Prediction of physical characteristics of the lamb carcass using in vivo bioimpedance analysis.

The increase of sheep meat competitiveness in international markets can be attributed to the rise of the quantity and the improvement of the quality of the edible portion of sheep carcasses. Usually, carcass yield is established after the slaughter of the animals. Yet, when carcass yield is determined in vivo, it can be both a costly and subjective method. This study proposes models for predicting the physical characteristics of lamb carcass using bioimpedance analysis (BIA) in live animals. Thirty-one Texel × Ile de France crossbreed ram lambs were slaughtered at 20, 26, 32 or 38 kg of BW. Before the slaughter, values of resistance (Rs) and reactance (Xc) were collected using a single-frequency BIA equipment (Model RJL Quantum II Bioelectrical Body Composition Analyzer). Then, BIA main variables such as body bioelectrical volume (V), phase angle (PA), resistive density (RsD) and reactive density (XcD) were calculated. After slaughter, cold carcass weight (CCW), cold carcass yield (CCY), subcutaneous fat thickness (SFT), soft tissue weight (STW) and soft tissue yield (STY) were also measured. Multiple regression analyses were carried out using the physical characteristics as dependent variables and the bioimpedance values as independent variables. Predictive performance of the models was assessed using leave-one-out cross-validation. The prediction model of CCW was obtained using the V, PA and RsD (R2 = 0.97), STW through the V, RsD and XcD (R2 = 0.97), CCY by Rs, Z and XcD (R2 = 0.69), STY by V and XcD (R2 = 0.67), and SFT only for XcD (R2 = 0.84). The results indicated that BIA has the potential to predict carcass characteristics of lambs at different body masses.

Carcaça e componentes corporais de cordeiros lactentes em pastejo de azevém suplementados com leguminosa ou concentrado / Carcass and body components of suckling lambs grazing ryegrass and supplemented with legume or concentrate

O objetivo desta pesquisa foi avaliar o efeito da suplementação privativa, com concentrado ou leguminosa, sobre as características da carcaça e dos componentes corporais de cordeiros lactentes mantidos em pastejo de azevém. Foram utilizados 27 cordeiros lactentes, distribuídos nos tratamentos que corresponderam aos sistemas de alimentação: cordeiros lactentes mantidos em pasto de azevém, cordeiros lactentes mantidos em pasto de azevém e suplementados com concentrado em comedouro privativo (creep feeding) e cordeiros lactentes mantidos em pasto de azevém e suplementados com leguminosa no pasto privativo (creep grazing). O concentrado era composto por milho, farelo de soja e calcário calcítico, e a leguminosa utilizada foi o trevo-branco. O delineamento experimental foi o inteiramente ao acaso. Os resultados foram submetidos à análise de variância, e as médias comparadas pelo teste de Tukey a 5% de significância. O conteúdo do trato gastrintestinal juntamente com bile e urina e a proporção de esôfago foram maiores (P<0,05) nos cordeiros mantidos em pasto de azevém. As proporções de fígado e intestino grosso foram maiores (P<0,05) nos cordeiros suplementados com concentrado. Os sistemas alimentares testados produzem carcaças com características semelhantes. A suplementação, com leguminosa ou concentrado, altera os componentes corporais de cordeiros lactentes mantidos em azevém.(AU)

This research objective was to evaluate the effect of private supplementation with concentrated or legumes, on the carcass characteristics and body components from suckling lambs kept on ryegrass pasture. Twenty seven suckling lambs were used, with about 17 days of age and weighing 9,91±0,594kg, which were distributed in the treatments that corresponded to feeding systems: suckling lambs kept on ryegrass pasture, suckling lambs kept on ryegrass pasture and supplemented with concentrated in private feeder (creep feeding) and suckling lambs kept on ryegrass pasture and supplemented with legume in the private pasture (creep grazing). The concentrate supplement was composed by corn, soybean meal and limestone, and was supplied ad libitum. The supplementary pasture was white clover legume. The experimental design was completely randomized, where the results were submitted to analysis of variance and means compared by Tukey test at 5% of significance level. The gastrointestinal+bile+urine content and the proportion of esophagus were higher (P<0,05) in lambs kept on ryegrass. The liver and large intestine proportions were higher (P<0,05) in lambs supplemented with concentrate. The tested alimentary systems produce carcasses with similar characteristics. The supplementation with legume or concentrate cause changes in body components of suckling lambs grazing ryegrass.(AU)

Resíduo úmido de cervejaria na terminação de cordeiros em confinamento e seus efeitos sobre as características da carcaça e dos componentes não carcaça / Wet brewery residue in the finishing of feedlot lambs and its effects on the carcass characteristics and non-carcass components

O objetivo deste trabalho foi avaliar o efeito de níveis crescentes de substituição da silagem de sorgo por resíduo úmido de cervejaria sobre as características da carcaça e dos componentes não carcaça de cordeiros terminados em confinamento. Foram utilizados 24 cordeiros machos, não castrados, oriundos de parto simples e mantidos em baias individuais. Os tratamentos foram constituídos por quatro níveis de substituição de silagem de sorgo por resíduo úmido de cervejaria, sendo: 0%; 33,5%; 66,5% e 100% de substituição. Utilizou-se uma relação volumoso:concentrado de 50:50, com base na matéria seca. O concentrado era constituído por milho desintegrado, farelo de soja e mistura mineral. As dietas eram isoproteicas, contendo 18,81% de proteína bruta. Os cordeiros foram abatidos quando atingiram o escore de condição corporal estabelecido em 3 (escala de 1 a 5). As características de carcaça analisadas não foram influenciadas significativamente (P>0,05) pela substituição de silagem de sorgo por resíduo úmido de cervejaria, sendo obtidos valores médios de 18,92kg para peso de carcaça quente, 18,22 kg para peso de carcaça fria, 47,03% para rendimento de carcaça quente, 45,29% para rendimento de carcaça fria e 3,41% para índice de quebra ao resfriamento. Quanto às proporções dos diferentes cortes comerciais avaliados, em relação ao peso de carcaça fria, foram verificados valores médios de 31,86% para perna, 18,12% para paleta, 39,46% para costilhar e 9,08% para pescoço. As proporções de diafragma, omaso cheio e omaso vazio, em relação ao peso vivo ao abate dos cordeiros, diminuíram linearmente (P≤0,05). As demais variáveis dos componentes não carcaça avaliadas não foram influenciadas (P>0,05) pelos níveis de resíduo úmido de cervejaria das dietas. Pode-se recomendar o uso de resíduo úmido de cervejaria como fonte exclusiva de alimento volumoso quando se utiliza uma relação volumoso:concentrado de 50:50, em base de matéria seca, para terminação de cordeiros em sistema de confinamento.(AU)

The objective of this study was to evaluate the effect of increasing levels of substitution of sorghum silage by wet brewery residue as forage food on carcass characteristics and non-carcass components of lambs finished in feedlot. Twenty four non castrated male lambs, Suffolk breed, single birth were maintained in individual stalls. The treatments consisted of four substitution levels of sorghum silage by wet brewery residue (0%; 33.5%; 66.5% or 100% of substitution). Roughage and concentrate were used at a 50:50 ratio, based on dry matter. The concentrate was composed of ground corn, soybean meal and mineral mixture. The diets were isoproteic containing 18.81% crude protein. The lambs were slaughtered when they reached the body condition score of 3 (ranging from 1 to 5). The carcass traits were not affected significantly (P>0,05) by substitution of sorghum silage by wet brewery residue. The average values of hot carcass weight, cold carcass weight, hot carcass yield, cold carcass yield and cooling weight losses were 18.92 kg, 18.22 kg, 47.03%, 45.29%, 3.41%, respectively. The proportion of commercial cuts in relation to the cold carcass weight, was 31.86% for leg, 18.12% for shoulder, 39.46% for ribs and 9.08% for neck. The proportion of diaphragm, omasum full and empty omasum in relation to body weight at slaughter decreased linearly (P≤0.05). The remaining variables of non-carcass components were not affected (P>0.05) by wet brewery residue inclusion. We recommend the use of wet brewery residue as exclusive source of roughage food when using roughage:concentrate at 50:50 ratio on a dry matter basis, for finishing feedlot lambs.(AU)

A new electro-optical approach for conductance measurement: an assay for the study of drugs acting on ligand-gated ion channels.

Ligand gated ion channels are involved in many pathophysiological processes and represent a relevant, although challenging, target for drug discovery. We propose an innovative electro-optical approach to their analysis able to derive membrane conductance values from the local membrane potential changes imposed by test current pulses and measured by fast voltage-sensitive fluorescent dyes. We exploited the potential of this proprietary method by developing a drug testing system called "ionChannel Optical High-content Microscope" (ionChannelΩ). This automated platform was validated by testing the responses of reference drugs on cells expressing different ligand-gated ion channels. Furthermore, a double-blind comparison with FLIPR and automated patch-clamp was performed on molecules designed to act as antagonists of the P2RX7 receptor. ionChannelΩ proved highly reliable in all tests, resulting faster and more cost-effective than electrophysiological techniques. Overall, ionChannelΩ is amenable to the study of ligand gated ion channels that are receiving less attention due to limitations in current assays.

Consumo e desempenho de cordeiros e borregos alimentados com dietas de alto concentrado de milho ou sorgo / Intake and performance of lambs and hoggets fed high concentrate corn or sorghum diets

Objetivou-se avaliar os consumos de matéria seca, nutrientes e desempenho na terminação de cordeiros e borregos submetidos a dietas de alto concentrado de grão de milho ou sorgo. Foram utilizados 32 animais, sendo 16 cordeiros (dente de leite) e 16 borregos (dois dentes) da raça Corriedale. As dietas eram constituídas de feno de aveia- branca (Avena sativa), grão de milho (Zea mays) ou de sorgo (Sorghum bicolor (L.) Moench), farelo de soja (Glycine Max), calcário calcítico, bicarbonato de sódio e monensina. Os cordeiros apresentaram superioridade (P≤0,05) quanto ao CMS (% do PV e g/kg PV0,75), ao CPB (kg/dia, % do PV e g/kg PV0,75), ao CEE (% do PV), ao CFDN e ao CFDA (% do PV e g/kg PV0,75), ao CNDT (% do PV), ao ganho de peso médio diário, à conformação in vivo e à conversão alimentar. Por outro lado, o CEE, o CCHT e o CCNE (kg/dia), o peso vivo inicial, o peso vivo final e o peso vivo ao abate foram superiores (P≤0,05) na categoria dos borregos. Em relação aos grãos avaliados, verificou-se maior (P≤0,05) CEE (kg/dia, % do PV e g/kg PV0,75) e menor (P≤0,05) CFDN (% do PV) e CFDA (kg/dia, % do PV e g/kg PV0,75) para os animais alimentados com dieta de alto concentrado à base de grão de milho em relação àqueles alimentados com grão de sorgo. Os cordeiros apresentam consumos relativos superiores aos borregos, porém sua resposta zootécnica é maior. O uso de dietas de alto concentrado de sorgo ou de milho proporcionou resultados semelhantes.(AU)

This study aimed to evaluate the dry matter, nutrient intake, and performance on feedlot of lambs and hoggets submitted to corn or sorghum high concentrate diets. Thirty-two Corriedale animals, being 16 lambs (milk teeth) and 16 hoggets (two teeth) were used. The diets were composed of white oat hay (Avena sativa), corn (Zea mays) or sorghum grain (Sorghum bicolor (L.) Moench), soybean meal (Glycine Max), limestone, sodium bicarbonate, and monensin. The lambs presented a superiority (P≤0.05) regarding the DMI (% of LW and g/kg LW0.75), CPI (kg/day, % of LW and g/kg LW0.75), EEI (% of LW), NDFI and ADFI (% of LW and g/kg LW0.75), TDNI (% of LW), daily average weight gain, conformation in vivo and feed conversion. On the other hand, the EEI, TCI, and NSCI (kg/day), the initial live weight, final live weight and the live weight at slaughter were superior (P≤0.05) in the hoggets category. In relation to the evaluated grains, a higher (P≤0.05) EEI (kg/day, % of LW and g/kg LW0.75) and a lower (P≤0.05) NDFI (% of LW) and ADFI (kg/day, % of LW and g/kg LW0.75) were verified for the animals fed with high concentrate diets based on corn grain in relation to those fed with sorghum grain. The lambs presented an intake relatively superior to the hoggets, however their zootechnical response is higher. The use of high concentrate diets of sorghum or corn provides similar results.

A novel environmental chamber for neuronal network multisite recordings.

Environmental stability is a critical issue for neuronal networks in vitro. Hence, the ability to control the physical and chemical environment of cell cultures during electrophysiological measurements is an important requirement in the experimental design. In this work, we describe the development and the experimental verification of a closed chamber for multisite electrophysiology and optical monitoring. The chamber provides stable temperature, pH and humidity and guarantees cell viability comparable to standard incubators. Besides, it integrates the electronics for long-term neuronal activity recording. The system is portable and adaptable for multiple network housings, which allows performing parallel experiments in the same environment. Our results show that this device can be a solution for long-term electrophysiology, for dual network experiments and for coupled optical and electrical measurements.

A new cross-correlation algorithm for the analysis of "in vitro" neuronal network activity aimed at pharmacological studies.

Modern drug discovery for Central Nervous System pathologies has recently focused its attention to in vitro neuronal networks as models for the study of neuronal activities. Micro Electrode Arrays (MEAs), a widely recognized tool for pharmacological investigations, enable the simultaneous study of the spiking activity of discrete regions of a neuronal culture, providing an insight into the dynamics of networks. Taking advantage of MEAs features and making the most of the cross-correlation analysis to assess internal parameters of a neuronal system, we provide an efficient method for the evaluation of comprehensive neuronal network activity. We developed an intra network burst correlation algorithm, we evaluated its sensitivity and we explored its potential use in pharmacological studies. Our results demonstrate the high sensitivity of this algorithm and the efficacy of this methodology in pharmacological dose-response studies, with the advantage of analyzing the effect of drugs on the comprehensive correlative properties of integrated neuronal networks.

Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals.

We have developed a semi-quantitative method for indirectly revealing variations in the concentration of second messengers (Ca(2+), cyclic AMP) in single presynaptic boutons by detecting the phosphorylation of the synapsins, excellent nerve terminal substrates for cyclic AMP- and Ca(2+)/calmodulin-dependent protein kinases. For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (at site 3) or synapsins I/II phosphorylated by either cAMP-dependent protein kinase or Ca(2+)/calmodulin-dependent protein kinase I (at site 1). Cerebellar granular neurones in culture were double-labelled with a monoclonal antibody to synapsins I/II and either of the polyclonal antibodies. Digitised images were analysed to determine the relative phosphorylation stoichiometry at each individual nerve terminal. We have found that: (i) under basal conditions, phosphorylation of site 3 was undetectable, whereas site 1 exhibited some degree of constitutive phosphorylation; (ii) depolarisation in the presence of extracellular Ca(2+) was followed by a selective and widespread increase in site 3 phosphorylation, although the relative phosphorylation stoichiometry varied among individual terminals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often occurred in specific nerve terminal sub-populations aligned along axon branches. In addition to shedding light on the regulation of synapsin phosphorylation in living nerve terminals, this approach permits the spatially-resolved analysis of the activation of signal transduction pathways in the presynaptic compartment, which is usually too small to be studied with other currently available techniques.

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.

Neurite extension occurs in the absence of regulated exocytosis in PC12 subclones.

We have investigated the process leading to differentiation of PC12 cells. This process is known to include extension of neurites and changes in the expression of subsets of proteins involved in cytoskeletal rearrangements or in neurosecretion. To this aim, we have studied a PC12 clone (trk-PC12) stably transfected with the nerve growth factor receptor TrkA. These cells are able to undergo both spontaneous and neurotrophin-induced morphological differentiation. However, both undifferentiated and nerve growth factor-differentiated trk-PC12 cells appear to be completely defective in the expression of proteins of the secretory apparatus, including proteins of synaptic vesicles and large dense-core granules, neurotransmitter transporters, and neurotransmitter-synthesizing enzymes. These results indicate that neurite extension can occur independently of the presence of the neurosecretory machinery, including the proteins that constitute the fusion machine, suggesting the existence of differential activation pathways for the two processes during neuronal differentiation. These findings have been confirmed in independent clones obtained from PC12-27, a previously characterized PC12 variant clone globally incompetent for regulated secretion. In contrast, the integrity of the Rab cycle appears to be necessary for neurite extension, because antisense oligonucleotides against the neurospecific isoform of Rab-guanosine diphosphate-dissociation inhibitor significantly interfere with process formation.

Parkinson's disease, pesticides, and glutathione transferase polymorphisms.

BACKGROUND: Parkinson's disease is thought to be secondary to the presence of neurotoxins, and pesticides have been implicated as possible causative agents. Glutathione transferases (GST) metabolise xenobiotics, including pesticides. Therefore, we investigated the role of GST polymorphisms in the pathogenesis of idiopathic Parkinson's disease. METHODS: We genotyped by PCR polymorphisms in four GST classes (GSTM1, GSTT1, GSTP1, and GSTZ1) in 95 Parkinson's disease patients and 95 controls. We asked all patients for information about pesticide exposure. FINDINGS: The distribution of the GSTP1 genotypes differed significantly between patients and controls who had been exposed to pesticides (controls vs patients: AA 14 [54%] of 26 vs seven [18%] of 39; AB 11 [42%] of 26 vs 22 [56%] of 39; BB 1 [4%] of 26 vs six [15%] of 39; AC 0 vs four [10%] of 39, p=0.009). No association was found with any of the other GST polymorphisms. Pesticide exposure and a positive family history were risk factors for Parkinson's disease. INTERPRETATION: GSTP1-1, which is expressed in the blood-brain barrier, may influence response to neurotoxins and explain the susceptibility of some people to the parkinsonism-inducing effects of pesticides.

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

Downregulation of MAP kinase activity signalled by HIV-1-gp120 coat protein in granular neurons and glial cells from rat cerebellum.

We have studied the effect of gp120 coat protein from HIV-1 on tyrosine phosphorylation processes in primary cultures of granular neurons or glial cells from the cerebellum of neonatal rats. The extracellular application of recombinant gp120 (200 pM) was able to reduce the phosphotyrosine content and the immunoreactivity for active form-specific antibodies of MAP kinase. Whereas in neurons MAP kinase appeared to be the only protein whose phosphotyrosine content was decreased, in glial cultures the inhibitory effect of gp120 on tyrosine phosphorylation processes appeared to be more widespread. In neuronal cultures, the effect of the viral protein was prevented by the concomitant treatment with depolarizing agents.

Tat protein from HIV-1 activates MAP kinase in granular neurons and glial cells from rat cerebellum.

We have investigated the effect of extracellularly applied Tat protein of the human immunodeficiency virus type 1 (HIV-1) on tyrosine phosphorylation processes, which represent a major signal transduction pathway of cells of the central nervous system. Primary cultures of rat cerebellar astrocytes or granule cells were incubated with synthetic Tat (10 ng/ml) for various periods of time and analyzed for their phosphotyrosine content by Western blotting. In both types of cultures Tat was able to induce the phosphorylation of mitogen-activated protein kinase (MAP kinase) on tyrosine residues, although with different kinetics and isoform specificity. In addition, in neuronal cells, but not in astrocytes, Tat increased the phosphotyrosine content of Shc, a protein involved in signal transduction downstream of receptor tyrosine kinase activation. This study shows that Tat applied extracellularly is able to induce the generation of intracellular signals in neuronal as well as glial cells.

Phosphorylation-dependent effects of synapsin IIa on actin polymerization and network formation.

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphorylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Hypertension-associated point mutations in the adducin alpha and beta subunits affect actin cytoskeleton and ion transport.

The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (Q529R) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: (a) actin assembly both in a cell-free system and within transfected cells; (b) topography of alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage.

Determination of ErbB2 protein in breast cancer tissues by different methods. Relationships with other biological parameters.

Four different methods to measure in parallel the erbB2 protein expression (p185neu) were evaluated in order to: a) compare two enzyme immunoassays with the immunohistochemical assays (IHC) and western blotting (WB) and b) extrapolate eventual relationships between erbB2 and biological parameters. Tissue samples from 248 patients with primary breast cancer were consecutively assayed. We used two different cut-off levels for WB, ELISA, and EIA, defined as follows: 1) the highest level of expression of non malignant tissue was chosen as the discriminant threshold between 'low' and 'elevated' samples: 2) the elevated group was further subdivided into two subgroups: 'intermediate' and 'high', according to their median value. According to the first cut-off, the results were considered 'elevated' in about 52% of cases with the three biochemical methods, while using the second cut-off the percentage lowered to about 26%. Considering this cut-off, the concordance rates between the paired biochemical methods ranged between: 78.4% (WB vs EIA), 93% (ELISA vs EIA), and 82.6% (ELISA vs WB). The comparison between biochemical and immunohistochemical methods gave these concordance rates: 82% (WB vs IHC), 90.5% (ELISA vs IHC), and 85.5% (EIA vs. IHC). According to the first cut off level, 27.5% of tumor samples showed IHC detectable p185 levels, in agreement with other immunohistochemical studies. The relationship between high erbB2 and estrogen and progesterone receptors showed an inverse association. No relationship was found between erbB2 and axillary lymph node positivity or tumor size. In short, the results of the four methods seem generally well correlated; nevertheless, it appears that different methodological approaches of measuring p185neu are not completely equivalent, and there is a need for an authoritative standardization and quality control for clinical applications.

Focal adhesion kinase in rat central nervous system.

Focal adhesion kinase (pp125FAK, FAK) is a 125 kDa non-receptor tyrosine kinase enriched in focal adhesions of various cell types, where it is thought to transduce signals triggered by contact with the extracellular matrix. We have studied the expression and localization of FAK in rat CNS. Immunoblotting, immunohistochemistry and in situ hybridization revealed the presence of FAK in all regions of the adult brain and demonstrated its enrichment in specific neuronal populations of the cerebral and cerebellar cortex, as well as in the hippocampus. During development, FAK protein levels were highest around birth in cerebral cortex and caudate putamen and decreased in the adult. In situ hybridization revealed enrichment of FAK mRNA in the ventricular germinative and external layers during the last period of embryonic growth. In primary cultures FAK immunoreactivity was localized in focal adhesions in astrocytes, whereas in developing neurons the highest levels were found in growth cones and perikarya. In the growth cone, FAK immunoreactivity colocalized with actin filaments. In mature neurons FAK appeared to be distributed in the whole cytoplasm, with no enrichment in any cellular compartment. Our results demonstrate the presence of high levels of FAK in rat CNS, maximal during development but persistent in the adult. Its enrichment in growth cones suggests that it may play a role in neurite outgrowth, as well as in plasticity in the adult.

HIV-1 gp120 glycoprotein induces [Ca2+]i responses not only in type-2 but also type-1 astrocytes and oligodendrocytes of the rat cerebellum.

Cultures of cerebellar cortex cells were exposed to the HIV-1 envelope glycoprotein, gp120, and investigated for cytosolic Ca2+ ion concentration ([Ca2+]i) changes by the fura-2 ratio videoimaging technique while bathed in complete, Na(+)-free or Mg(2+)-free Krebs-Ringer media. At the end of the [Ca2+]i experiments the cells were fixed and immunoidentified through the revelation of markers specific for neurons (microtubule associated protein-2), type-2 (A2B5) or all (glial fibrillary acidic protein) astrocytes, oligodendrocytes (galactocerebroside) or microglia (F4/80 antibody). In complete medium, rapid biphasic (spike-plateau) responses induced by gp120 (0.1-1 nM) were observed in a subpopulation of type-2 astrocytes. In addition, slow but progressive responses were observed in other type-2 cells and oligodendrocytes, whereas type-1 astrocytes showed small responses, if any, and granule neurons did not respond at all. Use of Na(+)-free medium (a condition that blocked another gp120-induced response, cytosolic alkalinization) resulted in an increase in [Ca2+]i response that was appreciable not only in type-2 but also in most type-1 astrocytes, possibly because of the inhibition of the Na+/Ca2+ exchanger and the ensuing decrease in Ca2+ extrusion. Granule neurons, including those in direct contact with responsive astrocytes, remained unresponsive, even when the experiments were carried out in Mg(2+)-free medium supplemented with glycine, a condition that favors activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastric carcinoma with osteoclast-like giant cells. Report of four cases.

Four cases of gastric carcinoma are described that are associated with an osteoclast-like giant cell (OGC) stromal component. The patients were all middle-aged men (range 53-63 years). Microscopically, the tumors were characterized by a bland cytologic appearance, and an either solid or cribriform pattern. Osteoclast-like giant cells were found adjacent to, or intimately intermixed with, the neoplastic cells in the primary gastric masses and in the lymph nodal metastases and were often associated with lymphocytes, histiocytes, and desmoplastic stroma. By immunohistochemistry, mononuclear cells and OGCs showed diffuse positivity for alpha-1-antichymotrypsin, alpha-1-antitrypsin, and CD68. Neoplastic cells that were positive for keratin and CEA, also showed reactivity for vimentin and the latent membrane protein of Epstein-Barr virus in one case. At follow-up, three patients had died at 13, 15, and 24 months after diagnosis, and one is still alive, without evidence of disease, after 120 months. This report describes a novel variant of gastric carcinoma with distinctive and histologic features.