Search PubMed⌕ Search

Biomedical subjects

A Cattaneo

Publications and source records attributed to A Cattaneo.

At least 55 records · Page 3Linked to original sources
PubMed1999

Functional blockade of tyrosine kinase A in the rat basal forebrain by a novel antagonistic anti-receptor monoclonal antibody.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

3T3 Cells↗
PubMed1999

Selection of antibodies for intracellular function using a two-hybrid in vivo system.

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.

Animals↗
PubMed1999

Single-chain variable fragments selected on the 57-76 p21Ras neutralising epitope from phage antibody libraries recognise the parental protein.

Phage antibodies have been widely prospected as an alternative to the use of monoclonal antibodies prepared by traditional means. Many monoclonal antibodies prepared against peptides are able to recognise the native proteins from which they were derived. Here we show that the same is also true for phage antibodies. We have selected a number of single-chain variable fragments (scFv) from a large phage scFv library against a peptide from the switch region II of p21Ras. This peptide is known to reside in a mobile area of the native protein and is the epitope of a well characterised monoclonal antibody. Selected scFvs were able to recognise native p21Ras in both ELISA and Western blots, indicating that peptides are also likely to be very useful in selecting from phage antibody libraries.

Amino Acid Sequence↗
PubMed1999

Increased density of M1 receptors in the hippocampus of juvenile rats chronically deprived of NGF.

Binding studies were used to assess the changes in affinity and/or number of M1 muscarinic receptors in hippocampi from juvenile rats chronically deprived of NGF. NGF deprivation was obtained by implanting into right ventricle at postnatal day 2 (P2) hybrydoma cells secreting high levels of monoclonal antibodies against NGF (alphaD11). Parenteral myeloma cells (P3U) were used as controls. Competition experiments were used to characterise the [3H]-PNZ binding sites in membrane preparations of hippocampi from rats sacrificed at P15. [3H]-PNZ bound M1 receptors both in P3U and alphaD11 group as shown by displacing potency order of antagonists: TLZ=4-DAMP>PNZ>p-F-HHSiD>MTC. The deprivation of NGF for two weeks significantly increased the number of M1 receptors without changing the Ki values of antagonists with exception of methoctramine which showed an increase in affinity in alphaD11 group. Similar changes in binding parameters were already observed after the first week of anti-NGF treatment. In contrast, a treatment for a week with implant at postnatal day 15 failed to produce any changes in M1 binding parameters. These results provide further physiological evidence for developmentally regulated modulatory role of NGF in the cholinergic function in the hippocampus.

Animals↗
PubMed1999

The selection of intracellular antibodies.

The intracellular expression of antibodies in mammalian cells is a strategy to inhibit the in vivo function of selected molecules but is limited by the unpredictable behaviour of antibodies when intracellularly expressed. Recent advances in the field of antibody expression in Escherichia coli show that the introduction of mutations can improve the properties of some antibody domains, but the general applicability of this approach to intracellular antibodies remains to be proved. As a complement to rational approaches, we describe selection schemes in which antibodies are selected on the basis of their performance in vivo as intracellular antibodies.

Animals↗
PubMed1999

Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula.

Allomyces arbuscula, an aquatic fungus, contains two Ca2+-dependent neutral cysteine proteases (CDP I and CDP II), eluting respectively, at 0.07 and 0.2 M NaCl from DEAE cellulose columns. The purified CDP I has a Mr of 39 kDa whereas CDP II appears as a doublet of 43 and 40 kDa. Both enzymes require free thiol, the same concentration of Ca2+ for half maximal activation, and are inactivated by thiol protease inhibitors. Our results show that despite these similarities the two enzymes are different because affinity-purified CDP II antibodies do not cross-react with CDP I antigen in Western blots. In contrast, there is a strong cross-reaction between the two 43 and 40 kDa CDP II peptides and their respective antibodies. Both enzymes cleave preferentially the carboxy terminus of Arg and to a limited extent Lys on the cleavage site. This primary specificity is governed by the nature of the amino acids in the P2 and P3 positions. In general either Pro or Gly in P2 is required, with preference for Pro and in P3 position, Gly over Val. CDP II has higher catalytic activity than CDP I. The sulfhydryl reagent NEM is a more potent inhibitor of CDP I than CDP II. Although the function of the phosphorylable site(s) is not clear, both CDP I and CDP II contain phosphorylable serine residue(s).

Calcium↗
PubMed1999

Dark rearing blocks the developmental down-regulation of brain-derived neurotrophic factor messenger RNA expression in layers IV and V of the rat visual cortex.

In this study, we describe the distribution of brain-derived neurotrophic factor messenger RNA in the binocular primary visual cortex of the rat during postnatal development, starting at postnatal day (P) 13. High-resolution non-isotopic in situ hybridization combined with Nissl staining were used to quantify the number of cells expressing brain-derived neurotrophic factor messenger RNA. At P13, most of the cells express brain-derived neurotrophic factor messenger RNA. After eye opening (P14-P15), the relative number of brain-derived neurotrophic factor messenger RNA-positive cells decreases by a factor of two in layer IV, i.e. that receiving the visual input, and in layer V. To verify the hypothesis that light could trigger this decrease, pups were kept in complete darkness from birth. At P22, pups reared in the dark were killed and the visual cortex processed for in situ hybridization and northern blotting. The results obtained in dark-reared animals prove that light deprivation can: (i) decrease the general levels of brain-derived neurotrophic factor messenger RNA, and (ii) increase the relative number of brain-derived neurotrophic factor messenger RNA-positive cells in layers IV and V with respect to control rats. Exposure to light for five days after the period of darkness restored the number of brain-derived neurotrophic factor messenger RNA-positive cells. We conclude that the expression of brain-derived neurotrophic factor messenger RNA in the rat primary visual cortex is regulated during development and that this process is under the control of visual input.

Animals↗
PubMed1999

Co-expression of TrkB and the N-methyl-D-aspartate receptor subunits NR1-C1, NR2A and NR2B in the rat visual cortex.

In the visual cortex, brain-derived neurotrophic factor expression is modulated through glutamate receptors, including the N-methyl-D-aspartate glutamate receptor. It has been proposed that the N-methyl-D-aspartate glutamate receptor subunit composition itself might be regulated by brain-derived neurotrophic factor. Here, we investigated the co-expression of the neurotrophin-4/brain-derived neurotrophic factor receptor TrkB with the N-methyl-D-aspartate glutamate receptor subunits NR1-C1, NR2A and NR2B, on postnatal days 10 and 22 and in the adult rat primary visual cortex. At both postnatal days 10 and 22, TrkB is co-expressed in all cortical layers with the studied N-methyl-D-aspartate glutamate receptor subunits. In the adult, in layers IV-V, co-expression is restricted to a subpopulation of neurons, while in layers II-III, VI nearly all neurons co-express TrkB with NR1-C1, NR2A and NR2B. We conclude that in layers IV-V, the co-expression of TrkB with subunits NR2B and NR2A is developmentally regulated.

Aging↗
PubMed1999

Differential regulation of brain-derived neurotrophic factor messenger RNA cellular expression in the adult rat visual cortex.

In this study, we report a comparative analysis of the distribution of brain-derived neurotrophic factor messenger RNA in the binocular primary visual cortex of rats analysed at the end of the critical period for monocular deprivation (postnatal day 35) and during adulthood (postnatal day 90). High-resolution non-isotopic in situ hybridization coupled with Nissl staining allowed to determine the relative number of neurons expressing brain-derived neurotrophic factor messenger RNA. In postnatal day 90 rats, the relative number of neurons positive for brain-derived neurotrophic factor messenger RNA significantly decreases in layer II/III with respect to postnatal day 35 animals, being constant in all the other cortical layers. Moreover, we demonstrate that dark rearing for 22 days, starting from postnatal day 90, determines: (i) a decrease of the overall level of brain-derived neurotrophic factor messenger RNA with a consequent reduction of labelling intensity in all cells throughout cortical layers II-VI; (ii) an increase of cell numbers expressing brain-derived neurotrophic factor messenger RNA in layers IV and V; and (iii) a decreased intensity of staining for brain-derived neurotrophic factor messenger RNA in dendrites after dark rearing. A re-exposure to light for 2 h after the period of darkness almost restores the number of brain-derived neurotrophic factor RNA-positive neurons. We conclude that the maturation of brain-derived neurotrophic factor messenger RNA in neurons of layer II/III goes beyond postnatal days 35-40, which can be considered the end of the critical period [Fagiolini M. et al. (1994) Vis. Res., 34, 709-720]. Moreover, we show that the cellular expression of brain-derived neurotrophic factor messenger RNA is regulated by light in adult rats as well as during development.

Animals↗
PubMed1999

Signs of severe bacterial infection in neonates.

The objective of this study was to identify a short list of valid signs for the development of standard case management guidelines for severe bacterial infection (SBI) in newborn infants, an important cause of neonatal deaths in low-income countries. The reported and observed signs of 83 sick neonates admitted during 12 consecutive months were recorded. At discharge, 50 cases were classified, using predefined criteria, as SBI, mostly pneumonia, and 33 as other disease. The neonates with other diseases were significantly younger than those with SBI. None of the reported and observed signs, when used alone, had a high sensitivity, an important feature for a severe disease amenable to effective treatment. The best sensitivity (74 per cent) was obtained when a doctor observed severe chest indrawing or fast breathing or 'not looking well'; the specificity was 67 per cent and the positive predictive value 77 per cent. The sensitivity of reported difficult breathing and of observed severe chest indrawing, when measured only for the diagnosis of pneumonia, improved to 77 per cent, with a specificity of 84 per cent and 66 per cent, respectively. Reported fever and the observation that the neonate was 'not looking well' were the best independent predictors of SBI on logistic regression analysis. Simple standard case management (SCM) guidelines based only on reported and observed clinical signs would not identify the majority of neonates with SBI at primary health care level.

Bacterial Infections↗
PubMed1999

Nipple care, sore nipples, and breastfeeding: a randomized trial.

Sore and cracked nipples are common and may represent an obstacle to successful breastfeeding. In Italy, it is customary for health professionals to prescribe some type of ointment to prevent or treat sore and cracked nipples. The efficacy of these ointments is insufficiently documented. The incidence of sore and cracked nipples was compared between mothers given routine nipple care, including an ointment (control group), and mothers instructed to avoid the use of nipple creams and other products (intervention group). Breastfeeding duration was also compared between the two groups. Eligible mothers were randomly assigned, after informed consent, to one of the two groups. No difference was found between the control (n = 96) and the intervention group (n = 123) in the incidence of sore and cracked nipples and in breastfeeding duration. However, several factors were associated with sore nipples and with breastfeeding duration. The use of a pacifier and of a feeding bottle in the hospital were both associated with sore nipples at discharge (p = 0.02 and p = 0.03, respectively). Full breastfeeding up to 4 months postpartum was significantly associated with the following early practices: breastfeeding on demand, rooming-in at least 20 hours/day, non-use of formula and pacifier, no test-weighing at each breastfeed. The incidence of sore and cracked nipples and the duration of breastfeeding were not influenced by the use of a nipple ointment. Other interventions, such as providing the mother with guidance and support on positioning and latching, and modifications of hospital practices may be more effective in reducing nipple problems.

Adult↗
PubMed1999

[Determinant factors for the use of screening for cervical cancer in Friuli Venezia Giulia].

BACKGROUND: To describe the determinants of missed or irregular Pap-test. METHODS DESIGN: cross-sectional descriptive study. SETTING: Trieste province (urban) and San Daniele district (mostly rural). SUBJECTS: 294 (89%) out of 332 (83%) women interviewed from a simple random sample of 400 women 25 to 64 years of age. INTERVENTION: none. DATA COLLECTION: planned telephone interview with a pre-tested questionnaire. RESULTS: 256 (87%) women had a Pap-test at least once in their life, 169 (57%) in the last two years. The 222 (76%) women with a Pap-test in the last five years know where and when, but not how often. The gynaecologist recommended the test in 63% of cases; only 23 women were informed by their general practitioner and only 8 decided the Pap-test based on this information. Fifty-three percent had the Pap-test always in the same health facility; the decision to change was related mainly with long waiting lists (18%) and distance (16%). In San Daniele district private health facilities were preferred to public ones more than in Trieste (48% vs 20%; p < 0.0005). Among women with regular Pap-test, 36% had higher education vs 23% among women with missed or irregular test (p = 0.05). The main reported reason for missed or irregular Pap test was inadequate information, followed by deficiencies in the quality of services. CONCLUSIONS: The Pap-test rate was higher than expected. The small number of women with missed or irregular Pap-test (66/294; 22%) does not allow to draw firm conclusions about the reasons for non use, though these seem to be similar to those already reported in the literature, i.e. lack of adequate information. A regional programme, including standard methods of assessment of coverage, would lead to increased regular use of this screening.

Adult↗
PubMed1998

A non-radioactive in situ hybridization method that does not require RNAse-free conditions.

This report describes a quick and versatile method to perform non-radioactive in situ hybridization in which none of the hybridization steps are performed under RNAse-free conditions. This study demonstrates that in situ hybridization can be performed without an RNAse-free environment provided that the concentration of RNAse introduced during the experiment does not reach 0.1 microg/ml, a concentration that is unlikely to be achieved through an accidental contamination. Moreover, evidence is provided that the only step sensitive to RNAse degradation is the pretreatment since degradation during the hybridization step can not occur due to a very efficient protective effect exerted by formamide. Finally, our data suggest that endogenous RNAse activity might be readily neutralized through paraformaldehyde fixation. A feature of this method is the strong fixation that ensures a perfect tissue preservation, even at level of the fine structure of the cell processes. The method allows a uniform tissue penetration by sodium periodate and sodium borohydride treatment and can be easily used in combination with diaminobenzidine immunohistochemistry for double labeling experiments.

Animals↗
PubMed1998

The mode of action of Y13-259 scFv fragment intracellularly expressed in mammalian cells.

The anti-p21ras Y13-259 single-chain Fv fragment (scFv) neutralizes the activity of p21-ras when intracellularly expressed in different systems. We have studied the mode of action of this inhibition in 3T3 K-ras fibroblasts and demonstrated that (i) this antibody fragment is highly aggregating when cytoplasmically expressed and (ii) the p21-ras antigen is sequestered in these aggregates in an antibody-dependent manner. This co-segregation leads to an efficient inhibition of DNA synthesis. These results suggest that an antigen can be diverted from its normal location inside the cells in an antibody mediated way, prospecting a new mode of action for intracellular antibodies in vivo.

3T3 Cells↗
PubMed1998

Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis.

Cerebellar granule cells undergo apoptosis in culture after deprivation of potassium and serum. During this process we found that tau, a neuronal microtubule-associated protein that plays a key role in the maintenance of neuronal architecture, and the pathology of which correlates with intellectual decline in Alzheimer's disease, is cleaved. The final product of this cleavage is a soluble dephosphorylated tau fragment of 17 kDa that is unable to associate with microtubules and accumulates in the perikarya of dying cells. The appearance of this 17 kDa fragment is inhibited by both caspase and calpain inhibitors, suggesting that tau is an in vivo substrate for both of these proteases during apoptosis. Tau cleavage is correlated with disruption of the microtubule network, and experiments with colchicine and taxol show that this is likely to be a cause and not a consequence of tau cleavage. These data indicate that tau cleavage and change in phosphorylation are important early factors in the failure of the microtubule network that occurs during neuronal apoptosis. Furthermore, this study introduces new insights into the mechanism(s) that generate the truncated forms of tau present in Alzheimer's disease.

Alzheimer Disease↗
PubMed1998

Cholinergic function in the hippocampus of juvenile rats chronically deprived of NGF.

Intracellular and extracellular recordings were used to assess the cholinergic function in hippocampal slices from juvenile rats chronically deprived of NGF. NGF was neutralised by implanting into the lateral ventricle of postnatal (P) day 2 rats, alphaD11 hybridoma cells (secreting monoclonal antibodies specific for NGF). Parental myeloma cells (P3U) were used as controls. At P15-P18, slow cholinergic EPSPs could be elicited in cells from both alphaD11- and P3U-treated rats. However, slices from alphaD11-implanted rats exhibited a 50% reduction in acetylcholine release following stimulation of cholinergic fibres. This effect was associated to a significant increase in the sensitivity of pyramidal cells to carbachol, as suggested by the shift to the left of the dose/response curve. This may reflect a compensatory mechanism for the reduced efficacy of cholinergic innervation in NGF-deprived rats. In both alphaD11- and P3U-treated rats, carbachol was able to induce a similar concentration-dependent depression of the field EPSPs, evoked by Schaffer collateral stimulation, suggesting that presynaptic muscarinic receptors were not altered. In rats implanted with alphaD11 cells at P15 and sacrificed at P21-P24, no changes in the sensitivity to carbachol were found. At this developmental stage, no differences in acetylcholine release were observed between P3U- and alphaD11-treated animals. These results provide physiological evidence for a regulatory role of NGF in the cholinergic function of the hippocampus during postnatal development.

Acetylcholine↗