Acid Sequence Research Articles

Bioactive peptide design using the Resonant Recognition Model

With a large number of DNA and protein sequences already known, the crucial question is to find out how the biological function of these macromolecules is "written" in the sequence of nucleotides or amino acids. Biological processes in any living organism are based on selective interactions between particular bio-molecules, mostly proteins. The rules governing the coding of a protein's biological function, i.e. its ability to selectively interact with other molecules, are still not elucidated. In addition, with the rapid accumulation of databases of protein primary structures, there is an urgent need for theoretical approaches that are capable of analysing protein structure-function relationships. The Resonant Recognition Model (RRM) [1,2] is one attempt to identify the selectivity of protein interactions within the amino acid sequence. The RRM [1,2] is a physico-mathematical approach that interprets protein sequence linear information using digital signal processing methods. In the RRM the protein primary structure is represented as a numerical series by assigning to each amino acid in the sequence a physical parameter value relevant to the protein's biological activity. The RRM concept is based on the finding that there is a significant correlation between spectra of the numerical presentation of amino acids and their biological activity. Once the characteristic frequency for a particular protein function/interaction is identified, it is possible then to utilize the RRM approach to predict the amino acids in the protein sequence, which predominantly contribute to this frequency and thus, to the observed function, as well as to design de novo peptides having the desired periodicities. As was shown in our previous studies of fibroblast growth factor (FGF) peptidic antagonists [2,3] and human immunodeficiency virus (HIV) envelope agonists [2,4], such de novo designed peptides express desired biological function. This study utilises the RRM computational approach to the analysis of oncogene and proto-oncogene proteins. The results obtained have shown that the RRM is capable of identifying the differences between the oncogenic and proto-oncogenic proteins with the possibility of identifying the "cancer-causing" features within their protein primary structure. In addition, the rational design of bioactive peptide analogues displaying oncogenic or proto-oncogenic-like activity is presented here.

S1 gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives

Infectious bronchitis virus CK/CH/LDL/97I was attenuated by serial passage in chicken embryos. Virus of passage 115 was attenuated as determined by clinical response to inoculation to 15-day-old specific pathogen free chickens. The vaccination-challenge test showed that the attenuated passage 115 virus could afford protection against the homologous pathogenic virus, passage 5, by the clinical response. Based on the sequence analysis and comparison of the S1 gene, both the pathogenic and attenuated CK/CH/LDL/97I viruses were populations that each included at least two subpopulations. Also, from analysis of the amino acid and nucleotide sequences of S1 gene, we speculate that recombination between the minor and dominant subpopulations plus accumulation of mutations in the S1 region of pathogenic passage 5 might lead to the formation of the embryo-passaged, attenuated passage 115.

RFamide Peptides Inhibit the Expression of Melanotropin and Growth Hormone Genes in the Pituitary of an Agnathan, the Sea Lamprey, Petromyzon marinus

Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates, and shown to have important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions. The present study identified RFamide peptides, which are teleost prolactin-releasing peptide (PrRP) homologs, in the sea lamprey, Petromyzon marinus and characterized their effect on the release of pituitary hormones in vitro. Two RFamide peptides (RFa-A and RFa-B) were isolated from an acid extract of sea lamprey brain, including hypothalamus by Sep-Pak C18 cartridge, affinity chromatography using anti-salmon PrRP serum, and reverse-phase HPLC on an ODS-120T column. Amino acid (aa) sequences and mass spectrometric analyses revealed that RFa-A and RFa-B consist of 25 and 20 aa, respectively, and have 75% sequence identity within the C-terminal 20 aa. The RFa-B cDNA encoding a preprohormone of 142 aa was cloned from the lamprey brain, and the deduced aa sequence from positions 48-67 was identical to the sequence of RFa-B. However, the preprohormone does not include an aa sequence similar to the RFa-A sequence. Cell bodies, which were immunoreactive to anti-salmon PrRP serum, were located in the periventricular arcuate nucleus, ventral part of the hypothalamus, and immunoreactive fibers were abundant from the hypothalamus to the brain. A small number of immunoreactive fibers were detected in the dorsal half of the rostral pars distalis of the pituitary, close to the GH-producing cells. In addition, anti-salmon PrRP immunoreactivities were observed in the pars intermedia, corresponding to melanotropin cells. Likewise, signal of RFa-B mRNA was detected not only in the brain but also in the pars intermedia. The synthetic RFa-A and -B inhibited GH mRNA expression in a dose-dependent fashion in vitro, which is comparable to the inhibitory effect of teleost PrRP on GH release. Both RFa-A and -B also inhibited the expression of proopiomelanotropin mRNA, but no effects were observed in the expression of proopiocortin and gonadotropin beta mRNAs. The results indicate that RFamide peptides, which are teleost PrRP homologs, are present in the hypothalamus and pituitary of sea lamprey, and may be physiologically involved in the inhibition of GH and melanotropin release in the sea lamprey pituitary.

Ensemble des gènes connus comme impliqués dans le développement cranio-facial

Many genes intervening in development, morphogenesis and craniofacial growth have been identified, primarily by the use of mice mutants. We can distinguish two families: the signalling factors and the transcription factors. The latter interact with DNA to activate or to inhibit the expression of other genes. Some of the transcription factors are called homeogenes because they interact with DNA by a sequence of amino acids known as homeobox that has been carefully conserved throughout the course of evolution. Those factors interact, and signalling cascades have been described. Current research projects seek to discern the exact role of each of these genes in craniofacial growth and to develop a better understanding of the interactions between them.

Encoding Type and Position in Peptide QSAR: Application to Peptides Binding to Class I MHC Molecule HLA‐A*0201

AbstractVarious QSAR approaches have evolved over time to find a relationship between chemical structure and biological response by translating the information in the chemical structure into a quantitative (Hansch approach) or qualitative (Free–Wilson approach) description followed by a statistical modeling. The nature and position of every amino acid in the peptide sequence dictate its binding to the receptor. We have investigated an approach to peptide QSAR where the position and type of every amino acid in the sequence are encoded in the QSAR paradigm, and along with the principles of the Hansch (descriptor QSAR) and the Free–Wilson (binary QSAR) methodologies we were able to arrive at the optimal sequence of amino acids in the peptide. As an example, we illustrate the procedure on two datasets of nonamer peptides that bind to the class I major histocompatibility complex molecule HLA‐A*0201. The models generated have statistically significant co‐relation coefficient (r2) and predictive r2 (r2pred). The cross‐validated r2 (q2) for “descriptor QSAR” approach and the “binary QSAR” approach are in an acceptable range. The QSAR analysis is able to identify the preferential and detrimental amino acids at every position of the nonamer peptide. This QSAR formalism as applied to peptides is a simple and effective way to explain structure–activity relationships as well as to optimize the binding affinity.

The in vitro anti-tumour activity of zoledronic acid and docetaxel at clinically achievable concentrations in prostate cancer

Bisphosphonates and chemotherapy have increasingly gained favour in the treatment of metastatic hormone resistant prostate cancer. We investigated whether zoledronic acid, at a concentration found at the bone, would enhance the anti-tumour activity of docetaxel in the hormone resistant prostate cancer cell line PC-3. Cells were exposed to zoledronic acid (1 mM) in combination or in sequence with docetaxel (3 nM). Cell viability, apoptosis and markers for inhibition of the mevalonate pathway were analyzed 48 or 72 hours after drug treatment. Reduction in cell viability and increased apoptosis levels were most pronounced with single agent zoledronic acid. Western blot analysis showed an overall reduction in the proliferation marker Mini chromosome maintenance protein 2 (MCM2) and reduction in caspase-3 precursor for all drug treatments and a marked reduction in Rho A levels with single agent zoledronic acid and zoledronic acid-docetaxel sequence. This study highlights the potency of zoledronic acid, when used at concentrations similar to those found at the bone, in reducing cell viability and causing apoptosis. Clinically, these findings suggest that in patients with bone metastases due to hormone resistance prostate cancer, who are not fit enough for systemic chemotherapy, single agent zoledronic acid may have a direct effect on viability of prostate cancer epithelial cells.

Genomic Diversity in the RegulatorynefGene Sequences in Indian Isolates of HIV Type 1: Emergence of a Distinct Subclade and Predicted Implications

The regulatory functional nef gene is known to mediate a cascade of events during pathogenesis in HIV infection. Variability in the nef gene sequences of HIV-1 A and B subtypes has been well documented. Reasonable data are also available on the pattern of genomic changes in the nef gene of African strains of HIV-1 subtype C, but very little is known about heterogeneity in the nef gene of Indian strains of HIV-1 subtype C, which accounts for 90% of the estimated 5.2 million cases of HIV infection in India. This is a huge number and, therefore, it is important to reveal the extent of sequence variability in the nef gene of HIV-1 subtypes circulating in different parts of India. We carried out full-length nef gene (approximately 620 bp) sequencing on a large number of clinical isolates of HIV-1 circulating in different geographic regions of India. Comparative and phylogenetic analysis revealed 88% (38/43) of cases was HIV-1 subtype C; four cases were diagnosed as subtype A and only one as subtype B. Although most of the crucial functional motifs of the nef gene were conserved, we did observe a few important variations in juxtapositions to functional domains. Interestingly, analyzed nef sequences showed an evolving pattern of segregation away from those reported from other parts of the world, to form a distinct Indian subclade. Deduced amino acid (aa) sequences used to predict HLA binding epitopes for consensus nef gene sequences of Indian strains of HIV-1 revealed two HLA subtype binding domains, GAFDLSFFL (at aa 83) and LTFGWCFKL (at aa 136), in high frequency. The findings from the present study may encourage use of nef gene in molecular diagnostics/genotyping, keeping track of the evolutionary trend and pinpointing the emergence of recombinant strains, and in the future, designing a multiepitope HIV vaccine suitable for the Indian population.

Prediction of Mutations in H5N1 Hemagglutinins from Influenza A Virus

In this study, we determine the mutation relation among 333 H5N1 hemagglutinins of influenza A viruses according to their amino acid and RNA codon sequences. Then, we calculate seven probabilistic numbers, which have been developed by us since 1999, for each amino acid in these hemagglutinins. With the seven numeric numbers as independents and the probability of occurrence of mutation at each hemagglutinin position as dependent, we use the logistic regression to model 967 missense point mutations from 333 hemagglutinins to get the population estimates. Thereafter, we predict the future mutation positions in H5N1 hemagglutinin. Finally, we use the translation probabilities between RNA codons and mutated amino acids to predict the would-be-mutated amino acids in H5N1 hemagglutinin.

Phase I trial results of PCK3145 in patients (pts) with castrate metastatic prostate cancer (PC)—The US experience

4650 Background: PCK3145 is a synthetic 15-mer peptide derived from the natural sequence of amino acids of Prostate Secretory Protein (PSCP94), a predominant protein found in seminal fluid. Its expression in PC is down-regulated in pts with metastatic disease and is believed to be a signal transduction inhibitor via multiple pathways in carcinogenesis. Recent data from a phase IIa trial in Great Britain demonstrated safety and suggested reduction in MMP-9 levels, an enzyme involved in angiogenesis, tumor invasion and metastasis. A phase I trial was performed to assess dosing and dosing schedule, pharmacokinetics, safety and impact on MMP-9 levels in pts with castrate metastatic PC who have failed multiple therapies including hormones and chemotherapy. Methods: Two cohorts of 10 pts received either 7.5 mg/m2 twice weekly or 15 mg/m2 weekly, respectively. Pts were treated for 4 weeks followed by a 2 week non-treatment period. Pts could receive addtional two-month cycles if PSAs and scans remained stable. MMP-9 levels, PSA, pharmacokinetics and imaging assessments were done at screening, and at weeks 2, 4 and 6. Results: Ten pts have been enrolled at each dose level. No adverse events were observed, however, declines in MMP-9 levels occurred in most pts by week #3 posttreatment. MMP-9 levels rose when drug was discontinued. Six pts were taken off study due to disease progression whereas 5 pts were discontinued due to rising PSAs. One pt at the 7.5 mg/m2 biweekly dosing schedule received 4 additional cycles with stable PSAs and scans with a PSA doubling time which increased from 4 months pretreatment to 8 months posttreatment. Conclusions: PCK3145 was well-tolerated without toxicity. While early, the weekly dosing schedule appears to result in greater declines in MMP-9 levels and will likely be used in a forthcoming phase II trial in chemotherapy naive pts. [Table: see text]

Shape of a Common Protein Module Suggests Role as Molecular Switch

A vital aspect of a neuron's job is deciding when to pass their cache of chemicals on to neighboring cells. To do this in a way that ensures effective communication, neurons must keep tight reins on their neurotransmitters—the chemical messengers they release to influence neighboring cells. Neurons quickly collect and then jettison these neurotransmitters, cycling through this process many times per second. Now a new study reveals the structure of a fragment of Munc-13—a key protein in this process—showing how it could act as a switch that neurons use to toggle quickly between storing and releasing their neurotransmitters. For this switching role, the key part of the protein is one of its C 2 domains (the C 2A domain). This domain is widespread, suggesting that other proteins may function by a similar mechanism. Neurons store their neurotransmitters in vesicles, tiny sacs that amass at the synapse between cells. There, they dock at the inside of the cell membrane and undergo one or more reactions that “prime” the vesicles to be ready to quickly unload their neurotransmitters outside the cell when the neuron fires. A cascade of protein interactions mediates these different steps. These protein cascades not only allow neurons to do their usual duty, but they also help neurons process information. If neurons react quickly or sluggishly, reliably or not, to a neuron firing, this affects the signal that gets passed on to neighboring neurons. And, if their reactions change with time, this plasticity can help the animal remember, learn, and adapt. To understand how the proteins interact, a key step is figuring out the shapes the proteins assume. Once they have the shapes, researchers can see how proteins, like pieces of a jigsaw puzzle, fit together. For example, this helps researchers make sense of chemical and biological data that suggest how proteins bind together and influence each other. Josep Rizo and colleagues targeted a protein called Munc13-1 since its family of proteins plays important roles in preparing vesicles to release neurotransmitters, and Munc13-1 itself is known to have a role in the plasticity of synapses. Rizo and colleagues homed in on one of the key zones of Munc13-1, called the C 2A domain. C 2 domains are well known for being able to bind to phospholipids, the fatty acids that make up cell and vesicle membranes. But the researchers found an unexpected role for the Munc13-1 C 2A domain: it can bind to other proteins as well. To figure out the shape of Munc13-1's C 2A domain, down to the atomic level, the researchers combined two methods: X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. X-ray crystallography is the classic method for “solving” the mystery of a protein's shape. But not all proteins crystallize well, and so are not amenable to the method. Or if they do crystallize, their shape may be distorted from the form it normally takes when floating in a cell's watery interior. Rizo and colleagues combined the two methods to create a detailed picture of the protein's shape. They used NMR spectroscopy to check how well the protein behaves in solution and to optimize the crystallization, aiding the X-ray studies. Also, data from the NMR spectroscopy on the shape of the protein helped them interpret the X-ray results. In the end, they were able to figure out the natural shape of the protein's C 2A domain to atomic resolution. These structural data also helped Rizo and colleagues make sense of their experiments designed to determine Munc13-1's binding partners. The researchers found that, in a pure solution, Munc13-1 proteins bound to each other in pairs. But when mixed with RIM2α (another protein that plays a key role in neurotransmitter release and neuron plasticity), then Munc13-1 formed pairs with RIM2α instead. Combining the structural and binding data, the researchers were able to piece together a picture of how Munc13-1 could work as a switch. They found that the C 2 domain of Munc13-1 has two adjacent and partially overlapping regions that can bind to other proteins. So two C 2 domains could bind each other using one part of that domain, and another part of the domain could bind to RIM2α. But the C 2 domain can't bind both at the same time. The researchers could see, once they had the shapes of the proteins, that they simply can't all fit together at once. Thus it seems that there is competition for Munc13-1 to bind either to itself or to other proteins. This mechanism could prepare the vesicles to be ready to unload the neurotransmitters when the neuron fires. Since the C 2 domain is widespread among proteins—and is highly similar among distantly related species—the findings could hold clues to understanding more roles for this common sequence of amino acids.

Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

BackgroundActinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking.ResultsHere we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galβ-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8–66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (>97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA.ConclusionThe genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.

Synthesis, characterization and calorimetric study of zinc group halide adducts with aniline

The adducts ZnCl 2·2an, ZnBr 2·1.5an, CdCl 2·2an, CdBr 2·2an, CdI 2·2an, HgCl 2·2an and HgBr 2·2an (where an = aniline) were synthesized and characterized by elemental analysis, infrared spectroscopy and thermogravimetry and solution calorimetry. Considering the Δ r H m θ as well as the D( M − N) values, the following acidity sequence can be established: Zn > Cd > Hg which can be also referring to the hardness of the metal halides. The Δ r H m θ values (kJ mol −1) for the reaction MX 2 (c) + nL (l) = MX 2·nL (c) are: −82.4 ± 3.6 (ZnCl 2·2an), −77.1 ± 0.4 (ZnBr 2·1.5an), −45.3 ± 1.4 (CdCl 2·2an), −45.2 ± 0.7 (CdBr 2·2an), −60.3 ± 0.1 (CdI 2·2an) and −41.4 ± 6.3 (HgCl 2·2an). In the same sequence of compounds, the D( M − N) values (kJ mol −1) are: 143.6, 157.3, 141.2, 126.2, 126.9 and 89.7, respectively.

Studies on the α-(1→4)- and α-(1→8)-fucosylation of sialic acid for the total assembly of the glycan portions of complex HPG-series gangliosides

A synthetic study on α-(1→4) and α-(1→8)-fucosylation of sialic acid is reported, with the ultimate aim being the total assembly of the glycan portion of HPG-series gangliosides. In both types of fucosylations, the combination of a phenylthio fucosyl donor and a 1,5-lactamized acceptor provided high-yielding glycosylations to afford α-fucosyl-sialic acid sequences. The obtained α-Fuc p-(1→8)-Neu pNAc glycan having a 1,5-lactam bridge has been successfully transformed into the corresponding glycosyl donor.

Actin Binding to the Central Domain of WASP/Scar Proteins Plays a Critical Role in the Activation of the Arp2/3 Complex

The Arp2/3 complex nucleates and cross-links actin filaments at the leading edge of motile cells, and its activity is stimulated by C-terminal regions of WASP/Scar proteins, called VCA domains. VCA domains contain a verprolin homology sequence (V) that binds monomeric actin and central (C) and acidic sequences (A) that bind the Arp2/3 complex. Here we show that the C domain binds to monomeric actin with higher affinity (K(d) = 10 microm) than to the Arp2/3 complex (K(d) > 200 microm). Nuclear magnetic resonance spectroscopy reveals that actin binds to the N-terminal half of the C domain and that both the V and C domains can bind actin independently and simultaneously, indicating that they interact with different sites. Mutation of conserved hydrophobic residues in the actin-binding interface of the C domain disrupts activation of the Arp2/3 complex but does not alter affinity for the complex. By chemical cross-linking the C domain interacts with the p40 subunit of the Arp2/3 complex and, by fluorescence polarization anisotropy, the binding of actin and the Arp2/3 complex are mutually exclusive. Our results indicate that both actin and Arp2/3 binding are important for C domain function but that the C domain does not form a static bridge between the two. We propose a model for activation of the Arp2/3 complex in which the C domain first primes the complex by inducing a necessary conformational change and then initiates nucleus assembly by bringing an actin monomer into proximity of the primed complex.

Isolation and identification of Japanese encephalitis virus in Liaoning Province

To study arboviruses carried by mosquitoes in Liaoning Province in 2002. Totally 4927 mosquitoes were collected from Liaoning Province in July 2002. Virus strains were isolated by inoculating BHK-21, C6/36 and Vero cells. The newly isolated strains were identified by serological (ELISA and IFA) and molecular methods (Real-Time PCR and RT-PCR). Two strains were isolated from mosquitoes causing cytopathogenic effect (CPE) on cells and were fatal to suckling mice. Serological tests showed that both were positive for the antibody to JEV. The PrM and E gene were cloned and sequenced. The phylogenetic analysis showed that the new isolates belonged to genotype I, JEV. Sequence analysis showed that the homology of nucleotide sequences and amino acid (AA) sequences between the two strains was 100%. Compared with the nucleotide sequences between the two strains and the standard JEV vaccine strain SA14-14-2, the difference was up to 4.11%, and the difference of AA was 0.6%. Two strains of JEV were isolated and identified in Liaoning province, both belonged to genotype I JEV.

Classification and phylogeny of Hexactinellida (Porifera)

Development of the present classification scheme of the class Hexactinellida was delayed because authors of the early species descriptions did not figure their specimens and few of them had access to compound microscopes necessary to visualize characters important in determining relationships. When microscopic information on spicule symmetry became available, Wyville Thomson in 1868 recognized the distinctive triaxial symmetry of the group and named it Vitrea. Schmidt's 1870 name, Hexactinellidae, was without the contamination associated with Thomson's Vitrea, and gained widespread support. Through a series of proposals and refinements, the present scheme recognizes two main lineages, Amphidiscophora and Hexasterophora, differing in shape and type of their microscleres. Since these structures are generally not retained in fossil material, paleontologists have long had an independent classification for hexactinellids, but the trend to unify the systems is now nearly complete. Hypotheses on phylogeny of the group remain virtually dependent upon scarce paleontological information, pending data from nucleic acid and protein sequence analyses that are not yet available for unravelling relationships among hexactinellids.

THERMAL STABILITY OF THE NEW SOLITON TRANSPORTED BIO-ENERGY UNDER INFLUENCE OF FLUCTUATIONS OF CHARACTERISTIC PARAMETERS AT BIOLOGICAL TEMPERATURE IN THE PROTEIN MOLECULES

The dynamic behaviors of the new soliton in the improved Davydov model in the protein molecules at biological temperature have been numerically simulated by utilizing the dynamic equations for the bio-energy transport and the Runge–Kutta way. In this simulation the influences of the temperature and structure disorders of the protein molecules on the soliton transporting the bio-energy have been completely considered. We find that the new soliton is quite stable in the cases of motion of a long time of 300 ps and of disorders of the structures of the proteins at biological temperatures of 300 K–320 K. The disorders of the structures contain the disorder of mass sequence of amino acids and the fluctuations of the coupling constant, force constant and dipole- dipole interaction constant and ground state energy of the proteins, designating the features of its structure and interactions between the particles in it. However, the soliton disperses in the cases of higher temperature of 325 K and larger structure disorders. The numerical results show that the new soliton is very robust against the influences of the thermal perturbation and structure disorders at biological temperature 300 K, its lifetime and critical temperature are at least 300 ps at 300 K and 320 K, respectively. These results are also consistent with analytical data.

Design and synthesis of 14C-labelled proteins as tools for protein digestion studies in fish larvae

In order to study the protein digestive capability in early stages of fish larvae, a method was developed to produce adequate amounts of pure, well-defined, high-quality, specifically 14C-labelled proteins. The proteins that were chosen were integral parts of the abundant cytoskeleton protein spectrin. The proteins were engineered by molecular biology technology to allow the specific incorporation of 14C-labelled leucine. The purified monodisperse proteins could be concentrated to more than 10 mg mL-1 without precipitation. These proteins were parts of the same 'mother protein' with similar amino acid compositions and sequences but different molecular weights. This made them suitable to study the influence of the molecular sizes of the proteins on digestion in fish larvae.

Single amino acid exchanges in FAD-binding domains of squalene epoxidase of Saccharomyces cerevisiae lead to either loss of functionality or terbinafine sensitivity

Squalene epoxidase (Erg1p) is an essential enzyme in the ergosterol biosynthesis pathway in yeast. For its enzymatic activity, Erg1p requires molecular oxygen, NAD(P)H and FAD. Amino acid analysis and sequence alignment with other squalene epoxidases revealed two highly conserved FAD-binding domains, FAD I and FAD II. By random PCR mutagenesis of the ERG1 gene, one erg1 allele was isolated that carries a mutation leading to a single amino acid exchange in the FAD I domain close to the N-terminus of Erg1p. This erg1 allele codes for functional squalene epoxidase and renders yeast cells hypersensitive to terbinafine. Amino acid exchanges of other conserved residues in the FAD I and FAD II regions either led to non-functional squalene epoxidase or to the formation of squalene epoxidase with wild-type properties. These results describe the importance of specific amino acids for enzymatic activity in the yeast squalene epoxidase Erg1p.

Acute Granulocytic Ehrlichiosis in a Rottweiler

Acute granulocytic ehrlichiosis was identified in a 6-year-old rottweiler that was presented for possible pancreatitis. Intracytoplasmic inclusion bodies were identified within neutrophils on a peripheral blood smear. Serology was ineffective in identifying the disease in the acute state. The diagnosis and identification of the organism were confirmed by polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencing. Based on elevations in amylase and lipase and the presence of right cranial-quadrant abdominal pain, concurrent pancreatitis was diagnosed. It is unknown if there was any association between the acute granulocytic ehrlichiosis and the pancreatitis. The dog recovered well following doxycycline therapy.