marrow cells showed that this antibody recognizes hematopoietic progenitors (particularly myelomonocytic) and can be used in murine models of bone marrow reconstitution.

Photolabeling of the phosphate binding site of chloroplast coupling factor 1 with [32P]azidonitrophenyl phosphate.

Chloroplast F1-ATPase (CF1) was photolabeled by a radiolabeled photoactivatable derivative of Pi, 4-azido-2-nitrophenyl [32P]phosphate (ANPP). The radioactivity was localized in the beta subunit of CF1. Upon cleavage of the beta subunit by cyanogen bromide, the predominantly labeled peptide was recovered, which was subsequently subjected to tryptic digestion. A tryptic peptide (spanning Ile312-Arg354), was found to contain nearly all the covalently bound radioactivity. By Edman degradation, the labeled amino acid residues were identified as Tyr328, Val329 and Pro330. The labeled beta-Tyr328 of CF1 is the equivalent of beta-Tyr311 of F1 from beef heart mitochondria, which was previously found to be photolabeled by ANPP [J. Garin et al. (1989) Biochemistry 28, 1442-1448].