G-6-PD Jalisco and G-6-PD Morelia: Two new Mexican variants

Summary Two new G-6-PD variants designated G-6-PD Jalisco and G-6-PD Morelia were identified in two unrelated Mexican families. An additional G-6-PD variant was found in each family: G-6-PD trinacria and G-6-PD A^-. In both families compound heterozygotes were identified. G-6-PD Jalisco and G-6-PD Morelia belong to Classes 3 and 4, respectively. G-6-PD Morelia is the first variant from its class with a high Km for NADP and a low Ki for NADPH.     

Haemophilus paragallinarum secretes metalloproteases

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH(4))(2)SO(4)) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4-9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 degrees C, but its activity diminished at 70 degrees C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.     

Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein

Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 kDa during its growth in brain heart infusion, tryptic soy broth, or Luria-Bertani glucose phosphate media, all lacking serum. Some of these proteins were recognized by sera from chickens experimentally infected with H. paragallinarum. A 110-kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae, and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza.     

Actinobacillus pleuropneumoniae metalloprotease: cloning and in vivo expression

The complete amino acid and nucleotide sequence of a secreted metalloprotease produced by Actinobacillus pleuropneumoniae serotype 1 is reported. A clone showing proteolytic activity in cell-free culture media was selected from a genomic library of A. pleuropneumoniae serotype 1 in pUC 19. The sequence obtained contained an open reading frame encoding a protein with 869 amino acids. This protein was identified as a zinc neutral-metalloprotease belonging to the aminopeptidase family, with a predicted molecular weight of approximately 101 kDa. This sequence showed high homology with other predicted or sequenced aminopeptidases reported for different Gram-negative bacteria. Expression of the protease was observed in lung tissue from pigs that died of porcine pleuropneumonia suggesting a role in pathogenesis.     

Electrophoretic pattern of sorbitol dehydrogenase (E.C.1.1.1.14) in human seminal plasma and spermatozoa

An electrophoretic study of sorbitol dehydrogenase (SORD) in sperm and seminal plasma (SP) was realized. In SP, three phenotypes (one slow band, one fast, and both bands) were observed, corroborating the electrophoretic variability of SP-SORD formerly reported by us, while, in sperm, SORD showed a phenotype of one band faster than the one of SP. Biochemical studies showed that thiol groups participate in the mobility of the SP fast band; furthermore, an interchange of the bands of SP-SORD was observed which suggests that the isozymes are due to conformational isomerism or to molecular aggregates.     

Recognition of HLA class I molecules by antisera directed to synthetic peptides corresponding to different regions of the HLA-B7 heavy chain

Antisera have been prepared in rabbits and in mice against different peptides corresponding to four hydrophilic and variable regions of HLA-B7 heavy chain (65-82, 99-118, 138-157, and 164-187). Specific antipeptide sera have been obtained with all synthetic peptides; for three of them which were more than 20 amino acids long, highly potent sera were elicited by injection of the free peptide. Three overlapping peptides included in region 138-157 have been used, and two different antigenic sites were detected in this region. HLA molecules solubilized in nonionic detergent were precipitated by antipeptide sera directed against regions 65-82, 138-157, and 164-187, but not by antipeptide serum directed against the less hydrophilic region 99-118. Analysis by two-dimensional electrophoresis of the isolated molecules confirmed the anti-HLA specificity of the antipeptide 65-82 and 138-157 sera. Variable numbers of HLA-related spots were found according to the antisera used. Antipeptide 138-157 serum precipitated numerous HLA molecules and therefore probably reacted with monomorphic determinants whereas antipeptide 65-82 appeared specific for a more limited number of HLA antigens. Such reagents directed against well-defined regions of the HLA class I heavy chain are of considerable interest, notably for the mapping of antigenic epitopes on the molecule and for the study of relationships between structure and function.     

Genetic heterogeneity of thalassemias in Mexican mestizo patients with hemolytic anemia

Twenty-eight Hb abnormalities (16 thalassemias, 10 Hb variants and 2 hereditary persistences of fetal hemoglobin) were detected in an etiopathogenic search in 131 patients with hemolytic anemia. The observed thalassemic genotypes [6 beta(0)/beta, 2 (delta beta) (0)/beta(0), 2 beta(0)/beta(+), 2 beta(0)/beta(s), 2 beta(+)/beta(+), 1 beta(+)/beta(s) and 1 (delta beta)(0)/beta] showed heterogeneity in the population studied. A native origin of some of these mutations is considered.     

Translocation and dissemination to target neurons of botulinum neurotoxin type B in the mouse intestinal wall

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis (botulism), which in most cases enter the organism via the digestive tract and then disseminate into the blood or lymph circulation to target autonomic and motor nerve endings. The passage way of BoNTs alone or in complex forms with associated nontoxic proteins through the epithelial barrier of the digestive tract still remains unclear. Here, we show using an in vivo model of mouse ligated intestinal loop that BoNT/B alone or the BoNT/B C‐terminal domain of the heavy chain (HCcB), which interacts with cell surface receptors, translocates across the intestinal barrier. The BoNT/B or HCcB translocation through the intestinal barrier occurred via an endocytosis‐dependent mechanism within 10–20 min, because Dynasore, a potent endocytosis inhibitor, significantly prevented BoNT/B as well as HCcB translocation. We also show that HCcB or BoNT/B specifically targets neuronal cells and neuronal extensions in the intestinal submucosa and musculosa expressing synaptotagmin, preferentially cholinergic neurons and to a lower extent other neuronal cell types, notably serotonergic neurons. Interestingly, rare intestinal epithelial cells accumulated HCcB suggesting that distinct cell types of the intestinal epithelium, still undefined, might mediate efficient translocation of BoNT/B.     

Use and trade of bitumen in antiquity and prehistory: molecular archaeology reveals secrets of past civilizations

Natural asphalt (or bitumen) deposits, oil seepage and liquid oil shows are widespread in the Middle East, especially in the Zagros mountains of Iran. Ancient people from northern Iraq, south-west Iran and the Dead Sea area extensively used this ubiquitous natural resource until the Neolithic period (7000 to 6000 BC). Evidence of earlier use has been recently documented in the Syrian desert near El Kown, where bitumen-coated flint implements, dated to 40,000 BC (Mousterian period), have been unearthed. This discovery at least proves that bitumen was used by Neanderthal populations as hafting material to fix handles to their flint tools. Numerous testimonies, proving the importance of this petroleum-based material in Ancient civilizations, were brought to light by the excavations conducted in the Near East as of the beginning of the century. Bitumen remains show a wide range of uses that can be classified under several headings. First of all, bitumen was largely used in Mesopotamia and Elam as mortar in the construction of palaces (e.g. the Darius Palace in Susa), temples, ziggurats (e.g. the so-called ''Tower of Babel'' in Babylon), terraces (e.g. the famous ''Hanging Gardens of Babylon'') and exceptionally for roadway coating (e.g. the processional way of Babylon). Since the Neolithic, bitumen served to waterproof containers (baskets, earthenware jars, storage pits), wooden posts, palace grounds (e.g. in Mari and Haradum), reserves of lustral waters, bathrooms, palm roofs, etc. Mats, sarcophagi, coffins and jars, used for funeral practices, were often covered and sealed with bitumen. Reed and wood boats were also caulked with bitumen. Abundant lumps of bituminous mixtures used for that particular purpose have been found in storage rooms of houses at Ra''s al-Junayz in Oman. Bitumen was also a widespread adhesive in antiquity and served to repair broken ceramics, fix eyes and horns on statues (e.g. at Tell al-Ubaid around 2500 BC). Beautiful decorations with stones, shells, mother of pearl, on palm trees, cups, ostrich eggs, musical instruments (e.g. the Queen''s lyre) and other items, such as rings, jewellery and games, have been excavated from the Royal tombs in Ur. They are on view in the British Museum. With a special enigmatic material, commonly referred to as ''bitumen mastic'', the inhabitants of Susa sculpted masterpieces of art which are today exhibited in the Louvre Museum in Paris. This unique collection is presented in a book by Connan and Deschesne (1996). Last, bitumen was also considered as a powerful remedy in medical practice, especially as a disinfectant and insecticide, and was used by the ancient Egyptians to prepare mixtures to embalm the corpses of their dead. Modern analytical techniques, currently applied in the field of petroleum geochemistry, have been adapted to the study of numerous archaeological bituminous mixtures found in excavations. More than 700 bituminous samples have been analysed during the last decade, using gas chromatography alone and gas chromatography coupled with mass spectrometry and isotopic chemistry (carbon and hydrogen mainly). These powerful tools, focused on the detailed analysis of biomarkers in hydrocarbon fractions, were calibrated on various well-known natural sources of bitumen in Iraq, Syria, Iran, Bahrain and Kuwait. These reference studies have made it possible to establish the origins of bitumen from numerous archaeological sites and to document the bitumen trade routes in the Middle East and the Arabo-Persian Gulf. Using a well-documented case history, Tell el ''Oueili (5800 to 3500 BC) in South Mesopotamia, we will illustrate in this paper how these new molecular and isotopic tools can help us to recognize different sources of bitumen and to trace the ancient trade routes through time. These import routes were found to vary with major cultural and political changes in the area under study. A second example, referring to the prehistoric period, describes bitumen traces on flint implements, dated from Mousterian times. This discovery, from the Umm El Tlel excavations near El Kown in Syria, was reported in 1996 in Boëda et al. At that time, the origin of the bitumen had not been elucidated due to contamination problems. Last year, a ball of natural oil-stained sands, unearthed from the same archaeological layer, allowed us to determine the source of the bitumen used. This source is regional and located in the Jebel Bichri, nearly 40 km from the archaeological site. The last case history was selected to illustrate another aspect of the investigations carried out. Recent geochemical studies on more than 20 balms from Egyptian mummies from the Intermediate, Ptolemaic and Roman periods have revealed that these balms are composed of various mixtures of bitumen, conifer resins, grease and beeswax. Bitumen occurs with the other ingredients and the balms studied show a great variety of molecular compositions. Bitumen from the Dead Sea area is the most common source but some other sources (Hit in Iraq?) are also revealed by different molecular patterns. The absolute amount of bitumen in balms varies from almost zero to 30% per weight.