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PubMed1990

Exhalation of malondialdehyde from the smoke of high grade cigarettes and effectiveness of filtering it.

It has been found that malondialdehyde penetrates during smoking through filters and exhales with smoke from cigarettes such as: "Pall Mall", "Winston", "Camel", "Lucky Strike", "Kansas", "Mustang", "Ronson", "Chesterfield", "Rally", "Oscar" and "Marlboro". These cigarettes exhale malondialdehyde in a different way, in free form in the amount from 8.5 to 23.5 mg/kg cigarettes on an average of 13.3 mg/kg for the whole group and totally (in free and bounded form) from 9.5 to 26.5 mg/kg (average 16.9 mg/kg). Moreover, it has been found that filters retain this aldehyde in free form on an average of 9.9 mg/kg and totally (in free and bounded form) of 19.0 mg/kg. Without filters these amounts would increase aldehyde contents in smoke by 42.7 and 52.9%, respectively. Low malonalogenic properties of tobacco, high effectiveness of cigarette filters, high degree of tar deposition before inhaling into lungs and slow (6 min and longer) smoking decide about low level of exhalation of malondialdehyde in cigarette smoke.

Filtration↗
PubMed2025

Coronavirus Cryptic Landscape and Draft Genome of a Novel CoV Clade Related to MERS From Bats Circulating in Northeastern Brazil.

We identified seven distinct coronaviruses (CoVs) in bats from Brazil, classified into 229E-related (Alpha-CoV), Nobecovirus, Sarbecovirus, and Merbecovirus (Beta-CoV), including one closely related to MERS-like CoV with 82.8% genome coverage. To accomplish this, we screened 423 oral and rectal swabs from 16 different bat species using molecular assays, RNA sequencing, and evolutionary analysis. Notably, gaps in the spike-encoding gene led us to design new primers and perform Sanger sequencing, which revealed high similarities to MERS-related (MERSr) CoV strains found in humans and camels. Additionally, we identified key residues in the receptor-binding domain (RBD) of the spike protein, suggesting potential interactions with DPP4, the receptor for MERSr-CoV. Our analyses also revealed evidence of recombination involving our laboratory-produced sequences. These findings highlight the extensive genetic diversity of CoVs, the presence of novel viral lineages, and the occurrence of recombination events among bat CoVs circulating in Brazil, underscoring the critical role bats play as reservoirs for emerging viruses and emphasizing the necessity of ongoing surveillance to monitor the public health risks associated with CoV spillover events.

Chiroptera↗
PubMed2002

Solution structure and backbone dynamics of an antigen-free heavy chain variable domain (VHH) from Llama.

Camelids, (dromedaries, camels, and llamas) produce heavy-chains antibodies, with their antigen recognition sites composed of a single VH-like domain, referred to as VHH. The solution structure of one of these VHHs domains (VHH-H14), raised against the alpha subunit of the human chorionic gonadotropin hormone (hCG), has been determined by (15)N heteronuclear three-dimensional NMR spectroscopy. The framework is well resolved within the set of 20 best-calculated NMR structures and is close to that of classical VH domains from vertebrate antibodies, consisting of two antiparallel beta-sheets organized in a beta-barrel. Loops display a lower precision, especially the Complementarity Determining Regions (CDRs), involved in antigen recognition. Comparison of the three-dimensional VHH-H14 solution structure with its previously solved crystal structure (Spinelli et al., Nature Struct. Biol. 1996;3:752-757) reveals a high similarity to the framework, whereas significant conformational differences occur on CDRs, leading to the assumption that the antigen recognition site is a more mobile part. In order to deepen our insights into the dynamics of VHH-H14 in solution, (15)N relaxation was measured with longitudinal R1 and transverse R2 self-relaxation rates, and (15)N steady-state heteronuclear nuclear Overhauser enhancements (NOE), making it possible to probe picosecond-to-millisecond internal motions. Determination of dynamic parameters (S(2), tau(e), and Rex) through the Lipari-Szabo Model-free approach enables the identification of several regions with enhanced dynamics. Especially, the mobility measurements from NMR confirm that the antigen recognition site is the most mobile part of the VHH-H14 domain on picosecond-to-nanosecond fast time scales. Several residues belonging to the three CDRs are submitted to chemical exchange processes occurring on slow microsecond-to-millisecond time scales, suggesting that the formation of the VHH/antigen complex should be accompanied by structural changes.

Amino Acids↗
PubMed1993

Molecular cloning and sequencing of zeta-crystallin/quinone reductase cDNA from human liver.

Zeta-crystallin is an enzyme-crystallin highly expressed in the lens of some hystricomorph rodents and camels. It has been shown to have a novel NADPH: quinone oxidoreductase activity and is present at enzymatic levels in a variety of tissues from various mammals. We report here the cDNA cloning of zeta-crystallin from a human liver library. One clone with the complete open reading frame was obtained. Ten nucleotides of the 5' and 796 of the 3' nontranslated regions are present in the clone including two possible polyadenylation signals. The deduced amino acid sequence is 328 residues long with a calculated molecular mass of 34910 daltons and isoelectric point of 8.73. It shows 84% identity with the guinea pig protein.

Amino Acid Sequence↗
PubMed2000

Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin.

The binding of [(125)I] orexin-A (Ox-A) to particulates from Chinese hamster ovary (CHO) cells expressing the cloned orexin-A receptor, or from rat forebrain areas, was sensitive to blockers of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) U-73122 and ET-18-OCH(3), little affected by phospholipase A(2) inhibitor quinacrine, and not sensitive to D609, a xanthate inhibitor of phosphatidylcholine-selective PLC. Interaction of the receptor with a PtdIns-PLC was further indicated by a large sensitivity of the binding to Ca(2+). Up to 50% of the binding was sensitive to the G-protein nucleotide site agonist GTP-gamma-S. Ligand attachment to the orexin-A receptor thus depends on an association with both PtdIns-PLC and G-protein alpha-subunits. In all paradigms examined, the binding of [(125)I]orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency similar to orexin-A (IC(50) range 30-100 nM). The rank order of potency for NPY-related peptides was hNPY > porcine peptide YY (pPYY) > (Leu(31), Pro(34)) human PYY > human PYY(3-36) > hNPY free acid > human pancreatic polypeptide. Among secretin-related peptides, the rank order of potency was porcine secretin > or = orexin-A > human pituitary adenylate cyclase-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Among opioid peptides, rat beta-endorphin and camel delta-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 microM. In view of high abundance of NPY in forebrain, the above cross-reactivity could indicate a significant contribution of NPY to signaling via orexin-A receptors.

Animals↗
PubMed2000

Neuroendocrine regulation of GnRH release in induced ovulators.

GnRH is the key neuropeptide controlling reproductive function in all vertebrate species. Two different neuroendocrine mechanisms have evolved among female mammals to regulate the mediobasal hypothalamic (MBH) release of GnRH leading to the preovulatory secretion of LH by the anterior pituitary gland. In females of spontaneously ovulating species, including rats, mice, guinea pigs, sheep, monkeys, and women, ovarian steroids secreted by maturing ovarian follicles induce a pulsatile pattern of GnRH release in the median eminence that, in turn, stimulates a preovulatory LH surge. In females of induced ovulating species, including rabbits, ferrets, cats, and camels, the preovulatory release of GnRH, and the resultant preovulatory LH surge, is induced by the receipt of genital somatosensory stimuli during mating. Induced ovulators generally do not show "spontaneous" steroid-induced LH surges during their reproductive cycles, suggesting that the positive feedback actions of steroid hormones on GnRH release are reduced or absent in these species. By contrast, mating-induced preovulatory surges occasionally occur in some spontaneously ovulating species. Most research in the field of GnRH neurobiology has been performed using spontaneous ovulators including rat, guinea pig, sheep, and rhesus monkey. This review summarizes the literature concerning the neuroendocrine mechanisms controlling GnRH biosynthesis and release in females of several induced ovulating species, and whenever possible it contrasts the results with those obtained for spontaneously ovulating species. It also considers the adaptive, evolutionary benefits and disadvantages of each type of ovulatory control mechanism. In females of induced ovulating species estradiol acts in the brain to induce aspects of proceptive and receptive sexual behavior. The primary mechanism involved in the preovulatory release of GnRH among induced ovulators involves the activation of midbrain and brainstem noradrenergic neurons in response to genital-somatosensory signals generated by receipt of an intromission from a male during mating. These noradrenergic neurons project to the MBH and, when activated, promote the release of GnRH from nerve terminals in the median eminence. In contrast to spontaneous ovulators, there is little evidence that endogenous opioid peptides normally inhibit MBH GnRH release among induced ovulators. Instead, the neural signals that induce a preovulatory LH surge in these species seem to be primarily excitatory. A complete understanding of the neuroendocrine control of ovulation will only be achieved in the future by comparative studies of several animal model systems in which mating-induced as well as spontaneous, hormonally stimulated activation of GnRH neurons drives the preovulatory LH surge.

Animals↗
PubMed1994

Organization of the human zeta-crystallin/quinone reductase gene (CRYZ).

zeta-Crystallin is a protein highly expressed in the lens of guinea pigs and camels, where it comprises about 10% of the total soluble protein. It has recently been characterized as a novel quinone oxidoreductase present in a variety of mammalian tissues. We report here the isolation and characterization of the human zeta-crystallin gene (CRYZ) and its processed pseudogene. The functional gene is composed of nine exons and spans about 20 kb. The 5'-flanking region of the gene is rich in G and C (58%) and lacks TATA and CAAT boxes. Previous analysis of the guinea pig gene revealed the presence of two different promoters, one responsible for the high lens-specific expression and the other for expression at the enzymatic level in numerous tissues. Comparative analysis with the guinea pig gene shows that a region of approximately 2.5 kb that includes the promoter responsible for the high expression in the lens in guinea pig is not present in the human gene.

Amino Acid Sequence↗
PubMed2001

Lateral recognition of a dye hapten by a llama VHH domain.

Camelids, camels and llamas, have a unique immune system able to produce heavy-chain only antibodies. Their VH domains (VHHs) are the smallest binding units produced by immune systems, and therefore suitable for biotechnological applications through heterologous expression. The recognition of protein antigens by these VHHs is rather well documented, while less is known about the VHH/hapten interactions. The recently reported X-ray structure of a VHH in complex with a copper-containing azo-dye settled the ability of VHH to recognize haptens by forming a cavity between the three complementarity-determining regions (CDR). Here we report the structures of a VHH (VHH A52) free or complexed with an azo-dye, RR1, without metal ion. The structure of the complex illustrates the involvement of CDR2, CDR3 and a framework residue in a lateral interaction with the hapten. Such a lateral combining site is comparable to that found in classical antibodies, although in the absence of the VL.

Amino Acid Sequence↗
PubMed2001

Degenerate interfaces in antigen-antibody complexes.

In most of the work dealing with the analysis of protein-protein interfaces, a single X-ray structure is available or selected, and implicitly it is assumed that this structure corresponds to the optimal complex for this pair of proteins. However, we have found a degenerate interface in a high-affinity antibody-antigen complex: the two independent complexes of the camel variable domain antibody fragment cAb-Lys3 and its antigen hen egg white lysozyme present in the asymmetric unit of our crystals show a difference in relative orientation between antibody and antigen, leading to important differences at the protein-protein interface. A third cAb-Lys3-hen lysozyme complex in a different crystal form adopts yet another relative orientation. Our results show that protein-protein interface characteristics can vary significantly between different specimens of the same high-affinity antibody-protein antigen complex. Consideration should be given to this type of observation when trying to establish general protein-protein interface characteristics.

Animals↗
PubMed2002

The genome of camelpox virus.

Camelpox virus (CMLV), a member of the Orthopoxvirus genus in the Poxviridae, is the etiologic agent of a disease of camels. Here we report the CMLV genomic sequence with analysis. The 205,719-bp CMLV genome contains 211 putative genes and consists of a central region bound by identical inverted terminal repeats of approximately 7 kb. A high degree of similarity in gene order, gene content, and amino acid composition in the region located between CMLV017 and CMLV184 (average 96% amino acid identity to vaccinia virus (VACV)) indicates a close structural and functional relationship between CMLV and other known orthopoxviruses (OPVs). Notably, CMLV contains a unique region of approximately 3 kb, which encodes three ORFs (CMLV185, CMLV186, CMLV187) absent in other OPVs. These ORFs are most similar to B22R homologues found in other chordopoxvirus genera. Among OPVs, CMLV is the most closely related to variola virus (VARV), sharing all genes involved in basic replicative functions and the majority of genes involved in other host-related functions. Differences between CMLV and VARV include deletion and disruption of a large number of genes. Twenty-seven CMLV ORFs are absent in VARV, including seven full-length homologues of NMDA-like receptor, phospholipase D, Schlafen, MT-4 virulence, kelch, VACV C8L, and cowpox (CPXV) B21R proteins. Thirty-eight CMLV ORFs, some of which are fragments of larger genes, differ in size from corresponding VARV ORFs by more than 10% (amino acids). Genome structure and phylogenetic analysis of DNA sequences for all ORFs indicate that CMLV is clearly distinct from VARV and VACV and, as it has been suggested for VARV, it may have originated from a CPXV virus-like ancestor.

Animals↗
PubMed1993

Carbonyl-metabolizing enzymes and their relatives recruited as structural proteins in the eye lens.

The refractive properties of the eye lens are determined by abundant soluble structural proteins known as crystallins. While some crystallins are common to most vertebrates, others are abundant only in groups of related species. These taxon-specific crystallins all turn out to be enzymes, apparently recruited by modification of gene expression without prior gene duplication. They include eta-crystallin, accounting for up to 25% of protein in elephant shrew lenses and apparently identical to cytoplasmic aldehyde dehydrogenase; rho-crystallin from frog lenses, a member of the same superfamily as aldose and aldehyde reductases; and zeta-crystallin, found in guinea pig and camel lenses, which is structurally related to alcohol dehydrogenase (ADH). Unlike ADH, zeta-crystallin requires NADPH rather than NAD+/NADH as cofactor. Molecular modelling of zeta-crystallin shows that amino-acid changes around the co-factor binding site are responsible for this change in affinity. Purified guinea pig lens zeta-crystallin has a substrate preference for orthoquinones which are reduced by a single electron transfer mechanism. cDNA sequencing of zeta-crystallin suggests that the expression in lens as a crystallin depends on a different gene promoter from that used predominantly in liver. The putative guinea pig zeta-crystallin lens promoter has now been assayed for function in transfection studies. Elements with positive and negative effects on transcription, at least one of which has tissue preferred function, have been defined. When introduced into transgenic mice this promoter exhibits tissue-specific expression in the lens. This is the first identification of a lens-specific, alternative promoter in an enzyme crystallin gene.

Aldehyde Dehydrogenase↗
PubMed1988

Dependence of O2 transfer conductance of red blood cells on cellular dimensions.

To estimate the significance of the dimensions of RBC on O2 transfer, the kinetics of O2 release from RBC into medium containing dithionite (40 mmol/l) was measured, by a stopped-flow technique, for nine different species with varying RBC size (man, llama, vicuna, alpaca, dromedary camel, pygmy goat, domestic hen, muscovy duck and turtle). The observed O2 transfer kinetics were found to be size-dependent, i.e. the O2 transfer conductance of the single RBC, gst, was lower, whereas the specific O2 transfer conductance of packed RBC, Gst, or of whole blood, theta st, was higher for smaller RBC. The ratio of surface area to effective diffusion path length which was found to be about one fourth of the mean cell thickness irrespective of cell size and cell shape, may be considered as the essential morphological factor determining O2 transfer efficiency of the single RBC.

Animals↗
PubMed1997

Molecular genetic analyses of parapoxviruses pathogenic for humans.

The current members of the genus parapoxvirus are orf virus (ORFV), bovine papular stomatitis virus (BPSV), pseudocowpoxvirus (PCPV) and parapoxvirus of red deer in New Zealand (PVNZ). BPSV and PCPV are maintained in cattle while ORFV is maintained in sheep and goats, but all three are zoonoses. Only the recently reported PVNZ has yet to be recorded as infecting humans. Tentative members of the genus are camel contagious ecthyma virus, chamois contagious ecthyma virus and sealpoxvirus. The separation of the parapoxviruses into 4 distinct groups has been based on natural host range, pathology and, more recently, on restriction endonuclease and DNA/DNA hybridisation analyses. The latter studies have shown that the parapoxviruses share extensive homology between central regions of their genomes, but much lower levels of relatedness within the genome termini. The high G + C content of parapoxvirus DNA is in contrast to most other poxviruses and suggests that a significant genetic divergence from other genera of this family has occurred. DNA sequencing of portions of the genome of ORFV, the type species of the genus, has allowed a detailed comparison with the fully sequenced genome of the orthopoxvirus, vaccinia virus (VACV). These studies have provided a genetic map of ORFV and revealed a central core of 88 kbp within which the genomic content was strikingly similar to that of VACV. This conservation is not maintained in the genome termini where insertions, deletions and translocations have occurred. The characterisation of specific ORFV genes may lead to the construction of attenuated vaccine strains in which genes such as those with the potential to interfere with the immune response of the host have been deleted. The current ORFV vaccines are living unattenuated virus and vaccination lesions produce virus which contaminates the environment in a manner similar to natural infection. The virus in scab material is relatively resistant to inactivation and this virus both perpetuates the disease in sheep and provides the most likely source of human infections. A vaccine which immunises animals without perpetuating the disease could be the best way of reducing the incidence of ORFV infection of humans. It is likely that protection against infection by ORFV is cell mediated and will require the endogenous production of relevant antigens. We have recently constructed a series of VACV recombinants each of which contains a large multigene fragment of ORFV DNA. Together the recombinants represent essentially all of the ORFV genome in an overlapping manner. Vaccination of sheep with the recombinant library provided protection against challenge with virulent ORFV. Further studies with this library may enable dominant protective antigens of ORFV to be identified and lead to their incorporation into a subunit vaccine.

Animals↗
PubMed1990

Cystic hydatid disease: aspects of the incidence in man in Kuwait, Arabian Gulf.

Cystic hydatid disease (CHD) is endemic in Kuwait, but the exact extent of the disease in man has yet to be determined. The incidence was calculated by serological testing of sera from patients with a presumptive clinical diagnosis of hydatidosis and follow-up until the final diagnosis was determined. During a one-year period, 123 sera were received from two District General Hospitals serving a population of approximately 500,000. Eighteen patients had confirmed CHD, 17 were serologically positive in two tests, indirect haemagglutination (IHA) and counterimmunoelectrophoresis (CIEP), while another positive patient was identified through surgery and histopathology. These cases represent an estimated incidence rate of 3.6 per 100,000. Various factors, however, indicate that this is an underestimation of the true incidence. The infection rate of camels with hydatid cysts and dogs harbouring the adult worm, a possible measure of the true incidence in man, is considered high. Because of the nature of the population structure, the majority of patients were, in fact, non-Kuwaitis. Kuwaitis, who form 40% of the population, constituted about 30% of the CHD patients. Females were in the majority, the 21 to 50 yr. age-group being the most symptomatic. Hydatid cysts were most commonly present in the liver. Finally, compared to other countries at the time when a control programme was instituted, the incidence rate in Kuwait is moderate to high. However, the number of individuals with CHD does not appear alarming as the available capacity of the medical services is adequate.

Adolescent↗
PubMed1995

Evolution of protamine P1 genes in mammals.

Protamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that protamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5-6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically.

Animals↗
PubMed1994

Restriction site patterns in the ribosomal DNA of camelidae.

The restriction map of rDNA from South American camelids and the Bactrian camel was analyzed by digestion of high-molecular-weight DNA with endonucleases EcoRI,BamHI and the two combined followed by Southern blot hybridization with probes for the 18S and 28S rDNA sequences. We scored a total of 17 restriction sites, six of which were mapped conserved in all the species. The other eleven corresponded to spacer regions and revealed variations between these taxa. The study showed that the two groups differ in the length of the internal transcribed spacer. Also they showed the existence of two regions of fast evolution on the opposite termini of the external spacer. A restriction site present at low frequency in the non-transcribed spacer of guanaco and llama was the only difference encountered within the South American group.

Animals↗
PubMed1990

Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization.

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus Kc of the membrane and of the cytoplasmic viscosity eta were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I0 passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of Kc/R3(0) and mu.R0 (R0 = equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of Kc = 2-3 x 10(-19) Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, Kc = 3.4 x 10(-20) Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (Kc = 5 x 10(-19) Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent Kc and eta values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocyte-stomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCl solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) Kc and eta are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent Kc and eta after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

[Biomechanical fragility of the infant skull].

Following previous experiments on postmortem skull fractures of infants, falls from 82-cm heights onto stone (A), carpet (B) and foam-backed linoleum (C), 35 further falling tests were carried out onto softly cushioned ground. In 10 cases a 2-cm thick foam rubber mat (D) was chosen and in 25 further cases a double-folded (8-cm-thick) camel hair blanket (E). Hence the results of altogether 50 tests could be evaluated. In test groups A-C on a relatively hard surface, skull fractures of the parietale were observed in every case; in test group D this fracture was seen in one case and in test group E in four cases. Measurements along the fracture fissures showed bone thickness of 0.1-0.4 mm. The fracture injuries originated in paper-thin single-layer bone areas without diploe, which can also be considered the preferred regions for skull fractures of older infants following falls from low heights. These results indicate that it is no longer possible to assume that the skull of infants is not damaged after falls from table height.

Battered Child Syndrome↗