Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser₆₅ to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.     

Failure of primary breast cancer neoangiogenesis to predict pattern of distant metastasis

In the present study, the primary tumor angiogenesis characteristics of 81 stage IV previously untreated breast cancers with synchronous metastasis to different distant sites (10 patients with soft tissue metastases, 31 with bone metastases, and 40 with visceral metastases) were analyzed. The primary intratumor microvessel density was assessed by immunohistochemical assay on paraffin-embedded primary tumor samples, using a monoclonal anti-CD34 antibody. The mean primary intratumor microvessel density (at 400× fields) was 78±39 (SD) microvessels per field. The microvessel density was not significantly related to the main clinical/pathological features of the tumor (age, cytohistological grade, DNA ploidy, diameter, and receptor status). The percentage of tumor cases with high primary intratumor microvessel density (cut-off median value of the series 73±39 microvessels/field) did not significantly differ in patients with bone, soft tissue, or visceral metastatic disease. Aanalysis of clinical outcome showed a significantly shorter time to progression and overall survival for patients with visceral metastases (P<0.001 and P<0.0002 by log-rank, respectively). Presence of visceral metastases was confirmed to be the only independent prognostic factor related to a worse TTP (hazard risk 2.15, 95% confidence interval 1.14–4.03, P<0.02) and overall survival (hazard risk 1.81, 95% confidence interval 0.98–3.35, P<0.06) by multivariate analysis. In conclusion, the assessment of neoangiogenesis of primary breast cancer by CD34 expression does not provide information predictive of different distant sites of metastasis. Received: 15 June 2001 / Accepted: 12 September 2001     

Evaluation of low serum vitamin B(12) in the non-anaemic pregnant patient

Low serum vitamin B(12) concentrations in pregnancy may not indicate true megaloblastic anaemia. In the present study we compared biochemical indices of vitamin B(12) deficiency (serum homocysteine and urine methylmalonic acid) in non-anaemic pregnant women with and without low serum vitamin B(12) concentrations. The groups were matched for age, parity and gestational age. No differences were found, and all values were within normal range. These results suggest that the measurement of low serum B(12) concentrations in pregnant women should be followed by analysis at the biochemical level before vitamin B(12) injections are started.     

Adaptive Wiener Filtering for Improved Acquisition of Distortion Product Otoacoustic Emissions

An innovative acoustic noise canceling method using adaptive Wiener filtering (AWF) was developed for improved acquisition of distortion product otoacoustic emissions (DPOAEs). The system used one microphone placed in the test ear for the primary signal. Noise reference signals were obtained from three different sources: (a) pre-stimulus response from the test ear microphone, (b) post-stimulus response from a microphone placed near the head of the subject and (c) post-stimulus response obtained from a microphone placed in the subjects nontest ear. In order to improve spectral estimation, block averaging of a different number of single sweep responses was used. DPOAE data were obtained from 11 ears of healthy newborns in a well-baby nursery of a hospital under typical noise conditions. Simultaneously obtained recordings from all three microphones were digitized, stored and processed off-line to evaluate the effects of AWF with respect to DPOAE detection and signal-to-noise ratio (SNR) improvement. Results show that compared to standard DPOAE processing, AWF improved signal detection and improved SNR. © 1998 Biomedical Engineering Society. PAC98: 4364Jb, 4360-c, 8790+y     

Metabolic polymorphisms and the micronucleus frequency in buccal epithelium of adolescents living in an urban environment

Micronuclei and other biomarkers were evaluated in oral cells from 11- to 16-year-old girls living in a foster home in the central area of México City. Variables analyzed for possible association with these biomarkers include smoking habits, body mass index, metabolic polymorphisms for NAT1 and GSTM1 and whether the cells were obtained from the cheek or pharynx. The results indicated that individuals having the NAT1*10 homozygous genotype showed a significant increase in chromatin buds and binucleated cells. When the damage in the cheek was compared with damage in the pharynx, a significant increase in micronuclei and binucleated cells was found for the latter tissue in all the individuals analyzed.     

Mechanisms of the ATP potentiation of hyposmotic taurine release in Swiss 3T3 fibroblasts

Reducing osmolarity by 35% increased ³H-taurine efflux from Swiss 3T3 fibroblasts from 0.5% to a peak of 5.7%. The presence of ATP (10–100 µM; EC₅₀ 1.5 µM) increased taurine efflux up to 10%, and decreased the set point for hyposmotically stimulated taurine release (HTR). ATP potentiation was mimicked by UTP, reduced by addition of suramin and pyridoxal phosphate-6-azophenyl-2,4-disulphonic acid (PPADS) and unaffected by ADP, ,-methylene-ATP (,-ATP) or 2-methylthio-ATP (Me-ATP), suggesting its mediation by purinergic P2Y₂ and P2Y₄ metabotropic receptors. Under isosmotic conditions ATP increased the cytosolic [Ca²⁺] ([Ca²⁺]_i) markedly, but did not increase taurine release. HTR was independent of external Ca²⁺ but was reduced (by 56–59%) by BAPTA-AM, thapsigargin-induced depletion of intracellular Ca²⁺ stores, or phospholipase C (PLC) inhibition. Blockade of calmodulin (CaM) or calmodulin kinase II (CaMKII) reduced HTR by 54% and 76%, respectively. The ATP-mediated potentiation was prevented fully by all these treatments. HTR was reduced by 30–50% by blockers of protein tyrosine kinases (AG18), phosphoinositide 3-kinase (PI3K) (wortmannin), p21rho (toxin B), p21rho-kinase (Y27632) and the stress-activated kinase p38 (PD169316). ATP-mediated potentiation was reduced similarly by these blockers. Simultaneous inhibition of PI3K and CaMKII abolished HTR. Altogether, these results suggest a modulatory effect of ATP, probably exerted by a potentiation of the Ca²⁺-dependent fraction of HTR. This fraction has as signalling elements a PLC-dependent [Ca²⁺]_i increase, resulting from Ca²⁺ released from thapsigargin-sensitive internal stores, followed by activation of CaM/CaMKII reactions. The Ca²⁺/ATP effect operates only when the Ca²⁺-independent, tyrosine kinase-mediated pathway is already activated. Suggested elements of cross-talk between the two pathways are PLC, PI3K and CaMKII.     

An evaluation of the inactive mouse X chromosome in somatic cell hybrids

The expression of mouseZfx, Rps4, Ube1x, andXist was evaluated in hamstermouse somatic cell hybrids containing either an active or an inactive mouse X chromosome using polymerase chain reaction of reverse transcribed RNA (RT-PCR). The results showed thatZfx, Rps4, andUbe1x are expressed exclusively from the active mouse X, whileXist is expressed exclusively from the inactive X. These findings confirm the pattern of X inactivation for these mouse genes reported previously based on expression in somatic tissues of F₁ females from interspecific crosses. These results demonstrate the existence of differences between human and mouse X inactivation, as the corresponding human genes,ZFX, RPS4X, andUBE1 escape X inactivation.     

The source and action of histamine in the isolated guinea-pig gallbladder

We have investigated the effects of histamine on motility of the gallbladder and characterized the receptor types involved. Histamine and the histamine H₁-receptor agonist, 2-thiazolylethylamine (2-TEA) contracted the isolated guinea-pig gallbladder strip in a dose dependent manner. The contractile response to histamine was shifted to the right by the H₁-receptor antagonist, mepyramine. In pre-contracted gallbladder strips, the H₂-receptor agonist dimaprit reduced the tension generated in a dose dependent fashion. The histamine H₂-receptor antagonist, ranitidine shifted the histamine concentration effect curve to the left and attenuated the dose dependent relaxations elicited at high concentrations. The histamine H₃-receptor agonist, (R)--methylhistamine (RMHA) elicited dose dependent contraction of the tissue which was significantly inhibited in the presence of mepyramine. The effects of electrical field stimulation (EFS) on the strips were not significantly altered by the presence of RMHA (10^–10–10^–7 M) indicating little pre-synaptic H₃ activity in this tissue. Histamine immunoreactivity (IR) was detected in gallbladder whole mount preparations of the mucosa and the muscularis/serosa. The histamine IR appeared cell bound in cells of varying morphological characteristics but no IR was detected in nerve fibres or cell bodies (ganglia). Alcian blue staining was consistent with the distribution of histamine IR cells as mast cells. The results indicate that histamine is distributed in the guinea-pig gallbladder and it can regulate contractile activity via activation of H₁ and H₂ but not H₃ receptors.