Microbial growth on C1 compounds: Uptake of [14C]formaldehyde and [14C]formate by methane-grown Pseudomonas methanica and determination of the hexose labelling pattern after brief incubation with [14C]methanol

1. A study has been made of the incorporation of carbon from [¹⁴C]formaldehyde and [¹⁴C]formate by cultures of Pseudomonas methanica growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compounds for periods of up to 1min., has been analysed by chromatography and radioautography. 3. Radioactivity was fixed from [¹⁴C]formaldehyde mainly into the phosphates of the sugars, glucose, fructose, sedoheptulose and allulose. 4. Very little radioactivity was fixed from [¹⁴C]formate; after 1min. the only products identified were serine and malate. 5. The distribution of radioactivity within the carbon skeleton of glucose, obtained from short-term incubations with [¹⁴C]methanol of Pseudomonas methanica growing on methane, has been investigated. At the earliest time of sampling over 70% of the radioactivity was located in C-1; as the time increased the radioactivity spread throughout the molecule. 6. The results have been interpreted in terms of a variant of the pentose phosphate cycle, involving the condensation of formaldehyde with C-1 of ribose 5-phosphate to give allulose phosphate.     

A revision of coregonine fish distribution and abundance in eastern James-Hudson Bay

Synopsis Recent sampling programs conducted in the estuaries of the Eastmain and La Grande rivers (James Bay) and the Great Whale, Little Whale, Innuksuac and Povungnituk rivers (Hudson Bay) revealed patterns of coregonine fish distribution that differ from previous observations. The relative abundance of cisco, Coregonus artedii, and lake whitefish, C. clupeaformis, varied among rivers but did not reveal a latitudinal cline. Previous sampling programs underestimated the abundance of cisco in the Little Whale River. In addition, cisco was the third most abundant species captured in the Povungnituk River, situated 200 km to the north of the previously proposed northern limit at Innuksuac River. As such, the low abundances of cisco in the Great Whale and Innuksuac rivers cannot be attributed to a physiological inability to cope with a reduced growing season. Immature cisco were almost totally absent from the estuaries of the Hudson Bay rivers following spring breakup whereas immature lake whitefish made up 100% of the catch in the Innuksuac River at the same time of year. Species-specific migration patterns in Hudson Bay that differ from those observed in James Bay and the existence of unique juvenile overwintering rivers are 2 hypotheses proposed to explain the discontinuous age-class distribution of cisco and lake whitefish observed in Hudson Bay.Contribution to the program of GIROQ (Groupe Interuniversitaire de Recherche Océanographique du Québec).     

Atrial natriuretic peptide-dependent phosphorylation of smooth muscle cell particulate fraction proteins is mediated by cGMP-dependent protein kinase

The atrial natriuretic peptide (ANP) stimulates cGMP production and protein phosphorylation in a particulate fraction of cultured rat aortic smooth muscle cells. Three proteins of 225, 132, and 11 kDa were specifically phosphorylated in response to ANP treatment, addition of cGMP (5 nM), or addition of purified cGMP-dependent protein kinase. The cAMP-dependent protein kinase inhibitor had no effect on the cGMP-stimulated phosphorylation of the three proteins but inhibited cAMP-dependent phosphorylation of a 17-kDa protein. These results demonstrate that the particulate cGMP-dependent protein kinase mediates the phosphorylation of the 225-, 132-, and 11-kDa proteins. The 11-kDa protein is phospholamban based on the characteristic shift in apparent Mr from 11,000 to 27,000 on heating at 37 degrees C rather than boiling prior to electrophoresis. ANP (1 microM) increased the cGMP concentration approximately 4-fold in the particulate fractions, from 4.3 to 17.7 nM, as well as the phosphorylation of the 225-, 132-, and 11-kDa proteins. In contrast, the biologically inactive form of ANP, carboxymethylated ANP (1 microM), did not stimulate phosphorylation of any proteins nor did the unrelated peptide hormone, angiotensin II (1 microM). These results demonstrate the presence of the cGMP-mediated ANP signal transduction pathway in a particulate fraction of smooth muscle cells and the specific phosphorylation of three proteins including phospholamban, which may be involved in ANP-dependent relaxation of smooth muscle.     

Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus

Cattle can be vaccinated against the tick Boophilus microplus by inducing an immunologic reaction against Ag in the tick gut. The uptake of antibody during feeding leads to severe damage to the parasite. One of the responsible tick gut Ag has now been purified and characterized: the Bm86 Ag. It is a membrane-bound glycoprotein present in very low abundance in extracts of partially engorged adult female ticks. It has an apparent m.w. of 89,000, an isoelectric point of 5.1 to 5.6 and an affinity for wheat germ lectin. Microgram amounts of this Ag are able to induce effective protection in cattle against the parasite, as shown by the decreased survival of ticks on vaccinated cattle and a reduction in engorgement weights and egg laying capacity of the survivors. Antisera to the Ag react with the surface of digest cells in the tick gut. As a result of the reaction with antibody, the endocytotic activity of these cells, which is a critical step in bloodmeal digestion in this tick, is strongly and rapidly inhibited. A number of peptides from this Ag, produced by digestion of the reduced and alkylated protein with endoproteinase lys-C, have been sequenced. One peptide has significant amino acid sequence homology with the epidermal growth factor precursor and a second peptide has homology with a putative protective antigen from Plasmodium falciparum.     

The herpes simplex virus protein Vmw65 can trans-activate both viral and cellular promoters in neuronal cells.

Somatostatin inhibited Ca2(+)-induced insulin secretion in permeabilized HIT-T15 cells, albeit with decreased sensitivity relative to intact cells. The inhibitory action required the presence of GTP, whereas GDP could not substitute for GTP. Pertussis-toxin treatment before cell permeabilization abolished the inhibition of secretion. Thus somatostatin, by activating a G-protein, interferes with exocytosis distal to the generation of soluble intracellular messengers.     

The smooth muscle 132 kDa cyclic GMP-dependent protein kinase substrate is not myosin light chain kinase or caldesmon.

Atrial natriuretic peptide (ANP) stimulates the phosphorylation of three cyclic GMP-dependent protein kinase substrate proteins of 225, 132, and 11 kDa (P225, P132 and P11 respectively) in the particulate fraction of cultured rat aortic smooth muscle cells [Sarcevic, Brookes, Martin, Kemp & Robinson (1989) J. Biol. Chem. 264, 20648-20654]. Vrolix, Raeymaekers, Wuytack, Hofmann & Casteels [(1988) Biochem. J. 255, 855-863] have reported the presence of a 130 kDa cyclic GMP-dependent protein kinase substrate protein in the membrane fraction of pig aorta or stomach, and suggested that it may be myosin light chain kinase (MLCK). The aim of the present study was to determine whether P132 from rat aorta was MLCK or caldesmon. Although P132 co-migrates with purified chicken gizzard MLCK on SDS/polyacrylamide gels, it is distinct from rat aortic MLCK. Partially purified MLCK from rat aorta migrated as a 145 kDa protein on SDS/polyacrylamide gels. Immunoblotting the partially purified rat aortic MLCK with antibody to bovine tracheal MLCK identified rat aortic MLCK (145 kDa) and a corresponding 145 kDa protein in the particulate fraction of cultured rat aortic smooth muscle cells, but did not detect the 132 kDa protein. Phosphopeptide maps of purified rat aortic MLCK prepared by digestion with Staphylococcus aureus V8 protease were distinct from those of P132. P132 was not caldesmon, since antibodies to caldesmon cross-reacted with 136 and 76 kDa proteins in the particulate fraction of rat aortic cells, but not with P132. Furthermore, caldesmon was partially extracted from the particulate into the soluble fraction by heating at 90 degrees C, whereas P132 was not. These results demonstrate that the ANP-responsive cyclic GMP-dependent protein kinase substrate of 132 kDa from rat aortic smooth muscle cells is not MLCK or caldesmon.     

No evidence for ‘skewed’ inactivation of the X-chromosome as cause of Leber's hereditary optic neuropathy in female carriers

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder of the optic nerves. It has been proposed that the specific mutations in the mitochondrial DNA (mtDNA) that are associated with LHON require and X-chromosomally encoded permissive factor in order to become expressed. This would explain both the preponderance of male patients and the fact that most carriers of specific mtDNA mutations remain unaffected. Although linkage studies have been negative so far, the existence of such a factor has not been ruled out. We investigated the genealogical data of 24 large LHON pedigrees and concluded that the presumed X-linked factor would be recessively inherited and that at least 57% of the affected females would be heterozygous. Therefore, these females must be the victim of nonrandom X-chromosomal inactivation (skewed lyonization). However, analysis of X-chromosomal methylation patterns in 16 LHON-affected females revealed substantial skewing in only 15%–20% of cases, which is not significantly different from the patterns in 49 controls. Moreover, we found the frequency of LHON in daughters of affected heterozygous females to be twice to three times as high as in daughters of unaffected heterozygous females, which cannot be explained by an X-chromosomally inherited factor. We conclude that the results of our investigations do not support the hypothesis that LHON is a digenic disease with an X-linked factor being the main cause of loss of vision in the presence of relevant mtDNA mutations. Received: 1 June 1995 / Revised: 20 September 1995     

Measles immunisation in children with allergy to egg.

OBJECTIVE--To examine the occurrence of adverse reactions to measles vaccine given as a single dose to children with egg allergy, and to determine if the administration of single dose to children with a positive result in an intradermal skin prick test with the vaccine is associated with adverse reactions. DESIGN--Review of results of immunisation and prospective study of 96 consecutively presenting children given intradermal skin testing with the vaccine. SETTING--Children''s allergy centre. SUBJECTS--410 children sensitive to egg referred to the allergy unit for advice about measles immunisation. MAIN OUTCOME MEASURES--Nature and severity of reactions associated with the administration of measles vaccine. RESULTS--All children had a positive result in a skin prick test with egg white, and five had a positive result in a skin prick test with vaccine. Of 96 consecutive children, 46 had a positive result in an intradermal test with vaccine. After immunisation with a full dose (0.5 ml) of vaccine adverse reactions were associated with a mild reaction in four children, none of whom required treatment. Only one of the 46 children with a positive result in an intradermal vaccine skin test had a reaction associated with vaccine administration. None of the children with a positive result in a skin prick test with measles vaccine reacted to the vaccine. The rate of minor reactions to the vaccine not requiring treatment was 0.98% (95% confidence interval 0.27% to 2.48%) and serious reactions requiring treatment was 0% (0% to 0.9%). CONCLUSION--Children with IgE mediated allergic reactions to egg protein should be investigated and managed by practitioners with special knowledge in this subject. Measles immunisation should be performed in a setting where any adverse reactions can be dealt with appropriately. Skin tests and measles vaccine and desensitisation are not necessary.