Topical anti-inflammatory drugs in the treatment of allergic pollinosic conjunctivitis: a comparative double-blind study. Anti-inflammatory drugs, i.e. glucocorticosteroids and NSAIDs (nonsteroidal anti-inflammatory drugs), are often included in the treatment of allergic conjunctivitis. The present study compares the clinical efficacy and safety of topical hydrocortisone, acetylsalicylic acid (ASA) and piroxicam compared to placebo. The trial, designed as a double-blind, randomized and parallel-group treatment, was carried out in a group of 40 patients suffering from seasonal allergic conjunctivitis due to Parietaria pollen, during the pollen season (June-July, 1990). Patients received hydrocortisone 0.1% solution, ASA 1% solution, piroxicam 0.5% solution or placebo as eye drops, all one drop in each eye q.i.d. for 14 days. The symptoms were evaluated at baseline and at the end of treatment by the clinician and by patients on a diary card. The hydrocortisone group showed a rapid and significant improvement during the first week of treatment, while ASA and piroxicam reduced symptomatology during the second week of treatment; this difference was statistically significant. At the end of the trial, the active drugs were comparable with regard to clinical efficacy. A statistically significant difference was observed between the active drugs and placebo, while no statistically significant difference was observed among the three drugs. No serious side-effects were observed. The results demonstrate the clinical efficacy of anti-inflammatory drugs in the treatment of pollen-induced allergic conjunctivitis; they also suggest the use of NSAIDs in long-term treatment, as their efficacy has been shown to be closely comparable to that of steroids, while avoiding the well-known side-effects of the latter in prolonged treatment.


Bronchial challenge with aspirin lysine in the diagnosis of asthmatics with sensitization to aspirin and its inhibition by aerosolized furosemide. We performed bronchial challenge with ASA lysine in 9 patients with a history of aspirin-induced asthma, 4 asthmatics with no history of hypersensitization to aspirin and 4 control subjects. The test consisted of successive inhalations of increasing concentrations of ASA lysine (11.25, 22.5, 45, 90, 180 and 360 mg/ml) and was interrupted when FEV1 showed a decrease of at least 20%. In order to determine the degree of bronchial hyperreactivity, we first carried out a bronchial challenge with histamine. All patients in the group sensitive to aspirin had a positive reaction to ASA lysine, while this was negative for patients in the two control groups. There was no significant correlation between PC20 to histamine and ASA lysine in any of the groups. On the other hand, 6 patients with sensitivity to ASA repeated the bronchial challenge with ASA lysine after previously inhaling furosemide, and in this second test, none of the 6 had a positive reaction. The variation of ASA lysine PC20 in both tests was positive for these patients (p < 0.001).


Interstitial pneumonitis induced in guinea-pigs by the antigens of Periplaneta americana. Data concerning the experimental induction in guinea-pigs of hypersensitivity pneumonitis with a cockroach antigen are presented. A glycoprotein obtained from the chitinous structures of Periplaneta americana was aerosolized daily to guinea-pigs during 12 weeks. The presence of specific antibodies (IgG) was detected by serological techniques; histopathological studies of the lungs showed interstitial infiltrates of macrophages and LT-CD8+ cells, as revealed by the MoAb used. Single non-necrotizing granulomas were characteristic from the 10th week to the end of the experiment. The results from this animal model suggest that this hypersensitivity pneumonitis is a typical delayed-type reaction due to chronic contact with the heterologous glycoproteins of P. americana.


Granulocyte migration in vivo and in vitro in healthy children of parents with infectious asthma. Our study was carried out on 28 healthy children aged 6-11 years (mean = 9 years) with at least one parent having infectious asthma, and 13 children aged 7-14 years (mean = 9 years), both of whose parents had infectious asthma. The age of the affected parents was 26-45 years (mean = 36 years). The control group comprised 20 healthy subjects aged 25-37 years (mean = 32 years) and 20 healthy children aged 6-14 years (mean = 9 years) with no family history of atopic diseases. In all subjects, the test of granulocyte migration in vitro was carried out using the method of Clausen, modified by us. The in vivo test was performed according to the Southam method. The results of our investigations demonstrate a defect of granulocyte migration in patients with infectious bronchial asthma. In healthy children, both of whose parents had infection, the migration of the granulocytes was impaired.


Rheumatoid factor activity in serum and bronchoalveolar lavage fluid from patients with acute hypersensitivity pneumonitis. The presence of rheumatoid factor was detected in both serum and bronchoalveolar lavage fluid (BALF) from patients with acute pigeon breeder's disease. IgM rheumatoid factor was positive in 14 of 20 serum samples and 6 of 20 BALF samples by ELISA. In contrast, negative results were found in 20 healthy subjects with a history of avian antigen exposure, as well as in the serum and BALF from 10 normal subjects. The simultaneous presence of rheumatoid factor in serum and BALF occurred in five patients, and four of them revealed high levels of rheumatoid factor in BALF in comparison with serum determinations. These abnormalities may play an important role during the acute inflammatory reaction in hypersensitivity pneumonitis.


Specific IgG subclasses in pollinosis.

Specific IgG subclasses in pollinosis. We studied IgG subclasses in pollinosis in order to determine the possible immunopathological role of these antibodies, especially antigen-specific IgG4. The selection of pollinosic patients allowed us to observe the influence natural antigenic exposure exerts upon antibodies. On the other hand, we intended to study the possible modifications immunotherapy produces on IgG subclasses. For this purpose, we selected 266 patients who were classified into the following groups: Group I, 65 patients without immunotherapy, 35 of whom were studied during the pollen season and 30 outside the pollen season; Group II, 40 nonpollinosic patients with hypersensitization to Dermatophagoides pteronyssinus were taken as atopic controls; Group III, 161 pollinosic patients with from 3 to 42 months of immunotherapy. For every patient, IgG1, IgG2 and IgG3 against Phleum pratense were determined by means of ELAST (Leti), and IgG4 against the same allergen by means of the ELISA technique developed by us. Our results confirm, first, that untreated pollinosic patients present higher concentrations of IgG4 against Phleum than nonpollinosic patients, and second, that immunotherapy produces significant modifications in the production of antigen-specific IgG.


Serum penicillin concentrations after intramuscular administration of benzathine penicillin G in children with rheumatic fever and controls. Serum and urine penicillin levels were determined in 11 children with rheumatic fever (RF) who were receiving benzathine penicillin G (BPG) prophylactically every 3 weeks and in 10 children without RF who received the drug for the treatment of other infections. The dose given was 600,000 units for children weighing less than 25 kg and 1,200,000 units for those with a weight above 25 kg. Blood and urine samples were collected from both groups before and on days 7, 14 and 21 after BPG administration. Our results showed that: minimum inhibitory concentrations (MICs) of BPG for group A beta-hemolytic streptococci were 0.02 IU/ml or 0.0125 microgram/ml; intramuscular BPG did not give adequate serum levels to block the growth of group A beta-hemolytic streptococci in approximately 24 and 62% of children included in the study on days 14 and 21 after its administration, respectively; BPG metabolism was similar in both groups and did not depend on the underlying disease; serum and urine levels did not vary according to sex and weight; and there was a small correlation between serum and urine levels.


Phylogenetic relationships among middle American pocket gophers (genus Orthogeomys) based on mitochondrial DNA sequences. Relationships among members representing each of the three subgenera of the Middle American rodent genus Orthogeomys (Rodentia: Geomyidae) were studied by comparing DNA sequence data from two regions of the mitochondrial genome. Results from 527 bp from the 16 S rDNA region and a 402-bp fragment of the cytochrome b gene indicate that the three subgenera are well differentiated genetically, with the subgenus Orthogeomys being distantly related to Macrogeomys and Heterogeomys, and Macrogeomys appearing as the most derived. Within the subgenus Macrogeomys, O. heterodus and O. cherriei form a distinct clade, as do O. dariensis and O. cavator. As with previous protein-electrophoretic studies, the placement of O. underwoodi could not be determined definitively within the subgenus Macrogeomys. We interpret our inability to determine phylogenetic relationships among these three clades as evidence for a rapid phyletic radiation within this subgenus. Sequence divergence estimates indicate that the Macrogeomys radiation took place following the time of completion of the Panamanian land bridge (1.9-2.9 mya). Additionally, the near identity of sequences of a newly described species, O. thaeleri, with those of O. dariensis (percentage sequence divergence = 0.3%) suggests that the two may be conspecific.


Uncommon serious complications in Stevens-Johnson syndrome: a clinical case. We present an extremely serious clinical case of Stevens-Johnson syndrome, the evolution of which has been followed for 3 years. The etiology was unknown, although it was related with the administration of amoxicillin. We found a type III immunity mechanism involving immune complexes. The serious complications affected the skin, mucosae and internal organs. The present sequelae are: esophageal stenosis, pneumopathy with a 50% deficit of pulmonary perfusion and bilateral trichiasis.


Correlation between blood eosinophils, T-helper cell activity markers and pulmonary function in patients with allergic and intrinsic asthma. We studied 21 asthmatic patients (17 with allergic asthma and 4 with intrinsic asthma) who on the date of admittance to our clinic presented eosinophil values higher than 400 eosinophils/mm3 and a marked alteration in pulmonary function. From the day of admittance, eosinophils/mm3 were followed-up for 28-30 days, along with T-helper cell activity markers (IL-2R+) and pulmonary function markers. In patients with allergic asthma with or without topical corticoid treatment, and in patients with intrinsic asthma under treatment with topical corticoids, we observed that the number of eosinophils/mm3 and the number of activity markers (IL-2R+) decreased progressively, with subsequent clinical recovery and improvement in pulmonary function. This was not observed in patients with intrinsic asthma not treated with topical corticoids. We conclude that, as previously demonstrated, the activation and proliferation of eosinophils bear a close relationship with the previous activation of T-helper cells (CD4).


Allergy to pollens from Betulaceae and Corylaceae in a Mediterranean area (Genoa, Italy)--a ten-year retrospective study. Allergy to pollens from Betulaceae and Corylaceae is becoming a leading problem in Genoa, a northern Mediterranean area in Italy. The results of a 10-year retrospective study on combined observations both on the aerobiological presence of Betulaceae and Corylaceae pollens (Alnus, Corylus, Ostrya) and on the incidence of positive SPT in the allergic population living in the same area are reported. Among 3473 patients suffering from seasonal respiratory allergy with positive SPT to one or more pollens, 558 (16.06%) showed SPT positivity to Betulaceae and/or Corylaceae pollens, both isolated and associated with other allergens. These patients suffered from winter or early spring hay fever, with a high incidence of bronchial asthma. A statistically significant increase (0.02 < p < 0.05) in the number of these sensitizations from 1981 to 1990 has been observed. Some possible explanations for this phenomenon, including an increase in the total amount of local airborne pollens, are suggested. About 24% of the patients with positive SPT to these pollens referred oral allergic syndrome associated with the ingestion of some foods, especially apples and nuts, with or without other additional clinical symptoms.


Hereditary angioedema type II--a study of two families. Hereditary angioedema (HA) is caused by a quantitative or qualitative deficiency of C1 esterase inhibitor (C1 INH). We present a study of nine patients with HA belonging to two different families. The symptoms started before 10 years of age in most cases (78%). Facial edema (lips, eyes) and of the extremities (feet, hands) were the most frequent complaints. Three patients presented edema of the glottis and one of them underwent a tracheostomy twice. Laboratory tests, outside the acute crisis, revealed low levels of C4 in all patients. The serum levels of C1 INH were normal in seven patients; however, functional activity was not observed in any of them. After the use of a modified androgen (danazol), control of symptoms was observed in all patients, although functional activity was re-established in only five patients.


Phylogenetic relationships of the endosymbionts of mealybugs (Homoptera: Pseudococcidae) based on 16S rDNA sequences. A portion of the gene coding for the 16S ribosomal RNA from the endosymbionts of three species of mealybugs [Pseudococcus longispinus (Targioni-Tozzetti), Pseudococcus maritimus (Ehrhorn), and Dysmicoccus neobrevipes (Beardsley)] was cloned, sequenced, and compared to a homologous fragment from bacteria representative of aphid endosymbionts as well as major subdivisions of the Proteobacteria. Parsimony analysis of the sequences indicated that the mealybug endosymbionts are related and belong to the beta-subdivision; in contrast, previous studies showed that aphid endosymbionts are part of the gamma-subdivision. These findings suggest that the endosymbiosis of mealybugs is a consequence of a single bacterial infection and indicate that this ancestor was different from the ancestor involved in aphid endosymbiosis.


Pollen calendar of Seville and its relation to allergies. We have carried out a study on the annual and weekly variation of airborne pollen in the air of Seville (Andalusia, Southern Spain) during 3 consecutive years using two Cour's collectors. We provide the pollen calendar of this city, which shows the annual distribution of the most important pollen types. Our results led us to check the validity of the pollen extracts usually used at the hospitals of the city to diagnose pollinosis.


Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an example from the compositae. The internal transcribed spacer (ITS) region of 18-26S nuclear ribosomal DNA was sequenced in 12 representatives of the Compositae subtribe Madiinae and two outgroup species to assess its utility for phylogeny reconstruction. High sequence alignability and minimal length variation among ITS 1, 5.8S, and ITS 2 sequences facilitated determination of positional homology of nucleotide sites. In pairwise comparisons among Madiinae DNAs, sequence divergence at unambiguously aligned sites ranged from 0.4 to 19.2% of nucleotides in ITS 1 and from 0 to 12.9% of nucleotides in ITS 2. Phylogenetic relationships among ITS sequences of Hawaiian silversword alliance species (Argyroxiphium, Dubautia, and Wilkesia) and California tarweed taxa in Adenothamnus, Madia, Raillardella, and Raillardiopsis are highly concordant with a chloroplast DNA-based phylogeny of this group. Maximally parsimonious trees from ITS and chloroplast DNA data all suggest (a) origin of the monophyletic Hawaiian silversword alliance from a California tarweed ancestor, (b) closer relationship of the Hawaiian species to Madia and Raillardiopsis than to Adenothamnus or Raillardella, (c) paraphyly of Raillardiopsis, a segregate of Raillardella, and (d) closer relationship of Raillardiopsis to Madia and the silversword alliance than to Raillardella. These findings indicate that the ITS region in plants should be further explored as a promising source of nuclear phylogenetic markers.


IgG4 levels in relation to Olea europaea immunotherapy. We determined olive pollen-specific IgG4 levels in 100 patients, 39 of whom had been subjected to no immunotherapy (IT) for Olea (31 allergic and 8 nonallergic individuals) and 61 of whom had been administered IT as extracts, including Olea pollen (29 extracts in BUs, 24 allergenic extracts polymerized with glutaraldehyde:Allergoid and 8 extracts standardized in PNUs). IgG4 levels were correlated to the clinical picture, type of extract and average cumulative dose (ACD). We found average IgG4 levels of 0.80 +/- 0.74, 8.60 +/- 13.07 (p < 0.01) and 1.42 +/- 2.5 micrograms/ml (n.s.) for the untreated group and those treated with BU and Allergoid, respectively. The difference between the IT-BU-treated and IT-Allergoid-treated patients was significant at p < 0.01. On the other hand, we found no significant differences among the average IgG4 levels of the three groups in relation to age or sex. The group of asthmatic patients treated with BU extracts had average IgG4 levels of 16 +/- 17.34 micrograms/ml, while those of the rhinoconjunctivitic, non-asthmatic group were 5.05 +/- 6.149 micrograms/ml, with p < 0.05 (Student's "t" test). Thus, patients treated with extracts polymerized with glutaraldehyde had IgG4 levels that were similar to those of the patients subjected to no IT. Also, the group treated with IT extracts standardized in BUs had increased IgG4 levels that were correlated with the cumulative dose, particularly in asthmatic patients.


The phylogenetic relationships of flies in the family Drosophilidae deduced from mtDNA sequences. The phylogenetic relationships of several taxa from representative genera, subgenera, groups, and subgroups in the Drosophilidae were examined using sequences from a 905-bp mtDNA fragment. Conventional cloning and sequencing techniques were used to obtain nucleotide sequences. In addition, polymerase chain reaction primers were designed for the rapid amplification and sequencing of this region for the species examined in the Drosophilidae. Phylogenetic analysis was done by cladistic techniques. Because of the coding nature of the 905-bp mtDNA fragment, several separate analyses of these sequences were performed. The genera Scaptomyza and Hirtodrosophila occupy ancestral branching positions in the molecular phylogeny. The genera Chymomyza and Zaprionus have intermediate branching positions, while the subgenera Drosophila and Sophophora are in the most derived position in the molecular phylogeny. Within the subgenus Sophophora, there is little resolution using these sequences, while within the subgenus Drosophila, D. melanica, D. funebris, and D. pinicola form a clade in a derived part of the phylogeny, with D. robusta and D. immigrans branching in an intermediate position in the phylogeny. D. mercatorum, a member of the repleta species group, occupies an ancestral position in the molecular phylogeny.


Serum sickness related to current procedures of in vitro fertilization--a case report. A variety of cutaneous and systemic adverse reactions related to procedures of in vitro fertilization (IVF) have been reported, among them skin reactions secondary to LHRH agonists--attributed to nonspecific histamine release--and a serum sickness-like syndrome which appears to be related to heterologous sera contained in the rinsing solution for graafian follicles. We report here the case of a 41-year-old woman who on two occasions suffered from clinical features consistent with serum sickness after being submitted to current IVF protocols. A specific IgG-mediated reaction against bovine serum albumin (BSA) was demonstrated. The avoidance of BSA or other heterologous sera in the rinsing solutions for graafian follicles is advisable in all IVF protocols, since it adds an unnecessary risk of developing immune complex-related illness.


Evolution of the mitochondrial cytochrome oxidase II gene among 10 orders of insects. We examine the complete nucleotide sequences of the mitochondrial cytochrome oxidase II gene of 13 species of insects, representing 10 orders. The genes range from 673 to 690 bp in length, encoding 226 to 229 amino acids. Several insertion or deletion events, each involving one or two codons, can be observed. The 3' end of the gene is extremely variable in both length and sequence, making alignment of the ends unreliable. Using the first 639 nucleotide positions, for which unambiguous alignments could be obtained, we examine the neighbor-joining trees based on nucleotide divergences and based on conserved subsets of that data, including transversion and amino acid and second codon position divergences. Each of these subsets produces different trees, none of which can be easily reconciled with trees constructed using morphology and the fossil record. Bootstrap analysis using second codon positions strongly supports affinities between the order Blatteria (cockroaches) and the order Isoptera (termites) and between a wasp and the published honeybee sequence (Order Hymenoptera). The divergence of insect orders is very ancient and may have occurred too rapidly for easy resolution using mitochondrial protein sequences. Unambiguous resolution of insect orders will probably require analysis of many additional taxa, using the COII gene and other conserved sequences.


Phylogenetic inference based on matrix representation of trees. Rooted phylogenetic trees can be represented as matrices in which the rows correspond to termini, and columns correspond to internal nodes (elements of the n-tree). Parsimony analysis of such a matrix will fully recover the topology of the original tree. The maximum size of the represented matrix depends only on the number of termini in the tree; for a tree derived from molecular sequences, the represented matrix may be orders of magnitude smaller than the original data matrix. Representations of multiple trees (which may or may not have identical termini) can readily be combined into a single matrix; columns of discrete-character-state data can be added and, if desired, weighted differentially. Parsimony analysis of the resulting composite matrix yields a hybrid supertree which typically provides greater resolution than conventional consensus trees. Use of this method is illustrated with examples involving multiple tRNA genes in organelles and multiple protein-coding genes in eukaryotes.


Convergence in ascospore discharge mechanism among pyrenomycete fungi based on 18S ribosomal RNA gene sequence. Fungi of the class Pyrenomycetes (Ascomycotina) form a morphological series ranging from those that shoot ascospores (sexual spores) forcibly from the ascus (spore sac) to fungi that ooze ascospores or have no obvious mechanism for ascospore release. Did forcible ascospore discharge evolve within these pyrenomycetes, or has it been lost in the group? We determined the sequences of the 18S ribosomal RNA gene from three fungi and used these, along with six sequences from our previous work and three sequences from GenBank, to infer the phylogeny of 12 ascomycetes with various ascospore discharge mechanisms. The 1720 base pairs of sequence data per fungus yielded 361 variable sites, 198 phylogenetically informative sites, and a single most parsimonious tree requiring 562 nucleotide changes. The tree shows that the capacity to shoot ascospores into the air has been lost or, less probably, gained repeatedly and independently. Species lacking forcible ascospore discharge are intercalated among three lineages of species with forcible discharge. In this tree, seven of the nine internal branches appeared in 95% or more of 500 bootstrap replicates. A tree uniting the fungi with forcible ascospore discharge into a monophyletic group required 45 additional steps and fit significantly less well with the data than the most parsimonious tree, based on a maximum likelihood test. Two of the fungi whose sequence we determined, Pseudallescheria boydii and Sporothrix schenckii, are not closely related to one another, even though both are human pathogens and both are from pyrenomycete lineages lacking forcible ascospore discharge. Using the well-resolved, most parsimonious tree, we inferred base substitution patterns in the 18S rRNA. The transition-to-transversion ratio was 1.9. Of all 12 possible substitutions, 29% were from U to C. At sites corresponding to yeast stem positions, A to G transitions were frequent, perhaps compensating for some of the U to C changes, and maintaining secondary structure base pairing (A to G:U to C = 3:4). In loop or bulge positions without secondary structure base pairing, U to C transitions were still frequent, but A to G transitions were rare (A to G:U to C = 1:5).


Structure and sequence of the Cu,Zn Sod gene in the Mediterranean fruit fly, Ceratitis capitata: intron insertion/deletion and evolution of the gene. We have cloned a 4-kb region encompassing the Cu,Zn superoxide dismutase (Sod) gene from a genomic library of the Mediterranean fruit fly, Ceratitis capitata, using a cDNA probe from Drosophila melanogaster. The coding sequence of 462 bases is equally as long as that in Drosophila species. The rate of amino acid replacement over the past 100 million years is approximately the same in the Diptera and in mammals, thus excluding the hypothesis (proposed to account for an apparent acceleration in rate of evolution of Sod over geological time) that the evolution of the SOD protein is much higher in the mammals than in other organisms. The coding region is interrupted by two introns in Ceratitis, whereas only one occurs in Drosophila. Phylogenetic comparisons indicate that the second intron was present in the common dipteran ancestor, but was lost shortly after the divergence of the Drosophila lineage from other Diptera. Analysis of the exon/intron structure of Sod in various animal phyla, plants, and fungi indicates that intron insertions as well as deletions have occurred in the evolution of the Sod gene.


Variation and the phylogenetic utility of the large ribosomal subunit of mitochondrial DNA from the insect order Hymenoptera. Nucleotide sequence variation from a 573-bp region of the mitochondrial 16S rRNA gene was determined for representative hymenopteran taxa. An overall bias in the distribution of A and T bases was observed from all taxa; however, the terebrants (parasitoids) displayed significantly lower AT ratios as well as a higher degree of strand asymmetry. Moreover, a strong positive correlation was observed between relative AT richness and sequence divergence, suggesting selection at the nucleotide level for A and T bases as well as functionality. Overall sequence difference ranged from 2.3 to 53.4%, with the maximum divergence between members of the two Hymenopteran suborders. These data were used in a phylogenetic analysis to illustrate the utility and degree of resolution provided by this information at various hierarchical levels within this taxonomically diverse order. Parsimony analysis revealed strong evidence for monophyly of the aculeates and the terebrants. Most noteworthy was a strongly supported clade containing the two terebrant superfamilies Icheumonoidea and Chalcidoidea. Conversely, high sequence divergence values resulted in instability at the base of the tree and limited resolution at the higher taxonomic levels. Nevertheless, these results do identify those taxonomic levels for which 16S rRNA sequences are phylogenetically informative.


A molecular perspective on mammalian evolution from the gene encoding interphotoreceptor retinoid binding protein, with convincing evidence for bat monophyly. The evolutionary relationships of the various orders of placental mammals remain an issue of uncertainty and controversy. Molecular studies of mammalian phylogeny at the DNA level that include more than just a few orders are still relatively meager. Here we report results on mammalian phylogeny deduced from the coding sequence of the single-copy nuclear gene for the interphotoreceptor retinoid binding protein (IRBP). Analysis of 13 species representing eight eutherian orders and one marsupial yielded results that falsify the hypothesis that megachiropteran bats are "flying primates," only convergently resembling microchiropteran bats. Instead, in agreement with more traditional views, as well as those from other recent molecular studies, the results strongly support a monophyletic Chiroptera (micro- and megabats grouped together). The IRBP results also offer some rare molecular support for the Glires concept, in which rodents and lagomorphs form a superordinal grouping. Also in congruence with other recent molecular evidence, IRBP sequences do not support the view of a superorder Archonta that includes Chiroptera along with Dermoptera (flying lemur), Scandentia (tree shrew), and Primates. IRBP was not however, without its shortcomings as a molecular phylogenetic system: high levels of homoplasy, evident in the marsupial outgroup, did not allow us to properly root the tree, and several of the higher level eutherian clades were only weakly supported (e.g., a Carnivora/Chiroptera clade and an Artiodactyla/Carnivora/Chiroptera clade). We suggest that these shortcomings may be diminished as the phylogenetic density of the data set is increased.


Molecular phylogeny of the prickly shark, Echinorhinus cookei, based on a nuclear (18S rRNA) and a mitochondrial (cytochrome b) gene. The classification of the sharks is unclear. This is particularly true for the superorder Squalomorphii. The relationships between the squalomorphs and other superorders of sharks and the relationships between the different orders within the squalomorphs are a matter of debate. Here, we report a molecular phylogeny for a little known member of this superorder, the genus Echinorhinus. Echinorhinus is most commonly classified in either the family Echinorhinidae (Squaliformes) or the family Squalidae (Squaliformes). However, some authors have suggested a closer relationship to the order Hexanchiformes. In an attempt to shed light on this controversy, we have cloned, sequenced, and compared two genes widely used in molecular phylogeny studies, the cytochrome b and the 18S rRNA from the rare prickly shark, Echinorhinus cookei, and two potential relatives, the spiny dogfish Squalus acanthias (Squaliformes), and the sevengill shark, Notorynchus cepedianus (Hexanchiformes). The sequences of these genes for the prickly shark, the dogfish, and the sevengill shark were found to be equally divergent, suggesting that the prickly shark is no closer to the order Squaliformes than to the order Hexanchiformes.


Gene conversion generates hypervariability at the variable regions of kallikreins and their inhibitors. The two mechanisms for generating hypervariability at the reactive center of serine proteases and their inhibitors are gene conversion followed by natural selection and natural selection for point mutation. One way to clarify the effects of these two mechanisms is to calculate separately the number of nonsynonymous substitutions and that of synonymous substitutions at the variable regions and at the conserved regions. Our data analysis shows that not only the number of nonsynonymous substitutions but also the number of synonymous substitutions at the variable regions exceed the corresponding numbers at the conserved regions. Thus gene conversion has provided needed variability at the variable regions of serine proteases and their inhibitors. Natural selection has helped perpetuate such variability.


A statistical method for detecting regions with different evolutionary dynamics in multialigned sequences. We describe a stochastic method for tracing the evolutionary pattern of multialigned sequences. This method allows us to detect gene regions with distinct evolutionary dynamics, e.g., regions that significantly deviate from the expected behavior. Accurate detection of hypervariable or hyperconstrained regions may provide useful information on the structure/function relationship of biosequences. This information can help localize functional constraints. In addition, the selection of distinct evolutionary dynamics may assist in the correct use of biosequences as reliable molecular clocks.


Reexamination of the African hominoid trichotomy with additional sequences from the primate beta-globin gene cluster. Additional DNA sequence information from a range of primates, including 13.7 kb from pygmy chimpanzee (Pan paniscus), was added to data sets of beta-globin gene cluster sequence alignments that span the gamma 1, gamma 2, and psi eta loci and their flanking and intergenic regions. This enlarged body of data was used to address the issue of whether the ancestral separations of gorilla, chimpanzee, and human lineages resulted from only one trichotomous branching or from two dichotomous branching events. The degree of divergence, corrected for superimposed substitutions, seen in the beta-globin gene cluster between human alleles is about a third to a half that observed between two species of chimpanzee and about a fourth that between human and chimpanzee. The divergence either between chimpanzee and gorilla or between human and gorilla is slightly greater than that between human and chimpanzee, suggesting that the ancestral separations resulted from two closely spaced dichotomous branchings. Maximum parsimony analysis further strengthened the evidence that humans and chimpanzees share the longest common ancestry. Support for this human-chimpanzee clade is statistically significant at P = 0.002 over a human-gorilla clade or a chimpanzee-gorilla clade. An analysis of expected and observed homoplasy revealed that the number of sequence changes uniquely shared by human and chimpanzee lineages is too large to be attributed to homoplasy. Molecular clock calculations that accommodated lineage variations in rates of molecular evolution yielded hominoid branching times that ranged from 17-19 million years ago (MYA) for the separation of gibbon from the other hominoids to 5-7 MYA for the separation of chimpanzees from humans. Based on the relatively late dates and mounting corroborative evidence from unlinked nuclear genes and mitochondrial DNA for the close sister grouping of humans and chimpanzees, a cladistic classification would place all apes and humans in the same family. Within this family, gibbons would be placed in one subfamily and all other extant hominoids in another subfamily. The later subfamily would be divided into a tribe for orangutans and another tribe for gorillas, chimpanzees, and humans. Finally, gorillas would be placed in one subtribe with chimpanzees and humans in another, although this last division is not as strongly supported as the other divisions.


Phylogenetic relationships in the honeybee (genus Apis) as determined by the sequence of the cytochrome oxidase II region of mitochondrial DNA. The complete nucleotide sequence of the mitochondrial cytochrome oxidase II (COII) gene was determined for five species of the honeybee (Genus: Apis): A. andreniformis, A. cerana, A. dorsata, A. florea, and A. koschevnikovi; these were then compared to the known sequence of the A. millifera gene from Crozier et al. (1989, Mol. Biol. Evol., 6: 399-411) and the wasp Excristes roborator (Liu and Beckenbach, 1992, Mol. Phylogenet. Evol., 1:41-52). Phylogenetic relationships were derived using the parasimony methods DNAPARS and PROTPARS of Felsenstein ("PHYLIP Manual Version 3.4, "University Herbarium, Univ. of California, Berkeley). The results suggest that A. dorsata is the most ancestral species, followed by the branching of A. florea/A. andreniformis and A. koschevnikovi, and then A. mellifera and A. cerana. This inference differs from the currently accepted view that considers the A. florea/A. andreniformis line to be the most ancestral.


Evolution of the salmonid mitochondrial control region. To explore the evolutionary nature of the salmonid mitochondrial DNA (mtDNA) control region (D-loop) and its utility for inferring phylogenies, the entire region was sequenced from all eight species of anadromous Pacific salmon, genus Oncorhynchus; the Atlantic salmon, Salmo salar; and the Arctic grayling, Thymallus arcticus. A comparison of aligned sequences demonstrates that the generally conserved sequence elements that have been previously reported for other vertebrates are maintained in these primitive teleost fishes. Results reveal a significantly nonrandom distribution of nucleotide substitutions, insertions, and deletions that suggests that portions of the salmonid D-loop may be under differential selective constraints and that most of the control region of these fishes may evolve at a rate similar to that of the remainder of their mtDNA genomes. Maximum likelihood and Fitch parsimony analyses of 9 kb of aligned salmonid sequence data give evolutionary trees of identical topology. These results are consistent with previous molecular studies of a limited number of salmonid taxa and with more comprehensive, classical analyses of salmonid evolution. Predictions from these data, based on a molecular clock assumption for the mtDNA control region, are also consistent with fossil evidence that suggests that species of Oncorhynchus could be as old as the Middle Pliocene and would have thus given rise to the extant Pacific salmon prior to about 5 or 6 million years ago.


Crocodilian evolution: insights from immunological data. The quantitative immunological technique of microcomplement fixation was used to examine serum albumin evolution among members of the order Crocodylia. The cross-reactivity of the albumin antisera and antigens employed in this study had been examined previously using the qualitative technique of immunodiffusion. The phylogenetic conclusions derived from these two data sets are highly congruent, including support of the families Alligatoridae and Crocodylidae, with the placement of Gavialis as the sister taxon of Tomistoma. Both methods provide similar information on the relative amounts of amino acid sequence divergence between albumin molecules; however, the data obtained from microcomplement fixation comparisons are more discriminating than those derived from immunodiffusion. The estimated divergence times within the Crocodylia derived from the fossil record are examined in light of divergence times predicted by the microcomplement fixation-based albumin clock. The traditional phylogenetic placement of Gavialis outside the remaining extant crocodilians is inconsistent with all molecular data sets and we suggest that a careful reexamination of both the extant and the fossil morphological data is warranted.


A phylogenetic study of the gibbons (Hylobates) using DNA obtained noninvasively from hair. Variation in a 252-nucleotide segment of the cytochrome b gene from 26 gibbons is described. DNA was extracted from hair, amplified, and directly sequenced. These sequences represent seven of the nine nominal species and three of the four hylobatid subgenera. Variation was observed at 55 sites, 42 of which are phylogenetically informative. Levels of transitional and transversional divergence between the taxa are similar to those reported for homologous mtDNA sequences in other mammals. Parsimony, maximum likelihood, and bootstrap analyses (1) support some traditional phylogenetic hypotheses (monophyly of the concolor gibbons), (2) suggest previously unrecognized affinities between the lar species group and Hylobates klossi and between H. lar and H. agilis unko, and (3) show that this segment does not contain information sufficient for completely resolving gibbon relationships at the subgeneric level. The study demonstrates the great potential of noninvasive DNA sampling for phylogenetic analyses of mammals.


Higher rates of amino acid substitution in rodents than in humans. An analysis of 54 protein sequences from humans and rodents (mice or rats), with the chicken as an outgroup, indicates that, from the common ancestor of primates and rodents, 35 of the proteins have evolved faster in the lineage to mouse or rat (rodent lineage) whereas only 12 proteins have evolved faster in the lineage to humans (human lineage). The average rate of amino acid substitution is significantly faster in the rodent lineage than in the human lineage. In addition, the average rate of insertion/deletion is also faster in rodents than in humans and there is a positive correlation between the rate of amino acid substitution and the rate of insertion/deletion in a protein sequence.


The evolutionary history of the sinica-group of macaque monkeys as revealed by mtDNA restriction site analysis. We estimated the phylogenetic relationships of mitochondrial DNA haplotypes within the sinica-group of macaques, which includes Macaca sinica, M. radiata, M. thibetana, M. assamensis, and possibly M. arctoides. Some effort was made to detect intraspecific variation by sampling individuals from different parts of the species' range or from different matrilines. In the case of M. assamensis, individuals were sampled from both subspecies (M. assamensis assamensis and M. assamensis pelops). Total genomic DNA was extracted from blood samples and cut with a battery of 16 restriction endonucleases. A total of 97 restriction sites were mapped for these enzymes in the sinica-group and M. nemestrina, which was used as an outgroup. Phylogenetic trees constructed by both the maximum parsimony method and the neighbor-joining method were highly congruent. A bootstrap analysis of the maximum parsimony tree indicated a high degree of confidence to the association of particular haplotypes within the 80% majority rule consensus tree. An exhaustive search of all possible trees also supported this topology, although one haplotype had to be eliminated from this analysis to save computer time. The results were also unaffected by weighting the character state changes in favor of site gains over site losses. The mtDNA phylogeny produced here differs from trees based on morphology and allozymes in three ways: M. sinica and M. radiata sit in two different branches of the tree; the two subspecies of M. assamensis are separated from one another; and M. arctoides consistently fell outside the rest of the sinica-group.(ABSTRACT TRUNCATED AT 250 WORDS)


Evolutionary relationships of the salmonid fish genus Salvelinus inferred from DNA sequences of the first internal transcribed spacer (ITS 1) of ribosomal DNA. DNA sequences of the ribosomal DNA (rDNA) first internal transcribed spacer region (ITS 1) of six species of the salmonid fish genus Salvelinus (alpinus, malma, confluentus, leucomaenis, fontinalis, and namaycush) and the closely related species Hucho perryi were determined. Phylogenetic analysis of the aligned sequences by both phenetic and cladistic methods with H. perryi as an outgroup generated one best topology which pairs S. alpinus with S. malma as the most recently derived species, and pairs S. confluentus with S. leucomaenis. Three other possible topologies favor the pairing of S. namaycush and S. fontinalis, with one tree placing them on separate branches, and vary the branching order of the interior groups. These results agree with previous studies based on comparisons of morphologies, isozymes, karyotypes, and restriction sites showing a close genetic relationship and possible hybridization between the members of this genus.


Evolutionary relationships within the fungi: analyses of nuclear small subunit rRNA sequences. Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.


Split decomposition: a new and useful approach to phylogenetic analysis of distance data. In order to analyze the structure inherent to a matrix of dissimilarities (such as evolutionary distances) we propose to use a new technique called split decomposition. This method accurately dissects the given dissimilarity measure as a sum of elementary "split" metrics plus a (small) residue. The split summands identify related groups which are susceptible to further interpretation when casted against the available biological information. Reanalysis of previously published ribosomal RNA data sets using split decomposition illustrate the potential of this approach.


18S rDNA sequences and the holometabolous insects. The Holometabola (insects with complete metamorphosis: beetles, wasps, flies, fleas, butterflies, lacewings, and others) is a monophyletic group that includes the majority of the world's animal species. Holometabolous orders are well defined by morphological characters, but relationships among orders are unclear. In a search for a region of DNA that will clarify the interordinal relationships we sequenced approximately 1080 nucleotides of the 5' end of the 18S ribosomal RNA gene from representatives of 14 families of insects in the orders Hymenoptera (sawflies and wasps), Neuroptera (lacewing and antlion), Siphonaptera (flea), and Mecoptera (scorpionfly). We aligned the sequences with the published sequences of insects from the orders Coleoptera (beetle) and Diptera (mosquito and Drosophila), and the outgroups aphid, shrimp, and spider. Unlike the other insects examined in this study, the neuropterans have A-T rich insertions or expansion regions: one in the antlion was approximately 260 bp long. The dipteran 18S rDNA evolved rapidly, with over 3 times as many substitutions among the aligned sequences, and 2-3 times more unalignable nucleotides than other Holometabola, in violation of an insect-wide molecular clock. When we excluded the long-branched taxa (Diptera, shrimp, and spider) from the analysis, the most parsimonious (minimum-length) trees placed the beetle basal to other holometabolous orders, and supported a morphologically monophyletic clade including the fleas+scorpionflies (96% bootstrap support). However, most interordinal relationships were not significantly supported when tested by maximum likelihood or bootstrapping and were sensitive to the taxa included in the analysis. The most parsimonious and maximum-likelihood trees both separated the Coleoptera and Neuroptera, but this separation was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)


Allozyme evidence for crane systematics and polymorphisms within populations of Sandhill, Sarus, Siberian, and whooping cranes. Electrophoretic analysis of proteins yielded evidence on the relationships of species of cranes and on genetic diversity within populations of some species. Diversity within the Greater Sandhill crane and a Florida population of the Florida Sandhill crane was similar to that of most other vertebrates, but diversity was low in the Mississippi Sandhill crane, in the Okefenokee population of the Florida Sandhill crane, and within the Siberian and Sarus cranes. Diversity was surprisingly high among whooping cranes, whose number dropped to less than 25 early in this century. Phylogenetic analysis, using both character state and distance algorithms, yielded highly concordant trees for the 15 species. The African crowned cranes (Balearica) were widely divergent from all other cranes. Species of Anthropoides, Bugeranus, and Grus clustered closely but sorted into two lineages: a Whooper Group consisted of the whooping, common, hooded, black-necked, white-naped, and red-crowned cranes of genus Grus; and a Sandhill Group included the Sandhill, Siberian, Sarus, and Brolga cranes of genus Grus, the wattled crane of genus Bugeranus, and the Demoiselle and blue cranes of genus Anthropoides.


The phylogeny of xantusiid lizards: the concern for analysis in the search for a best estimate of phylogeny. In the onrush of molecular-based phylogenetic hypotheses, previous morphological-based phylogenies are being ignored, discarded, or even treated with disdain. Coupled with this implicit superiority of molecular data is the sometimes tendency to construct a phylogeny from the molecular data with less than analytical rigor. This paper examines the phylogenetic relationships within the lizard family Xantusiidae employing both molecular and morphological data. The analysis focuses on four analytical points of the molecular data and on the phylogenetics synthesis of the two data sets. We conclude that the phylogeny of xantusiid lizards is not yet a robust hypothesis.


Molecular evidence linking hominid evolution to recent radiation of schistosomes (Platyhelminthes: Trematoda). Molecular phylogenies for seven species of schistosomes, including four species infecting man, were constructed from PCR-amplified sequences of two ribosomal genes: one nuclear (internal transcribed spacer 2 in the ribosomal multigenic family) and one mitochondrial (16S rDNA). The two phylogenies obtained are congruent, and the data suggest that the mitochondrial sequence evolves about three times faster than the nuclear sequence. We propose a calibration of the phylogenetic tree of schistosomes that dates "human capture" of these parasites from other animal hosts (rodents and ruminants) in Africa to 1-10 million years ago, when the first hominids invaded savanna areas, which are the favorable environment for parasite transmission.


A statistical examination of hypervariability in complementarity-determining regions of immunoglobulins. To determine the relative importance of gene conversion followed by natural selection and of natural selection for point mutation in generating variability in immunoglobulins, the numbers of synonymous and nonsynonymous substitutions in immunoglobulin sequences of various subgroups were estimated for complementarity-determining regions (CDRs) and for framework regions (FRs). Both the number of synonymous substitutions and the number of nonsynonymous substitutions in the CDR were found to exceed the corresponding numbers in the FR. Therefore, gene conversion is likely to be an important mechanism for providing variability in the CDR of immunoglobulins. The correlation coefficients between the number of synonymous substitutions and the number of nonsynonymous substitutions and between the substitution number in the CDR and that in the FR were found to be very low. Again, gene conversion is thought to be responsible for this finding.


Phylogenetic studies of ribosomal RNA variation in higher moths and butterflies (Lepidoptera: Ditrysia). The selection of exemplars has been shown both theoretically and empirically to affect tree topology, but the importance of the number and nature of taxa used to represent higher taxonomic lineages in molecular studies is rarely stressed. In our rRNA study of higher moths and butterflies (Lepidoptera: Ditrysia), the selection of different exemplars and outgroups caused major tree rearrangements. We also examined the effectiveness with which conserved rRNA regions track the diversification of Lepidoptera. Homoplasy is as prevalent at the few variable sites of conserved regions (18E, 18J, 28F) as at the many variable sites of a more rapidly evolving region (28B). Finally, 28B sequence variation differs qualitatively among lepidopteran superfamilies of presumed comparable age, the Papilionoidea (true butterflies) and Noctuoidea (cutworm moths and relatives).


Expression of members of the chromogranin family in primary neuroblastomas. Expression of the members of the chromogranin family [i.e., chromogranin A (CgA), chromogranin B, and secretogranin II (SgII)], the acidic proteins of the matrix of the chromaffin granules presently regarded as specific neuroendocrine markers, was investigated at gene and protein levels in a series of 14 cases of primary untreated neuroblastomas. Oligonucleotides and cRNA probes were employed for hybridization analysis of specific mRNAs (both by Northern blots and nonradioactive in situ hybridization); proteins were localized by immunocytochemistry. Expression of different amounts of each type of chromogranin was determined in all tumors. Cases found immunocytochemically negative were all positive by Northern blot and in situ hybridization. A better prognosis was associated with a higher relative expression of SgII; on the contrary, a worse outcome was observed in cases with a higher expression of CgA. These findings are consistent with the hypothesis that SgII is preferentially expressed in neuroblastomas undergoing neuronal differentiation. In cases of neuroblastomas, determination of expression levels of the different chromogranins in the tissues (and in the serum) can provide parameters of high diagnostic and prognostic value.


Molecular approaches for the analysis of chromogranins and secretogranins. Recent molecular analyses have contributed to our knowledge about the chromogranin/secretogranin (Cg/Sg) family and their utility in diagnostic pathology. The genes for five of these proteins have been cloned, and the deduced amino acid sequences have provided insights into the structure and possible functions of the Cgs/Sgs, including their role as prohormones. Northern hybridization and in situ hybridization histochemistry have provided a great deal of information about the tissue distribution of the Cg/Sg gene products. Some neoplasms such as small cell lung carcinomas, which have little stored Cg/Sg protein, have abundant cytoplasmic mRNAs that can be readily detected by hybridization studies. Some other neoplasms such as neuroblastomas have decreased CgA and increased SgII mRNAs during maturation to ganglioneuromas. There is also a differential expression of Cgs/Sgs in some endocrine neoplasms such as parathyroid adenomas, which express abundant CgA mRNA and little CgB mRNA, and in pituitary prolactinomas, which express CgB mRNA but not CgA mRNA. The mRNA for CgA has been found unexpectedly in some neoplasms such as 15% of colonic adenocarcinomas. Thus, molecular approaches in the analysis of Cgs/Sgs should contribute to the diagnosis of endocrine neoplasms and may provide support for a molecular classification of neoplasms in diagnostic pathology.


HPV DNA in intraepithelial neoplasia and carcinoma of the vulva and penis. Surgical specimens of 15 patients with early and 12 patients with advanced squamous cell carcinoma of the vulva and the penis were examined for the presence of human papillomavirus (HPV) type 6, 11, 16, and 18 DNA by Southern blotting (SB) and polymerase chain reaction (PCR) analysis. By SB, HPV type 16 DNA was detected in all early carcinomas and 2 of 12 cases of advanced squamous cell carcinoma (ISCC) of the vulva and penis. PCR revealed HPV DNA in four additional cases of vulvar and penile ISCC negative by SB. Three cases contained HPV16 and one HPV18. Two cases of vulvar and penile Buschke-Löwenstein (BL) tumor with malignancy and one case of vulvar verrucous carcinoma were also examined by both techniques. While BL tumors were associated with DNA of HPV6 or 11, no HPV association was found for verrucous carcinoma. Our results confirm that the detection rate of HPV DNA in early vulvar and penile carcinomas is much higher than in invasive carcinomas. In addition, we have shown that in the lower genital tract, 50% of cases of ISCC are HPV16 correlated. The absence of HPV DNA (types 6, 11, 16, and 18) in the remaining 50% of cases of ISCC thus suggests that vulvar and penile ISCC may have more than one pathogenetic pathway.


Detection of rearrangements of the BCL2 major breakpoint region in follicular lymphomas. Correlation of polymerase chain reaction results with Southern blot analysis. The translocation t(14;18)(q32;q21) occurs in 70% of follicular lymphomas and places the BCL2 proto-oncogene, normally located at 18q21, under the control of the immunoglobulin heavy chain (IgH) gene at 14q32. Its detection by the polymerase chain reaction (PCR), made possible by the close clustering of most of the BCL2 breakpoints in the major breakpoint region (MBR) of the gene, has numerous clinical applications. Since the PCR covers shorter lengths of DNA than Southern blotting, PCR-based tests may be more susceptible to microheterogeneity in breakpoint location. There have been no published studies of the impact of breakpoint microheterogeneity on the detection rate of this translocation by PCR. We studied 30 follicular lymphomas with the t(14;18), in which a BCL2 MBR rearrangement had previously been demonstrated and mapped by conventional Southern blotting, by PCR with the commonly used IgH and BCL2 MBR primers. Twenty-five cases (83%) had a junction fragment demonstrable by PCR. All three cases in which the MBR rearrangement mapped outside of the 4.3-kb HindIII fragment containing the MBR, as determined by Southern blotting, were negative by PCR. In addition, two cases with rearrangements within the HindIII fragment were also negative. All negative results were repeated at least once and were confirmed to be true negatives by actin PCR. Our results suggest that negative PCR results in this setting are attributable to small variations in BCL2 MBR breakpoint location and cannot be interpreted without the corresponding conventional Southern blotting data. With this caveat in mind, PCR analysis for the t(14;18) remains an extremely useful technique, especially in the follow-up and monitoring for minimal residual disease in previously characterized cases of follicular lymphoma.


p53 gene expression in medulloblastoma by quantitative polymerase chain reaction. The frequent cytogenetic abnormality--isochromosome 17q [i(17)q]--observed in medulloblastomas (MB) may result in altered expression of the oncosuppressor gene p53 that is located on 17p. p53 expression was therefore evaluated in five MBs and in one MB cell line derived from one of these tumors. Expression levels of p53 utilizing serially diluted unfractionated RNA from tumors and the cell line were assessed both by dot-blot and by reverse transcription (RT) followed by the polymerase chain reaction (PCR). The quality of RNA, efficiency of reaction, and transcript quantitation were determined by simultaneous transcription and amplification of a similarly sized fragment of the alpha-tubulin gene. All MBs showed low levels of expression of p53 compared to those found in normal tissues. p53 messenger RNA (mRNA) was significantly increased (two- to threefold) in the MB cell line compared to its tumor of origin and to the other MBs. Immunoperoxidase studies performed with monoclonal antibodies to the p53 protein product showed focal nuclear expression in one of five of the original tumors while most cells grown in vitro and in the xenograft were positive.(ABSTRACT TRUNCATED AT 250 WORDS)


High-specificity in-situ hybridization. Methods and application. We describe a technique of in-situ hybridization using oligonucleotide probes employing the expression of immunoglobulin VH genes as a model. Optimal conditions for hybridization with the 35S-labeled oligonucleotide probes were established with monoclonal B-cell lines that express VH genes of known nucleic acid sequence. The range of sensitivity and specificity achieved with this technique is documented. Under conditions of high stringency, this method can detect the expression of highly related VH hypervariable regions.


The polymerase chain reaction. History, methods, and applications. The polymerase chain reaction (PCR) uses in vitro enzymatic synthesis to amplify specific DNA sequences. PCR amplification can produce approximately 100 billion copies of one molecule of DNA in a few hours. PCR has revolutionized research in the biological sciences and medicine, and has influenced criminology and law. Several major scientific discoveries, including purification of DNA polymerase and elucidation of the mechanism of DNA replication, were essential for development of the present PCR technology. An overview of these discoveries and early work on in vitro DNA synthesis are presented. Basic PCR methodology, instrumentation, advanced PCR techniques, and applications are also discussed in this review. Several new amplification systems are mentioned. PCR is an extremely important and simple technology for research and diagnostic analyses of DNA and RNA. PCR technology and other amplification procedures will continue to produce novel applications in basic research and clinical medicine.


Role of the frozen tissue bank in molecular pathology. The new discipline of molecular pathology requires that high-quality, intact genomic DNA, mRNA, and proteins be available from frozen tissue samples. It is now necessary for pathology laboratories to establish consistent guidelines for the preparation and storage of frozen tissue samples in order to have properly preserved tissues available for diagnostic molecular techniques. Maintaining a frozen tissue bank requires a pathologist to oversee this program and to integrate it into the routine surgical pathology activities. A member of the laboratory technical staff can serve as a tissue bank coordinator and have responsibility for preparation of tissue samples, their systematic storage and retrieval, and routine maintenance of equipment and supplies. Tissue sampling must be done as soon as possible after excision of the specimen and is the responsibility of a qualified pathologist. The samples may be snap frozen without cryoprotection at -78 degrees C or colder for subsequent use in procedures requiring the extraction of genomic DNA, mRNA, or protein. To preserve tissue architecture and cytologic features for immunohistochemistry and in situ hybridization, the tissue should be frozen at -78 degrees C or colder with a cryoprotectant such as OCT. Long-term storage of the frozen tissue is recommended at -140 degrees C or colder in a locked liquid nitrogen freezer, and the record of sample inventory can easily be kept in a computerized database. Tissues sampled and stored under these conditions have been used successfully in a wide variety of molecular techniques. In addition to malignant tumor tissue, samples from benign lesions and normal tissues should be frozen.(ABSTRACT TRUNCATED AT 250 WORDS)


Epstein-Barr virus and carcinomas. Expression of the viral genome in an undifferentiated gastric carcinoma. The Epstein-Barr virus (EBV) is associated with undifferentiated nasopharyngeal carcinomas (NPCs). Recently, EBV DNA has been demonstrated also in some cases of gastric carcinoma with similar morphology as undifferentiated NPC. Here we report on the expression of EBV genes in a gastric undifferentiated carcinoma of nasopharyngeal type (UCNT). The small EBV-encoded nuclear RNAs, EBERs, were expressed in all tumor cells. The BZLF1 transactivator protein, which disrupts viral latency, was detectable in a small subset of tumor cells. The latent membrane protein and the nuclear antigen EBNA2 were not expressed. These results indicate that lytic EBV infection may be possible in undifferentiated epithelial cells. Our findings add to the growing body of evidence suggesting a strong association of gastric UCNT with EBV and point to the possibility of an involvement of the virus in the pathogenesis of this neoplasm.


Intratubular germ cell neoplasia in infantile yolk sac tumor. Verification by tandem repeat sequence in situ hybridization. The strong association of intratubular germ cell neoplasia (ITGCN) with adult germ cell testicular tumors is well known, but studies noting the absence of ITGCN in certain germ cell neoplasms such as spermatocytic seminoma, childhood teratoma, and infantile yolk sac tumor (YST) have raised the issue of whether these latter neoplasms follow a different path of tumorigenesis, accounting for their more benign behavior. A case study illustrating the association of ITGCN with infantile YST is presented to challenge this hypothesis. In addition to the usual characteristic features that included strong cytoplasmic glycogen deposits, and focal placental alkaline phosphatase immunoreactivity, the atypical intratubular germ cells manifested triploidy by in situ hybridization using as probe a telomeric tandem repeat sequence, p1-79, specific to chromosome 1. The invasive YST cells, in contrast, showed evidence of tetraploidy by both in situ hybridization and flow and image cytometric studies, excluding the possibility that the atypical intratubular germ cells represented intratubular invasion by adjacent YST. These findings challenge the belief that the infantile YST follows a different path of tumorigenesis than its adult germ cell counterpart and suggest other hypotheses that might better explain its more benign behavior.


A rapid and simple procedure for the routine detection of ras point mutations in formalin-fixed, paraffin-embedded tissues. The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 microns tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3-4 days.


Digoxigenin as an alternative probe labeling for in situ hybridization. In situ hybridization (ISH) techniques have proven to be invaluable tools for diagnostic molecular laboratories in the detection of gene expression and viral infection in cell and tissue samples. Radioactively labeled probes are still widely used for ISH because of their high sensitivity in detection. Among the non-isotopic systems only biotinylated probes for viral DNA detection found broader acceptance. Recently we introduced the digoxigenin probe labeling and detection system for ISH in our diagnostic molecular laboratory. Our experiences with digoxigenin-labeled probes are summarized in this report in the form of technical recommendations for probe choice, labeling, quantification, hybridization and detection. Several ISH protocols using RNA, DNA and oligonucleotide probes for the detection of mRNA and viral DNA are reported. Advantages, disadvantages and pitfalls of the digoxigenin system as well as comparisons with radiolabeled and other non-isotopic systems are discussed. Digoxigenin-labeled probes provide an attractive alternative to radiolabeled probes and are superior to other nonradioactive probes for ISH. Probe labeling, quantification, hybridization and detection can be performed with low biohazard risk. Working efforts and costs applying digoxigenin-labeled probes or radiolabeled probes for ISH are similar. Digoxigenin-labeled probes are stable for several months, provide an equal sensitivity in detection and a higher degree of cellular resolution than radiolabeled probes. The turnaround time of procedures is short and results of diagnostic ISH can be obtained much quicker than using radiolabeled probes.


In situ polymerase chain reaction detection of viral DNA, single-copy genes, and gene rearrangements in cell suspensions and cytospins. The study of low-copy viral or genomic DNA sequences by in situ hybridization (ISH) is often limited by sensitivity. Using the polymerase chain reaction (PCR) for the amplification of target DNA sequences in fixed cells [in situ PCR] (ISPCR) before ISH, we have been able to greatly improve the sensitivity of ISH. Viral DNA present in low copy number, single-copy genes, as well as immunoglobulin gene rearrangements (VH3 family genes), were successfully amplified in cells in suspension or on glass slides (cytospins). Single primer pairs were used in the in situ amplification step and 35S- or digoxigenin-11-dUTP-labeled region specific oligonucleotide probes were used for detection of amplificants by ISH. Artifacts, presumably resulting from leakage of in situ amplificants out of cells, may be a significant problem in selected instances. By optimal fixation and permeabilization of cells, limiting PCR cycle number, amplification of long DNA sequences, and/or incorporation of biotinylated dNTPs to produce bulkier amplificants together with washing of cells after ISPCR, diffusion artifacts were significantly reduced. Probe hybridization to single-stranded long PCR fragments or messenger RNA were excluded as a source for false-positive ISPCR results. The techniques reported dramatically increase the sensitivity of ISH in the detection of low-copy viral infection as well as in the study of gene rearrangements, and provide unique opportunities to study chromosomal translocations and point mutations at the cellular level.


Secretogranin II expression in Ewing's sarcomas and primitive neuroectodermal tumors. Production of chromogranins, the acidic components of the chromaffin granules regarded as specific neuroendocrine markers, was analyzed by immunocytochemistry and hybridization (Northern blotting and in situ hybridization) in primary lesions and cell lines of Ewing's sarcomas, primitive neuroectodermal tumors (PNETs), and neuroblastomas. Antibodies and probes specific for chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) were used. Ewing's sarcomas and PNETs, unlike neuroblastomas, were negative for CgA and CgB. Two primary Ewing's sarcomas, one primary PNET (an Askin tumor), and one PNET cell line (TC32) were found to strongly express the SgII gene, as shown by the presence of specific mRNA. This result supports the hypothesis that some Ewing's sarcomas represent a most primitive form of neuroectodermal tumor; in addition, it indicates a diagnostic role of SgII in cases of Ewing's sarcomas and PNETs.


A simplified method of detection of clonal rearrangements of the T-cell receptor-gamma chain gene. A series of T-cell proliferations in peripheral blood, bone marrow, or tissue samples, together with seven T-cell lines, were analysed for clonality. The technique used employs the polymerase chain reaction (PCR) to amplify rearranged T-cell receptor gamma genes, using primers recognising conserved sequences in the variable and joining gene segments. Of the 20 cases of T-cell leukaemia or lymphoma analysed, a clone was detected in 14 (70%): Of seven T-cell lines, a clone was detected in 6 (84%). No positive results were recorded in eight non-T-cell disorders (including nonlymphoid malignancies and reactive disorders). When the results of this technique were combined with the results of our previously published method for the detection of clonally rearranged T-cell receptor-beta (TCR-beta) genes using PCR, 9 of 10 (90%) T-cell tumours were detected. This method uses only four primer combinations in two tubes, and is therefore simple and rapid: it requires no radiolabelling, uses only a small amount of tissue, and can be performed on formalin-fixed, paraffin-embedded tissue.


DNA content measurement and in situ hybridization in condylomatous cervical lesions. The present study reports results obtained with DNA ploidy measurement by Feulgen cytophotometry and in situ hybridization (ISH) on the same sections of 36 cervical lesions containing human papillomavirus (HPV) DNA. Fifteen of 16 low grade lesions [11 flat condylomas and five condylomatous with intraepithelial neoplasias (CIN)-1, 1 with condylomatous features] were diploid. "Oncogenic" HPV (types 16 and 33) were detected in three of these cases, while the aneuploid case expressed HPV type 11. By contrast, in the 12 CIN-2 and eight CIN-3 with condylomas, there was a good correlation between presence of an aneuploid DNA pattern and detection of "oncogenic" HPVs. Demonstration of an aneuploid profile and "oncogenic" HPV as found in all our CIN-3, may indicative of a poor prognosis in low grade lesions. Hence, the combination of the two techniques (ISH and Feulgen cytophotometry) may provide significant prognostic information for condylomas and CINs.


Rapid detection and species identification of mycobacteria in paraffin-embedded tissues by polymerase chain reaction. The sensitivity and specificity of the polymerase chain reaction (PCR) in the detection of mycobacteria in paraffin-embedded tissues and in crude lysates of mycobacterial cultures were assessed. Sections of formalin-fixed, paraffin-embedded tissues were deparaffinized and then subjected to a simple proteinase K and boiling lysis procedure. These preparations were used directly for PCR amplification of the 383 bp segment of the gene encoding the 65 kDa mycobacterial surface antigen. Crude lysates of mycobacteria were used as positive controls. The specificity of the PCR products was confirmed by Southern blot using a region-specific digoxigenin-labeled oligonucleotide probe and chemiluminescent detection. The 383 bp diagnostic fragment was visualized in 11 of 12 acid-fast bacilli (AFB) stain/culture-proven-positive blocks. Crude lysates of mycobacteria were detected to a sensitivity of approximately 80 organisms. Amplified fragments from paraffin-embedded tissues and mycobacterial cultures of M. tuberculosis, M. avium-intracellulare, and saprophytic mycobacteria were distinguished by digestion with Nar 1 restriction endonuclease. These results suggest that PCR amplification followed by restriction enzyme digestion of the PCR product is a rapid, specific, and highly sensitive technique for the detection and speciation of mycobacteria in paraffin-embedded tissues.


An approach to definition of genetic alterations in prostate cancer. Growth patterns in prostatic cancer can reduce detectability of genetic alterations. Tumors show histologic grade heterogeneity, multifocality, interdigitation of benign and malignant glands, and varying amounts of stroma. These characteristics introduce sampling errors when one uses traditional methods for genetic analysis that depend on disaggregated cells [metaphase or interphase chromosome studies] or on tissue extracts [Southern blotting or polymerase chain reaction (PCR)] to detect molecular events. To circumvent these problems, we used two approaches to study paraffin-embedded tumors, which permit focused analysis of critical tissue components. Serial 4- to 5-microns sections are applied to slides in groups of three. Every second slide is hematoxylin and eosin stained to visualize areas of carcinoma, dysplasia, hyperplasia, and stroma; tumor-rich areas are circled with ink and used as templates to examine or excise the same areas from adjacent nonstained sections. PCR methods for quantitative and qualitative gene assay are effective in evaluating samples when alteration at a particular locus is suspected. Fluorescence in situ hybridization with chromosome-specific paracentromeric probes for detection of copy number of the relevant chromosome is applied to the adjacent section. Normal chromosome controls for both methods were demonstrated. This protocol enables us to correlate genetic alterations precisely with tumor extent and morphology.


Expression of stromelysin 3 in the stromal elements of human basal cell carcinoma. In basal cell carcinoma, release of proteolytic activity is implicated in extracellular matrix degradation and tumor infiltration. The stromelysin metalloproteinase family is a major candidate for the matrix proteolytic activity in infiltrative tumors. However, in murine models of basal cell carcinoma, neither stromelysin 1 nor 2 appears to play a role in tumor infiltration. We have analyzed the expression of the newly described stromelysin 3 in human basal cell carcinoma using Northern blot analysis and in situ hybridization. In 12 of 14 cases, levels of stromelysin 3 expression were more than tenfold above those observed in normal skin. In one of five cases of squamous cell carcinoma, stromelysin 3 expression was tenfold above levels seen in normal skin. Stromelysin 3 expression was either undetectable or extremely weak in all five cases of infiltrative malignant melanoma. In basal cell carcinoma, stromelysin 3 transcripts were localized by in situ hybridization to the stromal tissue immediately adjacent to basal cell carcinoma, the tumor cells themselves being negative. Therefore, expression of stromelysin 3 in stromal cells may be expected to play a significant role in destruction of the basal membrane zone and extracellular matrix in basal cell carcinoma invasion.


Molecular pathology of paraffin-embedded tissue. Current clinical applications. Molecular biology techniques have been adapted to analyze paraffin-embedded tissues, expanding their potential clinical utility. The isolation of intact nucleic acids from tissue blocks is fundamental to the molecular pathology of paraffin-embedded tissues. In vitro amplification with the polymerase chain reaction (PCR) promises to be the most useful means of retrospective analysis since it can be performed successfully on DNA that has partially degraded during fixation, paraffin embedding, and the extraction process. Four clinical situations in which DNA analysis of paraffin-embedded tissues can be helpful are: (a) gene rearrangement analysis in lymphoproliferative disorders, where fresh tissue has not been obtained at the time of surgery; (b) identification of infectious agents, particularly viruses; (c) genetic testing of families with a putative inherited disease where the affected member has died; and (d) specimen identification. The PCR and other techniques of genetic analysis are powerful in sensitivity and specificity when performed and interpreted with appropriate precautions and controls. DNA analysis of paraffin-embedded tissues will likely become a fixed part of the future pathologist's diagnostic armamentarium.


Analysis of BCL2 and MYC expression in non-Hodgkin's lymphomas by in situ hybridization: correlation with chromosome translocations. We have used an in situ hybridization method for analysis of expression of BCL2 and MYC on cytospun preparations of normal and malignant lymphoid cell lines and tissue sections of normal and malignant lymph nodes. The probes comprised 50-mer antisense oligonucleotides starting at the ATG codons of exon 3 of BCL2 and exon 2 of MYC. We studied the expression of these two genes in frozen tissue sections of biopsy specimens derived from normal and hyperplastic lymph nodes, B-cell lymphomas carrying the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, and T-cell lymphomas with clonal chromosome abnormalities. While all proliferating cells expressed both genes, BCL2 expression was increased two- to threefold in follicular lymphomas with t(14;18) and MYC expression was increased two- to four-fold in high-grade lymphomas with t(8;14). These results are consistent with previous data on deregulated expression of these genes obtained from study of lymphoma cell lines carrying the relevant translocations.


Rapid detection of MYCN gene amplification in neuroblastomas using the polymerase chain reaction. We have used the polymerase chain reaction (PCR) to detect amplification of the MYCN oncogene in neuroblastoma cell lines and to distinguish primary tumors with a single copy from those with MYCN amplification using DNA extracted from frozen sections. Two primer pairs were used to co-amplify a 428-bp fragment of the MYCN oncogene along with a 268-bp fragment of the beta-globin gene (a single-copy reference standard). After 30 cycles of PCR, the products were resolved by agarose gel electrophoresis. MYCN gene amplification was identified by visual comparison of the relative intensities of MYCN and beta-globin PCR product bands on the ethidium bromide-stained gel. This semiquantitative approach, while inadequate for precise determination of copy number, provided a simple, rapid, nonisotopic method for differentiating tumors with MYCN amplification from those with a single copy. Seventy-four primary tumors were classified as amplified or nonamplified by semiquantitative PCR. Twenty-two of 23 tumors known to carry MYCN gene amplification by Southern analysis were correctly identified by PCR. The single false-negative result was due to a sampling error: DNA was extracted from a block of tissue containing small foci of tumor surrounded by normal tissue. Fifty-one of 51 tumors with a single copy of MYCN were also correctly identified by PCR. We conclude that semiquantitative PCR is a reliable, non-isotopic alternative to Southern blotting for detection of MYCN gene amplification that can be performed rapidly on DNA extracted from frozen sections.


Amplification of the c-myc proto-oncogene in human chondrosarcoma. The genomic organization of four oncogenes, i.e., c-myc, c-myb, c-Ha-ras, and c-fms, was investigated in fresh surgical specimens from 10 patients with cartilaginous tumors. Among nine chondrosarcomas, six were primary lesions and three local recurrences. The remaining case was a chondroblastoma. Amplification of the c-myc proto-oncogene was the sole abnormality detected in this series, occurring in two chondrosarcomas (four- and eight-fold). No other genetic alteration such as oncogene rearrangement was found. Nor was there any amplification of the other oncogenes studied. Both c-myc-amplified tumors were primary lesions and histologically classified as grade II; according to flow DNA cytometry, one was diploid and the other aneuploid. In our limited series, there was no overall relationship between c-myc amplification, on the one hand, and histologic subtype, malignancy grade, surgical stage, or ploidy level, on the other. Our study shows that amplification of the c-myc oncogene, presumed to be involved in the development of malignancy, is encountered in occasional human chondrosarcomas, however, without any relationship to other well-known features of this tumor entity. The clinical significance of this gene amplification remains to be established.


A new nonisotopic detection of human papillomavirus DNA using polymerase chain reaction. A novel technique using a two-step polymerase chain reaction (PCR) with specific primers detecting human papillomavirus (HPV) DNA of types 6/11, 16, and 18 and a final nonisotopic colorimetric detection has been developed. Sixty formalin-fixed and paraffin-embedded sections were treated with this methodology and the results compared with those obtained with in situ hybridization (ISH). Twenty cases displaying HPV DNA with ISH were positive with PCR. Seven (35%) of 20 cases negative for ISH but evocative of HPV infection with classic histology displayed HPV DNA with the two-step PCR. Only one case (5%) of 20 normal tissues and/or inflammatory lesions not evocative of HPV infection and negative upon ISH showed HPV DNA. This original technique allows rapid, highly sensitive, and specific detection of HPV DNA and is suitable for most laboratories.


Description of an in situ hybridization methodology for detection of Epstein-Barr virus RNA in paraffin-embedded tissues, with a survey of normal and neoplastic tissues. The authors describe a highly sensitive and practical in situ hybridization method using an oligonucleotide probe for EBER1 RNA for the detection of Epstein-Barr virus (EBV) in formalin-fixed, paraffin-embedded tissue sections. Paraffin-embedded tissues from 793 cases of normal and neoplastic tissues were studied. Nuclear staining for EBV RNA was uniformly present in all or virtually all neoplastic cells in a variety of known EBV-positive tumors. We also demonstrate rare EBV-infected cells in normal lymphoid tissues. RNAase predigestion, competitive inhibition, and control probe studies confirmed the specificity of the staining. In addition, cross-reactivity of EBV RNA staining with other viruses was not present. Additionally, the distribution of EBV in a wide variety of other normal and neoplastic tissues is reported.


In situ analysis of Paget's disease of bone for measles-specific PCR-amplified cDNA. Paget's disease of bone is a disease of unknown etiology. The demonstration of viral-like particles on ultrastructural examination and the putative detection of viral antibodies and nucleic acids in the tissues suggest a possible viral association. The purpose of this study was to search for nucleic acid sequences homologous to measles virus using the recently described reverse transcriptase (RT) polymerase chain reaction (PCR) in situ hybridization (ISH) technique. After performing RT PCR ISH utilizing primers specific for the nucleocapsid region of the measles virus, an intense signal was evident in most measles-infected HeLa cells compared with a weak signal in few of these cells using standard cDNA-RNA ISH analysis. Amplified measles nucleic acid was detected in tissue from a patient who died of measles infection and was not detected in any of the 11 cases of Paget's disease of bone studied or in a giant cell tumor of bone that had tubuloreticular inclusions on electron microscopy. Therefore, these data suggest that infection by the measles virus is not associated with Paget's disease of bone.


[The status of and causes of change in the immunological reactivity of patients with iron-deficiency anemia]. The role of the immunity system in the pathogenesis of iron deficiency anemia and its complications was studied in 200 patients and in the experiment on 55 non-inbred white rats. The state of nonspecific resistance, humoral and cellular components of immunity was investigated. It has been shown that iron deficiency leads to imbalance in the immunity system. The degree of the affection and disorders in the integration between separate parameters of the immunity system determine the possibility of complications and influence the course of concomitant diseases. Ferrotherapy produces a positive effect on the immune status of patients with iron deficiency anemia.


[Cells inhibiting the formation of stromal CFU-F colonies in bone marrow cultures]. The presence of embryonal calf serum (ECS) in the cultural medium in necessary for the inhibition of CFUf colony growth in rabbit bone marrow cultures by hemopoietic and lymphoid cells. The inhibitory effect is sharply decreased when, besides ECS, the cultural medium contains sera autologous for the reacting cells (rabbit serum), and sera of mature animals of some other species (mouse, rat). The inhibitory activity depends on soluble factors penetrating millipore filters. It has been revealed that the cells inhibiting CFUf growth have the marker F480. T-cells, probably, are not involved in the effect studied.


[Therapeutic plasmapheresis in suppurative-septic surgery]. The results of the clinico-laboratory analysis conducted in 69 patients with varying forms of acute purulent surgical infections have shown that the therapeutic plasmapheresis is attended by the improvement of the disease clinical picture, and attenuation of endotoxicosis by day 3 after the perfusion. Indications and contraindications for plasmapheresis in critical patients are considered, the causes of unsuccessful plasmapheresis procedures are analyzed, the ways for the improvement of this operation are outlined.


[The physicochemical and biological properties of the bovine hemoglobin polymer]. A study was made of bovine hemoglobin polymer obtained by a new method: intraerythrocytic polymerization of hemoglobin followed by isolation of hemoglobin polymer. The polymer obtained had weighted mean and numerical average molecular mass of 388 kD and 134 kD, respectively, P 32 mm Hg, arteriovenous difference 5.1 vol%, Hill's coefficient 2.14, oxygen output rate constant 55 sec, and it retained its activity during long-term storage. Its solution in a concentration of 9-10 g/dl has a viscosity of 2.34, and an oncotic pressure of 240 mm water. Bovine hemoglobin polymer is nontoxic and capable of long-term circulation in the blood. Its safety and clinical effectiveness should be studied in an extended biological experiment.


[The action of karbogal on the complement system under model conditions and in an animal experiment]. The influence of dimethylperfluorocyclohexane (carbogal) on the hemolytic activity of human blood serum complement was studied in vitro. It has been show that different effects of carbogol on the complement depend on the serum lipid composition. Endotracheal administration of carbogal to rats results in a significant reduction of complement activity in the animal's blood. Significant changes in the hemolytic activity of the complement recorded 21 days after the endotracheal administration of the compound are, probably, associated with the complement depletion after its activation by carbogal particles or with disorders in the complement component synthesis by macrophages.


[The prostaglandin I2 and thromboxane A2 content of the blood of dogs in the dynamic development of hemorrhagic shock]. The content of prostaglandin I2 (PGI2) and thromboxane (Tx) A2 in the venous blood was studied in 5 dogs in the time course of hemorrhagic shock simulated by a single jet blood-letting from the femoral artery. PGI2 and TxA2 concentrations were estimated by the levels of their stable metabolites in RIA. It was found that acute hemorrhage stimulated the synthesis of prostanoids studied up to the animals' death. However, with the shock progressing, a shift took place in the ratio PGI2/TxA2 from 1:3.8 to 1:5.2, due to a higher rise of TxA2. Possible causes of PGI2 synthesis decrease and TxA2 production increase in hemorrhagic shock were considered. Basing on the data obtained a conclusion has been made on the necessity of exogenous PGI2 (prostacyclin) inclusion into the combined therapy of hemorrhagic shock.


[The hematological effects of the Chernobyl accident in children]. A total of 878 children from the regions with unfavourable radiation situation were investigated in varying periods after the catastrophe. The data obtained were compared with the results of the study of 317 children from "pure" regions. Morphological, metabolic and ultrastructural changes were detected in blood cells of the children from radioactively contaminated regions after the Chernobyl catastrophe. A tendency to normalization of these parameters was noted in the time course of the post-catastrophe period. No clear relationship was established between the dose and effect. However, significant correlations between the severity of disorders in blood cells and the intensity of free-radical processes in the body were recorded.


[Autolymphocytotoxins in the differential immunodiagnosis of chronic lympholeukemia and malignant non-Hodgkin's lymphoma at the leukemization stage]. The authors have investigated the possibility of using sera with cold auto-lymphocytotoxic antibodies for the differential diagnosis of chronic lymphocytic leukemia (CLL) and malignant non-Hodgkin's disease (MNHD) at the stage of leukemization. The new rapid immunologic method was approved in the diagnosis of 40 CLL and 52 MNHD patients. The high informative value of this method has permitted the authors to recommend its use as an additional procedure for the differential diagnosis of the above lympho-proliferative diseases.


[Biological role of metabolic pathways from L-arginine to nitric oxide]. This paper reviews recent developments in the biochemistry, pharmacology and physiology of the L-arginine/nitric oxide pathway. Nitric oxide accounts for the biological activity of endothelium-derived relaxing factor (EDRF) and its continuous release plays a crucial role in the regulation of vascular tone and platelet activity. In the nervous system nitric oxide is a neurotransmitter. In the peripheral nervous system, nitroxergic nerves form a part of the non-adrenergic, non-cholinergic innervation of the visceral organs. In the immune system, nitric oxide generated by activated macrophages has tumoricidal and antimicrobial activities. Growing evidence suggests that the alterations in the formation of NO in various tissues contribute to the pathogenesis of various diseases, including hypertension, atherosclerosis, diabetes, subarachnoid hemorrhage and septic shock. Therefore, the improvements in our understanding of the regulation of L-arginine/nitric oxide pathway on the molecular level may lead to the development of new drugs.


[Health care of the rural population in the Cracow region during 1985-1989]. The paper presents the structure and effects of the rural public health service in the Cracow region during the period of 1985-1989 with a short description of its demographic situation. The numbers and indexes concerning medical workers (especially general practitioners, stomatologists, pediatricians and gynecologists) as well as their services demonstrate a deterioration in the efficiency of the regional rural health service. This deterioration depends on the negative changes in the general condition of the country and Cracow region, of the population and public health service. The regional data are compared with the data of the whole of Poland and its other rural regions. The authors indicate principal directions in the reform of the public health service.


[Autotransplantation in lymphomas: is it effective?]. Recent data suggest that intensive chemotherapy, with or without radiation, followed by a bone marrow autotransplant is effective in advanced intermediate and high-grade non-Hodgkin lymphoma (predominantly large-cell lymphoma). We analyzed 12 studies of the autotransplants to determine outcomes. The results were compared to data from 29 chemotherapy studies. Complete remissions were reported in 53 percent of autotransplant recipients versus 17 percent of persons receiving chemotherapy. Two-year disease-free survival was 16 percent and 3 percent, respectively. It is uncertain whether these differences reflect subject selection or time to treatment (time censoring) biases in the autotransplant cohort. A randomized trial is needed to determine if autotransplants are superior to chemotherapy. Also unknown is whether similar results might not be obtained with higher doses of chemotherapy without an autotransplant.


[Effect of age and inductors on UDP-glucuronyltransferase activity]. This experiments were carried out on: 0.5-, 1-, 2-, 4-, 8-, 12-, 20- and 28-months old Wistar males rats. Before they were killed the animals received by intraperitoneal injection: phenobarbital (50 mg/kg in 0.9% saline 72- and 48-hr before the death), beta-naphthoflavone (20 mg/kg in sesame oil for 3 consecutive days prior to sacrifice), dexamethasone (10 mg/kg; see beta-naphthoflavone). All animals were decapitated after 24 hr of starvation. The livers were rinsed with cold 0.9% saline. Hepatic microsomes were prepared as described Dallner. The UDP-glucuronyltransferase activities with p-nitrophenol as aglycone were determined according to Burchell and Weatherill and protein concentration was measured as described Lowry et al. The activity of examined enzyme had maximal value in youngest rats, but in older one it was decreased. Minimum was observed in 8-month old animals and all older. Pretreatment of rats with beta-naphthoflavone significantly increased glucuronidation especially in sexually mature animals. The date obtained with phenobarbital and dexamethasone pretreatment rats suggested lower magnitude of stimulation of UDP-glucuronyltransferase than beta-naphthoflavone and lack of effect respectively.


[Effect of age and phenobarbital on liver activity of mixed function oxidase]. Effect of age and phenobarbital on the rat mixed function oxidase activity was studied. Male Wistar rats 0.5-, 1-, 2-, 4-, 8-, 12-, 20- and 28 month-old were used. In this study the levels of cytochrome P-450 and cytochrome b5. NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases activity were examined. Both cytochrome P-450 and NADPH-cytochrome P-450 reductase activity was induced by phenobarbital in all animals. Maximum was observed in 4- and 8-month-old for hemoprotein and 2-month-old rats for reductase activity but minimum in youngest and oldest one respectively. On the contrary, cytochrome b5 and NADH-cytochrome b5 reductase activity was inhibited after phenobarbital injection. The highest depression of cytochrome b5 content was found in youngest, but the enzyme activity in oldest rats.


[Prospective studies of the respiratory system in coke plant workers]. In standardized conditions of 24 hours clinical hospitalisation the group of men employed as cooking battery staff was examined twice in a 16 years interval. The examined men worked all the time at the same workshops and were exposed to harmful chemical and physical agents. All the subjects underwent routine medical examination, spirometric tests and the questionnaire of Medical Research Council extended to a smoking habit was gathered for each of men. After 16 years occupational exposition an incidence of chronic bronchitis increased from 6.1 to 24.6%. Also the significant decline of ventilatory parameters was noted. About 80% of examined workers showed greater than expected related to natural process of growing old, decline of ventilatory functions. Non significant differences between smokers and non-smokers were noted.