Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

alpha-N-acetyl-beta-endorphin1-26 from the neurointermediary lobe of the rat pituitary: isolation, purification, and characterization by high-performance liquid chromatography

The neurointermediary lobes from 190 rat pituitaries were homogenized in an acidic medium which inhibits peptidase activity and maximizes the solubilization of undamaged peptides. Octadecylsilyl-silica (ODS-silica) was used to extract the supernatant of the tissue homogenate. The ODS-silica eluate, now largely protein and salt free, was subjected to reversed-phase high-performance liquid chromatography (HPLC) employing 0.1% trifluoroacetic as counter ion. The column eluates were monitored for beta-endorphin immunoreactivity. Five immunoreactive components were observed. The most hydrophobic of these was repurified on the same HPLC column using 0.13% heptafluorobutyric acid as counter ion. Characterization of the purified peptide by gel permeation HPLC, amino acid analysis, and tryptic fragmentation indicated that it corresponded in structure to alpha-N-acetyl-beta-endorphin1-26. Amino acid analysis of the native peptide and its trypsin and carboxypeptidase fragments indicated that an alanyl residue occupies position 26. This finding is in contrast to the sequence predicted for the beta-lipotropin/corticotropin precursor by recombinant DNA techniques which suggests that the 26th residue of the beta-endorphin molecule should be valine.     

Demonstration of retinal afferents in the RCS rat,with reference to the retinohypothalamic projection and suprachiasmatic nucleus

In the Royal College of Surgeons (RCS) rat, characterized by inherited retinal dystrophy, retinal projections to the brain were studied using anterograde neuronal transport of cholera toxin B subunit upon injection into one eye. The respective immunoreactivity was found predominantly contralateral to the injection site in the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, medial terminal nucleus of the accessory optic tract, and bilateral hypothalamic suprachiasmatic nuclei. Although terminal density was somewhat reduced in dystrophic rats, the projection patterns in these animals appeared similar to those seen in their congenic controls and were comparable to the visual pathways described for the rat previously. In dystrophic rats, the number of cell bodies exhibiting immunoreactivity to vasoactive intestinal polypeptide, viz. a population of suprachiasmatic neurons receiving major retinohypothalamic input, was reduced by one-third, and some differences were observed in the termination pattern of the geniculohypothalamic tract, as revealed by immunoreactivity to neuropeptide Y in the suprachiasmatic nucleus.This study was supported by grants from the DFG (Re 644/2-1) and the NMFZ, Mainz (to S.R.).     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

Surface antigen changes occurring in short-term cultures of activated human T lymphocytes: analysis by flow cytometry

Surface antigens of activated and cultured human T cells were studied using peripheral blood lymphocytes activated with conditioned medium from phytohemagglutinin-activated leukocytes and maintained in liquid culture for 2 weeks with conditioned medium containing Interleukin 2. The ensuing cell population was tested for kinetic changes in cell size and for the expression of surface antigens by immunofluorescence staining with a panel of monoclonal antibodies and analysis by flow cytometry. Upon activation, the cell population progressively increased in size to large blasts, with the rapid appearance on all of the large dividing cells of the antigen recognized by OKT9, the transferrin receptor. Cells within the population continued to express the common peripheral T-cell antigens bound by OKT3 and UCHT1, and also the antigen bound by 3A1, but never the antigen bound by OKT6, a thymic cell marker. From the time of activation an increasing proportion of the T cells, up to 80%, expressed the antigen detected with OKIa and FMC4, which recognise nonpolymorphic Ia determinants. This sequence of events was followed by a general decrease in size of the cell population, a process accompanied by further phenotypic changes. The percentage of cells expressing Ia antigens decreased, but most striking was the rapid change in the OKT4:OKT8 ratio of cells within the population, from 60:40 to 40:60. Thereafter the proportions of OKT4⁺ to OKT8⁺ cells within the cultures remained relatively stable and it is suggested that these data provide evidence for a possible change in phenotype of cultured human T lymphoblasts, from OKT4 to OKT8.     

A method for preventing sorbitol interference with the determination of inorganic phosphate

A technique is described for preventing interference of sorbitol with the assay of P₁ by modifying the procedure of B. N. Ames (1966, in Methods in Enzymology, E. F. Neufeld and V. Ginsburg, eds., Vol. 8, pp. 115–118, Academic Press, New York). The new method relies on the ability of precipitated protein to bind phosphomolybdate and so allow separation of the P₁ from the soluble sorbitol. The conditions for the formation and precipitation of phosphomolybdate-protein complex and for the subsequent assay of P₁ are described. No unique set of conditions could be found which prevented interference at all sorbitol concentrations tested. Instead, conditions for the elimination of interference by particular sorbitol concentration ranges were established. The application of the procedure to samples containing 0–150 nmol of P₁ and 10–100 μmol of sorbitol is described. Complete recovery of P₁ was achieved after precipitation. Standard plots were linear. Coefficients of variation ranged from 9% with low amounts of P₁ (≤25 nmol) to 2.5% at higher levels (150 nmol). One hundred nanomoles of P₁ gave an absorbance at 700 nm of 0.87. Modifications are described to extend the technique to different sorbitol concentration ranges and other applications of the method are mentioned.     

Highly heterogeneous residual malaria risk in western Thailand

Over the past decades, the malaria burden in Thailand has substantially declined. Most infections now originate from the national border regions. In these areas, the prevalence of asymptomatic infections is still substantial and poses a challenge for the national malaria elimination program. To determine epidemiological parameters as well as risk factors for malaria infection in western Thailand, we carried out a cohort study in Kanchanaburi and Ratchaburi provinces on the Thailand-Myanmar border. Blood samples from 999 local participants were examined for malaria infection every 4 weeks between May 2013 and Jun 2014. Prevalence of Plasmodium falciparum and Plasmodium vivax was determined by quantitative PCR (qPCR) and showed a seasonal variation with values fluctuating from 1.7% to 4.2% for P. vivax and 0% to 1.3% for P. falciparum. Ninety percent of infections were asymptomatic. The annual molecular force of blood-stage infection (_molFOB) was estimated by microsatellite genotyping to be 0.24 new infections per person-year for P. vivax and 0.02 new infections per person-year for P. falciparum. The distribution of infections was heterogenous, that is, the vast majority of infections (>80%) were found in a small number of individuals (<8% of the study population) who tested positive at multiple timepoints. Significant risk factors were detected for P. vivax infections, including previous clinical malaria, occupation in agriculture and travel to Myanmar. In contrast, indoor residual spraying was associated with a protection from infection. These findings provide a recent landscape of malaria epidemiology and emphasize the importance of novel strategies to target asymptomatic and imported infections.     

Successful cryopreservation of honey bee drone spermatozoa with royal jelly supplemented extenders

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thawing quality of drone sperm. Semen samples were collected from sexually mature drones. Pooled semen was diluted with extender without RJ (control) or supplemented with different concentrations of RJ (1, 2, 4 or 8%). Sperm motility, plasma membrane functional integrity, and acrosomal integrity were evaluated. At post thaw, the highest sperm motility and acrosomal integrity rates were obtained in the RJ1 group. Functional integrity of sperm membrane was better preserved in the RJ1 and RJ2 groups compare to the other groups. The study shows that RJ supplemented extenders have beneficial effects on drone semen parameters. The results of the present study demonstrated advantage of using 1% RJ supplemented extender.     

Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats

Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats.S.-P.H. and P.-K.L. contributed equally to this work.