Properties of Chicken Lens MIP Channels Reconstituted into Planar Lipid Bilayers

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V ₀ = 18.5 mV, n= 4.5 and g _min/g _max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels. Received: 27 March 1996/Revised: 5 August 1996     

Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies

 Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percent- age of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA in the first patient’s cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation. Received: 16 April 1996/Accepted: 29 July 1996     

Resolution of pheromone pulses in receptor cells of Antheraea polyphemus at different temperatures

The ability of pheromone receptor cells of male Antheraea polyphemus (Saturniidae) to resolve stimulus pulses was determined at different temperatures (8°, 18°, 28°C). The cells were stimulated by repeated 20-ms puffs of the pheromone components (E, Z)-6, 11-hexadecadienyl acetate and (E, Z)-6,11-hexadecadienal. At higher temperatures, higher frequencies of stimulus pulses were resolved by the nerve-impulse response: about 1.25 pulses per second at 8°C, 2.5 pulses/s at 18°C and 5 pulses/s at 28°C. The decreased ability of receptor cells to resolve stimulus pulses at low temperatures may reduce the male moth's chance of reaching the pheromone source. The peak nerve-impulse frequency increased whereas the duration of nerve-impulse responses to single stimulus pulses decreased at higher temperatures. At a given temperature and stimulus intensity the peak nerveimpulse frequency decreased with shorter intervals between the stimulus pulses, but the duration of the responses remained almost constant. The time needed for recovery from adaptation caused by a single stimulus pulse was longer at lower temperatures. The aldehyde receptor cell recovered more quickly than the acetate cell. At low stimulus concentration, the resolution ability of the acetate cell was strongly decreased, whereas in the aldehyde cell it was only slightly impaired.     

Plasma and erythrocyte manganese concentrations

This study was carried out to assess manganese (Mn) status after an acute episode of myocardial infarction. Plasma and erythrocyte Mn concentrations were measured from admission to hospital to day 15 postadmission in 21 patients suffering from acute myocardial infarction and in three control groups. The determination of Mn in these biological fluids was performed by electrothermal atomic absorption spectrometry. Plasma Mn was higher (p<0.01) and erythrocyte Mn was similar in the acute myocardial infarction group compared to healthy age-matched control group. Plasma and erythrocyte Mn remained unchanged during the 2 wk after acute myocardial infarction and were not correlated to enzyme activities. A decrease of erythrocyte Mn with age, expressed in nmol/L, was noted (p<0.02). These results suggest that plasma and erythrocyte Mn do not provide an indication of myocardial damage. Nonetheless, Mn status in elderly merits further attention.     

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan^R) and -glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan^R calli. A high frequency of plants regenerated from Kan^R calli expressed both Kan^R and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.     

Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

胶孢镰刀菌产生串珠镰刀菌素的不稳定性

从陕北克山病病区分离到的两株串珠镰刀菌素产生菌株——胶孢镰刀菌(Fusarium subglutinans Wollenw.et Reinking)陕-6号和2-17号进行单孢分离,分别得到23和19个单孢分离株。这些单孢菌株可分为两种培养型:一种形态上与原始菌株相似,产生串珠镰刀菌素,产色素,具有大、小分生孢子,转管八次产毒量有下降;另一种则不产串珠镰刀菌素和孢子,无色素,后者在二株菌的单孢分离菌中所占比例分别为60.9％和15.8％。由此可见,胶孢镰刀菌产毒稳定性受异核体和该菌单核变异性共同影响。     

水稻主要病虫综合防治专家系统—系统外壳的研制

按照国家八五项目要求,以水稻病虫害管理知识为背景,笔者构建了水稻主要病虫综防专家系统外壳（ESRIPM）．并初步建立了珠江三角洲稻区主要害虫综合防治专家系统,系统中提出一种知识的组织方法,成功地把模型引入专家系统中．本系统具有知识编辑、咨询、专家系统;系统维护、4个一级子系统,系统中已装入三化螟,稻飞虱,稻纵卷叶螟等害虫综防管理的知识,并通过调试．本系统采用下拉式菜单,人机界面友好,操作简单．此专家系统外壳也适用建立其它各种害虫管理的专家系统．     

Comparative studies on the dynamics of crosslinked insulin

Molecular dynamics simulations were carried out on an insulin crosslinked between the N-terminal A chain and the C-terminal B chain to form a so-called mini-proinsulin: N -^A1-N -^B29-diaminosuberoyl insulin (DASI). To investigate the influence of crosslinking on the dynamics of the insulin moiety, the bridge was removed from a transient DASI structure and simulation was carried on independently with the then unlinked (ULKI) as well as with the crosslinked species. The effects of crystal packing and quaternary interactions were checked by simulating both types of monomers and dimers known from the hexamer structure. All simulations were compared to previous ones of native insulin. DASI shows general similarity to the native simulations in most parts of the structure. Deviations are visible in the segments to which the bridge is directly connected, i.e. their flexibility is reduced. Upon removal of the bridge the ULKI simulations reapproach those of native insulin. The influence of the bridge spreads over the whole molecule, but all of its main structural features remain intact. The simulations suggest that the displacement of the C-terminal B chain of native insulin, considered important for receptor interaction, is prevented by the bridge, which also partially shields some binding residues. This is in accordance with the poor biological potency of A1-B29-crosslinked insulins.Abbreviations DASI-insulin(DASI) bovineN -^A1-N -^B29-di-aminosuberoyl insulin - ULK-insulin (ULKI) Native beef insulin with the bridge of DASI removed     

Human RNaseP RNA and nucleolar 7-2 RNA share conserved ‘To’ antigen-binding domains

RNase P in both prokaryotes and eukaryotes is a ribonucleoprotein that cleaves tRNA precursors to generate the 5 termini of the mature tRNAs. Many patients with autoimmune diseases produce antibodies against a 40 kDa protein (designatedTo orTh antigen) which is an integral component of eukaryotic RNaseP as well as nucleolar 7-2 RNP which is identical to the mitochondrial RNA processing (MRP) RNP. Interestingly, theTo antigen found in human cells and the C5 protein, the only protein component ofE. coli RNaseP, are antigenically related. In this study, we show that a 56 nucleotide-long sequence, corresponding to nucleotides 20–75 near the 5 end of human RNaseP RNA, is sufficient to bind theTo antigen. We previously showed that the humanTo antigen binds to a short distinct structural domain near the 5 end of human 7-2/MRP RNA. There is no obvious primary sequence homology between theTo antigen binding sites in RNaseP RNA and 7-2/MRP RNA; however, these sequences are capable of assuming a similar secondary structure which corresponds to the recently proposed cage structure for RNaseP RNAs and 7-2/MRP RNA (Forster and Altman (1989) Cell 62: 407–409). These data are supportive of the idea that these two RNAs may have evolved from a common progenitor molecule.