Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA

We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognized by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).     

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Effects of N,N-dimethylformamide and extracellular matrix on transforming growth factor-beta binding to a human colon carcinoma cell line

Alterations in the binding of transforming growth factor-beta (TGF-beta) to the MOSER human colon carcinoma cell line caused by N,N-dimethylformamide (DMF) or extracellular matrix (ECM) were examined. DMF induced a more differentiated phenotype in the MOSER cells and resulted in a twofold increase in TGF-beta binding to the cells. This was due to an increase in receptor number with no significant alteration in the KD. The extent of increased TGF-beta binding was dependent on the dose and time of exposure to DMF. Upon removal of DMF, the receptor level returned to that of untreated cells within 6 hr. The binding of TGF-beta 1 and TGF-beta 2 to the cells was increased equally. Despite this increase in TGF-beta binding in the presence of DMF, the sensitivity of the MOSER cells to the growth inhibitory effects of TGF-beta was unaltered. Growth of the MOSER cells on ECM derived from a well-differentiated colon cell line increased the TGF-beta receptor number twofold without altering the KD. No change was observed if the MOSER cells were grown on ECM derived from a poorly differentiated cell line. While no alteration in sensitivity to TGF-beta was observed on cells grown in the presence of DMF, MOSER cells grown on the ECM derived from well-differentiated colon carcinoma cell lines were twofold more sensitive to the growth inhibitory effects of TGF-beta. These results indicated that growth conditions which resulted in a more differentiated phenotype resulted in an increase in the cellular receptors for TGF-beta.     

SLLP1, a unique,intra-acrosomal,non-bacteriolytic,c lysozyme-like protein of human spermatozoa

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.     

Biochemistry and ultrastructure of iridescent virus type 29

Physical and chemical parameters of iridescent virus type 29, isolated from the mealworm, Tenebrio molitor, have been analyzed. The icosahedral capsid is 130–135 nm in diameter and is surrounded by a fringe of coarse filaments. The virus has a buoyant density in CsCl of 1.31 g cm^?3 and contains 20 to 25 structural proteins as analyzed by isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The DNA has a buoyant density in CsCl of 1.6874 g cm^?3 indicating a G + C content of approximately 28%. The lipid components of this virus differ from those of the host cell; the virus contains about 80% cardiolipin and 20% phosphatidyl choline.     

Paternal contribution to small for gestational age babies: a multicenter prospective study

Our aims were to investigate whether men who fathered small for gestational age (SGA) infants themselves had lower birthweight, were more likely to be obese, have central adiposity and elevated blood pressure in adult life compared with men who fathered non-SGA infants. A total of 2,002 couples participating in the Screening for Pregnancy Endpoints (SCOPE) study were enrolled in early pregnancy and pregnancy outcome data collected prospectively. SGA was defined as birthweight <10th customized centile, obesity as BMI ≥30 kg/m(2), central adiposity as waist circumference >102 cm. Logistic regression was used to compare rates of obesity, and central adiposity between men who fathered SGA infants compared with those with non-SGA infants and the final model was adjusted for maternal and paternal confounders. The men who fathered an SGA infant (209 (10.4%)) themselves had lower mean birthweight (3,291 (530) g vs. 3,472 (584) g, P < 0.0001), were more likely to be obese (50 (24.8%) vs. 321 (18.3%), adjusted odds ratio (OR) 1.50, 95% confidence interval 1.05-2.16, adjusted for maternal and paternal factors) and to have central adiposity (52 (25.1%) vs. 341 (19.2%), adjusted OR 1.53, 95% confidence interval 1.06-2.20) compared with men who fathered a non-SGA infant. Elevated paternal blood pressure was not associated with SGA. In conclusion, we report a novel relationship between paternal obesity/central adiposity and birth of an SGA infant, which appears to be independent of maternal factors associated with fetal growth restriction.     

Computational and experimental analysis reveals a requirement for eosinophil-derived IL-13 for the development of allergic airway responses in C57BL/6 mice

Eosinophils are found in the lungs of humans with allergic asthma, as well as in the lungs of animals in models of this disease. Increasing evidence suggests that these cells are integral to the development of allergic asthma in C57BL/6 mice. However, the specific function of eosinophils that is required for this event is not known. In this study, we experimentally validate a dynamic computational model and perform follow-up experimental observations to determine the mechanism of eosinophil modulation of T cell recruitment to the lung during development of allergic asthma. We find that eosinophils deficient in IL-13 were unable to rescue airway hyperresponsiveness, T cell recruitment to the lungs, and Th2 cytokine/chemokine production in ΔdblGATA eosinophil-deficient mice, even if Th2 cells were present. However, eosinophil-derived IL-13 alone was unable to rescue allergic asthma responses in the absence of competence of other IL-13-producing cells. We further computationally investigate the role of other cell types in the production of IL-13, which led to the various predictions including early and late pulses of IL-13 during airway hyperresponsiveness. These experiments suggest that eosinophils and T cells have an interdependent relationship, centered on IL-13, which regulates T cell recruitment to the lung and development of allergic asthma.     

Crystal structure of the conserved subdomain of human protein SRP54M at 2.1 A resolution: evidence for the mechanism of signal peptide binding.

Protein SRP54 is an integral part of the mammalian signal recognition particle (SRP), a cytosolic ribonucleoprotein complex which associates with ribosomes and serves to recognize, bind, and transport proteins destined for the membrane or secretion. The methionine-rich M-domain of protein SRP54 (SRP54M) binds the SRP RNA and the signal peptide as the nascent protein emerges from the ribosome. A focal point of this critical cellular function is the detailed understanding of how different hydrophobic signal peptides are recognized efficiently and transported specifically, despite considerable variation in sequence. We have solved the crystal structure of a conserved functional subdomain of the human SRP54 protein (hSRP54m) at 2.1 A resolution showing a predominantly alpha helical protein with a large fraction of the structure available for binding. RNA binding is predicted to occur in the vicinity of helices 4 to 6. The N-terminal helix extends significantly from the core of the structure into a large but constricted hydrophobic groove of an adjacent molecule, thus revealing molecular details of possible interactions between alpha helical signal peptides and human SRP54.     

A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

Background

A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation.

Methods

A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique.

Results and Discussion

The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous.

Conclusion

The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.