Re-expression of the mannose 6-phosphate receptors in receptor-deficient fibroblasts. Complementary function of the two mannose 6-phosphate receptors in lysosomal enzyme targeting

We have previously generated primary embryonic fibroblasts lacking either the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR) or the cation-dependent MPR, two trans-membrane proteins that bind the mannose 6-phosphate (Man-6-P) recognition marker on soluble lysosomal enzymes (Ludwig, T., Munier-Lehmann, H., Bauer, U., Hollinshead, M., Ovitt, C., Lobel, P., and Hoflack, B.(1994) EMBO J. 13, 3430-3437). These two cell types partially missort phosphorylated lysosomal enzymes. Using two-dimensional gel electrophoresis, we show here that they secrete, in a large part, different phosphorylated ligands. In order to better understand the sorting function of the MPRs, we have re-expressed each MPR in MPR-negative fibroblasts. We show that the MPRs have similar capacities for transporting the bulk of the newly synthesized lysosomal enzymes and that they target individual ligands with various efficiencies. However, high levels of one MPR do not fully compensate for the absence of the other, demonstrating that the two MPRs have complementary targeting functions, perhaps by recognizing different features on lysosomal enzymes. The analysis of the phosphorylated oligosaccharides shows that the ligands missorted in the absence of the cation-dependent MPR are slightly but significantly depleted in oligosaccharides with two Man-6-P residues, when compared with those missorted in the absence of the cation-independent MPR. While these results could explain some differences between the structure and the sorting function of the two MPRs, they strongly suggest that the reason why cells express two different but related MPRs is to maintain an efficient Man-6-P-dependent targeting process that could be potentially regulated by MPR expression.     

An autosomal glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) polymorphism in human saliva

Glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) from human saliva has been demonstrated by the zymogram technique. Three phenotypes were found. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles Sgd-1 and Sgd-2.     

Biosynthesis, intracellular processing and secretion of haptoglobin in cultured rat hepatocytes

Biosynthesis, intracellular processing and secretion of the hetero-tetrameric (alpha 2 beta 2) glycoprotein, haptoglobin, were studied in primary cultured rat hepatocytes. The results obtained from pulse-chase experiments demonstrated that haptoglobin was initially synthesized as a larger precursor (pro-form), a single polypeptide chain comprising both the alpha- and beta-subunits, and immediately cleaved into subunits during intracellular transport, although about 8% of the newly synthesized haptoglobin was secreted as a pro-form. Monensin which impedes the secretory process at the Golgi complex blocked the complete glycosylation of beta-subunit but rather accelerated the conversion of the pro-form to subunits. These results indicate that the proteolytic processing of the haptoglobin precursor takes place at an early stage before the Golgi complex of the intracellular transport.     

Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP)

Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may participate in sphingolipid transport and signaling events. Re-uptake of the secreted precursor is mediated by high-affinity cell surface receptors that are apparently distinct from the M-6-P receptor. We found that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor that is expressed on most cells, can mediate cellular uptake and lysosomal delivery of SAP precursor. Additional in vivo experiments in mice revealed that the mannose receptor system on macrophages also participates in precursor internalization. We conclude that SAP precursor gains entry into cells by at least three independent receptor mechanisms including the M-6-P receptor, the mannose receptor and LRP.     

Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells

We have previously reported that plasminogen activator inhibitor type-1 (PAI-1) expression in endothelial cells (ECs) can be modulated differently by smooth muscle cells depending on their origin. Human pulmonary artery smooth muscle cells (HPASMCs) strongly downregulated PAI-1 expression in ECs. Fibroblasts (FBs) are another cell type that could come in close contact with ECs. Therefore, it was the aim of this study to investigate whether FBs could also influence the fibrinolytic potential of ECs. As in the case of HPASMCs, PAI-1 antigen produced by human umbilical vein ECs (HUVECs) cocultured with human skin FBs (HSFBs) was significantly lower as compared with the sum of PAI-1 secreted by the respective cell types cultured separately. Not only HUVECs but also human skin microvascular ECs (HSMECs) responded in a dose-dependent way to serum-free conditioned media (CM) from HSFBs from one individual donor. Similar results were obtained when CM from HSFBs from four other individual donors were used. PAI-1 mRNA decreased in HUVECs incubated for 6 hours with HSFB-CM to 24% to 55% of control, depending on the preparation of HSFBs used. A significant PAI-1 downregulatory effect was only observed when CM from low-passage HSFBs (up to passage no. 5) was used, whereas no reduction in EC PAI-1 production was observed with CM obtained from HSFBs in passage no. 8. This PAI-1 downregulatory activity present in HSFB-CM was heat-labile and had a molecular mass of approximately 5 kD. When CM from HPASMCs was analyzed in the same way, an almost identical elution profile was found. In conclusion, our data showed that FBs can decrease the expression of PAI-1 in ECs. Such an effect could be operative during wound-healing and at other capillary sites where FBs could render ECs profibrinolytic, thereby facilitating processes requiring an increase in proteolytic activity such as EC migration and proliferation.     

The alternatively spliced Kunitz protease inhibitor domain alters amyloid beta protein precursor processing and amyloid beta protein production in cultured cells

The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a approximately 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (betaAPP). During normal processing the betaAPP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPPbeta) and a COOH-terminal fragment beginning at Abeta1, which is subsequently cleaved by gamma secretase releasing secreted Abeta. Most secreted Abeta is Abeta1-40, but approximately 10% of secreted Abeta is Abeta1-42. Alternative betaAPP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPPalpha) and a COOH-terminal fragment beginning at Abeta17, which is subsequently cleaved by gamma secretase releasing a approximately 3-kDa secreted form of Abeta (P3). Several of the betaAPP isoforms that are produced by alternative splicing contain a 56-amino acid Kunitz protease inhibitor (KPI) domain known to inhibit proteases such as trypsin and chymotrypsin. To determine whether the KPI domain influences the proteolytic cleavages that generate Abeta, we compared Abeta production in transfected cells expressing human KPI-containing betaAPP751 or KPI-free betaAPP695. We focused on Abetas ending at Abeta42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Abeta1-42 and total Abeta ending at Abeta42 (Abeta1-42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by alpha secretase (sAPPalpha) and beta secretase (sAPPbeta). In mouse teratocarcinoma (P19) cells expressing betaAPP695 or betaAPP751, expression of the KPI-containing betaAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPalpha and a correspondingly higher percentage of Abeta1-42 and sAPPbeta. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces alpha secretase cleavage so that less P3 and relatively more full-length Abeta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing betaAPP produced may be an important factor influencing Abeta deposition.     

Binding and degradation of lipoprotein(a) and LDL by primary cultures of human hepatocytes. Comparison with cultured human monocyte-macrophages and fibroblasts

Although lipoprotein(a) (Lp[a]) has structural similarities to low-density lipoprotein (LDL) that include the presence of apolipoprotein B100, there is some disagreement over the strength of its interaction with the LDL receptor and its cellular catabolism by the LDL receptor-mediated pathway. To clarify this subject we evaluated LDL receptor-mediated binding and degradation of Lp(a) and LDL in three human cell lines. The binding of 50 nmol/L Lp(a) at 37 degrees C to the LDL receptor of primary hepatocytes, macrophages, and fibroblasts was only 10%, 29%, and 29% of the respective value obtained with 50 nmol/L LDL. Analysis of 4 degrees C binding curves indicated that Lp(a) and LDL had equal affinities for the LDL receptor of fibroblasts, whereas maximal binding of Lp(a) was remarkably lower than that of LDL. LDL receptor-mediated degradation of 50 nmol/L Lp(a) in hepatocytes, macrophages, and fibroblasts was only 17%, 22%, and 26%, respectively, of the value obtained with 50 nmol/L LDL and varied greatly among the cells in that it was lowest in hepatocytes, an order of magnitude greater in macrophages, and two orders of magnitude greater in fibroblasts. In contrast, the nonspecific degradation rate of Lp(a) was similar to that of LDL in each of the three tested cell lines. However, the proportion of the degradation of Lp(a) that was nonspecific varied greatly, being 76%, 58%, and 33% in hepatocytes, macrophages, and fibroblasts, respectively. These studies indicate that not only is Lp(a) recognized by the LDL receptor but also that, in fibroblasts, Lp(a) and LDL have equal affinities for the LDL receptor, although Lp(a) has a much lower receptor occupancy than LDL. Additionally, they show that there are great cellular differences in the LDL receptor-mediated degradation of Lp(a). If these results can be extrapolated in vivo, where normal LDL levels are 40- to 50-fold higher than those of Lp(a), it would be unlikely that the hepatic LDL receptor is significantly involved in the degradation of Lp(a).     

The effect of human herpesvirus-6 (HHV-6) on cultured human neural cells: oligodendrocytes and microglia

Human herpesvirus-6 (HHV-6) is a betaherpesvirus that has been frequently associated with pediatric encephalitis. In 1995 Challoner et al reported that HHV-6 variant B (HHV-6B) was linked to multiple sclerosis (MS) due to the presence of viral DNA and antigen in the oligodendrocytes surrounding MS plaques. These findings led us to examine HHV-6B's in vitro tropism for primary neural cells. HIV-6B mediated cell-to-cell fusion in cultured adult oligodendroglia. Infection of oligodendrocytes was further confirmed by transmission electron microscopy (EM), which showed the presence of intracellular HHV-6 particles, and by PCR for HHV-6 DNA. However, the release of infectious virus was low or undetectable in multiple experiments. Microglia were also susceptible to infection by HHV-6B, as demonstrated by an antigen capture assay. We did not detect infection of a differentiated neuronal cell line (NT2D). Our findings suggest that HHV-6B infection of oligodendrocytes and/or microglia could potentially play a role in neuropathogenesis.     

Co-expression of beta-amyloid precursor protein (betaAPP) and apolipoprotein E in cell culture: analysis of betaAPP processing

Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimer's disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Abeta), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Abeta production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Abeta, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of betaAPP and the secretion of Abeta in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Abeta at concentrations like those in human cerebrospinal fluid. betaAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on betaAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of betaAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Abeta aggregation or clearance under physiologically relevant conditions.     

Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells

There is currently no effective therapy for human prion diseases. However, several polyanionic glycans, including pentosan sulfate and dextran sulfate, prolong the incubation time of scrapie in rodents, and inhibit the production of the scrapie isoform of the prion protein (PrPSc), the major component of infectious prions, in cultured neuroblastoma cells. We report here that pentosan sulfate and related compounds rapidly and dramatically reduce the amount of PrPC, the non-infectious precursor of PrPSc, present on the cell surface. This effect results primarily from the ability of these agents to stimulate endocytosis of PrPC, thereby causing a redistribution of the protein from the plasma membrane to the cell interior. Pentosan sulfate also causes a change in the ultrastructural localization of PrPC, such that a portion of the protein molecules are shifted into late endosomes and/or lysosomes. In addition, we demonstrate, using PrP-containing bacterial fusion proteins, that cultured cells express saturable and specific surface binding sites for PrP, many of which are glycosaminoglycan molecules. Our results raise the possibility that sulfated glycans inhibit prion production by altering the cellular localization of PrPC precursor, and they indicate that endogenous proteoglycans are likely to play an important role in the cellular metabolism of both PrPC and PrPSc.     

Effects of the M1 agonist xanomeline on processing of human beta-amyloid precursor protein (FAD, Swedish mutant) transfected into Chinese hamster ovary-m1 cells

1. Three experiments were conducted to investigate the diurnal variation of blood viscosity in broilers. In experiment 1 food and water were supplied freely at 20 degrees C (20-FW). In experiment 2 food and water were withdrawn at 20 degrees C (20-NFW), while in experiment 3 food and water were withdrawn at 30 degrees C (30-NFW). 2. Blood sampling time points were 09.00 h, 15.00 h, 21.00 h, 03.00 h and 09.00 h the next day in each experiment. 3. In all experiments, whole blood viscosity (WBV), red blood cell count (RBC) and haematocrit (HCT) were greater during the dark (21.00 h and 03.00 h) than during the light period. During the dark period, there were no differences in WBV, RBC and HCT between 20-FW and 20-NFW, or between 20-NFW and 30-NFW. At 09.00 h, WBV and HCT were higher in 20-FW than in 20-NFW. At 15.00 h and 09.00 h (day 2), WBV and HCT were greater in 20-NFW than in 30-NFW. 4. There were no light-dark differences in plasma viscosity (PV), plasma protein concentration (PPC) or mean corpuscular volume (MCV) in any experiment. However, 20-NFW birds had a lower PPC and higher MCV compared with 20-FW, and a higher PPC and lower MCV compared with 30-NFW, while no difference was found in PV. 5. WBV increased linearly with RBC and HCT. PV increased with PPC, while MCV decreased. 6. These results indicate that there is diurnal variation in whole blood viscosity, which is greater during the dark than during the light period. During the light period it is strongly influenced by high environmental temperature and food and water withdrawal.     

Urokinase-type plasminogen activator and plasminogen activator inhibitors (PAI-1 and PAI-2) in extracts of invasive cervical carcinoma and precursor lesions

BACKGROUND: HFE mutations are associated with hereditary haemochromatosis. However, a simple method capable of demonstrating the cis/trans arrangement of alleles is lacking, and linkage disequilibrium between HFE alleles and classic HLA loci is unknown. These are important issues as the pathogenic role of the mutations is not known. AIMS: To develop a simple method of genotyping HFE mutations suitable for clinical use in addition to large disease studies. PATIENTS: A total of 330 Caucasoid cadaveric organ donor controls were examined. Ten individuals previously HLA-H genotyped by polymerase chain reaction using restriction fragment length polymorphism (PCR-RFLP) were also examined to validate the method. METHODS: A simple polymerase chain reaction using sequence specific primers (PCR-SSP) capable of haplotyping the mutations was developed. HFE allele and haplotype frequencies and linkage disequilibrium with eight HLA class I and II loci were examined in the control population. RESULTS: 27% and 19.7% of patients were positive for the 63D and 282Y alleles, respectively. No chromosome carried both 63D and 282Y. Linkage disequilibrium between 282Y and HLA-A*03 was confirmed, but was not straightforward: some A*03-associated alleles (DRB1*15, DQB1*06), but not all (B*07, Cw*0702), were associated with 282Y. CONCLUSIONS: Linkage disequilibrium data suggest that an HLA-B*07 containing haplotype contains an element affording protection from haemochromatosis and may suggest the timing of the founder 282Y mutation.     

Deletion of an endosomal/lysosomal targeting signal promotes the secretion of Alzheimer's disease amyloid precursor protein (APP)

Alzheimer's disease amyloid precursor protein (APP) generates a beta-amyloid protein (A beta) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloidogenic pathway which produces A beta, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a G0-protein binding sequence. We constructed two mutants, 695 deltaNPTY and 695 deltaGYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP.     

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.     

Production of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) in human brain tumours

We investigated the role of plasminogen activators (PAs) and their inhibitor (plasminogen activator inhibitor-1, PAI-1) in human brain tumours. The amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1), and the activity of u-PA and t-PA were determined by enzyme-linked immunosorbent assay (ELISA), and u-PA and PAI-1 were immunolocalized using monoclonal antibodies in human brain tumours and normal brain tissues. The tissues were surgically removed from 64 patients; normal brain tissue (5 cases), low-grade glioma (4 cases), high-grade glioma (17 cases), metastatic tumour (9 cases), meningioma (benign 12 cases, malignant 6 cases), acoustic schwannoma (11 cases). u-PA activity and u-PA and PAI-1 antigen levels were significantly elevated in malignant brain tumours (malignant meningiomas, high-grade gliomas, and metastatic tumours) and acoustic schwannomas but very low in benign meningiomas, low-grade gliomas and normal brain. There was no difference in t-PA antigen levels among normal and malignant tissues, however levels of t-PA activity were markedly decreased in metastastic tumours. All malignant brain tumour tissues showed positive immunostaining for u-PA and PAI-1, however, some tumour cells showed negative intensity while others showed strong intensity for these antibodies. This contrasts to the homogeneous staining pattern found in acoustic schwannoma. These findings indicate that malignancy in human brain tumours is associated with elevated levels of u-PA and PAI-1 and that an imbalance between these proteins in a micro-environment contributes (ascribes) to tumour cell invasion.     

The FML (Fucose Mannose Ligand) of Leishmania donovani. a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with alpha-amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca2+. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.     

Tumor necrosis factor-alpha and interleukin-1 induce activation of MAP kinase and SAP kinase in human neuroma fibroblasts

Two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), which are released by macrophages during the early inflammatory phase of nerve injury, are known to induce activation of mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK), which locate at different signal transduction pathways and are involved in cell cycle G0/G1 transition and cellular proliferation in human fibroblasts. Activation of these two protein kinases by the cytokines may stimulate fibroblast proliferation in damaged nerves and thereby play a role in the formation of a neuroma, a disorganized mass of tissue that interferes with neural regeneration and repair. To investigate the possibility that this mechanism is operative in neuroma formation, we used cultured, serum-starved fibroblasts from surgically removed human neuromas stimulated with TNF-alpha and/or IL-1 alpha and IL-1 beta, and measured the activation of MAPK and SAPK using myelin basic protein (MBP) and human c-Jun (1-169) glutathione S-agarose transferase (GST) fusion protein as substrates. For comparison, neuroma fibroblast cultures were also stimulated with phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor-AB (PDGF-AB), a potent activator for MAPK. TNF-alpha and both forms of IL-1 produced a rapid activation of MAPK, with a peak at 15 min for TNF-alpha stimulation, and a peak at 30 min for IL-1 stimulation. TNF-alpha combined with either IL-1 alpha or IL-1 beta produced a synergistic effect on the activation of MAPK. The increases in MAPK induced by TNF-alpha and IL-1 were similar to the increases induced by PMA and PDGF-AB. To confirm the presence of MAPK, immunoprecipitation and immunoblotting were carried out on experimental and control lysates. TNF-alpha and IL-1 also increased activation of SAPK, but to a lesser extent than MAPK. PMA and PDGF-AB were also much less effective in stimulating activation of SAPK. Our findings indicate that TNF-alpha and IL-1 activate parallel signal transduction pathways in human neuroma fibroblasts, and that they are relatively stronger activators of MAPK than of SAPK. Previous studies have convincingly demonstrated that MAPK and SAPK are involved in human fibroblast proliferation. The results of our study suggest that TNF-alpha and IL-1 may play a role in frustrating functional nerve regeneration after injury by stimulating these two kinases, which, in turn, leads to fibroblast proliferation and formation of neuromas.     

Age-related alteration of proline hydroxylase and collagen-binding heat shock protein (HSP47) expression in human fibroblasts

It is now well recognized that a disorder of left ventricular filling can be sufficient to account for congestive heart failure. Furthermore, evaluation of heart disease would not be complete if it did not include assessment of left ventricular filling, improvement of which probably ensures better control of the heart disease. An efficient and reliable tool for the study of diastolic function is therefore essential. The authors review the current state of knowledge and the more recent developments in Doppler echocardiography in the evaluation of left ventricular diastolic function. After revising the pathophysiology, the methods of studying ventricular filling are described. The recording technique is described, taking into account recent developments in transthoracic and transoesophageal approaches. This investigation provides parameters allowing semiquantitative estimation of filling pressures (mean left atrial pressure, end-diastolic pressure) and reliable evaluation of overall diastolic performance.     

HLA antigens and erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in Sardinia

Wind enhances the carcinogenic effect of chronic Iltraviolet radiation (UVL). This was demonstrated in hairless mice that were irradiated for 42 weeks with mercury are lamps. One group of animals was exposed to continuous wind flow of 2.7 m/s except for the daily I-2 min time interval when they were removed from the wind tunnel and irradiated. Another group of animals received identical irradiation but were protected from wind. The first tumour appeared in the UVL and wind group after 105 days of irradiation, and at 164 days of irradiation all surviving mice in the group had developed tumours. The group of mice receiving identical irradiation but protected from wind had their first tumour appear at 154 days of irradiation, and by 164 days of irradiation only 40% of the mice had developed tumours.