Selective inhibition of Zn(2+)-glycerophosphocholine cholinephosphodiesterase by tellurium tetrachloride. A Zn(2+)-glycerophosphocholine cholinephosphodiesterase (EC 3.1.4.38) purified from mouse brain was found to be reversibly inhibited by tellurium tetrachloride. This effect was characterized by a competitive pattern of inhibition, with apparent Ki values of 0.7 microM and 1.5 microM for the hydrolysis of p-nitrophenylphosphocholine and glycerophosphocholine respectively. Interestingly, the inhibitory effect of tellurium tetrachloride was found to be greatly potentiated by tetramethylammonium salt, indicative of a synergistic interaction between the two compounds. Additionally, it was observed that the effect of tellurium tetrachloride was not affected by a number of other metal ions, and was more pronounced at neutral pH, suggesting that the inhibitory role of the tellurium tetrachloride may be of importance under physiological conditions. Thus Zn(2+)-glycerophosphocholine cholinephosphodiesterase is proposed to be one of the target enzymes which is susceptible to the inhibitory effect of tellurium tetrachloride.


The herpes simplex virus immediate-early protein ICP27 stimulates the transcription of cellular Alu repeated sequences by increasing the activity of transcription factor TFIIIC. Infection with herpes simplex virus (HSV) results in an increase in the transcription of the endogenous Alu repeated sequence by RNA polymerase III. This effect is also observed in uninfected cells stably transformed with a plasmid expressing the HSV immediate-early protein ICP27 or in cells transfected with the gene encoding this protein. Both uninfected cells expressing ICP27 and cells infected with virus producing functional ICP27 display increased activity of the cellular transcription factor TFIIIC when compared with untreated cells. This increase is not observed, however, in cells infected with a mutant strain of virus which does not produce ICP27. Hence ICP27 induces elevated Alu transcription by activating transcription factor TFIIIC, which is the limiting factor for such transcription. This is the first report of increased activity of a cellular transcription factor during HSV infection, when most cellular gene activity is inhibited.


Anaerobic reactions of Rhus vernicifera laccase and its type-2 copper-depleted derivatives with hexacyanoferrate(II). Anaerobic reactions of Rhus vernicifera laccase and its type-2 copper-depleted derivatives with hexacyanoferrate(II) were investigated by absorption and e.s.r. spectroscopy. When native laccase was treated with excess hexacyanoferrate(II), the type-1 and type-2 coppers were immediately reduced and the e.s.r. signal due to type-3 copper was transiently observed. After incubation, a novel e.s.r. signal (g parallel = 2.31, g perpendicular = 2.08) developed together with the type-1 copper signal. Only the novel e.s.r. signal was left after the sample had been treated with ascorbate. In the corresponding absorption spectrum, a new band was observed at around 490 nm. A similar new e.s.r. signal did not appear for the type-2-copper-depleted (T2D) laccase, in which the type-3 copper had been reduced during the procedure to deplete the type-2 copper. On the other hand, the novel e.s.r. signal emerged when the type-3 copper in T2D laccase had been previously reoxidized with H2O2. The novel e.s.r. signal was not significantly saturated even by 200 mV microwave power at 4 K. Quantitative estimations and a small molecule study for CuII-FeII(CN)6 and CuII-FeIII(CN)6 systems suggested that the novel e.s.r. signal corresponds to some sort of adduct involving the type-3 copper and hexacyanoferrate(II).


Release and fixation of CO2 by guinea-pig kidney tubules metabolizing aspartate. 1. The metabolism of L-[U-14C]aspartate, L-[1-14C]aspartate and L-[4-14C]aspartate was studied in isolated guinea-pig kidney tubules. 2. Oxidation of C-1 plus that of C-4 of aspartate accounted for 90-92% of the CO2 released from aspartate, whereas oxidation of the inner carbon atoms of aspartate (which occurs beyond the 2-oxoglutarate dehydrogenase step) represented only 8-10% of aspartate carbon oxidation. 3. The formation of [1-14C]glutamine and [1-14C]glutamate from [1-14C]aspartate and [4-14C]aspartate indicated that about one-third of the oxaloacetate synthesized from aspartate underwent randomization at the level of fumarate. 4. With [U-14C]aspartate as substrate, the percentage of the C-1 of glutamate and glutamine found radiolabelled after 60 min of incubation was 92.7% and 47.5% in the absence and the presence of bicarbonate respectively. 5. That CO2 fixation occurred at high rates in the presence of bicarbonate was demonstrated by incubating tubules with aspartate plus [14C]bicarbonate; under this condition, the label fixed was found in C-1 of glutamate, glutamine and aspartate, as well as in C-4 of aspartate, demonstrating not only randomization of aspartate carbon but also aspartate resynthesis secondary to oxaloacetate cycling via phosphoenolpyruvate carboxykinase, pyruvate kinase and pyruvate carboxylase. 6. The importance of CO2 fixation in glutamine synthesis from aspartate is discussed in relation to the possible role of the guinea-pig kidney in systemic acid-base regulation in vivo.


A nucleotide receptor in vascular endothelial cells is specifically activated by the fully ionized forms of ATP and UTP. Extracellular ATP causes an increase in the concentration of cytoplasmic free calcium ([Ca2+]i) in bovine pulmonary-artery endothelial (BPAE) cells that results in the synthesis and release of prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation. We show here that PGI2 release in BPAE cells correlates with the concentration of the fully ionized form of extracellular ATP (ATP4-) and not with the concentration of other ionic forms of ATP. Concentrations as low as 10 nM-ATP4- elicited an increase in PGI2 release [EC50 (concn. giving half-maximal stimulation) 3 microM] in BPAE cells incubated in an iso-osmotic medium, pH 7.4, lacking Ca2+ and Mg2+. When the pH or the Mg2+ concentration of the medium was varied so as to maintain a constant level of ATP4-, while varying the concentration of proton-ATP (HATP3-) or MgATP2- respectively, PGI2 release remained constant. An inhibitory effect of extracellular Mg2+ on PGI2 release could be attributed solely to a decrease in the concentration of ATP4-. In contrast with Mg2+, extracellular Ca2+ stimulated PGI2 release induced by ATP. Several results suggest that extracellular Ca2+ modulates PGI2 release by increasing Ca2+ uptake through an ATP(4-)-activated plasma-membrane channel. In BPAE cells incubated in Ca(2+)-free medium, ATP elicited a transient increase in [Ca2+]i that declined to the basal level within 60 s. In cells incubated in Ca(2+)-containing medium, ATP caused an increase in [Ca2+]i that had two components: a transient peak in [Ca2+]i (0-60 s) and a sustained increase in [Ca2+]i that was maintained for several minutes after ATP addition. Increasing the concentration of extracellular calcium from 0.25 mM to 10 mM had no effect on the transient rise in [Ca2+]i induced by ATP, but significantly enhanced the magnitude of the sustained increase in [Ca2+]i. Alterations in the magnitude of the sustained increase in [Ca2+]i would likely modulate PGI2 release, which was not complete until 2 min after ATP addition. Extracellular Ca2+ also stimulated PGI2 release induced by bradykinin. Bradykinin caused a sustained increase in [Ca2+]i in BPAE cells in the presence of extracellular Ca2+. Finally, the magnitude of PGI2 release induced by UTP, a more potent agonist than ATP, correlated with the concentration of extracellular fully ionized UTP (UTP4-). These findings support the hypothesis that nucleotide receptors in BPAE cells recognize the fully ionized form of ATP and UTP and are coupled to signal-transduction pathways involving the mobilization of intracellular Ca2+, the influx of extracellular Ca2+ and the subsequent release of PGI2.


Effect of treatment in vivo of rats with bacterial endotoxin on fructose 2,6-bisphosphate metabolism and L-pyruvate kinase activity and flux in isolated liver cells. The effect of treatment of rats with bacterial endotoxin on fructose 2,6-bisphosphate (Fru-2,6-P2) metabolism was investigated in isolated liver cells prepared from 18 h-starved animals. The results obtained support the hypothesis that a stimulation of 6-phosphofructo-1-kinase (PFK-1) activity and an inhibition of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) may be one mechanism underlying the inhibition of gluconeogenesis from lactate and pyruvate by endotoxin. We suggest that the stimulation of PFK-1 and inhibition of Fru-1,6-P2ase activity is the result of a 2-3-fold increase in Fru-2,6-P2. The latter is not due to changes in the total activity or phosphorylation state of the bifunctional 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase, but appears to be the result of a decrease in the cytosolic concentration of phosphoenolpyruvate (PEP), an inhibitor of PFK-2 activity. The effect of endotoxin is resistant to the presence of glucagon, which has comparable effects in cells prepared from both control and endotoxin-treated animals. The mechanism by which endotoxin treatment of the rat decreases PEP and gluconeogenesis remains to be established. However, it does not involve alterations in either the total activity or the phosphorylation state of pyruvate kinase, nor does it involve increased flux through this enzyme in the intact cell, which is in fact decreased in this model of septic shock. It is suggested that the decreased flux may result from a lower rate of formation of PEP, suggesting that the prime lesion in sepsis is an inhibition of one or more of the steps leading to PEP formation.


Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.


Correction of human mucopolysaccharidosis type-VI fibroblasts with recombinant N-acetylgalactosamine-4-sulphatase. A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.


Purification and partial identification of bone-inducing protein from a murine osteosarcoma. A highly purified fraction with bone-inducing potential was obtained from a murine osteosarcoma using initial fractionation, gel filtration on Sephacryl S-200, and cation-exchange, hydroxyapatite and reverse-phase h.p.l.c. Protein sequencing of the sample derived from this active fraction revealed the N-terminal amino acid sequence to be Ala-Ala-Leu-Arg-Pro-Leu-Val-Lys-Pro, which is identical to the N-terminal sequence of mouse, human and rat ribosomal protein L32, except for an additional methionine residue at the N-terminus of the three latter proteins. Amino acid analysis of the sample also showed its similarity to L32. Two lines of evidence using an antibody specific for the above peptide strongly suggest that this L32-related protein from a murine osteosarcoma has the potential to induce ectopic bone formation. First, the protein specifically detected by the antibody co-distributes with the bone-inducing active fraction. Secondly, a highly purified sample obtained using the antibody was shown to induce the formation of bone with active haematopoiesis.


Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein. The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology. These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli. As a result, the DHQ synthase domain is overproduced in E. coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol. The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A. nidulans. The qutB gene of A. nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E. coli. The quinate dehydrogenase is not overproduced, but is active in E. coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E. coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.


Caldesmon-phospholipid interaction. Effect of protein kinase C phosphorylation and sequence similarity with other phospholipid-binding proteins. Recently published data [Vorotnikov & Gusev (1990) FEBS Lett. 277, 134-136] indicate that smooth muscle caldesmon interacts with a mixture of soybean phospholipids (azolectin). Continuing this investigation, we found that duck gizzard caldesmon interacts more tightly with acidic (phosphatidylserine) than with neutral (phosphatidylcholine) phospholipids. A high concentration of Ca2+ (50 microM) decreased the interaction of caldesmon with phosphatidylserine. Among chymotryptic peptides of caldesmon, only those having molecular masses of 45, 40, 23, 22 and 20 kDa were able to specifically interact with phospholipids. These peptides, derived from the C-terminal part of caldesmon, contained the sites phosphorylated by Ca2+/phospholipid-dependent protein kinase, and phosphorylation catalysed by this enzyme decreased the affinity of these peptides for phospholipids. In the presence of Ca2+, calmodulin competed with phospholipids for the interaction with the caldesmon peptides. The C-terminal part of caldesmon contains three peptides with a primary structure similar to that of the calmodulin- and phospholipid-binding site of neuromodulin. These sites may be involved in the interaction of caldesmon with calmodulin and phospholipids.


Recombinant human interleukin-1 beta-induced increase in levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid. To evaluate the effects of intraarticular injection of recombinant human interleukin-1 beta (IL-1 beta) on levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid (SF), and to determine the effects of leukocyte depletion on SF proteoglycan and stromelysin levels. Levels of leukocytes and of proteoglycans, stromelysin, and collagenase were evaluated 12 hours after the intraarticular injection of various doses of IL-1, and over a 24-hour period after injection at a single dose level. We used a monoclonal antibody (MAb) against leukocyte integrins, which markedly depressed leukocyte accumulation in SF, to evaluate the role of synovial leukocytes on IL-1-induced increases in SF proteoglycan and stromelysin levels. Levels of both proteoglycans and stromelysin increased in the IL-1-injected joints between 4 hours and 24 hours after the injection of a single 200-ng dose of IL-1. The highest levels of stromelysin and proteoglycans were achieved with IL-1 doses greater than or equal to 100 ng. Infiltration of polymorphonuclear cells (PMN) into the joint fluid of the IL-1-injected rabbits also increased, in a dose-dependent manner. Treatment of rabbits with MAb 1B4 markedly reduced infiltration of PMN into the joint, without affecting either stromelysin or proteoglycan levels. Taken together, the data suggest that there is a coordinate increase in SF stromelysin and proteoglycan levels in rabbits injected with IL-1, and that leukocytes play a minimal role in the accumulation of proteoglycans and stromelysin in the SF.


Multiple observations on a single unrestrained cat of long-lasting facilitation of transmission through the lateral geniculate nucleus. The field potential responses of the lateral geniculate nucleus (LGN) to test electrical stimuli delivered to the optic tract (OT) were recorded in an unanaesthetised, freely moving cat. The response consisted of two waves, one attributable to depolarisation of the OT terminals (t), and one attributable to the postsynaptic response (r) of LGN neurones. A train of 300 electrical stimuli at 0.2 Hz to the OT alone did not influence the amplitude of these waveforms. In contrast after a train of 150 pulses had been delivered to both the OT (at 0.2 Hz) and LGN (pseudo-random with mean intervals of 0.2 Hz), the amplitude of both the t and r waves to test OT electrical stimuli was markedly enhanced for the following 6 days. Enhancement of the t wave followed that of the r wave.


Diet and breast cancer.

Diet and breast cancer. It is a general opinion that the Western diet plays a significant role in increasing the risk of breast cancer in the Western World. Recently some likely mechanisms involved in increasing the risk have been disclosed. It has been found that a Western-type diet elevates plasma levels of sex hormones and decreases the sex hormone binding globulin concentration, increasing the availability of these steroids for peripheral tissues. The same diet results in low formation by intestinal bacteria of mammalian lignans and isoflavonoid phyotestrogens from plant precursors. These diphenolic compounds seem to affect hormone metabolism and production and cancer cell growth by many different mechanisms making them strong candidates for a role as cancer protective substances. The sex hormone pattern found in connection with a Western-type diet combined with low lignan and isoflavonoid excretion was found particularly in postmenopausal breast cancer patients and omnivores living in high-risk areas, and to a lesser degree in areas with less risk. However, the pattern observed was not entirely due to diet.


Intra-oral salivary gland tumours in the West Midlands. Intra-oral salivary gland tumours reported from the West Midlands over the years 1975-88 were examined as to a number of epidemiological variables; the question as to their increasing incidence was also addressed. The findings were largely in agreement with those of most previous authors in that most of the tumours occurred in the palate, but whereas most of these were benign, a large proportion of those in other sites were malignant. However, firm conclusions are difficult to draw since there is much disagreement over reporting criteria. An international workshop and comprehensive tumour registries are required in order to determine more accurate data and conclusions. Similar cooperation is necessary in order to substantiate or refute the suggestion that the incidence of salivary gland tumours is increasing.


A new liver functional study using Tc-99m DTPA-galactosyl human serum albumin: evaluation of the validity of several functional parameters. Several parameters calculated with a new functional imaging agent for the liver, Tc-99m DTPA-galactosyl human serum albumin, were evaluated in 9 patients with liver cirrhosis, one with hepatocellular carcinoma, and five with both liver cirrhosis and hepatocellular carcinoma. LU3, which represents the cumulative uptake of the tracer from 3 to 4 minutes after injection, showed a strong correlation (r = 0.858, p = 0.0001) with LHL15, which represents the count ratio for the liver to sum for the liver and heart 15 minutes after injection of the tracer. It also showed a strong correlation (r = -0.896, p = 0.0001) with the indocyanine green retention rate (ICGR15). Regional ICGR15 is therefore calculable from the regional LU3. GSAR15, which represents the radioactivity of the tracer retained in the blood 15 minutes after injection, showed a strong correlation (r = 0.878, p = 0.0001) with HH15, which represents the count ratio for the heart 15 minutes after injection of the tracer divided by the count for the heart 3 minutes after injection. In conclusion, LU3 and GSAR15 are interesting and promising parameters for assessing liver function.


Asparagine-linked sugar chains of plasma membrane glycoproteins from healthy and common variable immunodeficiency B lymphoblastoid cell lines. Asparagine-linked sugar chains of plasma membrane glycoproteins, which are formed by glycosylation during B cell maturation, were examined with B lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus derived from healthy controls and patients with common variable immunodeficiency (CVI). Both two patients with CVI showed hypogammaglobulinemia and impaired B cell functions. LCLs from healthy controls and the patients showed CD19+ and HLA/DR+ in the cell surface and secreted IgM. In both healthy controls and the patients, the main oligosaccharide in asparagine-linked sugar chains of the membrane glycoproteins of LCLs was biantennary sugar chain with bisected GlcNAc (Gal2-GlcNAc2-Man3-GlcNAc-GlcNAc-Fuc-GlcNAcOT). Biantennary sugar chain with an alpha-fucosyl residue linked at the proximal GlcNAc was seen but biantennary sugar chain without an alpha-fucosyl residue at the proximal GlcNAc was little detected in each LCL. There was no difference in quality and quantity of asparagine-linked sugar chains between healthy controls and the patients. These results suggest that glycosylation during B cell maturation may not be impaired in patients with CVI.


Effects of cigarette smoke on the clearance of short asbestos fibres from the lung and a comparison with the clearance of long asbestos fibres. Long asbestos fibres are generally considered to have greater disease-producing potential than short asbestos fibres. However, recent reports have suggested that short fibre asbestos appears to be as effective an inducer of macrophage growth factors and toxic oxygen species as long fibre asbestos, but that short fibres are readily removed from lung and do not gain access to tissues. Because smoke is believed to impair the clearance of asbestos fibres from lung, we examined the clearance of a short (geometric mean length 1.3 microns) amosite preparation administered by intratracheal instillation to guinea-pigs. Half the animals in each group were exposed to the smoke of 10 cigarettes daily. Animals were sacrificed 1 day, 1 week, or 1 month later, the macrophages recovered by lavage, and fibre concentrations and sizes determined by analytical electron microscopy in macrophages and lung tissue. A 30-fold increase was seen in total numbers of fibres retained in macrophages in smokers compared to non-smokers by 1 month, and there was an eightfold increase in retention of short fibres in the lung tissue by 1 month. By contrast, a long fibre amosite preparation (geometric mean length 8.9 microns) showed approximately the same increase in fibre retention in macrophages, but only a twofold increase in tissue retention. We conclude that (1) cigarette smoke markedly impairs the clearance of short amosite fibres from the lung with enhanced retention of fibres in both macrophages and tissue; (2) the effects of smoke on short fibre tissue retention appear to be greater than those on long fibre retention; (3) with the long fibre preparation, smoke causes increased tissue retention of relatively shorter fibres; (4) for both fibre size experiments, the increase in total fibres in macrophages in smoke-exposed animals reflects an increase in the total number of fibre-containing macrophages, rather than an increase in the number of fibres phagocytized per macrophage; (5) enhanced short fibre retention markedly increases total fibre surface area, a parameter which has been suggested as a measure of fibre toxicity, to the point where short fibres might under some circumstances have roughly the same potential toxicity as long fibres. These observations suggest that short asbestos fibres could play an important role in the pathogenesis of some types of asbestos-related disease in cigarette smokers.


Proteoglycan synthesis and osteophyte formation in 'metabolically' and 'mechanically' induced murine degenerative joint disease: an in-vivo autoradiographic study. We investigated the in-vivo proteoglycan synthesis in specific areas of murine knee joint articular cartilage after the induction of degenerative joint disease by means of 35S-sulphate autoradiography. Degenerative joint disease was induced either by direct interference with cartilage metabolism (papain and iodoacetate), or by the induction of joint instability (collagenase). Injection of iodoacetate and papain led to inhibition of proteoglycan synthesis mainly in the central parts of the patellae, patellaris femoris and the central part of the medial tibial plateau. Articular cartilage adjacent to the strongly inhibited areas frequently showed a significantly enhanced synthesis of proteoglycans. A strong inhibition of proteoglycan synthesis was observed in the central part of the medial plateau after collagenase injection while other cartilage sites and joint structures such as the capsule and ligaments were stimulated in their proteoglycan synthesis. This study shows that the localization of changes in cartilage metabolism in degenerative joint disease of the knee might be related to differences in the pathogenetic mechanism in different variants of this common joint disorder.


Classification of tumour 1H NMR spectra by pattern recognition. 1H spectra of tumours or normal tissues, which include signals from all hydrogen-containing metabolites, are too complex for the human eye to interpret. We have studied 58 1H spectra from perchloric acid extracts of three normal tissues (liver, kidney and spleen) and five rat tumours (GH3 pituitary, fibrosarcoma, Morris Hepatomas 7777 and 9618a and Walker carcinosarcoma). Instead of editing them or quantifying individual metabolites, we have used statistical pattern recognition techniques to classify them into groups. This automatic, objective method differentiated spectra from normal and malignant rat tissue biopsies, and from different types of cancer. It seems likely that this technique can be applied to human tissues and thus used for cancer diagnosis.


Identification of lactate, threonine and alanine in rat thymus and tumorigenic lymphoid cells using 1H 2-D COSY NMR spectroscopy. One- and two-dimensional 1H NMR spectra were obtained for normal murine thymus and malignant lymphoma tissue, as well as for the supernatant fractions from high speed centrifugal separations. Crosspeaks in the two-dimensional spectra resembled those reported by others for adenocarcinoma and leukemic lymphoblast cells, assigned tentatively to the carbohydrate fucose. However, for the present systems, spectral analysis and the spectral response to addition of known compounds led to assignment of the crosspeaks as follows: 1.33-4.12 ppm, lactate anion; 1.33-4.26 ppm, threonine; 1.48-3.78 ppm, alanine. Differences between the NMR data for the normal and malignant specimens were only in the relative intensities of the peaks. No peaks characteristic of fucose were found in spectra of cytosol, tissue or membrane lipids. Thus, the NMR data for malignant lymphoma cells are significantly different from those for adenocarcinoma and leukemic lymphoblasts. The NMR characteristics of different types of cancer cell must be individually determined.


An assessment of the ability of human bone marrow cultures to generate osteoclasts. Several groups have successfully generated osteoclasts in cultures of murine haemopoietic cells. This approach would clearly be useful in the analysis of mechanisms of regulation of human osteoclast formation if analogous results could be obtained in cultures of human bone marrow. This communication describes independent attempts by three groups to generate unequivocally defined osteoclasts from bone marrow obtained from human iliac crest, femoral neck, rib, and from foetuses. The haemopoietic tissue was incubated using techniques described by others for production of osteoclast-like cells, and with variants of this technique using strategies based on our experiences with murine osteoclastogenesis. Haemopoietic cells were incubated with calcium regulating hormones, cytokines, osteoblastic supernatants, and osteoblastic or bone marrow stromal cell layers. Formation of cells capable of excavation of bone slices was rarely seen. Despite the paucity of bone resorbing cells, multinucleate cells (MNCs) developed with similar characteristics to the MNCs that have been interpreted as osteoclast-like in human bone marrow cultures. The MNCs were, however, calcitonin-receptor (CTR) negative, and did not show the typical pattern of reactivity with osteoclast-specific antibodies. They possessed instead an antigenic profile characteristic of macrophage polykaryons. We conclude that the MNCs which consistently generate in human bone marrow cultures do not possess phenotypic characteristics specific for osteoclasts and appear to be macrophage polykaryons. The conditions required for osteoclast generation in cultures of human haemopoietic cells remain to be defined.


Readthrough suppression in the mammalian type C retroviruses and what it has taught us. Mammalian type C retroviruses use translational suppression to synthesize the enzymes which function in virus replication. The UAG termination codon at the end of the coding region for the viral core proteins is translated as glutamine at a frequency of approximately 5%, allowing synthesis of the enzymes as part of a large fusion protein. This unusual mechanism has several benefits for the virus: first, it modulates the relative levels of synthesis of the core proteins and the enzymes. This is essential for the proper assembly of the virus particle, since the fusion protein alone is apparently unable to assemble into particles. Second, the presence of the core protein moiety in the fusion protein probably provides a mechanism for targeting the enzymes to the virus particle. The mechanism of the suppression phenomenon is now under investigation. Recent studies have revealed that suppression in the viral context is dependent upon a complex cis-acting signal in the viral mRNA, including a pseudoknot beginning 9 nucleotides 3' of the termination codon. In addition, studies with viral mutants have shown that UAA and UGA, like UAG, are efficiently suppressed in the presence of this signal, and have identified the amino acids used in the suppression of these termination codons in reticulocyte lysates. In several cases, this analysis revealed the existence of previously unknown suppressor tRNAs. One important question which has not been answered is whether the suppression mechanism used by the virus has a parallel in the synthesis of host proteins.


The secondary structure of the regulatory region of the transferrin receptor mRNA deduced by enzymatic cleavage. The secondary structure of the portion of the transferrin receptor mRNA responsible for the regulation of the transcript's half-life has been deduced by ribonuclease H cleavage directed by antisense oligodeoxyribonucleotides as well as with other ribonucleases sensitive to RNA secondary structure. The data indicate that both a synthetic 252-nucleotide RNA and the comparable portion of a 2.7-kb cellular mRNA contain three stem-loops referred to as iron-responsive elements (IREs). This secondary structure appears to be relatively static, with little interconversion with another possible structure having a similar calculated free energy but involving longer-range base pairing. Deletion of a selected cytosine residue from each of the IRE loops has been shown to yield an unregulated, unstable mRNA. This altered RNA has a secondary structure similar, if not identical, to that of the RNA that is competent in regulation.


Molecular cloning and characterization of Na,K-ATPase from Hydra vulgaris: implications for enzyme evolution and ouabain sensitivity. We have used molecular and biochemical techniques to analyze Na,K-ATPase from a simple metazoan, Hydra vulgaris. First we isolated and characterized cDNA clones encoding the Na,K-ATPase alpha subunit from a Hydra lambda gt11 cDNA library. The open reading frame predicts a protein of 1031 amino acids that bears a high degree of primary sequence and secondary structure similarity to mammalian, avian, and arthropod alpha subunits. The predicted Hydra alpha subunit contains charged residues at the termini of the H1-H2 extracellular domain, suggesting that the Hydra alpha subunit may be resistant to cardiac glycoside inhibition. Biochemical analysis of partially purified Hydra Na,K-ATPase reveals both high- and low-affinity components of ouabain-inhibitable ATPase activity. Our results suggest that the evolutionary ancestor of all metazoans possessed a Na,K-ATPase alpha subunit that was highly conserved with respect to its vertebrate counterparts. Further, expression of a ouabain-resistant Na,K-ATPase activity in Hydra suggests that cardiac glycoside resistance arose randomly during evolution of the Na,K-ATPase.


Detection of Caenorhabditis transposon homologs in diverse organisms. Although transposons that move via DNA intermediates are common in bacteria, invertebrates, and plants, none have been clearly documented in vertebrates and certain other classes of organisms. One such family of transposons includes invertebrate elements related to Caenorhabditis elegans Tc1. Blocks of aligned protein segments derived from this family were used to search a nucleotide sequence databank. Among the relatives detected were known bacterial insertion elements, revealing the ancient origin of the family. Furthermore, a Tc1-like homolog was detected in a catfish, raising the possibility that this valuable tool of C. elegans genetics can be used with vertebrate genomes. This study illustrates the use of multiple protein blocks for detection and evaluation of distant relationships.


Treatment failure in a case of fungal keratitis caused by Pseudallescheria boydii. A case is presented of Pseudallescheria boydii fungal keratitis in an agricultural welder. Treatment with azole antifungal drugs (miconazole and itraconazole) and with penetrating keratoplasty was unsuccessful in eradicating the infection, and eventually the eye was eviscerated.


The effect of the alpha 2-adrenergic agonist, guanfacin, on the energy metabolism of steers fed on low-quality-roughage diets. The effect of the alpha 2-adrenergic agonist, guanfacin, on the energy metabolism, feed intake and live weight (LW) change of steers was studied in three experiments. In the first, the metabolic rate of twelve steers was measured after a 72 h fast. The next day, after a 96 h fast, six steers were injected intramuscularly with 15 mg guanfacin in sterile saline (9 g sodium chloride/l) and six with sterile saline alone, and metabolic rate was measured again. Treatment significantly (P less than 0.01) lowered metabolic rate by approximately 20% (53.9 v. 66.8 kJ/kg per d). In the second experiment twelve steers were fed on long-chopped, low-quality roughage (Pangola grass (Digitaria decumbens) hay) ad lib. for 6 weeks. Six steers were continuously infused through a jugular catheter with 15 mg guanfacin/d (about 40 micrograms/kg LW) in sterile saline. The other six served as controls. There was no significant effect of treatment on feed intake (g dry matter (DM)/kg LW) or the rate of LW loss. Treatment significantly (P less than 0.05) increased the retention time of fluid (17.9 v. 22.1 h) in the alimentary tract. In the final experiment twenty-three steers were divided into four treatment groups and fed on long-chopped, low-quality roughage (Pangola hay). Treated animals were continuously infused with guanfacin at the rate of 20, 40 or 80 micrograms/kg LW per d. Control steers were not infused. At the end of the 6-week feeding period metabolic rate was measured after a 72 h fast. Regardless of dose, guanfacin significantly (P less than 0.01) lowered metabolic rate.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of diet, level of intake, sodium bicarbonate and monensin on urinary allantoin excretion in sheep. The present experiment was designed to study the effects of factors likely to alter microbial purine yield from the rumen on urinary excretion of allantoin-nitrogen (UAN). Sixteen mature Clun Forest-Welsh crossbred wethers were used in a 2 x 2 x 2 x 2 factorial design to investigate the effects of (1) level of intake, (2) wheat:nutritionally improved straw (NIS) ratio, (3) sodium bicarbonate inclusion and (4) monensin inclusion on diet digestibilities, fractional outflow rates of solids and liquids from the rumen and urinary allantoin excretion. Each treatment occurred in each of two experimental periods. The treatments were designed to influence microbial purine yield via changes in rumen outflow rate and microbial maintenance coefficient. Increasing the proportion of NIS and increasing feeding level decreased digestibility and increased the fractional outflow rate of solids. Increasing the level of intake increased the fractional outflow rate of liquids. Urinary allantoin excretion (/kg live weight0.75 per d) was significantly increased by an increased proportion of wheat in the diet and increased level of intake, and significantly reduced by NaHCO3. There was a significant interaction effect such that increasing level of intake did not increase UAN with the high-NIS diet, despite an increased fractional outflow of solids from the rumen, in contrast to the increase observed with the high-wheat diet. Taken together with other observations it is suggested that high sodium concentrations in the diet reduce the efficiency of microbial synthesis, probably by increasing the energy cost of maintaining osmolarity.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of dietary fibre and tannins from apple pulp on the composition of faeces in rats. The present study was undertaken to explore the effect of apple pulp on weight and composition of faeces. This material is rich in dietary fibre (DF; 620 g dry matter/kg) and contains appreciable amounts of polyphenols. Recent reports indicate that both condensed tannins (CT) and soluble polyphenols form cross-links with protein and inhibit digestive enzymes, affecting the protein digestibility, and may produce a stimulation of endogenous nitrogen excretion. Two groups of male Wistar rats were fed on either a control diet free of DF or a diet containing 100 g apple pulp DF/kg during 7 d after a 4 d adaptation period. Body-weight and food intake were monitored daily and faeces and urine were collected once daily. DF, water content and polyphenolic compounds were measured in faeces, and N content in both faeces and urine. Faecal weight increased in the fibre group by 280 and 240% when compared with wet and dry faecal weights of animals fed on the fibre-free diet. Soluble dietary fibre (SDF) excreted in faeces was 10.9% of the SDF ingested, which suggested a low resistance to fermentation of this fraction. Of the insoluble DF, 43% of the ingested fibre was fermented. Polyphenols were degraded in the intestinal tract. Of the ingested CT, 68.6% was recovered in faeces, while the soluble polyphenols were extensively degraded (85.7% of that ingested). On the other hand, a higher faecal N excretion was observed for the fibre-fed group, suggesting a decrease in the digestibility of the dietary protein and lower apparent digestibility of the dietary protein and lower apparent digestibility and N balance indices.


Determination of flavin contents in neutrophils by a sensitive chemiluminescence assay: evidence for no translocation of flavoproteins from the cytosol to the membrane upon cell stimulation. A sensitive and specific chemiluminescence (CL) method with bacterial luciferase was adapted for accurate measurement of the flavins FAD and FMN in the membrane and cytosolic fractions of neutrophils prepared from pig and human blood. The FAD and FMN contents (FAD/FMN = 100:2) in the membranes were essentially the same in resting (R) and myristate-stimulated (S) cells, although O2(-)-generation was markedly enhanced exclusively in S membranes. The O2(-)-forming activity of S samples remained unchanged or even increased after washing the membranes with buffer, although one-third of the FAD was lost during washing (a decrease from 140 to 95 pmol/10(8) cell-equivalent (CE) during washing). The cytosol is known to contain at least three components that are essential for O2- production (p47-phox, p67-phox, and a G-protein), and that are translocated to membranes upon activation, but its flavin content was one tenth of that of the membranes. The cytosol was treated with fatty acids in the absence of membranes to induce substantial precipitation of p47-phox, p67-phox and a protein of 32 kDa. No difference relative to a solvent-control was noted in the low flavin content of the precipitate indicating that these cytosolic components are not flavoproteins. These results do not support the possibility of translocation of a cytosolic flavoprotein to the membrane upon activation of the respiratory burst.


The source of non-heme iron that binds nitric oxide in cultivated macrophages. In cultured macrophages (J 774 line) a decrease in iron-sulfur centers (ISC) was not observed after 5 min treatment with nitric oxide (NO) (10(-7) M NO/10(7) cells). The content of these centers was measured by electron spin resonance (ESR) spectroscopy at 16-60 K. However, the appearance of a characteristic ESR signal at g(av) = 2.03 indicated the formation of dinitrosyl iron complex (DNIC) in these cells. These findings suggest that loosely bound non-heme iron (free iron) but not iron from ISC is mainly involved in DNIC formation. ISC might release iron for DNIC formation after their destruction induced by the products of NO oxidation (NO2, N2O3, etc).


Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium. Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.


Prolonged cytomegalovirus infection with viremia is associated with development of cardiac allograft vasculopathy. Several reports have suggested an association between cytomegalovirus infection and the subsequent development of cardiac allograft vasculopathy. The difficulties in interpreting these studies include the variety of methods used for the diagnosis of cytomegalovirus infection and variable criteria for the diagnosis of cardiac allograft vasculopathy. To determine whether specific aspects of cytomegalovirus infection are risk factors for cardiac allograft vasculopathy, the patient population of the Oregon Cardiac Transplant Program was analyzed for the following variables: cytomegalovirus infection, primary cytomegalovirus infection, and persistent cytomegalovirus infection for 4 or 6 months documented by either blood or urine cultures and persistent cytomegalovirus viremia for 4 months. In the 129 patients available for analysis, there was no higher incidence of cardiac allograft vasculopathy in patients with or without cytomegalovirus infection, nor was there a higher incidence of cardiac allograft vasculopathy in primary cytomegalovirus infection. There was a nonstatistically significant trend toward an increased incidence of cardiac allograft vasculopathy in patients with persistent cytomegalovirus infection as assessed by cultures positive for infection in either blood or urine. There was, however, a significant increase in the incidence of cardiac allograft vasculopathy in patients who had persistent viremia for at least 4 months compared with those without this finding (47% vs 18%, respectively; p = 0.012). In our population persistent cytomegalovirus viremia and presumably long-term exposure of the allograft coronary tree to cytomegalovirus is associated with cardiac allograft vasculopathy.


Inhibitory effects of ATP and other nucleotides on atrial natriuretic peptide (ANP) binding to R1-type ANP receptors in human neuroblastoma NB-OK-1 cell membranes. ATP dose-dependently inhibited rat 125I-ANP-(99-126) binding to membranes from the human neuroblastoma cell line NB-OK-1 by increasing the KD value for the hormone without altering the Bmax value. After a 20 min preincubation with 37.5 pM 125I-ANP-(99-126) and 0.5 mM ATP, followed by the addition of 0.3 microM unlabelled ANP-(99-126), the proportion of rapidly dissociating receptors was 4-times higher than in the absence of ATP. The other nucleotides ADP, AMP, AMP-PNP, ATP gamma S, GTP, GDP, GMP, GMP-PNP and GTP gamma S were also inhibitory but with a lower potency and/or efficacy. Binding equilibrium data were satisfactorily simulated by a computer program based on partially competitive binding of ANP-(99-126) and the nucleotides, and this, together with the data on dissociation kinetics, strongly suggests that several nucleotides, when added at concentrations up to 1 mM, form a ternary ANP-receptor-nucleotide complex.


Cytomegalovirus and other herpesviruses: do they have a role in the development of accelerated coronary arterial disease in human heart allografts? In conclusion, a great deal of indirect and inferential data point to herpesviruses as having a role in atherogenesis. It has been shown that the herpesviruses are able to remain within vascular tissue in a latent state, allowing for reactivation to occur with subsequent sequelae of an active infection. Herpesviruses affect the cellular metabolic activity of cells, induce the accumulation of lipids, and inhibit the production of matrix proteins. They have the ability to inhibit endothelial cell binding to the basement membrane. It is also known that the herpesviruses, particularly CMV, can initiate a variety of immunologic responses that may contribute to endothelial damage, precipitating atherogenesis. We are only beginning to understand how CMV may participate in ACAD. Greater attention must be focused on the exact cause-and-effect relationship between CMV infection and ACAD. Even the presence of CMV genomes in arterial walls of allografts must be viewed conservatively in the knowledge of CMV ubiquity and other probable contributions to ACAD. If CMV is involved in the development of ACAD, as an active or latent infection, directly or indirectly, it probably involves numerous coexistent mechanisms (Figure 5).


Identification of epidermal growth factor Thr-669 phosphorylation site peptide kinases as distinct MAP kinases and p34cdc2. A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent Km approximately 0.45 mM) for phosphorylation by purified p44mpk, a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42mapk. Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42mapk was measured. However, Mono Q chromatography of A431 cells extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a approximately 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42mapk, but it immunoreacted as a 40 kDa protein only with anti-p44mpk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42mapk. These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.


Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin. Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.


Specific inhibition of NGF receptor tyrosine kinase activity by K-252a. An involvement of protein tyrosine kinase in the transduction of the signals initiated by nerve growth factor (NGF) was investigated. A tyrosine kinase inhibitor, herbimycin, inhibited neurite outgrowth of rat pheochromocytoma PC12 cells induced by NGF but not that by dibutyryl-cAMP. Herbimycin and genistein blocked NGF-dependent activation of ras p21 whose essential function in neuronal differentiation has been reported. These observations suggested that tyrosine kinase activity is involved in the signaling pathways. K-252a, by contrast, inhibited NGF-induced but not EGF-dependent activation of ras p21. Tyrosine kinase activity of gp140trk, a constituent of NGF receptor, is activated by NGF for much a longer period compared to the activation of EGF receptor autokinase activity by EGF. We further demonstrated that autophosphorylation of gp140trk is selectively inhibited by K-252a.


Changes in plasma levels of thrombomodulin during haemodialysis. We studied the changes in plasma levels of thrombomodulin (TM) known to be present on vascular endothelial cells and released into circulating blood following damage to endothelial cells, during haemodialysis in 49 patients with end-stage chronic glomerulonephritis maintained on haemodialysis. After a single haemodialysis, plasma levels of TM were significantly elevated from 38.4 +/- 13.8 ng/ml to 46.8 +/- 14.6 ng/ml (p less than 0.05). Plasma levels of TM before haemodialysis were significantly higher in the cases of long-term haemodialysis than in those of short-term haemodialysis (p less than 0.01). From these observations, increased plasma levels of TM may be due to decreased clearance of TM by renal insufficiency, or damage to endothelial cells induced by the recurrent haemodialysis. In those patients with a greater residual blood volume in a dialyser, the changes in plasma levels of thrombin-antithrombin III complex and TM during a single haemodialysis were significantly higher. From these findings, it was suggested that endothelial cell damage is more severe in patients with greater residual blood volume.


Comparison of the effects of fibrin binding on the biochemical properties of single-chain tissue-type plasminogen activator (t-PA) and single-chain chimeric plasminogen activator (t-PA/scu-PA). The effect of the binding of the single-chain chimeric plasminogen activator t-PA/scu-PA, which contains amino acids 1 to 274 of tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of single-chain urokinase-type plasminogen activator (scu-PA), to fibrin on its biochemical properties was investigated in a purified system. In contrast to the binding of single-chain tissue-type plasminogen activator (sct-PA) on fibrin, which causes an increase in its intrinsic activity, t-PA/scu-PA enzyme activity is not elevated. In contrast to sct-PA which retains its single-chain form during fibrin-binding, t-PA/scu-PA is converted to its more active two-chain form. The activating process of t-PA/scu-PA is accelerated by increasing fibrin concentrations. With constant concentrations of fibrin monomer, the activation velocity also increases with time. Since this effect is inhibited by epsilon-aminocaproic acid and by a monoclonal antibody directed against the fibrin-binding site of t-PA, the activation process depends on the fibrin-binding of the molecule. The results point to the fact that t-PA/scu-PA is autocatalytically converted to its two-chain form during fibrin-binding. The conspicuous differences of the effect of the fibrin-binding on the biochemical properties of sct-PA and t-PA/scu-PA are caused obviously by small differences in the structures of the protease-domains and/or by different communications between the identical A-chains and the protease domains of the enzymes.


Reduction of plasma levels of prothrombin fragments 1 and 2 during thromboprophylaxis with a low-molecular-weight heparin. In a randomized double-blind placebo-controlled study, plasma levels of prothrombin fragment 1 and 2 and total fibrin/fibrinogen degradation products were measured preoperatively and on days 1, 3, 5 and 7 postoperatively in 131 patients undergoing total hip replacement. Patients received a subcutaneous injection of either a low molecular weight heparin (Logiparin) or placebo once daily. Postoperative deep vein thrombosis was diagnosed by bilateral phlebography 7 to 10 days after operation. In the placebo group postoperative levels of prothrombin fragments 1 and 2 and total fibrin/fibrinogen degradation products were significantly higher in patients with postoperative thromboembolic complications, whereas in the low-molecular-weight heparin group no statistical differences were observed. Compared with placebo the administration of a low-molecular-weight heparin was associated with a significant reduction in the levels of prothrombin fragments 1 and 2 and total degradation products. Our observations suggest that the postoperative thrombin generation is moderated by thromboprophylaxis with Logiparin.


Identification of structured peptide segments in folding proteins. Prediction of the structures of long polypeptides and small proteins has been carried out using conformational energy calculations. These calculations can be applied to large proteins if structured regions of their sequences can be identified. Three different approaches to identifying such sequences are presented. First, sequences of five or more contiguous hydrophobic residues tend to nucleate alpha-helices. Second, peptide sequences from parent proteins that have the same biological activities as the parent proteins are highly structured. Third, structured synthetic peptide segments from proteins inhibit the folding of the parent proteins by competing with the corresponding segment of the protein chain for associating with complementary regions. Examples of each of these approaches are presented.


NMR studies of structure and dynamics of isotope enriched proteins. Structural studies of globular proteins by nmr can be enhanced by the use of isotope enrichment. We have been working with proteins enriched with 15N, and with both 15N and 13C. Due to the isotope enrichment we could assign several large proteins with up to 186 residues and could address structural questions. Furthermore, we can accurately measure heteronuclear and homonuclear vicinal coupling constants. This involves in part multidimensional multiple resonance experiments. This is important for characterization of minor conformational changes caused by mutations. We have also made use of isotope enrichment to study the internal mobility of proteins. We also have developed novel methods for measuring accurately 15N relaxation parameters, in particular transverse relaxation rates. This has led us toward a method for directly mapping spectral density functions of the rotational motions of N-H bond vectors in proteins. The protein systems that are discussed include the unlabeled proteins kistrin and cytochrome c551, and the labeled proteins eglin c, a flavodoxin, and human dihydrofolate reductase.


Peptidomimetics as receptors agonists or peptidase inhibitors: a structural approach in the field of enkephalins, ANP and CCK. Stabilization of biologically active conformations of native peptides by cyclization or introduction of hindering residues led to peptidominetics endowed with high affinity and selectivity for one class of receptors and able to cross the blood brain barrier. This is the case of BUBU, Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) and BUBUC, Tyr-D-Cys-(OtBu)-Gly-Phe-Leu-Thr(OtBu) for the opioid delta receptors and of BC 254, Boc-gamma-D-Glu-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-PheNH2 and of BC 264, Boc-Tyr(SO3H)gNle-mGly-Trp-MeNle-Asp-PheNH2 for central CCK-B receptors. Inhibition of metabolizing peptidases such as aminopeptidase N and endopeptidase 24.11 (NEP) for enkephalins and of NEP and ACE for atrial natriuretic peptide and angiotensin I by mixed inhibitors such as kelatorphan and RB 101 or ES14, rationally designed by taking into account the structural differences in the active site of these zinc-metallopeptidases, led to potent analgesics devoid of the major morphine side effects or to new antihypertensives.


ACTH in the pigeon vestibular ganglion.

ACTH in the pigeon vestibular ganglion. Adrenocorticotropic hormone (ACTH) is a member of the opiocortin family of peptides. Within the central nervous system, ACTH-synthesizing neurons are generally considered to be located within the hypothalamus and are known to be involved in the neural circuitry underlying the integration of stress responses. We now report the existence of ACTH-positive perikarya within the pigeon vestibular ganglion. This result makes it likely that ACTH may act in other systems as a transmitter/modulator outside a stress-related context. Since the vestibular ganglia of mammals are known to be glutaminergic, the present data further extends possibility that ACTH is co-localized with glutamate.


GABAA receptor immunoreactivity in the white matter. Using immunohistochemical methods with polyclonal antibodies directed against a specific sequence of the beta 1-subunit of the GABAA receptor, we found strong immunoreactivity in the white matter of cat brain. The immunopositive products were present primarily on processes of glial cells, especially astrocytes. Immunoreactivity appeared also on the cell bodies of astrocytes and on the cytoplasmic membranes of neurons. The abundant immunostaining in the white matter suggests that (1) GABAA receptors are present on glial cells in vivo, (2) GABAA receptors may be localized on non-synaptic membranes in the white matter and (3) activation of GABAA receptors may have some trophic effects on preservation of the structure and functional properties of the white matter.


Long-term depression is induced in Ca2+/calmodulin kinase-inhibited visual cortex neurons. To elucidate a role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in induction of long-term potentiation (LTP), KN-62, a selective inhibitor for CaMKII, was injected into layer 2/3 neurons of sliced visual cortex obtained from young rats. Tetanic stimulation (5 Hz, 1 min) applied to the white matter after the KN-62 injection induced long-term depression (LTD) of excitatory postsynaptic potentials (EPSPs) evoked by test stimulation of the white matter in 9 of the 14 cells tested. However, EPSPs evoked by test stimulation of the non-tetanized site were not changed, indicating that the induction of LTD was input-specific. Simultaneously, recorded field potentials which were derived from neurons with intact CaMKII showed LTP. These results suggest that postsynaptic CaMKII plays a role in the induction of LTP/LTD in visual cortex.


In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells. The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.


Conserved sequence blocks 5' to start codons of plant mitochondrial genes. Three sequence blocks of 10-12 bp are conserved in sequence and order 5' to putative start codons of several higher-plant mitochondrial genes. At least 25 examples were found, primarily associated with coxII, atp6, and orf25, in monocotyledons and dicotyledons. The proximal block can be 9 bp from start codons, and the three blocks generally occur within 100 bp 5' of start codons. In three examples 5' termini of the blocks represent recombination breakpoints, resulting in conservation of the blocks in resultant configurations. The two proximal blocks can form a secondary structure motif. The occurrence of the blocks near start codons, and conserved sequence and order, is consistent with a possible role in translation initiation or regulation.


PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues. Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.


Transformation of cauliflower (Brassica oleracea L. var. botrytis)--an experimental survey. The paper compares different approaches for the genetic transformation of cauliflower (Agrobacterium-mediated, PEG-mediated and/or electroporation). Transient expression of the neomycin phosphotransferase II (NPTII) gene could be detected after direct gene transfer. Stable transformation was achieved using both Agrobacterium-mediated and direct gene transfer. Expression as well as incorporation of the NPTII sequence could be demonstrated.


Aspects of gap junction structure and assembly. The development of ideas about gap junction structure is summarized, including some recent results obtained by use of atomic force microscopy. Particular attention is paid to novel aspects of the biosynthesis and assembly of connexons and to the formation of new junctions.


Biophysics of gap junctions.

Biophysics of gap junctions. Gap junction channels, now known to be formed of connexins, connect the interiors of apposed cells. These channels can be opened and closed by various physiological stimuli and experimental treatments. They are permeable to ions and neutral molecules up to a size of about 1 kDa or 1.5 nm diameter, including second messengers and metabolites. The processes of gating and of permeation are the subject of this review. Voltage is a readily applied stimulus, and transjunctional voltages, or those between cytoplasm and exterior, affect most junctions. Single channel transitions between open and closed states are rapid and presumably involve a charge movement as occurs with channels of electrically excitable channels of nerve and muscle. Identification of gating domains and charges by domain replacement and site-directed mutagenesis is being pursued. Raising cytoplasmic H+ or Ca2+ concentrations rapidly reduces junctional conductance, and this action is generally reversible, at least in part. A number of lipophilic alcohols, fatty acids and volatile anesthetics have similar actions. Phosphorylation also modulates junctional conductance, and in several cases, sites of phosphorylation are known. These gating processes appear similar to those induced by voltage. Permeability measurement indicates that the channel is aqueous and that permeation is by diffusion with only minor interactions with the channel wall. Differences among junctions are known, but further characterization of connexin and cell specificity is required.


Molecular biology and genetics of gap junction channels. Gap junctional communication between cells provides a mechanism for the movement of molecular information between cells via the unit of gap junction structure and function, the gap junctional channel. In the past five years, there has been rapid progress in identifying and characterizing a multigene family that is responsible for producing the gap junction polypeptides that are responsible for generating gap junctional channel oligomers between cells. The products of these genes have been referred to as connexins, and the multigene family can be categorized into two classes at present, the alpha class and the beta class. Members of these two classes can be distinguished on the basis of their primary sequence and overall predicted topological organization. The gap junction genes map to different chromosomes in both mice and humans, and these genes are utilized on a cell specific basis. Furthermore, these genes can be developmentally regulated, and multiple genes can be co-expressed simultaneously by the same cell type. Efforts to understand the precise structure-function relationship of the products of these different genes is now being approached by utilizing various expression systems. Criteria that can be used as a basis for determining membership in the multigene family is presented and discussed, as well as the rationale for using a nomenclature system for the gap junction multigene family that is based on genetic and structural relationships rather than the molecular size of the deduced protein products.


The crystalline lens. A system networked by gap junctional intercellular communication. The vertebrate eye lens is a solid cyst of cells which grows throughout life by addition of new cells at the surface. The older cells, buried by the newer generations, differentiate into long, prismatic fibers, losing their cellular organelles and filling their cytoplasms with high concentrations of soluble proteins, the crystallins. The long-lived lens fibers are interconnected by gap junctions, both with themselves and with an anterior layer of simple cuboidal epithelial cells at the lens surface. This network of gap junctions joins the lens cells into a syncytium with respect to small molecules, permitting metabolic co-operation: intercellular diffusion of ions, metabolites, and water. In contact with nutrients at the lens surface, the epithelial cells retain their cellular organelles, and are able to provide the metabolic energy to maintain correct ion and metabolite concentrations within the lens fiber cytoplasms, such that the crystallins remain in solution and do not aggregate (cataract). Gap junctions are formed by a family of integral membrane channel-forming proteins called connexins. Gap junctions between lens epithelial cells are composed of a connexin which is common between many different cell types, notably myocardial cells and connective tissue fibroblasts. The gap junctions between epithelial cells and lens fibers have not yet been biochemically characterized. The gap junctions formed between lens fibers are composed of at least two different connexins, one of which has not been detected between other cell types. The unusual physiology and longevity of the lens fibers may require the special set of connexins which are found joining these cells.


Gap junctions in development--a perspective. The status of the gap junction as a pathway for cellular interactions during development is reviewed. Current evidence suggests that gap junctions play an important part in ensuring normal development, although the precise role of gap junctional communication remains to be defined. Communication through gap junctions acts alongside cellular interactions achieved by the release of growth factors during embryogenesis. Differences between groups of developing cells may be reflected in, and possibly controlled by, alterations in the selectivity of the gap junctions. It seems likely that gap junctional communication is involved in the control of embryonic patterning rather than phenotypic differentiation.


Leukocyte integrins: structure, function and regulation of their activity. Adhesion is a crucial requirement for the correct regulation of immune and inflammatory responses. In the immune system, leukocytes can interact with each other and with vascular endothelium as well as with extracellular matrix components, changing rapidly and transiently from circulating non-adherent to adherent states. Most of these interactions are mediated by integrins. This review will focus mainly on the structure and function of integrins expressed by leukocytes. The mechanisms for regulating the functional activity of these adhesion receptors, as well as the intracellular signals transduced through integrins, are described.


The neurobiology of tumours and head injury, and the neuroimmunology of central nervous system malignancy. The application of modern investigative techniques in molecular biology, cytogenetics and immunocytochemistry has yielded much new information on central nervous system tumours both in vitro and in vivo. Although the clinical potential of these findings is yet to be fully realized, preliminary clinical studies based on this new information are now in progress. The mechanisms and consequences of brain damage in head injury have been studied both clinically and experimentally for many years; new investigative techniques focusing on the cellular and subcellular basis of brain damage have provided information which is interesting not only in terms of basic neurobiology but also in the identification of new areas for possible therapeutic intervention.


Headache and migraine.

Headache and migraine. That the 'primary' phenomenon in migraine with aura is neuronal ('spreading depression') seems increasingly probable, but the relationship of migraine with and without aura and of both to tension headache remains uncertain. Depression and migraine are related, but other psychological correlates are controversial. Two new therapeutic agents, with action on 5-hydroxytryptamine (5-HT; serotonin) receptors are of promise.


Metabolic and toxic peripheral neuropathies including diabetes. Current literature on neuropathies caused by metabolic defects and exogenous toxins is reviewed. Recent work on the molecular basis of hereditary amyloidosis is initially considered followed by various aspects of diabetic neuropathy and then the effects of ciguatoxin and podophyllin on peripheral nerves.


Excitatory amino acid neurotransmission, excitotoxicity and excitotoxins. Exciting recent developments have begun to define the molecular basis for excitatory amino acid (EAA) receptor diversity and function. Clarification of the roles of these receptors will require identification of the entire repertoire of EAA subunit genes, the subunit composition of each receptor subtype and the mapping of subunits within the central nervous system (CNS). This may allow the development of selective receptor targeting by therapeutic agents. Further evidence is emerging about the molecular processes underlying excitotoxic injury and the importance of free radical formation acting in concert with calcium-dependent processes is being increasingly recognized. There are many clues indicating that primary or secondary excitotoxic mechanisms may play a part in the pathogenesis of some chronic human neurodegenerative disorders. Further work is needed to clarify the mechanisms of selective vulnerability of particular neuronal types given the widespread distribution of EAA receptors within the CNS.


A four-proton-families model for pH-dependent enzyme activation: application to intestinal brush border sucrase. Current concepts of pH-dependent enzyme function are expanded to consider enzymes with up to four key proton families. In an earlier paper the authors extended classical theory to explain the existence, in the acid ionization reaction, of two functionally distinct, V and K, proton families, exemplified by the 1988 sucrase three-proton-families model of Vasseur et al. They now propose that enzymes having two distinguishable proton families at each side of the pH-activity curves exist in nature although there is no previously published evidence of their existence. The resulting, more general, four-proton-families model is treated as a useful framework from which submodels can be derived by simplification, the simplest being the 1911 linear model of Michaelis and Davidsohn, which took into account two proton families out of the theoretical maximum of four proposed here. It is shown that whether a three-proton-families or a four-proton-families model can explain sucrase better is not merely a question of theory but also involves the practical question of having enough data, at each side of the pH spectrum, to permit making an unequivocal choice between the two alternatives. The paper concludes with a discussion of substrate-induced pK shifts according to both models.


Recombinant antigens in viral diagnosis. DNA-fragments coding for a variety of different viral antigens have been cloned and expressed in E. coli. Selected purified recombinant antigens were used for detection of specific antibodies by the means of ELISA technique. This approach has been used for the development of four different ELISA's for the detection of HIV- and EBV-specific antibodies.


Opiate receptor blockade by naltrexone and mood state after acute physical activity. Acute mood changes occur with various forms of physical activity. Increased levels of endogenous opioids (endorphins) in response to exercise may mediate activity-induced shifts in mood state. Thirteen female and six male aerobics class participants aged 20-46 years received the opiate receptor antagonist naltrexone and a placebo in randomized, double-blind crossover fashion on two separate occasions at the same 75-min high-intensity aerobics class. Mood states were assessed before and after each class, which were spaced 5 days apart, using the Profile of Mood States questionnaire (POMS), a mood adjective checklist, and a Visual Analogue Scale (VAS) which measured mood in relation to several emotional extremes. Mood changes over the course of each aerobics class were compared in the naltrexone and placebo groups. For men and women, significant differences between conditions were observed in overall mood by both the POMS (P less than 0.005) and VAS (P less than 0.02). There were significant differences between conditions for most subscales of each mood instrument (P less than 0.05); with the placebo, mood states became calmer, more relaxed and pleasant, tending away from depression, anger and confusion. Positive mood shifts did not occur when subjects were preloaded with naltrexone, suggesting that activity-generated mood changes are mediated through endorphinergic mechanisms.


Spinal cord compression in prostate cancer. A 10-year experience. Of 478 patients treated at a single institution for prostate cancer, 29 developed spinal cord or cauda equina compression. In 5 patients, spinal cord compression was the first evidence of malignancy. Clinical features were predominantly pain, weakness, sensory and sphincter disturbance. The median duration of symptoms was 2 weeks, although the diagnosis was made rapidly at presentation. Clinical diagnosis correlated well with myelographic findings. Only 1 patient suffered neurological deterioration as a consequence of myelography. The functional outcome was dependent on the ability to walk prior to treatment. The median survival in those who were bedridden following treatment was 6 weeks (range 3.5-13) and 21 weeks (range 7-110+) in those who were ambulant following therapy.


Adrenalectomy increases phosphoinositide hydrolysis induced by norepinephrine or excitatory amino acids in rat hippocampal slices. Phosphoinositide hydrolysis induced by norepinephrine, quisqualate, or trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), but not by carbachol, was approximately 50% greater in hippocampal slices from adrenalectomized (14 days) rats compared with controls. These changes appeared to be selective for the hippocampus because no effects of adrenalectomy on phosphoinositide hydrolysis were detected in cortical or striatal slices. The enhanced response to norepinephrine in hippocampal slices after adrenalectomy was observed throughout the effective concentration range of norepinephrine, was not influenced by in vitro addition of corticosterone, was not mimicked or altered by incubation with dibutyryl cyclic adenosine 3',5'-monophosphate (AMP), and did not appear to be due to impaired inhibition of the response to norepinephrine which was elicited by activation of protein kinase C or by inclusion of an inhibitory concentration of quisqualate. These findings indicate that adrenalectomy either removes an inhibitory influence of glucocorticoids on the phosphoinositide system in the hippocampus or that the neurodegeneration of granule cells in the dentate gyrus following adrenalectomy is associated with neurotransmitter-selective increases in phosphoinositide hydrolysis. These data provide further evidence that glucocorticoids modify signal transduction in the brain and extends their known influence to the phosphoinositide second messenger system.


Location of nicotinic and muscarinic cholinergic and mu-opiate receptors in rat cerebral neocortex: evidence from thalamic and cortical lesions. In vitro receptor binding techniques were used to identify the cellular location of nicotinic and muscarinic cholinergic and mu-opiate receptors in the fronto-parietal region of rat cerebral neocortex. Changes in the normal pattern of receptor binding of ligands for these 3 receptors were examined in a series of adjacent sections after unilateral thalamic fiber or cortical cell lesions. Thalamocortical fibers were destroyed by making either electrolytic lesions or kainic acid injections centered in the region of the thalamic ventrobasal complex. These lesions reduced cortical labeling of nicotinic ([3H]nicotine) and mu-opiate ([3H]DAGO) receptors while they did not affect cortical muscarinic ([3H]quinuclidinyl benzilate ([3H]QNB)) labeling. Intracortical injections of quinolinic acid (QA) were used to destroy cortical neurons and spare extrinsic fibers. Cortical QA lesions markedly reduced muscarinic and mu-opiate labeling, but had no significant effect on nicotinic binding at short survivals. Our results suggest that a subset of nicotinic receptors is located presynaptically on the specific thalamo-cortical fibers, while muscarinic receptors are located primarily on cortical neurons. Receptors of the mu-opiate type appear to be located both presynaptically on thalamo-cortical terminals and on intrinsic cortical neurons. The differences in the location of these receptor types suggest that each one modulates discrete aspects of cortical processing.


Alterations in spinal cord excitatory amino acid receptors in amyotrophic lateral sclerosis patients. Excitatory amino acids (EAA) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We have analyzed the distribution of the N-methyl-D-aspartate (NMDA) 1-(1-(2-thienyl)-cyclohexyl) piperidine (TCP), kainate and alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA) quisqualate subtypes of EAA receptors using quantitative receptor autoradiography in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. We observed that in control spinal cords [3H]TCP/NMDA binding sites were located both in the ventral and dorsal horns with the highest densities being situated in lamina II. [3H]AMPA and [3H]kainate binding sites were present almost exclusively in the substantia gelatinosa of the dorsal horn. In ALS, the distribution of these 3 types of receptors was unchanged, but [3H]TCP/NMDA binding was decreased both in the dorsal and ventral horns. [3H]kainate binding was possibly decreased in substantia gelatinosa, of ALS cords. However, the limited sample size available for [3H]kainate binding did not permit statistical analysis. [3H]AMPA binding sites were unaltered in ALS. These results indicate that there is a preferential reduction in NMDA receptors in ALS. We suggest that should an excitotoxic mechanism be involved in the pathogenesis of ALS, then NMDA receptors may be the target of this effect.


Activation of 5-HT1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord. Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and alpha-methyl-5-HT (alpha-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 microM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or alpha-Me-5-HT (both 100 microM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. alpha-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT1C/2 antagonist ketanserin and the 5-HT1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of a novel cell line from pleomorphic adenoma of the parotid gland with myoepithelial phenotype and producing interleukin-6 as an autocrine growth factor. A cell line was obtained from a primary culture of a pleomorphic adenoma of the parotid gland in a 24-year-old woman. The cells of the line (PA 16/23) grew spontaneously in minimal culture conditions and showed stable morphologic characteristics over 30 passages. PA 16/23 cells had immunophenotypic and ultrastructural features similar to those of transformed myoepithelial cells, which are regarded as the precursors of pleomorphic adenomas. Furthermore, these cells have been demonstrated immunocytochemically to contain interleukin-6 (IL-6) on light and electron microscopic examination. IL-6 also has been found by enzyme-linked immunosorbent assay in the culture supernatant and has been proven to be capable of stimulating growth of the PA 16/23 cells.


Chemoembolization of hepatocellular carcinomas. A study of the biodistribution and pharmacokinetics of doxorubicin. This study evaluated the effects of an association of ethiodized oil (Lipiodol Ultra Fluide, Laboratoires Guerbet, Aulnay-sous-Bois, France), with or without gelatin sponge, with doxorubicin (Adriamycin, Adria Laboratories, Columbus, OH) on the biodistribution and kinetics of doxorubicin during intraarterial injection. Eighteen patients with hepatocellular carcinoma on cirrhotic liver received a therapeutic injection into the hepatic artery of 50 mg of doxorubicin alone (Group 1; n = 4), or emulsified in 10 ml of ethiodized oil and 2.5 ml of ioxaglate (Hexabrix, Laboratoires Guerbet) with (Group 2; n = 7) or without (Group 3; n = 7) gelatin sponge embolization. Before treatment, the absence of intrahepatic shunts was verified by an injection of technetium-labeled albumin macroaggregates. The biodistribution of doxorubicin was studied on two fronts: (1) pharmacokinetic--by measurement of the doxorubicin blood level during the 48 hours after injection; and (2) scintigraphic (2 mg of doxorubicin were labeled with 2 mCi of iodine 131)--by examination of the scintigrams and calculation of the following parameters: tumours liver/nontumorous liver binding ratio (T/NT ratio), liver/liver+lungs+abdomen binding ratio, and doxorubicin half-life in tumorous tissue. Pharmacokinetics results showed the following: the peak plasma concentration was significantly higher in Group 1 as compared with Groups 2 or 3 (Group 1: 2.1 +/- 0.9 mg/ml; Group 2: 0.9 +/- 0.3 mg/ml; Group 3: 0.5 +/- 0.2 mg/ml); the area under curve calculated from time zero to 1 hour was lower in Groups 2 and 3 compared with Group 1. Examination of the scintigrams showed the following: diffuse activity throughout the organism (Group 1), diffuse activity with strong hepatic and tumorous binding (Group 2), and mostly hepatic and tumoral binding (Group 3). The liver/liver+lungs+abdomen binding ratio was 28% +/- 1% in Group 1, 36% +/- 5% in Group 2, and 63% +/- 7% in Group 3. The T/NT ratios were 1.0 +/- 0 (Group 1), 1.5 +/- 0.1 (Group 2), and 4.7 +/- 0.5 (Group 3). The doxorubicin half-lives in tumourous tissue were 0.7 +/- 0.1 days (Group 1), 1.8 +/- 0.2 days (Group 2), and 2.6 days (n = 1; Group 3). This study shows (1) that the association of ethiodized oil with doxorubicin lowers the peak concentration of doxorubicin and increases the intratumoral concentration and half-life of doxorubicin, and (2) that these kinetic ameliorations are even more pronounced after embolization. Therefore, from a kinetic standpoint, the doxorubicin-ethiodized oil-gelatin sponge association is the best.


Detection of large cell component in small cell lung carcinoma by combined cytologic and histologic examinations and its clinical implication. In the classification recently proposed by the Pathology Committee of International Association for the Study of Lung Cancer, small cell lung carcinoma (SCLC) was divided into three subtypes: pure SCLC, mixed small cell/large cell carcinoma (mixed SC/LC), and combined SCLC. To examine the clinical utility of this classification, histologic specimens, cytologic specimens obtained by brushing or fine-needle aspiration, and sputum cytologic specimens from 430 patients with SCLC were reviewed. When the subtype of SCLC was determined from the biopsy specimen, cytologic specimen obtained by brushing or fine-needle aspiration, and sputum cytologic specimen, the frequency of mixed SC/LC was 25 of 299 (8.4%), 75 of 400 (18.8%), and 8 of 232 (3.4%), respectively. Whatever the diagnostic method, patients with mixed SC/LC showed a poorer response to treatment and worse prognosis than those with pure SCLC: a median survival of 144 days versus 285 days when classified with the use of biopsy specimens; 160 days versus 275 days with cytologic specimens obtained by brushing or fine-needle aspiration; and 47 days versus 259 days with sputum cytologic specimens, respectively. These findings showed that mixed SC/LC should be separated from pure SCLC as a distinctive group and that cytologic studies of specimens obtained by brushing or fine-needle aspiration were sensitive and useful procedures for this purpose.


Immunohistochemical study of bone GLA protein in primary bone tumors. The immunoreactivity of bone GLA protein (BGP) in primary bone tumors, including osteosarcoma, chondrosarcoma, malignant fibrous histiocytoma of bone (MFH), and giant cell tumor of bone (GCT), was investigated with anti-BGP rabbit serum and peroxidase-antiperoxidase complex. As to intracellular localization, BGP antigenicity was detected in 33 of 35 cases of osteosarcoma and 12 of 25 cases of chondrosarcoma. However, there were no positive findings in all 15 cases of MFH or 20 cases of GCT. In chondrosarcoma, the frequency of positively stained cases increased according to pathologic grading (i.e., 3 of 14 cases of Grade 1, 7 of 9 cases of Grade 2, and 2 of 2 cases of Grade 3). Although the multinucleated cells in MFH or GCT were not immunostained, BGP antigenicity was observed in the multinucleated cells of osteosarcoma (12 of 15 cases). In the matrix of osteosarcoma, BGP immunoreactivity of the tumorous osteoid was observed in 28 of 32 cases. However, in the matrices of chondrosarcoma, MFH, and GCT, BGP immunoreactivity was not observed. These results suggest that the immunohistochemical study of BGP is useful for the differential diagnosis of bone tumors.


Relaxin influences the growth of MCF-7 breast cancer cells. Mitogenic and antimitogenic action depends on peptide concentration. The effects of relaxin (RLX) on the human breast adenocarcinoma cell line MCF-7 have been evaluated. The cells were maintained in culture with Dulbecco's modified Eagle's medium with 1% and 10% fetal calf serum. Highly purified porcine RLX was added at concentrations ranging between 10(-11) and 10(-6) M, and, after 96-hour incubation in the presence of the peptide, cell proliferation, intracellular cyclic adenosine monophosphate (cAMP) content, percent cycling cells, and structural and ultrastructural pattern were studied. The results indicate that RLX is a direct modulator of MCF-7 cell proliferation, stimulating growth at low concentrations and inhibiting growth at high concentrations. Determinations of percent cycling cells and intracellular cAMP accumulation agree with the results of the growth studies. Addition of different concentrations of 8-Br-cAMP to the culture medium results in a dose-related stimulation of MCF-7 cell proliferation. Morphologic examination shows that, in the current experiments, RLX does not induce any clear-cut signs of differentiation of MCF-7 cells in terms of activation of secretion or intracellular lipid deposition. These findings indicate that RLX should be regarded as a novel agent involved in the control of growth of human breast cancer cells.


Angiosarcoma after tylectomy and radiation therapy for carcinoma of the breast. Angiosarcoma (AS) is an uncommon tumor that rarely develops after external beam radiation therapy (EBRT). Thirty-six cases have been reported in the literature. The authors present two additional cases. Each of these patients received breast-conserving treatment for breast carcinoma that consisted of tylectomy and EBRT. In each case, AS developed in the field of prior irradiation. Currently, seven cases of AS after radiation therapy for breast-conserving treatment of breast carcinoma have been reported. The average time interval between the administration of radiation therapy and the development of AS is 8.6 years. This complication is rare and should not influence the decision to offer breast-conserving therapy to patients. However, patients should be informed that AS may develop as a result of radiation therapy.


Microinvasive germ cell neoplasia of the testis. The term microinvasive germ cell neoplasia denotes the presence of neoplastic germ cells in the tubuli and interstitium of the testis, unaccompanied by clinically detectable tumor. Testicular biopsy specimens from three patients (age range, 26-38 years) without clinical evidence of tumor showed microinvasive germ cell neoplasia. The indications for biopsy were gynecomastia and testicular atrophy in Patient 1, infertility in Patient 2, and nonseminomatous cancer in the contralateral testicle in Patient 3. In all three cases, orchiectomy specimens disclosed multifocal intratubular and extratubular growth of neoplastic germ cells, occasionally confluent in seminoma-like infiltrates. In Cases 1 and 2, no malignant cells were found at biopsy of the contralateral testis. In contrast to intratubular (in situ) germ cell neoplasia, microinvasion constitutes a definitive malignancy and the starting point of differentiation into seminoma or nonseminomatous tumor. Inguinal orchiectomy is recommended as primary therapy. The necessity of complementary therapy is an issue that must be investigated.