Endocrinology and paracrinology: Roles of growth factors during peri-implantation development

Several growth factor ligand and receptor gene products havebeen shown to play roles during preimplantation mammalian development.Genes for insulin-like growth factors (IGFs), transforming growthfactors (TGFs), fibroblast growth factor (FGF), platelet-derivedgrowth factor (PDGF) and receptors for insulin, IGF, PDGF, TGFand epidermal growth factor (EGF) are expressed by early embryosof several species including mouse, rat, cow and sheep. Rolesof growth factors during early development have been demonstratedby addition of purified growth factors to culture medium orby molecular genetic techniques that interfere with gene expression.In this way, it has been shown that successful development ofthe blastocyst is dependent on the action of epidermal growthfactor (EGF) and leukaemia inhibitory factor (LIF). Recent experimentsshow that both LIF and EGF stimulate secretion of urokinase-typeplasminogen activator (uPA) and gelatinase B/ matrix metalloproteinase-9(MMP-9) in day 7 mouse blastocyst outgrowths. At the same time,tissue inhibitors of MMPs (TIMPs) are also expressed by embryonic,decidual and uterine tissues during the implantation process.It appears that LIF may act directly or indirectly, by inducingthe expression of other cytokines, to regulate the temporaland spatial production and activity of proteases and proteaseinhibitors to create a favourable environment for implantation.     

Study of the HLA gene and antigen frequency from a Saudi Arabian hospital

The HLA-A, -B, -C and DR loci antigen frequencies were determined, respectively, on 1145, 558 and 352 healthy nonrelated Saudi family members. B21, CW4, CW7, and Dr7 showed the highest gene frequencies, of 14.6%, 28.3%, 7.4% and 19.5%, when compared to other populations. Haplotypes A2-B5, A32-B51, A26-B8, A2-B21, A28-B35, Aw19-B21 and B21-CW4 showed the highest frequencies when compared to other populations. Gene frequencies of 14.6% and 20.2% for B21 and Aw19 antigens, respectively, are highest among Middle East populations. Gene frequencies for A1 (10.5%), A2 (24.9%), A3 (8.9%), A9 (16.7%) and A28 (7.9%) are similar to the 10.1%, 24.9%, 8.3%, 16.8% and 7.7%, respectively, reported for the Turkish population. Also, gene frequencies for B5 (18.5%), B21 (14.6%) and B35 (10.2%) are very close to 17.1%, 14.0% and 10.2%, respectively, reported in the Yemenite population. The above results suggest some influence of other populations on the 'pure' Arab population.     

Hydroxyurea increases in vitro, antigen-independent B cell development in bone marrow.

The fluorescence-activated cell sorter and Coulter counter were used to study the effect of hydroxyurea in vitro in B cell development in 24 hr cultures of adult mouse bone marrow. This agent, which kills cells in the S phase of the cell cycle, caused a 40-70% increase in the absolute number of B cells in bone marrow cultures as compared with untreated cultures. There was also an increased response to the B cell mitogen, bacterial lipopolysaccharide, as measured by the stimulation index for 3H-thymidine incorporation. Hydroxyurea in vitro increased B cells numbers in fetal liver which contains pre-B cells, but not in lymph nodes or spleen which lack these cells.     

The glycogenolytic response to stimulation of the splanchnic nerves in adrenalectomized calves, sheep, dogs, cats and pigs

1. The effects of stimulation of the peripheral ends of one or both splanchnic nerves have been investigated in calves, sheep, dogs, cats and pigs after removal of both adrenal glands.2. Stimulation of both splanchnic nerves produced comparable hyperglycaemic and glycogenolytic effects in sheep, dogs and cats; the mean liver glycogen concentration was reduced by between 7.0 and 10.5 mg/g, five min after stimulation was discontinued, at which time the mean plasma glucose concentration had risen by between 126 and 137 mg/100 ml.3. In five 3-5 week-old calves which were tested under identical conditions the mean liver glycogen concentration was reduced by 13.3 +/- 1.9 mg/g and the plasma glucose concentration raised by 216 +/- 1.9 mg/100 ml., 5 min after stimulation was terminated.4. In pigs, stimulation of both splanchnic nerves invariably produced a rise in the plasma glucose concentration, even when the concentration of glycogen in the liver was less than 5 mg/g before stimulation. The response was, nevertheless, considerably smaller in these animals than in any of the other species investigated.5. Splanchnic nerve stimulation also caused a rise in mean aortic blood pressure and blood haematocrit during the period of stimulation; changes of approximately the same order of magnitude were encountered in all five species.6. Dogs were found to resemble calves in that the hyperglycaemic response to stimulation of a single splanchnic nerve did not differ significantly from that obtained when both were stimulated simultaneously; furthermore, either nerve was found to be equally effective.7. In cats the change in plasma glucose concentration in response to stimulation of a single splanchnic nerve was always less than that which occurred in response to bilateral stimulation although comparable changes in blood haematocrit occurred in both groups of animals.8. It is concluded that stimulation of the splanchnic nerves causes break-down of glycogen in the livers of various unrelated species of adult animals but that the magnitude of the hyperglycaemic response in the young calf provides further evidence of the importance of the sympathetic system in the control of metabolism in the young animal.     

Notes on the large scale preparation and on the properties of anti-lymphocyte serum for use in mice

Mouse spleen and thymus cells have been used in the preparation of horse anti-mouse anti-lymphocyte serum (HAMLS). The cells were used either separately or in a mixture and three types of immunization schedules were used, viz. two-pulse, extended and chronic.

Antisera of marked immunosuppressive activity, as measured by the ability to prolong the life of skin homografts in mice, were obtained using all three schedules, the median survival time being, at best, 27·5 days for the chronic schedule, 26·7 days for the two-pulse schedule and 25·8 days for the extended schedule. The two-pulse and the extended schedules produced non-toxic antisera in a relatively short period of time but were uneconomic in terms of antigen and horses. The chronic schedule was preferred but after 10 weeks the development of unwanted antibodies precluded the further useful immunization of the horses.

     

Essential light chain of Drosophila nonmuscle myosin II

Summary We have cloned and sequenced a cDNA encoding the essential (alkaline) light chain of nonmuscle myosin from Drosophila melanogaster. The protein predicted from the cDNA matches partial amino acid sequence derived from essential light chain protein that copurifies with native nonmuscle myosin heavy chain. This completes the sequence of the three myosin subunits, two of which have been shown genetically to be required for morphogenesis and cytokinesis (the heavy chain encoded by zipper and the regulatory light chain encoded by spaghetti squash). The essential light chain protein is 147 amino acids in length and is 53% identical to human smooth muscle essential light chain. The sequence is consistent with the presence of four helix-loop-helix domains seen in crystallographic structures of the striated muscle myosin light chains and their close relative, calmodulin. We identified the most conserved residues among essential light chain sequences from multiple phyla and present their locations on the crystallographic structure of striated muscle essential light chain. This highlights several conserved contacts among the myosin subunits that may be important for the structure and regulation of the myosin motor. The gene encoding Drosophila nonmuscle essential light chain (Mlc-c) localizes to cytological position 5A6 and we discuss prospects for genetic analysis in this region.     

Prenatal diagnosis in Treacher Collins syndrome using combined linkage analysis and ultrasound imaging.

Treacher Collins syndrome is an autosomal dominant disorder of facial development, the features of which include conductive hearing loss and cleft palate. In the current investigation, linkage analysis has been used to make first trimester diagnostic predictions in a pregnancy at high risk of producing an affected child. The results of this analysis predicted that the child would be affected. As predictions of the severity of the disease were not possible, the pregnancy was also assessed by ultrasound imaging. This confirmed the affected diagnosis and predicted that the child would be severely affected.     

Competitive ELISA for the measurement of tau protein in Alzheimer's disease.

Tau protein is a major component of paired helical filaments (PHFs) which constitute the characteristic neurofibrillary tangle lesions observed in Alzheimer's disease. Two tau mAbs have been produced which show distinct patterns of immunoreactivity with intact human tau and with tau incorporated in PHFs. The mAb 423 recognises PHFs but not human tau on immunoblots whereas mAb 7/51 reacts with human tau but its epitope is buried within the PHF and is only exposed after formic acid treatment. A competitive ELISA has been developed for both of these mAbs and these have been used to quantify the two distinct tau epitopes in PHFs. Samples containing antigen are incubated with horseradish peroxidase-conjugated mAb at 4 degrees C for 16 h and non-adsorbed antibody then measured by binding, at 37 degrees C for 1 h, to a fragment of tau coated on microtitre plates. Bound enzyme-labelled antibody is measured kinetically using a spectrophotometer capable of automatically mixing the samples throughout a 2-min incubation with substrate and chromogen. The interfacing of the plate reader with a computer permits competitive curves to be plotted automatically using Softmax. Curves are fitted using a 4-parameter logistic algorithm which allows one to determine the relative immunoreactivity for different samples. The application of these assays to monitoring biochemical fractions and quantifying distinct immunochemical presentations of tau protein with these two mAbs is described.     

Diadenosine polyphosphates induce intracellular Ca2+ mobilization in human neutrophils via a pertussis toxin sensitive G-protein.

The diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) and diadenosine 5',5"'-P1,P6-hexaphosphate (Ap6A) all stimulated increases in intracellular Ca2+ in human neutrophils. Maximal increases in intracellular Ca2+ of 650 nM were obtained at dinucleotide concentrations of 500-700 microM. These increases in intracellular, Ca2+ were completely abolished by pre-treatment of the neutrophils with pertussis toxin and were hardly affected when the extracellular buffer was devoid of Ca2+. On the other hand, adenosine triphosphate (ATP) could stimulate much greater increases in intracellular Ca2+ (up to 1.1 microM) at much lower concentrations (half maximal responses obtained at around 5 microM ATP). Receptor de-sensitization experiments indicate that human neutrophils may possess two types of P2-purinoceptors. The first of these may bind ATP (but not the dinucleotides) with high affinity whilst the second may bind the dinucleotides with lower affinity and also bind ATP.