The dipeptide alanyl-glutamine prevents intestinal mucosal atrophy in parenterally fed rats. This study was performed to determine whether the addition of alanyl-glutamine (Ala-Gln) can prevent intestinal mucosal atrophy induced by standard solution of total parenteral nutrition (S-TPN). Forty-one male Sprague-Dawley rats weighing 250 g were randomly divided into four groups: group I was killed after overnight fasting; group II received S-TPN. The other groups received S-TPN supplemented with amino acids other than glutamine (group III) or supplemented with Ala-Gln 2 g/100 mL (group IV); both solutions were isocaloric and isonitrogenous. After 1 week of TPN the rats were killed, and the duodenum, proximal jejunum, mid-small bowel, and distal ileum were obtained for morphologic and functional analysis. Weight gain did not differ significantly among these four groups, and there was no difference in nitrogen balance between groups III and IV. Serum glutamine in group IV (102.8 +/- 13.3 mumol/dL) was significantly increased (p less than .05) compared with groups I, II, and III (66.2 +/- 3.9, 55.7 +/- 7.8, and 61.3 +/- 10.8 mumol/dL, respectively). Mucosal wet weight, protein, RNA, sucrase, and maltase of group IV were significantly increased (p less than .05) compared with groups II and III. Villus height was significantly increased (p less than .05) in the jejunum of group IV rats compared with groups II and III, but not in any other segments of the intestine. No significant changes were observed in crypt depth among all groups. Diamine oxidase in groups II, III, and IV was significantly decreased (p less than .05) compared with group I in all segments except for the ileum.(ABSTRACT TRUNCATED AT 250 WORDS)


Treatment of ventricular arrhythmias after CAST. The primary objective of this article is to review the management of ventricular arrythmias in the light of the unfavourable results reported in the Cardiac Arrhythmia Suppression Trial (CAST). CAST tested the hypothesis that suppression of ventricular arrhythmias recorded on a Holter monitor in patients with myocardial infarction would lead to a decrease in subsequent mortality, presumably by preventing sudden death. In the trial, patients with a myocardial infarction which occurred six days to two years previously and with asymptomatic ventricular premature beats which could be suppressed by one of the antiarrhythmic agents flecainide, encainide or moricizine, were randomised to treatment with one of these agents or placebo. Over a mean follow-up period of 10 months, mortality was significantly higher in those patients receiving flecainide or encainide than in those receiving placebo. On the recommendation of the Data and Safety Monitoring Board the trial in these groups was terminated. More recently CAST II in which moricizine was compared to placebo was also terminated, again because of a higher mortality in the patients receiving active treatment. It is likely that much of the excess mortality can be attributed to proarrhythmic effects of the agents. Current management of ventricular arrhythmias are considered in the light of these findings. CAST suggests that specific treatment should be dictated by the presence of associated symptoms and as much by associated structural heart disease as the arrhythmia per se. In particular, specific treatment of ventricular premature beats alone should be avoided. In those with potentially lethal ventricular arrhythmias, referral for appropriate investigation and consideration of non-pharmacological measures is necessary.


The surveillance challenge: final stages of eradication of poliomyelitis in the Americas. Current levels of surveillance have contributed to substantial reductions in morbidity and mortality due to poliomyelitis in the Americas. Despite the success of the poliomyelitis eradication initiative, it has become critical that surveillance be intensified so that the absence of wild poliovirus circulation can be verified with confidence in countries not reporting confirmed cases of poliomyelitis. Cases of acute flaccid paralysis continue to be classified as compatible with poliomyelitis, because investigations of such patients do not provide sufficient information to rule out wild poliovirus as the cause of paralysis. At this stage of the eradication initiative, the presence of compatible cases in some countries in Latin America indicates a failure of the surveillance system. The greatest challenge for the eradication initiative may be correcting the remaining deficiencies of the existing surveillance system that hinder efforts to verify that wild poliovirus is no longer being transmitted in the Americas.


Light-regulated expression of the psbD gene family in Synechococcus sp. strain PCC 7942: evidence for the role of duplicated psbD genes in cyanobacteria. The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains two psbD genes encoding the D2 protein of the photosystem II reaction center: psbDI, which is cotranscribed as a discistronic message with psbC (the gene encoding CP43, a chlorophyll-a binding protein), and psbDII, which is monocistronic. Northern blot analysis of psbD transcripts showed that the two genes responded differently when wild-type cells were shifted from moderate to high light intensity. Whereas psbDII transcripts increased 500% relative to unshifted control cells, psbDI-psbC transcripts remained unchanged. The beta-galactosidase activities expressed from translational fusions between the psbD genes and the Escherichia coli lacZ reporter gene displayed responses similar to those seen in the RNA. D2 protein levels in thylakoid membranes from wild-type cells increased to 250% of those of the unshifted control cells 12 h after a shift to high light intensities. In contrast, in a mutant strain (AMC016) that carries an inactive psbDII gene, D2 levels decreased by 50% under identical conditions. These results suggested that induction of psbDII gene expression by light can serve as a supplementary system for maintaining a functional photosystem II reaction center at high light intensity. This hypothesis was corroborated by mixed-culture experiments, in which AMC016 cells competed poorly with wild-type cells at high light intensity. These data suggest for the first time that differential expression of members of a cyanobacterial gene family serves to maintain a functional PSII reaction center under diverse environmental conditions.


The coxII gene in carrot mitochondria contains two introns. The gene for cytochrome oxidase subunit II (coxII) in carrot is encoded by a unique locus in the mitochondrial genome. In contrast to the coxII genes in the numerous other plant species investigated to date, the coding region is interrupted by two group II introns. The carrot 5' intron is the homologue of the single intervening sequence found in several monocot and dicot coxII genes. Sequences similar to the 3' intron of the carrot coxII gene have not been reported previously and are not detectable by hybridization with Oenothera mtDNA. Northern hybridizations indicate complex precursor transcript patterns with mRNA molecules up to 10 kb length. The excised intron sequences appear to be stably maintained in the mRNA pool. Amino acid sequence comparisons suggest that the carrot coxII mRNA needs to be edited by numerous C to U transitions.


Sequence and length heterogeneity of alpha Fc gamma R transcripts in AKR mice. The murine low-affinity receptors for IgG, Fc gamma RIII and Fc gamma RII, are encoded by the alpha and the beta Fc gamma R genes, respectively. By contrast to the sequence and the molecular polymorphism of human Fc gamma RIII, no heterogeneity of the murine Fc gamma RIII has been reported and a single alpha Fc gamma R transcript was observed. We describe here a double heterogeneity of alpha Fc gamma R transcripts. First, by S1 mapping of alpha transcripts and by cloning of cDNA coding for Fc gamma RIII, we found a strain-related sequence heterogeneity: four amino acids in the coding region and two stretches of nucleotides in the 3' untranslated sequences differ between alpha transcripts of AKR and BALB/c mice. Second, in AKR mice, we found a cell-dependent length heterogeneity: a short 0.9 kb alpha transcript was present in peritoneal thioglycolate-elicited cells (PEC) from AKR mice. This transcript was present neither in mast cells and NK cells from AKR and BALB/c mice nor in PEC from BALB/c mice. A short cDNA, with a deletion of all the 3' untranslated sequences, has been cloned from AKR PEC, and corresponds to the short alpha transcript. All the differences found in the 3' untranslated sequences of AKR alpha transcripts are located within the fifth exon of the mouse alpha Fc gamma R gene.


Characterization of interactions involving anti-metatype antibodies and immune complexes. Immunizations of high affinity anti-fluorescein monoclonal antibody 4-4-20 affinity labeled with fluorescein 5-isothiocyanate into a rabbit elicited antibodies specific for the liganded conformation of 4-4-20 (termed "anti-metatype" antibodies). Reaction of liganded 4-4-20 with anti-metatype antibodies caused significant delay (up to 23-fold) in the rate of dissociation of fluorescein ligand from the active site. In this study, structural analogues of fluorescein, including fluorescein 5-isothiocyanate, fluorescein 6-isothiocyanate, 5-dichlorotriazinyl aminofluorescein and 5-carboxyfluorescein, were bound by monoclonal antibody 4-4-20 and anti-metatype antibody reactivity was observed through delay in the dissociation rate of ligand from Mab 4-4-20. Significant delays (ranging from 5- to 242-fold) were observed for all structural analogues examined indicating that 4-4-20 maintained similar but not necessarily identical conformations upon binding fluorescein structural analogues. Additionally, fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate were conjugated to carrier molecules of increasing mol. wt (ranging from 225 to 14,600 D) in an attempt to sterically interfere with "metatopes" at the mouth of the active site and localize regions of anti-metatype antibody binding. These fluorescein-conjugated compounds were reacted with 4-4-20, and binding of anti-metatype antibodies delayed dissociation rates from 24- to greater than 1500-fold. These results indicated that the mechanism whereby anti-metatype antibodies delay the release of fluorescyl ligands from the active site probably does not solely involve steric hindrance of the ligand due to binding of anti-metatype antibodies at the mouth of the active site. Studies with 4-4-20 Fab fragments and a single chain derivative of 4-4-20 (consisting of the variable regions tethered by a 14 amino acid linker) indicated that anti-metatype reactivity was specific for the immunoglobulin variable region.


C1-inhibitor prevents PEG fractionation-induced, EDTA-resistant activation of mouse complement. Fractionation of mouse serum by precipitation with a critical amount of polyethylene glycol 6000 (PEG; 11% w/v) results in a classical and alternative pathway-independent activation of the terminal complement route. The activation can take place after the separation of an activating principle together with the terminal route components from a natural regulator. The isolation and identification of the regulatory component preventing this activation in serum, is subject of this paper. The regulator was purified by fractionated PEG-precipitation (15-25%), followed by heparin-Sepharose affinity, Mono Q anion-exchange, and Superose 12 gel filtration chromatography. The regulator appeared to be a single-chain protein with a Mr of 96 k. A protein with similar activity purified from human serum had a Mr of 104 k and was functionally and antigenically indistinguishable from C1-INH. The mouse 96 k protein inhibited C1-esterase activity indicating that this protein is indeed C1-INH. Mouse C1-INH regulates the PEG fractionation-induced bypass activation of complement, but does not interfere with the assembly or the lytic activity of membrane attack complexes. alpha 2-Macroglobulin appeared also to be capable of inhibiting the PEG-precipitation-induced activation process, but with lower efficiency.


Functional analysis of interferon-alpha subtypes using monoclonal antibodies to interferon-alpha 4a--subtype reactivity, neutralisation of biological activities and epitope analysis. A panel of monoclonal antibodies (mAb) to a major human interferon-alpha (IFN-alpha) subtype, -alpha 4a, have been produced, characterised and used for studies of structure/function relationships of IFN-alpha subtypes. The mAb were tested for effects on receptor binding of IFN-alpha 4a, reactivity with other major subtypes -alpha 1, -alpha 2b and -alpha 14 by competitive ELISA and western immunoblotting, and for neutralisation of antiviral and antiproliferative activities of the four subtypes. The mAb could be grouped according to reactivity with IFN-alpha subtypes, group I (designated I-4-A) reacted with -alpha 4a and -alpha 2b, group II (I-4-C and I-4-F) reacted with -alpha 4a and -alpha 1, group III (I-4-D), I-4-G and I-4-H) reacted with -alpha 4a only, whereas group IV (I-4-I) reacted with -alpha 4a, -alpha 1 and -alpha 2b. No mAb reacted with IFN-alpha 14. Sequence comparisons of reactive and non-reactive IFN-alpha subtypes, and reactivity patterns with IFN-alpha fragments obtained by Lys-C digestion indicated that the epitopes were located in the N-terminal region (group I), in two regions of the middle of the molecule (group III and IV) and in the C-terminal region (group II). Binding of mAb to any of these four distinct epitopes neutralised the biological activities of IFN-alpha 4a, and in all cases, except I-4-A, inhibited receptor binding. Only the group III mAb bind to an epitope proposed to be in the vicinity of residues 30-40 which are implicated, from in vitro mutagenesis studies, in receptor binding. Binding of mAb to the other 3 epitopes neutralises biological activities by indirect mechanisms. These results emphasise the antigenic diversity between highly homologous IFN-alpha subtypes, which may have a wider functional significance. Individual mAb will have practical applications in the purification and detection of several IFN-alpha subtypes and so facilitate their further characterisation. By virtue of their different mechanisms of neutralisation, this panel of mAb will be useful in further studies of receptor interaction and signal transduction by IFN-alpha, and illustrate principles which are relevant to immunochemical studies of the receptor interactions of other cytokines.


Continuing V gamma 2 to J gamma 2 rearrangements of murine T cell receptor gamma genes in a B-committed immature cell line. In SPL2-1-2, a murine B-committed immature cell line transformed with a temperature-sensitive mutant of Abelson virus, T cell receptor (TCR) gamma gene rearrangements, together with IgH gene rearrangements, were induced during culture at a non-permissive temperature (39 degrees C). During 11-12 months of culture, TCR gamma gene rearrangements occurred in all cells. In contrast to TCR gamma genes, neither TCR beta or TCR delta were detected even after 11-12 months of culture at a non-permissive temperature. The majority of the TCR gamma gene rearrangements observed here were V gamma 2 to J gamma 2 joinings and the remaining rearrangements were J gamma 1-linked. V gamma 1 to J gamma 4 and V gamma 3 to J gamma 3 joinings were not detected. Approximately 70% of cells with TCR gamma gene rearrangements produced normal-sized transcripts from the rearranged TCR gamma genes. Cloning and sequencing analysis of a cDNA from the transcripts demonstrated that the whole structure of the cDNA was similar to that of T-lineage cells. These results showed that TCR gene rearrangements were restricted to the gamma genes and that V gamma 2 to J gamma 2 joinings occurred preferentially in this B-committed immature cell line. Furthermore, TCR gamma gene rearrangements also occurred in intracytoplasmic mu-chain producing cells. This indicated that the existence of intracytoplasmic mu-chains did not prevent TCR gamma gene rearrangements, although the existence of mu-chains is known to inhibit further productive IgH gene rearrangements, (allelic exclusion). These results should provide many implications for the mechanism of TCR gene rearrangements, especially that of cross-lineage rearrangements.


Identification and characterization of a partial cDNA expressing interleukin-1-like activity in keratinocytes. A partial cDNA has been isolated from the human keratinocyte cell line COLO-16 which is distinct from either IL-1 alpha or beta and encodes a protein which displays some of the biological properties associated with IL-1. The 720 bp partial cDNA hybridized on Northern blots of COLO-16 mRNA to a 1.6 kbp message of low abundance. Expression of the partial cDNA in COS-1 cells resulted in activity in three IL-1 assays: thymocyte co-stimulation, D10.G4.1 T-cell stimulation and fibroblast proliferation. Antisera generated against synthetic peptides based on inferred protein sequence from the cDNA reacted with a 20 kDa and 30 kDa species in both the COLO-16 cell line and PMA-stimulated normal human keratinocytes. These novel species were also present in PMA-stimulated and unstimulated human dermal fibroblasts and human T-cell lines.


Permanent bruxism as a manifestation of the oculo-facial syndrome related to systemic Whipple's disease. We report here a new case of oculomasticatory syndrome related to systemic Whipple's disease. The patient presented typical ophthalmoparesis associated with ocular myorhythmia consisting of 1 Hz convergent oscillations of both eyes. The masticatory involvement was remarkable and consisted of a permanent bruxism leading to severe tooth abrasions. Possible pathophysiology of such a disorder is discussed.


Myasthenogenicity of a human acetylcholine receptor alpha-subunit peptide: morphology and immunology. Each of 10 rats inoculated with a synthetic peptide comprising residues 125-147 (without a disulfide bond) of human acetylcholine receptor (AChR) alpha-subunit (H alpha) had deposits of IgG and C3 (immune complexes) and showed morphological changes in the fine structure at the motor end-plates 5 weeks after a single immunization. Antibody to the H alpha peptides was elevated 1 week after immunization, but, antibody levels to solubilized human or rat AChR were very low in 8 of the 10 rats. These results suggest that the immune response to peptide H alpha is the myasthenogenic site, which induces morphological change at the end-plates.


Chromogranin A epitopes: clues from synthetic peptides and peptide mapping. Chromogranin A (CgA) is a 48 kDa acidic protein in neuroendocrine secretory vesicles whose primary structure is now known. We used synthetic peptides, synthetic peptide antisera, intact molecule antisera, chymotryptic peptide mapping, microsequencing, immunoblotting, and immunoprecipitation to probe the location of immunodominant domains within the CgA molecule. Polyclonal anti mid-molecule, anti N-terminal and anti C-terminal antibodies specifically visualized CgA (both bovine and human) in one and two dimensional immunoblots of adrenal chromaffin vesicles, and the stain CgA fragments further suggested bidirectional (both N- and C-terminal) cleavage or processing of CgA. Anti intact CgA immunoblotting of HPLC-separated peptides from chymotrypsin-digested bovine CgA revealed several strongly immunoreactive internal peptides, two of which were positioned by N-terminal amino acid sequencing: CgA91ff. and CgA197ff.. A single synthetic peptide (CgA79-113) was recognized by three antibodies developed against the intact CgA molecule: two polyclonal rabbit antisera as well as a monoclonal mouse antibody. Not all antigenicity algorithm-predicted domains were immunogenic, suggesting that some of these predicted domains may not be accessible. Polyclonal anti mid-molecule, anti N- and anti C-terminal synthetic peptide antisera specifically immunoprecipitated 125I-labeled bovine CgA from aqueous solution; mid-molecule antisera precipitated substantially greater amounts than terminal antisera. The immunoprecipitation results suggested exposed terminal as well as interior hydrophilic epitopes in the molecule in its intact, native conformation. 125I-human CgA was best precipitated by anti N-terminal antisera, consistent with greatest interspecies sequence conservation at the N-terminus of CgA. The terminal antisera reacted immunohistochemically in a granular pattern with adrenal medullary chromaffin cells (but not adrenal cortical cells) and pancreatic islet cells (but not pancreatic exocrine acini). Thus, synthetic and chymotryptic peptides yielded novel and specific insights into the structure, conformation, vesicular processing and interior immunodominant domains of CgA.


Evidence of NK1 and NK2 tachykinin receptors and their involvement in histamine release in a murine mast cell line. Binding of [3H]substance P (SP) and histamine release were examined using a cloned mouse mast cell line. SP binding was saturable and specific. In the presence of 30 mM Na2SO4/50 mM Tris buffer, SP interacted with two types of binding sites with Kd values of 0.3 and 40 nM. High-affinity SP binding was blocked by the inclusion of 0.5 uM of the NK1 receptor selective ligand septide in the binding mixture. Neurokinin A (NKA) evoked concentration-dependent histamine release. At concentrations in the nanomolar range, the NK1 preferring agonists SP, SP methylester and physalaemin evoked less than or equal to 5% net release of histamine, which was substantially less than the maximum effect of NKA (+37%) in the micromolar range. Pretreatment of the cells with the NK2 antagonist peptide A reduced NKA-induced histamine release. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P, a putative SP antagonist, also elicited histamine release in the micromolar range, apparently acting as an agonist at the NK2 site. Compound 48/80, N-terminal SP fragments, neurokinin B and the two selective NK2 receptor antagonists cyclo(Gln-Trp-Phe-(R)-[ANC-2]Leu-Met) (peptide A) and cyclo(Gln-Trp-Phe-Gly-Leu-Met) (peptide B) were ineffective. Although the results suggest the coexistence of functional NK1 and NK2 receptors, it appears that in this mast cell line neurokinin-induced histamine release is primarily mediated by the NK2 receptor, characterized biochemically as a low affinity binding site with a Kd value of 40 nM for SP.


Is the morphology of ventricular premature beats a marker for left ventricular dysfunction? The morphology of ventricular premature beats has been described as a marker for the presence or absence of myocardial disease. Furthermore, the premature beat has been reported to be a potential marker for a dilated and hypokinetic left ventricle. To verify this previously tested hypothesis, 37 healthy patients with ventricular premature beats on an electrocardiogram (ECG), Holter monitor ECG, or a stress test ECG were classified according to the ventricular premature beat morphology. Group 1 had ventricular premature beat QRS complexes with a smooth contour or with narrow (less than 40 msec) notching. Group 2 had ventricular premature beats with broad (greater than 40 msec) notching or shelves. All of these patients had normal or borderline normal ECGs and normal multiple-gated acquisition (MUGA) scans. Nine patients had type 1 ventricular premature beats, 20 patients had type II ventricular premature beats, and eight patients had both type I and type II ventricular premature beats. We conclude that the presence of group 2 ventricular premature beats is so frequent in patients with normal left ventricular ejection fractions that the use of this marker in identifying abnormal left ventricular function is suspect.


[Treatment of carcinoid syndrome with a somatostatin analogue]. Octreotide, a long-acting somatostatin analogue has recently been introduced in the therapy of gastroenteropancreatic endocrine tumors, but home experience has been lacking. With the aim of drawing attention to this therapeutic possibility, a case of malignant carcinoid syndrome treated with octreotide for 18 months is reported. Despite the therapeutic attempts preceding the octreotide administration a gradual progression in clinical symptoms was observed and cardiac failure due to fibrotic and valvular heart disease developed. Cytotoxic chemotherapy, serotonin antagonists or repeated selective embolisation of the hepatic artery only resulted in a short transitional improvement. Octreotide in a dose of 100 micrograms three times daily by subcutaneous injection provided effective and rapid relief from episodic flushing and serious diarrhoea. Plasma level of serotonin and 24-hour urinary excretion of 5-hydroxyindolacetic acid decreased from 6 micrograms/ml to 2 micrograms/ml and from 800 mumol/day to 70 mumol/day, respectively. No changes in the number and extension of liver metastases could be seen after introducing the octreotide treatment. The patient's compensated cardiac status could be preserved and continuous therapy provided an acceptable quality of life.


A multiple-source method for studying the prevalence of developmental disabilities in children: the Metropolitan Atlanta Developmental Disabilities Study. The Metropolitan Atlanta Developmental Disabilities Study is the first US, population-based epidemiologic study of the prevalence of mental retardation, cerebral palsy, hearing impairment, and visual impairment among school-age children. The study population consisted of children who were 10 years of age between 1985 and 1987 and whose mothers were residents of the five Georgia counties of Clayton, Cobb, DeKalb, Fulton, and Gwinnett at the time of the child's birth. Since children with developmental disabilities are identified by and receive services from various health, social service, and education systems, a multiple-source case identification method was used. This study is unique in that individual school records were used to identify children with the four disabilities. Use of a multiple-source method made it possible to confirm specific conditions and to classify subtypes of disabilities. About 95% of the children with one or more of these four disabilities were initially identified through the school systems. This approach is much less costly than conducting medical and psychologic assessments on populations of children. In addition, this method made it possible to estimate accurately the "administrative prevalence" of these disabilities (ie, the number of children previously identified with these disabilities for the purpose of providing services). The prevalence rates found in this study, per 1000 10-year-old children, were as follows: mental retardation, 10.3; cerebral palsy, 2.0; hearing impairment, 1.0; and visual impairment, 0.6.(ABSTRACT TRUNCATED AT 250 WORDS)


Origin of raised maternal serum alpha-fetoprotein levels in second-trimester oligohydramnios. Concanavalin A (Con A) subtyping of alpha-fetoprotein (AFP) revealed higher concentrations of AFP non-reactive with Con A in sera of 12 pregnant women with second-trimester oligohydramnios and raised total serum AFP levels than in sera of 42 pregnant women with raised total serum AFP levels and a normal amniotic fluid volume. This suggests that in oligohydramnios the origin of excess AFP in the maternal compartment is amniotic fluid. It is proposed that oligohydramnios and the associated raised maternal serum AFP levels are caused by damage of the fetal membranes prior to 16 weeks of gestation resulting in leakage of amniotic fluid to the decidual tissue and resorption in the maternal circulation.


Can gestational dates routinely derived from very early ultrasound be used to interpret maternal serum alpha-fetoprotein measurements? This study assesses the reliability of estimating gestational age via crown-rump or gestational sac measurements obtained at 8 weeks' gestation or earlier as part of routine physician office practice. To accomplish this, we studied 88 pregnancies managed at 45 different sites in which both an early first-trimester ultrasonically-derived gestational age estimate and a second-trimester biparietal diameter (BPD) estimate were available. The first- and second-trimester determinations were highly correlated, but the first-trimester determinations were, on average, 0.43 weeks earlier than the second. The first-trimester estimates were satisfactory for use in interpreting maternal serum alpha-fetoprotein (MSAFP) screening measurements, but second-trimester BPD measurements obtained prior to MSAFP screening should be the method of choice for interpreting MSAFP values, due to the increased sensitivity for detecting open spina bifida.


[Mediators and neuromediators in asthma]. The physiopathological mechanisms underlying the multifactorial syndrome that is asthma are very complex and protean. Most probably, they are genetically determined, but they are largely modulated by the environment and by the inflammation of bronchi in which allergy occupies a special place. Chemical mediators of cellular origin interact with each other and with the cells that live or are recruited in the airways. Among these mediators histamine and arachidonic acid metabolites seem to play a predominant role, but the clinical use of antagonists has not confirmed the data obtained in vitro and in vivo in animals and even man. Cytotoxic mediators (cationic proteins, free oxygen radicals) are though to exert their noxious effect directly on the bronchial epithelium. No single neuromediator of the adrenergic and cholinergic system can explain the dysfunctions observed in asthma. Mediators of the non-adrenergic non-cholinergic system seem to be more interesting owing to their potential interaction with cells and with chemical mediators which contribute to the development of a true neurogenic inflammation.


Adherence of pathogenic and non-pathogenic Entamoeba histolytica strains to neutrophils. The adherence of polymorphonuclear leukocytes (PMNs) to eight pathogenic and nine nonpathogenic strains of Entamoeba histolytica was examined. No difference between pathogenic and nonpathogenic strains was found. The addition of different carbohydrates confirmed the importance of the 170-kDa lectin of E. histolytica in binding to PMNs, corroborated by the finding that treatment of PMNs with galactosidase inhibited adherence. Inhibition of the microfilament system of E. histolytica using cytochalasin B resulted in a loss of adherence to PMNs. Inhibition of the microtubule system using nocodazole did not affect adherence. Preincubation of the trophozoites with serum resulted in enhanced adherence, but the serum factor responsible for this effect could not be identified. Fibronectin, vitronectin, integrins (CD11/CD18 molecules), complement, and mannose-binding protein did not seem to mediate adherence between E. histolytica and PMNs. In summary, these results indicate that defective adherence mechanisms are not a common feature of nonpathogenic E. histolytica strains.


Perfusion with anti-insulin gamma globulin indicates a B to A to D cellular perfusion sequence in the pancreas of the rhesus monkey, Macaca mulatta. The cellular sequence of intraislet vascular perfusion has been shown to be important in the regulation of islet hormone secretion in the rat and dog islet. In order to test whether a B to A to D sequence of islet cellular perfusion is also present in a nonhuman primate, pancreata from the rhesus monkey, Macaca mulatta, were isolated and perfused in vitro in the presence and absence of anti-insulin gamma globulin. In the presence of the insulin antibody, efflux concentration of insulin decreased rapidly (-95 +/- 1.8%), whereas glucagon and somatostatin concentrations increased (111 +/- 28% and 239 +/- 38%, respectively). These results suggest the presence of a B-A-D cellular sequence of vascular perfusion within the monkey islet. The present results also strongly support the hypothesis that a B-A-D sequence of islet perfusion is important in the regulation of islet hormone secretion and further emphasize the central role of the B-cell in intraislet cellular interactions. The results also suggest that, despite differences in islet anatomy, a B-A-D order of islet cellular perfusion may be the preferred functional sequence among mammalian species.


A method for determining the positions of polar hydrogens added to a protein structure that maximizes protein hydrogen bonding. An automated method for the optimal placement of polar hydrogens in a protein structure is described. This method treats the polar, side chain hydrogens of lysine, serine, threonine, and tyrosine and the amino terminus of a protein. The program, called NETWORK, divides the potential hydrogen-bonding pairs of a protein into groups of interacting donors and acceptors. A search is conducted on each of the local groups to find an arrangement which forms the most hydrogen bonds. If two or more arrangements have the same number of hydrogen bonds, the arrangement with the shortest set of hydrogen bonds is selected. The polar hydrogens of the histidyl side chain are specifically treated, and the ionization state of this residue is allowed to change, if this change results in additional hydrogen bonds for the local group. The program will accept Protein Data Bank as well as Biosym-format coordinate files. Input and output routines can be easily modified to accept other coordinate file formats. The predictions from this method are compared to known hydrogen positions for bovine pancreatic trypsin inhibitor, insulin, RNase-A, and trypsin for which the neutron diffraction structures have been determined. The usefulness of this program is further demonstrated by a comparison of molecular dynamics simulations for the enzyme cytochrome P-450cam with and without using NETWORK.


Serotype conversion in Vibrio cholerae O1. Vibrio cholerae O1 exists as two major serotypes, Inaba and Ogawa, which are associated with the O antigen of the lipopolysaccharide and are capable of unequal reciprocal interconversion. The 20-kilobase rfb regions encoding O-antigen biosynthesis in strains 569B (Inaba) and O17 (Ogawa) have been cloned in Escherichia coli K-12 and the nucleotide sequences have been determined. Besides several base substitutions and a small deletion in the 569B sequence relative to O17, there is a single nucleotide change resulting in a TGA stop codon within the gene for the 32-kDa RfbT protein. We have demonstrated that rfbT is responsible for serotype conversion (Inaba to Ogawa). The construction of a specific rfbT mutation in the Ogawa strain O17, and the ability of the gene from O17 to complement Inaba strains to Ogawa, confirmed rfbT as the gene required for the Ogawa serotype. By Southern hybridization and sequencing of PCR products of a number of strains, we have shown that the changes observed in one Inaba strain (569B) are not conserved in other Inaba strains. This may explain why some Inaba strains are able to convert to Ogawa whereas others are not. The protein encoded by rfbT has been identified and expressed in E. coli K-12 using a phage T7 expression system. Amino-terminal analysis of partially purified protein has identified the translational start of the protein. Primer extension studies have enabled the 5' end of the mRNA to be defined. It exists as a separate transcript from the rest of the rfb region, and the distinctive G + C content of rfbT suggests that it has been acquired from a non-Vibrio source.


In vivo T-cell ablation by a holo-immunotoxin directed at human CD3. We have evaluated the in vivo efficacy of anti-CD3-CRM9, a holo-immunotoxin constructed with a diphtheria toxin binding-site mutant. Eighty percent of established human T-cell subcutaneous tumors in nude mice completely regressed following intraperitoneal injection of immunotoxin at a dose set at half the minimum lethal dose assayed in toxin-sensitive animals. Similar regressions produced by a 137Cs source required a dose in excess of 500 cGy. The high degree of in vivo T-cell ablation produced by this immunotoxin is apparently due to maintenance of the toxin translocation function provided by CRM9 and a necessary intracellular routing function supplied by CD3. This immunotoxin may be useful in treating conditions caused by pathologic oligoclonal T-cell expansion such as graft-versus-host disease, autoimmune diseases, and possibly AIDS.


Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate and kainate in cultured cerebral cortical neurons. By using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene protects against cytotoxicity induced by the excitatory amino acids quisqualate (QA) and N-methyl-D-aspartate (NMDA), whereas no effect was observed on cell damage mediated by kainate (KA) or 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA). In parallel it was shown that KA and AMPA increased the concentration of intracellular free calcium ([Ca2+]i) mainly by influx, whereas the increase in [Ca2+]i stimulated by NMDA and QA predominantly was caused by release of Ca2+ from intracellular stores, which for NMDA seemed to be mediated at least partly by Ca2+ influx. In accordance with the effects on cytotoxicity, dantrolene blocked the increase in [Ca2+]i elicited by QA and NMDA leaving the increase induced by KA and AMPA unaffected. The finding that 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate, which regarding toxicity is a selective KA antagonist, only reduced the KA-stimulated increase in [Ca2+]i by 30% may suggest that the elevation of [Ca2+]i is not the only element in KA-induced cytotoxicity. On the other hand, the present study underlines the importance of Ca2+ for cytotoxicity induced by some excitatory amino acids (glutamate, NMDA, and QA) and supports the current proposal that multiple mechanisms are operating, even concerning calcium homeostasis. Because excitatory amino acid-induced cytotoxicity is thought to be involved in neuropathological conditions such as ischemia, it is possible that dantrolene might be of therapeutic interest.


A uridine-rich sequence required for translation of prokaryotic mRNA. Binding of 30S ribosomal subunits to mRNA during the initiation of prokaryotic translation is known to be influenced by the initiation codon and the Shine-Dalgarno sequence. Site-directed mutagenesis of rnd, the Escherichia coli gene encoding RNase D, has now shown that a U8 sequence upstream of the Shine-Dalgarno region is also essential for expression of this mRNA. Alteration of two to five uridine residues within this sequence has no effect on mRNA levels but decreases RNase D protein and activity by as much as 95%, indicating that the U-rich sequence acts as an enhancer of translation. Moreover, mutant transcripts bind to 30S ribosomes in vitro with lower affinity than their wild-type counterparts, suggesting that the role of the U8 sequence is in the initial binding of ribosomes to the translation initiation region of the message. These data demonstrate that sequences other than those previously recognized can be essential for translation initiation.


What is the optimum size for the genetic alphabet? An important question in biology is why the genetic alphabet is made of just two base pairs (G.C and A.T). This is particularly interesting because of the recent demonstration [Piccirilli, J. A., Krauch, T., Moroney, S. E. & Benner, S. A. (1990) Nature (London) 343, 33-37] that the alphabet can in principle be larger. It is possible to explain the size of the present genetic alphabet as a frozen character state that was an evolutionary optimum in an RNA world when nucleic acids functioned both for storing genetic information and for expressing information as enzymatically active RNA molecules--i.e., ribozymes. A previous model [Szathmáry, E. (1991) Proc. R. Soc. London Ser. B 245, 91-99] has described the principle of this approach. The present paper confirms and extends these results by showing explicitly the ways in which copying fidelity and metabolic efficiency change with the size of the genetic alphabet.


Enhanced expression of the developmentally regulated extracellular matrix molecule tenascin following adult brain injury. Tenascin is an extracellular matrix molecule synthesized and released by young astrocytes during embryonic and early postnatal development of the nervous system, and it is concentrated in boundaries around emerging functional neuronal units. In the adult nervous system, tenascin can be detected only in very low levels. Distinct spatial and temporal distributions of tenascin during developmental events suggest a role in the guidance and/or segregation of neurons and their processes within incipient functional patterns. We show here, using in situ hybridization and immunocytochemistry, that stab wounds of the adult mouse cerebellar and cerebral cortices result in an enhanced expression of tenascin in a discrete region around the lesion site that is associated with a subset of glial fibrillary acidic protein-positive astrocytes. Tenascin up-regulation in the lesioned adult brain may be directly involved in failed regeneration or indirectly involved through its interactions with other glycoconjugates that either inhibit or facilitate neurite growth.


sar: a genetic mouse model for human sarcosinemia generated by ethylnitrosourea mutagenesis. A mouse mutant with sarcosinemia was found by screening the progeny of ethylnitrosourea-mutagenized mice for aminoacidurias. Paper chromatography, column chromatography, and gas chromatography-mass spectrometry identified high levels of sarcosine in the urine of the mutant mice. While sarcosine cannot be detected in the urine of plasma of normal mice, the urinary sarcosine level of 102 +/- 58 mmol per g of creatinine in the mutant mice was at the upper range of the urinary levels (1.5-4.5 mmol of sarcosine per g of creatinine) observed in humans with sarcosinemia. Similarly, the plasma sarcosine level of 785 +/- 153 mumol/liter in the sarcosinemic mice was at the upper range of the plasma sarcosine levels (53-760 mumol/liter) observed in affected humans. Sarcosine dehydrogenase [sarcosine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.1] activity was deficient in sarcosinemic mice. The sarcosinuria phenotype in these mice was inherited as an autosomal recessive trait. This mouse mutant provides a useful genetic model for human sarcosinemia and for development of therapeutic approaches for genetic disease.


Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of approximately 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.


Retinoic acid mimics transforming growth factor beta in the regulation of human immunodeficiency virus expression in monocytic cells. Retinoic acid (RA) exerts potent suppressive and upregulatory effects on human immunodeficiency virus (HIV) expression in mononuclear phagocytes, strikingly similar to the effects of the cytokine transforming growth factor beta (TGF-beta). RA significantly inhibited phorbol ester-mediated, but not tumor necrosis factor alpha-mediated, induction of HIV transcription in the chronically infected promonocytic U1 cell line. RA and TGF-beta also completely suppressed the induction of virus production in U1 cells by interleukin 6 alone or in combination with glucocorticoids, which predominantly upregulate virus expression at the posttranscriptional level. Despite the close parallel to TGF-beta-induced effects, no evidence was obtained that RA mediated its effect by inducing secretion of active TGF-beta 1, -beta 2, or -beta 3. As with chronically infected U1 cells, similar inhibitory effects were also observed in primary monocyte-derived macrophages previously infected with HIV and then exposed to either RA or TGF-beta. In contrast, stimulation of monocyte-derived macrophages or U937 cells (the parental cell line of U1) with either RA or TGF-beta prior to in vitro infection resulted in the enhancement of virus production. Given the already successful use of retinoids in the treatment of several malignancies and the present demonstration of their capability of blocking the induction of HIV expression in infected mononuclear phagocytes, it would be of interest to pursue the potential role of this class of compounds in the development of strategies aimed at the pharmacologic regulation of HIV expression.


Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor alpha clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia. The characteristic t(15;17) of acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR-alpha) gene on chromosome 17 to a gene on chromosome 15 called PML, a putative transcription factor. This distinct translocation results in a fusion mRNA detected by Northern analysis. Two cDNAs have been isolated that differ in the extent of 3' PML nucleic acid sequence contained. This study describes a reverse transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha fusion transcript, which amplifies PML/RAR-alpha mRNA from APL cells with either reported breakpoint. DNA sequencing of the predominant RT-PCR products from 6 patients showed identical RAR-alpha exonic breakpoints and two PML breakpoints. This RT-PCR assay was positive in leukemic cells from 30/30 APL patients with the molecular rearrangement confirmed by cytogenetics or Northern analysis. In leukemic cells of patients with a morphologic diagnosis of APL lacking the t(15;17) by routine cytogenetics, a positive RT-PCR assay predicted clinical response to all-trans-retinoic acid (RA) therapy. Dilutional studies with leukemic cells that express (NB4) or do not express (HL-60) a PML/RAR-alpha fusion mRNA reveal that this RT-PCR assay detects the transcript from as little as 50 pg of total RNA. In APL cells from 5/6 patients treated with RA alone, a complete response by clinical and cytogenetic criteria accompanied a persistently positive RT-PCR assay. This preceded relapse by 1-6 months. RT-PCR for PML/RAR-alpha mRNA provides a more-sensitive test for the t(15;17) than routine cytogenetics or Northern analysis. This molecular rearrangement detected by RT-PCR best defines this RA-responsive malignancy. The RT-PCR assay for the PML/RAR-alpha transcript yields important diagnostic and prognostic information in the management of APL patients.


A common antiviral cytotoxic T-lymphocyte epitope for diverse major histocompatibility complex haplotypes: implications for vaccination. Of nine established murine haplotypes, mice of three types (H-2d, H-2u, and H-2q) possess major histocompatibility complex class I glycoproteins able to present an identical viral peptide for recognition and lysis by virus-specific cytotoxic T lymphocytes. Incorporation of this viral epitope into a recombinant vaccinia vaccine and administration of a single dose protects mice with these three haplotypes from an ordinarily lethal challenge of virus. Hence, a common epitope can exist. The sharing of the ability to bind such epitopes among different MHC haplotypes underscores the feasibility of developing an effective cytotoxic T-lymphocyte vaccine for outbred populations like humans.


Stimulation of protein-tyrosine phosphorylation in rat striatum after lesion of dopamine neurons or chronic neuroleptic treatment. Even though the short-term actions of dopamine on postsynaptic receptors are well-characterized, the molecular bases for long-term trophic interactions between dopamine neurons and their targets remain unclear. Since protein-tyrosine phosphorylation plays a key role in the action of trophic factors, we have investigated its possible involvement in the interactions between dopamine neurons and their striatal targets. Lesioning rat nigrostriatal dopamine neurons by using 6-hydroxydopamine increased the phosphorylation on tyrosine of several proteins, including a major 180-kDa protein (pp180) in the ipsilateral striatum. Protein-tyrosine kinase activity was also increased in the striatum ipsilateral to the lesion, whereas no change in phosphotyrosine phosphatase activity was detected. The stimulation of pp180 phosphorylation was observed 1, 2, and 8 weeks after 6-hydroxydopamine lesion, was selective for the destruction of dopamine neurons, and was mimicked by chronic blockade of dopamine receptors with neuroleptics. Additional lesion experiments and subcellular fractionation showed that pp180 is located in neuronal postsynaptic densities, suggesting that pp180 is a postsynaptic component of corticostriatal synapses. Our results indicate that lesion of specific afferent fibers can activate tyrosine phosphorylation in central neurons and suggest that tyrosine phosphorylation is involved in the long-term consequences of dopamine deficiency and may play a role in synaptic plasticity.


Isolation of a candidate human hematopoietic stem-cell population. We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.


CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle. Critical cell cycle transitions are controlled by the coordinate actions of the p34cdc2 protein kinase and its regulatory subunits, cyclins. Recently we identified another human p34 homolog, cyclin-dependent kinase 2 (CDK2) by complementation of a cdc28-4 mutation in Saccharomyces cerevisiae using a lambda YES human cDNA expression library. CDK2 is 66% identical to CDC2Hs and 89% identical to the Xenopus Eg1 gene, forming a distinct subfamily of CDC2-related protein kinases. We have found that CDK2 encodes a 33-kDa cyclin A-associated protein kinase that contains phosphotyrosine, two characteristics it shares with CDC2Hs. However, we show that the subunit composition of these two protein kinase complexes can vary in different cell types, that they have different in vitro substrate preferences, and that CDK2 mRNA is observed much earlier than CDC2Hs mRNA when lymphocytes are stimulated to enter the cell cycle. We suggest that cells in different developmental or transformed states may have different mechanisms of cell cycle regulation.


p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15. p107wee1 is a protein kinase that functions as a dose-dependent inhibitor of mitosis through its interactions with p34cdc2 in Schizosaccharomyces pombe. To characterize the kinase activity of p107wee1, its carboxyl-terminal catalytic domain was purified to homogeneity from overproducing insect cells. The apparent molecular mass of the purified protein (p37wee1KD) was determined to be approximately 37 kDa by gel filtration, consistent with it being a monomer. Serine and tyrosine kinase activities cofiltered with p37wee1KD, demonstrating that p107wee1 is a dual-specificity kinase. In vitro, p107wee1 phosphorylated p34cdc2 on Tyr-15 only when p34cdc2 was complexed with cyclin. Neither monomeric p34cdc2 nor a peptide containing Tyr-15 was able to substitute for the p34cdc2/cyclin complex in this assay. Furthermore, the phosphorylation of p34cdc2 by p107wee1 in vitro inhibited the histone H1 kinase activity of p34cdc2. These results indicate that p107wee1 functions as a mitotic inhibitor by directly phosphorylating p34cdc2 on Tyr-15 and that the preferred substrate for phosphorylation is the p34cdc2/cyclin complex.


Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase. The raf genes encode a family of cytoplasmic proteins with intrinsic protein-serine/threonine kinase activity. The c-raf gene is the cellular homolog of v-raf, the transforming gene of murine sarcoma virus 3611. The constitutive kinase activity of the v-Raf protein has been implicated in transformation and mitogenesis. The activity of Raf-1, the protein product of the c-raf gene, is normally suppressed by a regulatory N-terminal domain. Activation of various tyrosine-kinase growth factor receptors results in activation of Raf-1 and its hyperphosphorylation. Further, Raf-1 has been shown to act either downstream or independently of the p21ras protein, as indicated by experiments involving microinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that either pp60v-src or p21c-ras can independently activate the autokinase activity of Raf-1, but only to a limited extent. Surprisingly, both pp60v-src and p21c-ras are required to fully activate Raf-1. Analysis of the Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylation sites are distributed equally on serine and threonine residues. When Raf-1 is analyzed by immunoblotting, as previously reported for mammalian cell experiments, a marked increase in the apparent molecular weight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.


Molecular associations between the T-lymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5. The T-cell antigen receptor (TCR) complex is the key structure involved in signal transduction in T cells. To analyze associations between the TCR complex and other molecules, immunoprecipitations were carried out, followed by phosphorylation of molecules in vitro by tyrosine kinases associated with the precipitated molecules. This provided a sensitive assay for molecular complexes, and associations were demonstrated between the TCR complex and the surface antigens CD2, CD4, or CD8 and CD5 in normal rat T cells. The complexes were readily seen in immunoprecipitates from Brij 96 but not Nonidet P-40 detergent extracts. The multimolecular complexes are associated with the internal tyrosine kinases p56lck and p59fyn. The presence of p56lck associated with CD4 or CD8 was also examined in early thymocytes, natural killer cells, and macrophages. The kinase was present in all cases except that of normal macrophages.


Coordinated synthesis and degradation of cdc2 in the mammalian cell cycle. The product of the cdc2 gene (cdc2 or p34cdc2), the catalytic subunit of M phase-promoting factor (MPF), is held at a constant steady-state level throughout the cell cycle. In this report, we show that the constant concentration is maintained by a coordinated regulation of protein synthesis and degradation. At the end of each mitosis, cdc2 transcription is shut off, and the mRNA is rapidly degraded. A 12-fold activation of cdc2 gene transcription occurs every round of the cell cycle at the G1/S transition, in a growth factor-dependent manner. The increase in mRNA correlates with the accumulation of newly synthesized cdc2 during S and G2 phases. At the onset of mitosis, the translation of cdc2 mRNA is shut off. During G1 phase, the cdc2 protein has a relatively long half-life of 18 hr, so cdc2 made in the previous cell cycle is maintained. Once synthesis is activated at G1/S, a concurrent mechanism of degradation is activated, and the protein half-life is reduced to 7.5 hr. By the end of interphase, new cdc2 accounts for 75-85% of the total cdc2 pool. In addition, we show that greater than 75% of the new cdc2 complexes with cyclin, suggesting that a majority of the new cdc2 functions as MPF.


Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages. Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.


A supportive role of neural cell adhesion molecule (NCAM) in adhesion between leukaemic blasts and cytotoxic lymphocytes. The expression and function of neural cell adhesion molecule (NCAM, CD56, Leu19) on leukaemic blasts was investigated. The expression of NCAM was frequent (64%) in 14 studied cases of acute myeloid leukaemia (AML) but not in chronic lymphocytic leukaemia (CLL: 1/3 cases positive) or immunocytoma (IC: no positivity). No correlation with the expression of other AM (intercellular adhesion molecule-1 (ICAM-1), leucocyte function antigen-1 (LFA-1). VLA beta chain) or with AML type, according to FAB classification, was observed. Blocking of NCAM with anti-Leu 19 MoAb on AML targets resulted in a significant decrease of their susceptibility to LAK killing and in inhibition of conjugate formation. In the case of B prolymphocytic leukaemia (B-PLL) which did not express ICAM-1 or LFA-1 but was NCAM+, a complete resistance to LAK activity and lack of conjugate formation was observed. Blocking of NCAM on LAK effectors did not decrease their cytotoxic activity. Our results suggest that NCAM, in the presence of other AM, may have a supportive role in adhesion of leukaemic targets to LAK effectors.


Preferential interaction of the CD4+29+/45RA-subset of human CD4+ T lymphocytes with an antibody against the cell-membrane ganglioside GD3. This study shows that the two major subpopulations of CD4+ T lymphocytes defined on the basis of differential expression of the CD29 and CD45RA antigens, show significant differences in reactivity with a monoclonal antibody against the GD3 ganglioside. Double staining studies showed that the GD3 ganglioside is predominantly expressed on cells from the CD4+29+/45RA subsets. The preferential interaction of the CD4+29+/45RA cell subset with the anti-GD3 MoAb was further confirmed by the proliferative and calcium-flux studies. Accordingly, the reciprocal, CD4+(29-)/45RA+ subset was unable to proliferate in response to the anti-GD3 MoAb alone. Although it did show a significant mobilization of calcium ions and proliferation to IL-2 when stimulated with the anti-GD3 MoAb, these response were much less pronounced than the responses of the CD4+29+/45RA- subset. Finally, when two T-cell stimulating monokines, IL-1 and IL-6, were tested for the ability to modulate the anti-GD3 mediated proliferation, only the former, but not the latter was able to enhance the proliferation. Although the natural ligand for the GD3 ganglioside remains unknown, the data presented here provide further evidence in support of the notion that the T-cell surface molecules different from the T-cell receptor MHC-antigen complex may contribute to the preferential activation of one of the CD4+ T lymphocyte subsets.


Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. In this report we have analysed the kinetics of modulation of human peripheral blood T lymphocyte membrane molecules upon activation with optimal amounts of phytohaemagglutinin (PHA) and concanavalin A (ConA). The following activation-related and differentiation/adhesion molecules were selectively and concomitantly investigated on CD4+ and CD8+ subsets by dual colour flow cytometry: CD69, CD25 and CD71; CD2, CD45RA and L-selectin. Cultures were assayed after 24, 48, 72, 120 and 168 h of incubation with PHA and ConA. This approach allowed a comprehensive evaluation of membrane phenomena occurring during activation of normal resting human T lymphocytes. Data show that the kinetics of expression of these molecules follows a precise and consistent time-course with no major differences between CD4 and CD8 subsets. CD69 expression peaked at 24 h, whereas CD25 and CD71 expression peaked at 48/72 h with some differences between PHA and ConA activation. L-selectin expression started an evident decrease in step with culture time whose magnitude was dependent on the lectin used, being higher with PHA than with ConA. Conversely, the expression of CD45RA remained stable for 72 h and then briskly decreased with no major differences between PHA and ConA activation. CD2 molecules increased with time in number and density, although the percentage of positive cells remained essentially constant (greater than 85%). After 48/72 h of stimulation about 10% of cells co-expressed CD4 and CD8 molecules. To ascertain whether the phenomenon was restricted to cells in a particular activation state, the phenotype of cells in the diverse phases of the cell cycle was established. Results obtained show that only actively proliferating cells, that is cells in S and G2-M phases, co-expressed the two molecules, suggesting that such a phenomenon reflects a momentary dysregulation of the normal sequence of gene expression. The present data are also discussed in the light of the dynamic role of T lymphocyte activation and adhesion molecules in regulating cell-cell interactions, tissue localization and eventual immunological function.


Cancer cell invasion and metastasis.

Cancer cell invasion and metastasis. The most life-threatening aspect of cancer is the undetected spread of tumor cells throughout the body. Improved understanding of how these cells invade tissues is leading to new treatments.


Chemotherapeutic strategies in metastatic colorectal cancer: an overview of current clinical trials. 5-Fluorouracil (5-FU) is still the mainstay of chemotherapy in patients with metastatic colorectal cancer. A prolonged infusion of 5-FU is more active than any other schedule of 5-FU used to date. Cisplatin does not improve treatment results compared with 5-FU alone and is not recommended outside clinical trials. Biomodulation of 5-FU is a major step forward in the treatment of colorectal cancer patients and as the standard chemotherapy for advanced colorectal cancer. Two schedules of folinic acid daily for 5-day (low and high doses) and weekly high dose in combination with daily or weekly 5-FU are the most widely used schedules. Although the response rates to either schedule are comparable, the profile of toxicity is different, being stomatitis for the daily schedule and diarrhea for the weekly schedule as the dose-limiting toxicity. Modulation of 5-FU by methotrexate is time dependent. An interval of 24 hours between methotrexate and 5-FU is necessary for effective modulation. Other modulators, like interferon and N-phosphonoactyl-L-aspartate (PALA), are promising treatment options currently under investigation in randomized trials. The data from phase II and III trials using modulation of 5-FU by folinic acid, PALA, or methotrexate, or using continuous infusion 5-FU indicate that all of these strategies are active. Randomized trials are currently underway to further investigate these therapeutic approaches and whether a specific modulation offers more therapeutic advantages.


Principles in the biomodulation of cytotoxic drugs by interferons. The interferons (IFNs) were identified as novel, endogenous antiviral agents in 1957. Shortly thereafter, antiproliferative and immunostimulatory activities were identified for these compounds. Based on these observations, partially purified IFNs entered clinical trials in the 1970s and recombinant IFNs in 1980. IFNs have demonstrated important clinical activity in hairy cell leukemia, melanoma, renal cell carcinoma, and Kaposi's sarcoma as monotherapy. Shortly after their introduction into clinical trials, however, preclinical studies demonstrated synergistic interactions between IFNs and cytotoxic drugs. Numerous preclinical trials have demonstrated a broad spectrum of interactions between IFNs and at least 20 cytotoxic agents both in vitro and in vivo. Early clinical trials suggest a benefit to combinations of fluorouracil and recombinant interferon alfa in refractory gastrointestinal malignancies. Combinations of IFNs and cytotoxic agents deserve further investigations; however, different principles apply for combining IFNs with cytotoxic drugs than for the design of combination chemotherapy regimens.


Synergistic antitumor interactions between newly synthesized ruthenium complexes and cytokines in human colon carcinoma cell lines. The purpose of these studies was to assess the antiproliferative properties of newly synthesized, heterocyclic ruthenium complexes alone and in combination with cytokines (tumor necrosis factor-alpha, interferon alfa, beta, and gamma) against various human colon carcinoma cell lines. To determine whether any of these ruthenium compounds possesses antitumor activity and reveals synergistic interaction with cytokines six new ruthenium complexes were studied. All six compounds exerted dose-dependent antitumor effects in all colon cancer cell lines tested. The most effective compounds were trans-indazolium[tetrachloro(2H-indazole)ruthenate (III, N1)] and trans-indazolium[tetrachlorobis(1 H-indazole)ruthenate (III, N2)]. Interferon alfa, beta, and gamma as well as tumor necrosis factor-alpha exerted only minimal antiproliferative effects in colon carcinoma cell lines. The data were further analyzed to determine whether preincubation with cytokines altered sensitivity of the cells to synergistically potentiating growth-inhibitory effects. Although simultaneous incubation of ruthenium complexes and interferon did not result in synergistic or additive interactions, 24-hour preincubation with interferon alfa, beta, and gamma significantly enhanced antitumor activity. We conclude from these data that two of six newly synthesized ruthenium complexes possess antiproliferative activity against a panel of human colon carcinoma cell lines. Moreover, biological modulation with interferon using 24-hour preincubation resulted in synergistic interactions. We are planning a phase I trial, since antitumor activity of these ruthenium compounds has been demonstrated in vitro and in vivo, and toxicity as well as stability data are now available.


Hypertonic solutions in the treatment of hypovolemic shock: a prospective, randomized study in patients admitted to the emergency room. The infusion of small volumes of hypertonic saline solution or hypertonic saline plus dextran 70 is remarkably effective in restoring adequate hemodynamic conditions after hypovolemic shock. This prospective double-blind study compares the immediate hemodynamic effects of a bolus infusion of 7.5% NaCl or 7.5% NaCl plus 6% dextran 70 (both 2400 mOsm/L) in severe hypovolemia. One hundred five adult patients admitted in hypovolemic shock (systolic blood pressure less than 80 mm Hg) were revived on arrival to the emergency room and administration of a 250 ml intravenous bolus of hypertonic saline solution (n = 35), hypertonic saline plus dextran (n = 35), or isotonic saline solution (n = 35). This infusion was immediately followed by standard crystalloid and blood replacement until systolic pressure reached 100 mm Hg. Mean arterial pressure (MAP) was measured every 5 minutes, and all intravenous infusions were registered. Plasma volume expansion was calculated from plasma protein concentration measurements. Patients were followed up throughout their hospital course, and results of treatment were recorded. At the end of the infusion period, and 5 and 10 minutes after infusion, MAP was significantly higher in patients receiving either hypertonic solution, compared with the group receiving isotonic solution. All groups showed similar trends toward restoration of hemodynamic parameters thereafter. The calculated plasma volume expansion, immediately after the bolus infusion, was significantly higher (24.1% +/- 1.8% and 24.9% +/- 1.1%) in the hypertonic groups, compared with isotonic groups (7.9% +/- 1.3%). Significantly greater volumes of fluids were required to restore systolic pressure in the patients receiving isotonic saline solution than in the groups receiving hypertonic solution. There were no significant differences between the groups receiving hypertonic solutions. The incidence of complications was low, and the mortality rate was similar in all groups. Infusion of 250 ml hypertonic saline solution in patients with severe hypovolemia was not related to any complications, nor did it affect mortality rates; it improved MAP significantly, acutely expanded plasma volume by 24%, and reduced significantly the volumes of crystalloids and blood required in their resuscitation.


Capillary and venule proliferation in the healing process of Dacron venous grafts in rats. A woven, noncrimped graft (made of Dacron and coated with an elastomer) that was 1.5 mm in diameter and 15 mm long was capable of replacing the vena cava in rats. The elastomer mixture consisting mainly of silicone rubber was necessary to bond the woven Dacron fibrils of the woven graft to prevent fraying at the anastomoses and bleeding through the interstices of the lightly woven, fairly high porosity (500) conduit. Sequential histologic studies showed that patency was associated with the ingrowth of small venules and the spreading of endothelial cells from each anastomosis toward the center. Small venules appeared in loose connective tissue, forming a pseudointima 12 days after grafting. This process occurred in the midportion of the graft before pannus endothelial growth covered the endothelial surface. Various thicknesses of the external polymer coat were studied for their influence on healing. None of the grafts developed a thrombus in these latter studies, and regardless of the thickness of the external elastomer, endothelial resurfacing was complete at 30 days. However, the graft with the thinnest external elastomer coating had the best-formed vasa vasorum, and the intima at both the anastomoses and midportions of the graft was significantly thinner than intima found in grafts of other composition. We conclude that this woven Dacron polygraft provides a surface resistant to early thrombosis; that healing occurs mainly by pannus ingrowth, but external and interstitial factors are also important; and that properties inherent in the polygraft wall determine the size of the residual lumen.


[Volume therapy in traumatology].

[Volume therapy in traumatology]. Circulatory failure is the most dominating problem in the multitraumatized patient in hospital. This article describes the most important pathophysiological events from a compensated stage to the final irreversible stage. The practical consequences for both the doctor and the patient are the amount of bleeding and the duration of the stage of shock. When there are technical problems with venous access we recommend surgical cut-down of peripheral veins. The article also describes the principal quality of the most commonly used volume substitutes. Finally, the author discusses the use of sodium bicarbonate in the event of lactacidosis.


Immunochemical properties of Naja naja atra (Taiwan cobra) phospholipase A2 using polyclonal and monoclonal antibodies. The immuno-chemical properties of Naja naja atra phospholipase A2 (NNA-PLA2) were studied by using the chemically modified PLA2 derivatives and the PLA2 homologues toward anti-NNA-PLA2 polyclonal and monoclonal antibodies. Anti-NNA-PLA2 polyclonal antibodies inhibited the enzymatic activity of NNA-PLA2 and Hemachatus haemachatus DE-I by 87% and 68%, respectively. However, the enzymatic activities of Naja nigricollis CMS-9 and notexin were not significantly affected by the polyclonal antibodies. Competitive enzyme immunoassay revealed that the affinity of NNA-PLA2 for polyclonal antibodies was 330-fold higher than that of Hemachatus haemachatus DE-I. Naja nigricollis CMS-9 and notexin failed to inhibit the binding of NNA-PLA2 to polyclonal antibodies. This implies that the epitope(s) of NNA-PLA2 might comprise some substituted residues in the sequence of PLA2 homologues. Three monoclonal antibodies against NNA-PLA2 were prepared by a hybridoma technique. Two of these monoclonal antibodies inhibited the enzymatic activity of NNA-PLA2, but the other did not. Removal of the N-terminal octapeptide affected the epitope interacting with these monoclonal antibodies. Selective modification of tyrosine residues at positions 3 and 63 or lysine residues at positions 6 and 65 induced a substantial reduction in affinity of NNA-PLA2 for polyclonal and monoclonal antibodies. The three monoclonal antibodies failed to recognize PLA2 homologues. The comparison of the sequence of NNA-PLA2 to those of PLA2 homologues showed that most of the amino acid substitutions of PLA2 homologues occur in the spatially nearby region of the N-terminal region and residues at positions 63 and 65.(ABSTRACT TRUNCATED AT 250 WORDS)


Balloon dilatation of prostate: keys to sustained favorable results. Seventy-seven patients with small prostates (less than 40 g) and significant obstructive symptomatology were treated using balloon dilatation of the prostate (BDP) with a sized-to-fit intra-prostatic balloon. Eighty-seven percent of the patients experienced a 50 percent or greater decrease in symptom score at longest follow-up (12.0 months, average: 3-24 months, range). Forty-nine percent of the patients had a 50 percent or greater improvement in peak uroflow. An anterior commissurotomy through the fibromuscular stroma was consistently found and is the hallmark of a technical and therapeutic success. No cases of incontinence, retrograde ejaculation, or impotence developed. When properly performed, BDP has a place in the spectrum of BPH treatment alternatives.


Transurethral resection of prostate immediately after renal transplantation. The subject of transurethral resection of the prostate (TURP) after renal transplantation has not been evaluated in the urologic literature. We retrospectively compared the outcome of renal transplantation in 8 patients who underwent transurethral resection of the prostate within ten days of renal transplantation with 8 patients who did not undergo prostate surgery. Patients were computer-matched for seven parameters. There was no statistically significant difference in patient survival (6 vs 7) and graft survival (56% vs 88%) between the two groups. However, there was a 25 percent incidence of major perioperative complications (including one mortality) in the TURP group directly attributable to the procedure. Transurethral resection of the prostate can be safely performed immediately after renal transplantation only if urine is sterile, antibiotics and steroids are carefully administered perioperatively, low-gravity irrigation is used, and hemostasis is meticulous.


Detection of residual prostate cancer after radiotherapy by sonographically guided needle biopsy. Detection of persistent or recurrent prostate cancer by digital rectal examination (DRE) after definitive radiotherapy is difficult. With the availability of transrectal ultrasonography (TRUS), the detection of prostate cancer has improved substantially. Since 1987 we have used TRUS to evaluate the prostate after definitive radiotherapy. A hypoechoic lesion suggestive of cancer was identified in 45 of 56 patients (80%) studied. Sonographically directed transrectal needle biopsies were performed in 27 of these (60%), and 16 (59%) were positive for cancer. The presence of a palpable nodule suggestive of cancer (present in 7 patients) was not predictive of a positive biopsy specimen. In 14 patients ultrasound-guided and digitally-guided biopsies were performed at the same time; 8 (57%) of the ultrasound-guided biopsy specimens were positive compared with only 4 (29%) of the digitally-guided biopsy specimens. In all 7 patients with an elevated serum level of prostate-specific antigen (PSA) an ultrasound-guided biopsy result was positive. Random biopsies of sonographically normal (isoechoic) areas of the prostate were performed in 8 patients, but only 2 specimens (25%) were positive for cancer. Ultrasound-guided transrectal biopsy of hypoechoic lesions was a safe and effective technique for identifying residual cancer in the irradiated prostate, regardless of the palpable findings. In the presence of an elevated PSA level, such biopsies invariably identified residual cancer. The use of TRUS, ultrasound-guided biopsy, and the measurement of PSA, in addition to DRE, may aid in the detection of residual cancer after definitive radiotherapy.


Unusual extraskeletal myxoid chondrosarcoma. We describe an unusual soft tissue tumor occurring within the rectus femoris muscle of a 64-year-old woman. The site, size, macroscopic, and histological appearances were all consistent with an extraskeletal myxoid chondrosarcoma. However, the present case differs significantly from previous reports of this uncommon tumor in that electron microscopy did not show any evidence of chondroblastic differentiation. Furthermore, the cells failed to stain for vimentin while labeling intensely for neuron-specific enolase, contained large numbers of cytolysosomes having a multivesicular appearance, and focally produced an external lamina. Based on the typical histological appearances we conclude that this is an unusual variant of extraskeletal myxoid chondrosarcoma in which there is evidence of nerve sheath differentiation.


Merkel cell carcinoma with pagetoid spread. A case of Merkel cell carcinoma showing epidermotropism is presented. The neoplastic cells displayed dotlike immunoreactivity for cytokeratins and strong immunoreactivity for neuron-specific enolase. Although no neuroendocrine granules were found, characteristic paranuclear fibrous bodies were present.


Murine cross-reactive T-cell epitopes of Neisseria meningitidis outer membrane proteins. Five non-covalent vaccines of outer membrane proteins (OMPs) complexed to capsular polysaccharide were prepared from Neisseria meningitidis serogroup B strains. Each vaccine contained distinct serotype (class 2/3) and subtype (class 1) OMPs. The cross-reactivity of the T-cell response to the meningococcal vaccine-associated proteins was examined in an in vitro T-cell proliferative assay, following antigenic priming of mice with one of these vaccines (MB6:P1.6) or with its purified class 1 (subtype P1.6) and class 2 (serotype 6) proteins. Cross-reactive T-cell epitopes were found in all five vaccine preparations on both the class 1 and class 2/3 OMPs. Priming of mice with the subtype P1.6 N-terminal peptide led to a significant but small increase in T-cell proliferation with the MB6:P1.6 vaccine.


A comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in Alzheimer's disease. Several studies have demonstrated that the accurate visualization and quantification of pathological lesions in neurodegenerative disorders depend on the reliability of staining methods. In an attempt to gain a better assessment of the density and distribution of the neuropathological markers of Alzheimer's disease, we compared the staining efficiency of a modified thioflavine S protocol for neurofibrillary tangles (NFT) and senile plaques (SP) to different argentic impregnation techniques (Bielchowsky, Gallyas, Globus, Campbell-Switzer-Martin) and to immunohistochemical stainings obtained with two different antibodies against the amyloid beta protein A4 and the microtubule-associated tau protein. The modified thioflavine S technique (MTST) detects up to 60% more SP and up to 50% more NFT than the Bielschowsky and Globus methods, respectively. The results obtained with the specific antibodies are comparable to those obtained with the MTST, but these immunotechniques are more expensive and time consuming for routine neuropathological evaluation, and the appropriate antibodies are not always commercially available. Furthermore, the morphological appearance of NFT and SP with MTST is greatly improved when compared to the classical thioflavine S and the increased signal-to-noise ratio between specifically stained structures and background permits an accurate semi-automatic quantification.


Specific monoclonal antibodies against normal microtubule-associated protein-2 (MAP2) epitopes present in Alzheimer pathological structures do not recognize paired helical filaments. We have developed monoclonal antibodies that detect normal microtubule-associated protein-2 (MAP2) epitopes in routinely fixed, paraffin-embedded tissue. The somatodendritic distribution of MAP2 in bovine and human nervous tissue was confirmed with several of these antibodies. Furthermore, some of these antibodies immunohistochemically labeled certain pathological structures in Alzheimer brain, especially neurites in senile plaques. Electron microscopic observations, however, indicate that these MAP2 epitopes are not located in the Alzheimer paired helical filaments themselves, but in amorphous granular structures coexistent with them. While the pathological nature of these structures is undetermined, they may represent artefactual modifications of normal cytoskeletal components.


Autopsy findings in two siblings with infantile Refsum disease. Recognition of adrenal atrophy during a review of autopsy findings in two sisters who died at 8 months and 3 1/2 years prompted estimation of very long chain fatty acids, phytanic acid and pristanic acid on wet liver fixed in formalin for 12 years. These were shown to be markedly increased and defects in multiple peroxisomal functions and decrease in particulate catalase were shown in cultured fibroblasts, confirming an abnormality of peroxisomal biogenesis. The patients had presented with failure to thrive, recurrent diarrhoea and vomiting, poor mental development, retinal pigmentation, blindness and in the older patient deafness, with only mild dysmorphic features. Autopsy in the older patient showed adrenal atrophy, cirrhosis, and foamy histiocytes in multiple organs. The brain showed no demyelination, little cytoarchitectural abnormality, occasional perivascular histiocytes in the grey matter and meninges and prominent Purkinje cells in the molecular layer of the cerebellum. In the younger patient the changes were very subtle in spite of the marked clinical similarity. Despite the young age at death the clinicopathological features are most suggestive of infantile Refsum disease. In many situations anatomical pathology can be very useful in the recognition and study of peroxisomal disorders.


Directional and compartmentalised drainage of interstitial fluid and cerebrospinal fluid from the rat brain. Pathways for drainage of interstitial fluid and cerebrospinal fluid from the rat brain were investigated by the injection of 2-5 microliters Indian ink into cerebral white and grey matter and into the subarachnoid space over the vertex of the left frontal lobe. Animals were killed by formalin or glutaraldehyde perfusion 5 min-2 years after injection, and the distribution of ink over the surface of the brain, in 2-mm slices of brain cleared in cedar wood oil, in paraffin sections and by electron microscopy was documented. These investigations showed that carbon particles were distributed diffusely through the interstitial spaces of the white matter whereas they spread selectively along perivascular spaces in the grey matter outlining both arteries and veins and extending to surround capillaries within 1 h. Carbon particles were rapidly ingested by perivascular cells and, to some extent, by meningeal cells surrounding the larger vessels. Very little movement of carbon-labelled perivascular cells and perivascular macrophages was seen after 2 years. Carbon particles entering the subarachnoid space over the vertex of the cerebral hemispheres drained along selected paravascular and subfrontal pathways in the subarachnoid space to the cribriform plate and thence into nasal lymphatics and cervical lymph nodes. These studies demonstrate the diffuse spread of fluidborne tracers through cerebral white matter in the rat, the perivascular spread of tracer in grey matter and the compartmentalised directional flow or tracer through the subarachnoid space to the cribriform plate and nasal lymphatics. Furthermore, particulate matter selectively injected into perivascular spaces in rat grey matter is rapidly and efficiently ingested by perivascular cells.


SP-40,40 is a constituent of Alzheimer's amyloid. Cerebrospinal fluid (CSF), serum and seminal plasma contain a small amount of SP-40,40, a modulatory protein of the human complement system. The SP-40,40 in each body fluid was different in molecular size on SDS-PAGE, and glioblastoma cells, hepatoma cells and testicular tumor cells produced SP-40,40, while neuroblastoma cells did not. Therefore, it was estimated that CSF SP-40,40 originated in glia cells, serum SP-40,40 in liver cells and seminal plasma SP-40,40 in testicular cells. SP-40,40 concentrations in CSF of the patients with Alzheimer's disease and the patients with cerebral tumor were higher than those of normal donors. beta-Amyloid deposits in the brains of the patients with Alzheimer's disease were stained with an anti-SP-40,40 monoclonal antibody (mAb) but not with an anti-S-protein mAb, while cellular processes around beta-amyloid were stained with an anti-S-protein mAb but not with an anti-SP-40,40 mAb. Therefore, beta-amyloid contained SP-40,40 in a form different from that in the soluble membrane attack complex (SMAC, SC5b-9) of the complement, which contains S-protein as well as SP-40,40.


Selective loss of nigral neurons in Alzheimer's disease: a morphometric study. Loss of neurons from the substantia nigra (SN), which is sometimes observed in Alzheimer's disease (AD), was quantitatively analyzed in 10 cases of presenile AD and 19 age-matched controls. On sections from the upper and lower portions of the SN, the pigmented zone (zona compacta) and the non-pigmented zone (zona reticulata) were delineated, and these zones were partitioned into quarters: medial, mid-medial, mid-lateral and lateral. This approach clarified topographical preference of neuronal depletion in the SN of AD; namely (1) pigmented neurons were more severely affected than non-pigmented neurons, (2) neuronal depletion was more marked in the lower SN (-38%, P less than 0.001), where the pigmented neurons in the medial quarter were most severely affected (-51%, P less than 0.001), (3) in the upper SN (neuronal loss: -21%, P less than 0.01), the pigmented neurons in the mid-medial quarter were most severely affected (-43%, P less than 0.01). These findings suggest that some groups of nigral neurons are primarily involved in presenile AD. Gallyas staining after bleaching of melanin pigments uncovered a large number of neurofibrillary tangles (NFTs) mainly in the pigmented zone, especially in the medial quarter. A large number of NFTs, scarse senile plaques, and substantial depletion of neurons form an unique combination of Alzheimer pathology in the SN not well recognized so far.


Expression of oligodendroglia and Schwann cell markers in human nervous system tumors. An immunomorphological study and western blot analysis. Sixty tumors of the central and peripheral nervous system and seven brain metastases of extracranial carcinomas were examined using the peroxidase-antiperoxidase method to study the expression of myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and the HNK-1/Leu-7 epitope. The immunocytochemical findings were compared with and correlated to Western blot results. None of the tumor types, including oligodendrogliomas, neurinomas and neurofibromas, expressed MAG and MBP, whereas myelin sheaths and their remnants within the tumors yielded specific immunoreactions. In contrast, the HNK-1/Leu-7 antibodies labelled the majority of the tumors tested including oligodendrogliomas and Schwann cell tumors. As demonstrated by Western blot experiments the HNK-1/anti-Leu-7 antibodies exhibited positive reactions with diverse polypeptides both in tumors and in non-neoplastic brain tissue at positions not corresponding to MAG. This suggests that the epitope recognized by HNK-1/Leu-7 antibodies is shared by a variety of unrelated proteins in normal and neoplastic tissues. Our results strongly indicate the absence of detectable amounts of MBP and MAG in oligodendrogliomas and Schwann cell tumors. The immunomorphological and immunochemical findings clearly showed the wide distribution of the HNK-1 epitope within different tumor types of the central and peripheral nervous system. In conclusion, the data demonstrate that specific cell markers for human oligodendrogliomas and Schwann cell tumors are still lacking.


Senile plaques, amyloid beta-protein, and acetylcholinesterase fibres: laminar distributions in Alzheimer's disease striate cortex. The laminar distributions of senile plaques and amyloid beta-protein (A beta P) within the striate cortex of patients with Alzheimer's disease (AD) were studied with enhanced Bielschowsky (roughly equivalent to the Campbell technique) and immunohistochemical methods. The laminar distribution of acetylcholinesterase (AChE) fibres within the striate cortex of both AD patients and control patients was studied with an enzyme histochemical method. Quantification of Bielschowsky-stained plaque numbers along intersect lines drawn parallel to laminar boundaries revealed a significant aggregation of plaques at the interface of layers IVc and V. Lines drawn through layer VI intersected significantly fewer plaques than lines through other laminae. Immunoperoxidase staining for A beta P revealed a similar distribution of senile plaques, and addition, prominent, diffuse deposits of A beta P within layers I and IVc. AChE fibres were markedly depleted in the striate cortex of AD cases. In control cases, AChE fibres were, like A beta P immunoreactivity, concentrated within layer I and IVc. The results indicate that enhanced silver methods may not reveal the complete distribution of A beta P. The codistribution of A beta P-immunoreactive diffuse amyloid deposits and AChE fibres to the same cortical laminae is consistent with the possibility that these deposits may be formed from degenerating cholinergic elements. The formation of a line of senile plaques at the interface of two cortical laminae within the striate cortex, in an anatomically analogous situation to a similar line of plaques within the dentate gyrus, suggests that formation of well-defined plaques may be accelerated by the interaction of specific neuronal systems.


Midline brain tumors in MSV-SV 40-transgenic mice originate from the pineal organ. Adult transgenic mice expressing the large T-antigen of the Simian virus 40 (SV 40) under the control of the Moloney murine sarcoma virus (MSV) enhancer and the SV 40 promoter develop inheritable uniform midline brain neoplasms showing features of primitive neuroectodermal tumors. The origin and histogenesis of these tumors were investigated in the present study. The brain and pineal organ of fetal and young transgenic mice less than 3 months old displayed normal macroscopic and microscopic features. In 3.5-month-old animals, the pineal organ was considerably enlarged due to hyperplasia, finally leading to tumor formation. Immunocytochemical demonstration of large T-antigen showed that this oncoprotein was already expressed in the nuclei of certain cells in the pineal organ of fetuses (16 and 18 days old) and newborn animals, but was absent from all other parts of the brain. The immunocytochemical demonstration of S-antigen (arrestin), a highly characteristic marker for pinealocytes, was used for further characterization of the large T-antigen-immunoreactive cells. The fetal pineal organ did not contain immunoreactive S-antigen. This first occurred in certain pinealocytes of newborn mice. Double immunostaining revealed that in newborn and older transgenic mice the immunoreactive large T-antigen was exclusively found in nuclei of cells containing S-antigen immunoreaction in their cytoplasm. Thus, transformed pinealocytes appear as stem cells of the experimental tumors. The results of this study suggest that primitive neuroectodermal tumors and the normal tissue from which they originate share certain molecular and immunocytochemical features.


Melanotic cerebral ganglioglioma: evidence for melanogenesis in neoplastic astrocytes. A composite melanotic glial-ganglionic tumor was resected from a 17-year-old girl who presented with a 5-year history of epilepsy. Grossly, the tumor was partly cystic, partly solid, located superficially in the temporal lobe. Histologically, its glial component was composed of spindle and pleomorphic cells, including tumor giant cells, which were associated with Rosenthal fibers, eosinophilic granular bodies and marked desmoplasia. The cells had immunohistochemical and ultrastructural features of astrocytes, and some were invested by incomplete basal lamina. Thus, the tumor had many features in common with pleomorphic xanthoastrocytoma. However, its most striking feature was the presence of melanin pigment in numerous neoplastic cells. Immunoelectron microscopy revealed glial fibrillary acidic protein-positive intermediate filaments in tumor cells bearing melanosomes and premelanosome, proving their astrocytic nature. This case demonstrates, for the first time, melanosomal melanogenesis in human cells with astrocytic phenotype, and provides additional evidence for the ability of central neuroepithelial cell derivatives to produce melanin.


alpha B-crystallin is present in reactive glia in Creutzfeldt-Jakob disease. alpha-Crystallin is a major eye lens protein, composed of two types of subunits, alpha A and alpha B. The alpha A subunit is restricted to the lens, but alpha B-crystallin has recently also been detected in non-lenticular tissues, including the nervous system. With the use of a polyclonal antiserum directed against a synthetic C-terminal peptide of human alpha B-crystallin, the presence of alpha B-crystallin could be demonstrated immunohistochemically in astrocytes in the brains of patients with Creutzfeldt-Jakob disease (CJD). Most intensive localization was observed in the spongiotic tissue representing abundant progressively changed astrocytes in CJD. In age-matched control brains weak positive reaction was located in individual oligodendroglia cells and subpial astrocytes. Prominent increase of alpha B-crystallin in pathological glia in CJD may represent a response to stress.


[Volar plate osteosynthesis in typical and atypical distal radius fractures]. 28 patients with a fracture of the distal end of the radius were treated by a T-plate osteosynthesis through the volar approach. There were 7 unstable distal metaphyseal fractures and 21 dislocated intra-articular fractures. 21 patients were investigated 6 months to 8 years after operation according to the scheme of Sarmiento. 17 patients had a good or excellent result, 4 patients a fair or poor result. 2 patients developed a Sudeck's dystrophy (Algodystrophy), one of them with a radial-ulnar bone bridge. The volar application of the plate is indicated for flexion and extension fractures. In cases with compression of the dorsal cortex a bone graft is indicated to improve a stable osteosynthesis. A conventional tomography on two views helps to diagnose exactly an intra-articular fracture and to decide whether to use a plate or pins and external fixation after open reduction. Remanipulation or an operation 2 weeks after trauma increases the risk of a Sudeck's dystrophy and leads to a poor result.