Further studies on the lipid metabolism of the normal and vitamin B12-deficient chick embryo

1. The triglyceride, cholesterol ester and total phospholipid fractions were isolated from the livers and yolk sacs of normal and vitamin B₁₂-deficient chick embryos after 13, 15, 17, 19 and 21 days of incubation, and the fatty acid compositions were determined. 2. At all stages of incubation, the concentration of cholesterol ester in the livers of the normal embryos were greater, and on days 15 and 17 the concentrations of triglyceride were considerably less, than the corresponding concentrations in the livers of the deficient embryos. 3. Between day 13 and day 21 of incubation the concentration of oleic acid in the liver triglycerides of the normal embryos increased, whereas the concentrations of palmitic acid and docosahexaenoic acid decreased. Vitamin B₁₂ deficiency resulted in higher concentrations of palmitic acid in the liver triglycerides on days 15, 17 and 19, higher concentrations of C₁₈ polyunsaturated acids on days 13 and 15 and lower concentrations of oleic acid on days 13, 15, 17 and 19. 4. At all stages of development, cholesterol oleate accounted for almost 80% of the total liver cholesterol esters in both normal and deficient embryos. 5. As development of the normal embryos progressed, the concentrations of palmitic acid and arachidonic acid in the liver phospholipid decreased, whereas the concentrations of stearic acid and docosahexaenoic acid increased. Vitamin B₁₂ deficiency resulted in markedly higher concentrations of stearic acid and palmitic acid and markedly lower concentrations of arachidonic acid and docosahexaenoic acid in the liver phospholipids. 6. Vitamin B₁₂ deficiency did not influence the fatty acid composition of the triglyceride, cholesterol ester and phospholipid fractions either in the yolks of fertile unincubated eggs or in the yolks obtained from eggs that had been incubated for 13, 15, 17, 19 and 21 days.     

Equilibrium,kinetic and structural properties of hemoglobin Cranston,an elongated β chain variant

Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston³ at 20 °C in the absence of phosphate revealed a high affinity (P₅₀ = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P₅₀ = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′₄, of ～ 6 × 10⁶m^?1s^?1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ～ 0·5 × 10⁶m^?1s^?1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k₄～ 11 s^?1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α₂β^Aβ^Cranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.     

The Staphylococcal Insertion Sequence IS257Is Active

The plasmid pJ3356 confers high-level mupirocin resistance on a strain of Staphylococcus aureus isolated from a hospital. The plasmid also carries two copies of IS257. Recombination of an IS257-containing plasmid conferring erythromycin resistance, pOX7-IS, into either of the IS257s of pJ3356 has been observed. The co-integration of pJ3356 and a small plasmid, pOX7, is also reported and involves duplication of one of the IS257s from pJ3356 together with 8 bp of pOX7 at the site of integration. Thus IS257 has been shown to be an active mobile genetic element.     

Differential Regulation of D1 Dopamine Receptor- and of A2a Adenosine Receptor-Stimulated Adenylyl Cyclase by μ-, δ1-, and δ2-Opioid Agonists in Rat Caudate Putamen

Abstract: Inhibition and stimulation of adenylyl cyclase by opioid and D₁ dopamine or A_2a adenosine agonists, respectively, were characterized in the caudate putamen of rats. D₁ dopamine receptors have been reported to be localized preferentially on striatonigral neurons and A_2a adenosine receptors on striatopallidal neurons. The aim of the present study was to evaluate the effects of μ-[Tyr-d -Ala-Gly-(N-Me)Phe-Gly-ol (DAMGO)], δ₁-[Tyr-d -Pen-Gly-Phe-d -Pen (DPDPE)], and δ₂- ([d -Ala²]deltorphin-II [DT-II]) opioid agonists on the D₁ dopamine receptor- and A_2a adenosine receptor-stimulated adenylyl cyclase in membranes from rat caudate putamen. The results show that DAMGO, DPDPE, and DT-II inhibit forskolin-stimulated adenylyl cyclase [selectively antagonized by d -Phe-Cys-Tyr-d -Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; μ antagonist), 7-benzylidenenaltrexone (BNTX; δ₁ antagonist), and naltriben (NTB; δ₂ antagonist), respectively], but only μ- and δ₂-opioid agonists inhibit D₁ dopamine-stimulated adenylyl cyclase (antagonized by CTOP and NTB, respectively). Furthermore, DT-II and DPDPE inhibit A_2a adenosine-stimulated adenylyl cyclase (antagonized by NTB and BNTX, respectively), whereas DAMGO did not inhibit A_2a adenosine-stimulated adenylyl cyclase activity. These results suggest that μ-, δ₁-, and δ₂-opioid receptors display differential localization and provide neurochemical evidence suggesting the differential location of the δ₁ and δ₂ subtypes. μ-Opioid receptors may be preferentially expressed by striatonigral neurons, δ₁- by striatopallidal neurons, and δ₂- by these two striatal efferent neuron populations.     

Prolonged P300 latency in children with the D2 dopamine receptor A1 allele.

Previous studies have indicated the presence of a hereditary component in the generation of the P300, or P3, a late positive component of the event-related potential. Moreover, the dopaminergic system has been implicated in the P3. In the present study, 98 healthy Caucasian boys, mean age of 12.5 years and of above-average intelligence, were studied. The sample was composed of 32 sons of active alcoholic (SAA) fathers, 36 sons of recovering alcoholic (SRA) fathers, and 30 sons of social drinker (SSD) fathers, with none of them having yet begun to consume alcohol or other drugs. TaqI A D2 dopamine receptor alleles (A1 and A2) were determined. A significant difference in the frequency of the A1 allele was found among these three groups of boys, with the SAA group having the highest A1 allele frequency (.313), followed by the SRA (.139) and the SSD (.133) groups. The relationship of the A1 and A2 alleles to P3 amplitude and latency was also determined. The results showed no significant difference in P3 amplitude between boys with the A1 and A2 allele. However, P3 latency was significantly longer in the total sample of boys with the A1 allele compared with those carrying the A2 allele. These findings suggest that polymorphism of the D2 dopamine receptor gene is an important determinant of P3 latency.     

Effect of relative humidity on fungal colonization of fiberglass insulation.

Fiberglass duct liners and fiberglass duct boards from eight buildings whose occupants complained of unacceptable or moldy odors in the air were found to be heavily colonized by fungi, particularly by Aspergillus versicolor. Unused fiberglass was found to be susceptible to fungal colonization in environmental chambers dependent upon relative humidity. No colonization was observed at relative humidities below 50%.     

Fungal colonization of fiberglass insulation in the air distribution system of a multi-story office building: VOC production and possible relationship to a sick building syndrome

Complaints characteristic of those for sick building syndrome prompted mycological investigations of a modern multi-story office building on the Gulf coast in the Southeastern United States (Houston-Galveston area). The air handling units and fiberglass duct liner of the heating, ventilating and air conditioning system of the building, without a history of catastrophic or chronic water damage, demonstrated extensive colonization withPenicillium spp andCladosporium herbarum. Although dense fungal growth was observed on surfaces within the heating-cooling system, most air samples yielded fewer than 200 CFU m^–3. Several volatile compounds found in the building air were released also from colonized fiberglass. Removal of colonized insulation from the floor receiving the majority of complaints of mouldy air and continuous operation of the units supplying this floor resulted in a reduction in the number of complaints.     

Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein lambda1.

A previously identified nucleoside triphosphatase activity in mammalian reovirus cores was further characterized by comparing two reovirus strains whose cores differ in their efficiencies of ATP hydrolysis. In assays using a panel of reassortant viruses derived from these strains, the difference in ATPase activity at standard conditions was genetically associated with viral genome segment L3, encoding protein lambda1, a major constituent of the core shell that possesses sequence motifs characteristic of other ATPases. The ATPase activity of cores was affected by several other reaction components, including temperature, pH, nature and concentration of monovalent and divalent cations, and nature and concentration of anions. A strain difference in the response of core ATPase activity to monovalent acetate salts was also mapped to L3/lambda1 by using reassortant viruses. Experiments with different nucleoside triphosphates demonstrated that ATP is the preferred ribonucleotide substrate for cores of both strains. Other experiments suggested that the ATPase is latent in reovirus virions and infectious subviral particles but undergoes activation during production of cores in close association with the protease-mediated degradation of outer-capsid protein mu1 and its cleavage products, suggesting that mu1 may play a role in regulating the ATPase.     

Proton Nuclear Magnetic Resonance Spectroscopy of Primary Cells Derived from Nervous Tissue

Abstract: Cell culture techniques, high-resolution in vitro ¹H nuclear magnetic resonance (NMR) spectroscopy, and chromatographic analyses were used to compare the properties of purified cell populations derived from the PNS and cortical neurones. Cell cultures were immunocytochemically characterised with specific antibodies to ensure purity of the individual cultures. Spectra of perchloric acid extracts of cultured Schwann cells, perineural fibroblasts, dorsal root ganglion neurones, and cortical neurones displayed several common features. However, statistically significant differences were found by ¹H NMR spectroscopy in most metabolites among the cell types studied. In addition, cells could be distinguished by the presence or absence of certain amino acids. For example, N -acetylaspartate was present in dorsal root ganglion neurones and cortical neurones, γ-aminobutyric acid was present in large amounts in cortical neurones, and Schwann cell spectra displayed a large signal from glycine. These results extend our earlier findings that different cell types of the CNS exhibit highly characteristic metabolite profiles to now include the major cell types of the PNS. These latter cell types also exhibit characteristic metabolite compositions, such that even Schwann cells and oligodendrocyte type 2 astrocyte (O-2A) progenitor cells—precursors of the myelinating cells of the CNS and PNS, respectively—can be readily distinguished from each other.