An allelic series of mutations in the kit ligand gene of mice. I. Identification of point mutations in seven ethylnitrosourea-induced Kitl(Steel) alleles.

An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced Kitl(Sl) alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved alpha-helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.

An allelic series of mutations in the Kit ligand gene of mice. II. Effects of ethylnitrosourea-induced Kitl point mutations on survival and peripheral blood cells of Kitl(Steel) mice.

The ligand for the Kit receptor tyrosine kinase is Kit ligand (Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is encoded at the Steel (Sl) locus of mice. Previous studies revealed that Kitl(Sl) mutations have semidominant effects; mild pigmentation defects and macrocytic, hypoplastic anemia occur in heterozygous mice, and more severe pigmentation defects and anemia occur in homozygotes. Lethality also occurs in mice homozygous for severe Kitl(Sl) mutations. We describe the effects of seven new N-ethyl-N-nitrosourea (ENU)-induced Kitl(Sl) mutations and two previously characterized severe Kitl(Sl) mutations on pigmentation, peripheral blood cells, and mouse survival. Mice heterozygous for each of the nine mutations had reduced coat pigmentation and macrocytosis of peripheral blood. In the case of some of these mutations, however, red blood cell (RBC) counts, hemoglobin concentrations, and hematocrits were normal in heterozygotes, even though homozygotes exhibited severely reduced RBC counts and lethality. In homozygous mice, the extent of anemia generally correlates with effects on viability for most Kitl(Sl) mutations; i.e., most mutations that cause lethality also cause a more severe anemia than that of mutations that allow viability. Interestingly, lethality and anemia were not directly correlated in the case of one Kitl(Sl) mutation.

UNC-119 suppresses axon branching in C. elegans.

The architecture of the differentiated nervous system is stable but the molecular mechanisms that are required for stabilization are unknown. We characterized the gene unc-119 in the nematode Caenorhabditis elegans and demonstrate that it is required to stabilize the differentiated structure of the nervous system. In unc-119 mutants, motor neuron commissures are excessively branched in adults. However, live imaging demonstrated that growth cone behavior during extension was fairly normal with the exception that the overall rate of migration was reduced. Later, after development was complete, secondary growth cones sprouted from existing motor neuron axons and cell bodies. These new growth cones extended supernumerary branches to the dorsal nerve cord at the same time the previously formed axons retracted. These defects could be suppressed by expressing the UNC-119 protein after embryonic development; thus demonstrating that UNC-119 is required for the maintenance of the nervous system architecture. Finally, UNC-119 is located in neuron cell bodies and axons and acts cell-autonomously to inhibit axon branching.

Drosophila alpha- and beta-spectrin mutations disrupt presynaptic neurotransmitter release.

Spectrins are plasma membrane-associated cytoskeletal proteins implicated in several aspects of synaptic development and function, including presynaptic vesicle tethering and postsynaptic receptor aggregation. To test these hypotheses, we characterized Drosophila mutants lacking either alpha- or beta-spectrin. The Drosophila genome contains only one alpha-spectrin and one conventional beta-spectrin gene, making it an ideal system to genetically manipulate spectrin levels and examine the resulting synaptic alterations. Both spectrin proteins are strongly expressed in the Drosophila neuromusculature and highly enriched at the glutamatergic neuromuscular junction. Protein null alpha- and beta-spectrin mutants are embryonic lethal and display severely disrupted neurotransmission without altered morphological synaptogenesis. Contrary to current models, the absence of spectrins does not alter postsynaptic glutamate receptor field function or the ultrastructural localization of presynaptic vesicles. However, the subcellular localization of numerous synaptic proteins is disrupted, suggesting that the defects in presynaptic neurotransmitter release may be attributable to inappropriate assembly, transport, or localization of proteins required for synaptic function.

Gestational diabetes: is a higher cesarean section rate inevitable?

OBJECTIVE: To determine the rate of and indication for cesarean section for women with gestational diabetes mellitus (GDM) compared with glucose-tolerant women. RESEARCH DESIGN AND METHODS: From a consecutive series of women with GDM seen over a 9-year period for medical management, women who had had a cesarean section were identified and the reason for the section determined from a review of the medical record. A control group of women who had had a section were obtained from an existing database of glucose-tolerant women. RESULTS: The section rate for women with GDM was higher at 19.8% than the 15.6% for glucose-tolerant women. However, after adjustment for age and parity, no significant differences were found. There were also no differences found for the primary indication for section. CONCLUSIONS: In our health area of New South Wales, Australia, women with GDM do not have a higher section rate compared with glucose-tolerant women. Concerns about the diagnosis of GDM leading to an increased rate of obstetric intervention should not be generalized.

Mutations in beta-spectrin disrupt axon outgrowth and sarcomere structure.

beta-Spectrin is a major component of the membrane skeleton, a structure found at the plasma membrane of most animal cells. beta-Spectrin and the membrane skeleton have been proposed to stabilize cell membranes, generate cell polarity, or localize specific membrane proteins. We demonstrate that the Caenorhabditis elegans homologue of beta-spectrin is encoded by the unc-70 gene. unc-70 null mutants develop slowly, and the adults are paralyzed and dumpy. However, the membrane integrity is not impaired in unc-70 animals, nor is cell polarity affected. Thus, beta-spectrin is not essential for general membrane integrity or for cell polarity. However, beta-spectrin is required for a subset of processes at cell membranes. In neurons, the loss of beta-spectrin leads to abnormal axon outgrowth. In muscles, a loss of beta-spectrin leads to disorganization of the myofilament lattice, discontinuities in the dense bodies, and a reduction or loss of the sarcoplasmic reticulum. These defects are consistent with beta-spectrin function in anchoring proteins at cell membranes.

UNC-13 is required for synaptic vesicle fusion in C. elegans.

We analyzed the synaptic physiology of unc-13 mutants in the nematode C. elegans. Mutants of unc-13 had normal nervous system architecture, and the densities of synapses and postsynaptic receptors were normal at the neuromuscular junction. However, the number of synaptic vesicles at neuromuscular junctions was two- to threefold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses was almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses had morphologically docked vesicles, these vesicles were not competent for release as assayed by spontaneous release in calcium-free solution or by the application of hyperosmotic saline. These experiments support models in which UNC-13 mediates either fusion of vesicles during exocytosis or priming of vesicles for fusion.

Drosophila UNC-13 is essential for synaptic transmission.

The UNC-13 protein family has been suggested to be critical for synaptic vesicle dynamics based on its interactions with Syntaxin, Munc-18 and Doc 2alpha. We cloned the Drosophila homolog (Dunc-13) and characterized its function using a combination of electrophysiology and ultrastructural analyses. Dunc-13 contained a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression was restricted to neurons. Elimination of dunc-13 expression abolished synaptic transmission, an effect comparable only to removal of the core complex proteins Syntaxin and Synaptobrevin. Transmitter release remained impaired under elevated calcium influx or application of hyperosmotic saline. Ultrastructurally, mutant terminals accumulated docked vesicles at presynaptic release sites. We conclude that Dunc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion.

The stoned proteins regulate synaptic vesicle recycling in the presynaptic terminal.

The Drosophila stoned locus was identified 25 years ago on the basis of stress-sensitive behavioral mutants (Grigliatti et al., 1973). The locus is dicistronic and encodes two distinct proteins, stoned A and stoned B, which are expressed specifically in presynaptic terminals at central and peripheral synapses. Several stoned mutant alleles cause embryonic lethality, suggesting that these proteins are essential for synaptic function. Physiological analyses at the stoned synapse reveal severe neurotransmission defects, including reduced and asynchronous neurotransmitter release and rapid fatigue after repetitive stimulation. At the EM level, stoned synapses show a depletion of synaptic vesicles and a concomitant increase in membrane-recycling intermediates. Mutant terminals also display a specific mislocalization of the synaptic vesicle protein synaptotagmin. These results suggest that the stoned proteins are essential for the recycling of synaptic vesicle membrane and are required for the proper sorting of synaptotagmin during endocytosis.

Gestational diabetes: do lean young caucasian women need to be tested?

OBJECTIVE: The American Diabetes Association has recommended that pregnant women with low-risk factors need not be tested for gestational diabetes mellitus (GDM). The aim of this study was to determine the prevalence of GDM in women with low-risk factors and to see if the pregnancy outcomes of women with GDM from a low-risk group were different from the outcomes of other women with GDM. RESEARCH DESION AND METHODS: Over an 18-month period, all pregnant women were offered a test for GDM using a 75-g glucose tolerance test at the beginning of the 3rd trimester. GDM was diagnosed if the 2-h glucose level was > or =8.0 mmol/l. The prevalence of GDM was determined in women with defined low-risk factors (Caucasian ethnic origin, age <25 years, and BMI <25 kg/m2). The pregnancy outcomes of women with GDM from a low-risk group were compared with those of other women with GDM. RESULTS: From a tested population of 2,907 women, 573 were identified as coming from a low-risk group. The prevalence of GDM in this low-risk group was 2.8%. The pregnancy outcomes of women with GDM from a low-risk group were no different from the pregnancy outcomes of other women with GDM, with respect to frequency of insulin use, units of insulin per day, morbidity, emergency caesarian section, and the percentage of both large- and small-for-gestational-age babies. In our population, if low-risk women were excluded, 80% of women would still require testing and nearly 10% of all cases of GDM would be missed. CONCLUSIONS: Women from a low-risk group have a 2.8% prevalence rate of GDM. The pregnancy outcomes of women with GDM from a low-risk group are similar to the outcomes of other women with GDM. Concerning the use of the 75-g glucose tolerance test in pregnancy, the recommendation not to test women from a low-risk group requires further evaluation in different populations before it can be endorsed.

Responsiveness of vein bypass grafts to stimulation with norepinephrine and 5-hydroxytryptamine.

The in vitro reactivity of vein bypass grafts to norepinephrine (NE) and 5-hydroxytryptamine (5-HT) was studied in 20 rabbits undergoing bypass grafting. In these animals the right external jugular vein was grafted into the right carotid artery. The cumulative dose-response to NE and 5-HT of rings of vein grafts 2, 4, and 6 weeks after insertion was compared with that of rings from the normal contralateral jugular vein by means of an organ bath to measure changes in isometric tension. With NE there was no significant difference in the response of vein grafts harvested at 2 weeks and control veins. However, at 4 and 6 weeks there was a progressive decrease in the sensitivity of the grafts to NE. The difference in ED50 values (ED50 defined as concentration of agonist required to elicit 50% of the maximal response) between control veins and vein grafts at 4 weeks was twofold and at 6 weeks it was fivefold. None of the control veins responded to 5-HT stimulation. However, the vein grafts contracted with 5-HT, exhibiting sigmoid dose-response curves. The vein grafts showed intimal hyperplasia, which was maximal after 4 weeks. These results show that vein grafts undergo a progressive decrease in their sensitivity to norepinephrine and develop a marked, sustained increase in sensitivity to 5-HT. This finding is in contrast to previous observations in intimal hyperplastic rabbit aorta, suggesting a fundamental difference in the functional expression of arterial and venous intimal hyperplasia.