Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

The role and amplification of the HS Alu subfamily founder gene

A recently identified Alu element (Leeflang et al. J. Mol. Evol. 1993, 37:559–565), referred to as the putative founder of the HS (PV) subfamily, was found to be present at orthologous loci in the human, chimpanzee, gorilla, and gibbon lineages. The evolution of this Alu suggested that it is a source gene in the evolution of Alu family repeats for one of the most recent subfamilies, HS. We have determined that this putative founder of the HS subfamily was not present at the orthologous loci in older primates, including old world and new world monkeys. Thus, this particular Alu locus has only been responsible for the establishment of a very small subfamily of Alu sequences. We have further demonstrated that this putative founder Alu was not responsible for the de novo Alu insertion into the neurofibromatosis-1 gene of an individual causing neurofibromatosis. Our data demonstrate that although the putative founder of the HS subfamily found by Leeflang et al. (1993) probably gave rise to one of the most recent subfamilies of Alu sequences, it has not been very active in retroposition. Correspondence to: T.H. Shaikh     

Evolution of the master Alu gene(s)

Summary A comparison of Alu sequences that comprise more recently amplified Alu subfamilies was made. There are 18 individual diagnostic mutations associated with the different subfamilies. This analysis confirmed that the formation of each subfamily can be explained by the sequential accumulation of mutations relative to the previous subfamily. Polymerase chain reaction amplification of orthologous loci in several primate species allowed us to determine the time of insertion of Alu sequences in individual loci. These data suggest that the vast majority of Alu elements amplified at any given time comprised a single Alu subfamily. We find that, although the individual divergence relative to a consensus sequence correlate reasonably well with sequence age, the diagnostic mutations are a more accurate measure of the age of any individual Alu family member. Our data are consistent with a model in which all Alu family members have been made from a single master gene or from a series of sequential master genes. This master gene(s) accumulated diagnostic base changes, resulting in the amplification of different subfamilies from the master gene at different times in primate evolution. The changes in the master gene(s) probably occurred individually, but their appearance is clearly punctuated. Ten of them have occurred within an 15-million-year time span, 40–25 million years ago, and 8 changes have occurred within the last 5 million years. Surprisingly, no changes appeared in the 20 milion years separating these periods.     

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

A preliminary phylogeny for the NeotropicalRubiaceae

The sequence of subfamilies,Cinchonoideae, Antirheoideae andRubioideae, attemps to show their natural affinities and phylogeny. The subfamilies are those ofVerdcourt, and the order in which they are presented is that ofBremekamp. A list is presented of the subfamilies, tribes and genera of theRubiaceae to be utilized in the Catálogo Ilustrado de las plantas de Cundinamarca, Colombia.     

Biogeochemical Processes of Methane Cycle in the Soils,Bogs, and Lakes of Western Siberia

The biogeochemical processes of methane production and oxidation were studied in the upper horizons of tundra and taiga soils and raised bogs and lake bottom sediments near the Tarko-Sale gas field in western Siberia. Both in dry and water-logged soils, the total methane concentration (in soil particles and gaseous phase) was an order of magnitude higher than in the soil gaseous phase alone (22 and 1.1 nl/cm³, respectively). In bogs and lake bottom sediments methane concentration was as high as 11 l/cm³. Acetate was the major precursor of the newly formed methane. The rate of aceticlastic methanogenesis reached 55 ng C/(cm³day), whereas that of autotrophic methanogenesis was an order of magnitude lower. The most active methane production and oxidation were observed in bogs and lake sediments, where the ¹³C values of CO₂were inversely related to the intensity of bacterial methane oxidation. Methane diffusing from bogs and lake bottom sediments showed ¹³C values ranging from –78 to –47, whereas the ¹³C value of carbon dioxide ranged from –18 to –1. In these ecosystems, methane emission comprised from 3 to 206 mg CH₄/(m²day). Conversely, the dry and water-logged soils of the tundra and taiga took up atmospheric methane at a rate varying from 0.3 to 5.3 mg CH₄/(m²day). Methane consumption in soils was of biological nature. This was confirmed by the radioisotopic method and chamber experiments, in which weighting of methane carbon was observed (the ¹³C value changed from –51 to –41).     

β-Thalassemia major resulting from a compound heterozygosity for the β-globin gene mutation: further evidence for multiple origin and migration of the thalassemia gene

Summary We describe in a Japanese family -thalassemia resulting from a compound heterozygosity for a -globin gene mutation. One mutation is a C-to-T transition at IVS-2 nucleotide position 654 on the background of Mediterranean haplotype IX. Another mutation is a G-to-A transition at IVS-2 nucleotide position 1, associated with a novel haplotype XL The occurrence of these mutations on various chromosomal backgrounds provides strong evidence for an interplay of gene migration, interallelic gene conversion, and multiple origins of the same mutation.     

5′ and 3′ SINE-PCR allows genotyping of pig families without cloning and sequencing steps

A microsatellite-containing clone, isolated from a pig Chromosome (Chr) 1-specific library was characterized by sequencing and computer analysis. The (CA)₁₇ microsatellite motif was located at the 3 end of a short interspersed element (SINE) sequence at the position normally occupied by the oligo (A) stretch. Further computer analysis indicated that 12% of published pig SINE sequences contain dinucleotide repeat motifs adjacent to their 3 ends. By performing PCR with a single SINE primer in combination with a panel of arbitrarily selected unique primers, we have demonstrated that, as in human, polymorphisms can be detected and typed in pig family DNAs. A large number of SINE primer x unique primer combinations have been screened for the ability to detect polymorphisms in pig reference family DNAs. This approach does not require prior sequence information other than that of the pig SINE. We have also found polymorphisms at the 5 ends of pig SINE sequences by similar methods, but with a primer facing out to the 5 end of the SINE.     

Restriction fragment length polymorphisms of the mouse T-cell receptor gene families

We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cell receptor genes of 25 inbred Mus musculus strains and 8 wild Mus species. Included in the inbred mice tested were several strains which spontaneously develop systemic autoimmune disease. Extensive polymorphism was evident for the variable (V) gene segments of the gene family for both the inbred strains and wild mouse species. Changes in the total number of bands hybridizing with probes for V gene segments suggest that members of a V gene segment subfamily are not closely linked, but are interspersed with members of other subfamilies; that expansion and contraction of the multimembered subfamilies may be an important diversifying factor. Our data obtained with gene probes revealed genomic diversity that is much more limited than that seen for the locus. Analysis of inbred mice with probes for the gene locus revealed some RFLPs, but little evidence of expansion or contraction in the numbers of gene segments. Among the autoimmune mice, NZW, NZB, and BXSB/MpJ all display distinctive differences with gene probes. NZW mice have a large deletion of the gene family, which has been reported previously. We found no differences to distinguish the MRL/MpJ lpr/lpr mice from non-autoimmune strains.     

Atomic structures of two nitroxide spin labels complexed with human thrombin: comparison with solution studies

Crystal structures of thrombin complexed with two spin labels called para-V, 4-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine, and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 Å resolution, respectively. Previous electron spin resonance studies with these labels gave rise to a low-resolution topography map of thrombin's extended active site. These labels monitor two distinct areas of the thrombin active site: (1) an apolar binding site which manifests itself in an biphasic activation/inhibition effect on thrombin activity and (2) a region sensitive to -thrombin autoproteolytic cleavage(s) to -thrombin (Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150, chymotrypsin numbering). Para-V was found to bind along the substrate binding cleft, while meta-V was found to bind both at the substrate primary specificity pocket and at a site which interacts with the -cleavage loop. These studies reaffirm that accurate information may be gained from solution studies and indicates the complementarity of solid-state studies.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

M?nnchen-überschu? bei Schwarzspechtnestlingen (Dryocopus martius)?

Zusammenfassung Von 1979 bis 1991 wurde auf einer etwa 200 km² großen Mischwaldfläche in Süddeutschland bei 445 Schwarzspechtnestlingen (140 Bruten; Alter der Jungvögel: 14 bis 20 Tage) eine Geschlechtsbestimmung vorgenommen. Das Gesamtgeschlechterverhältnis (0,92 :1 ; alle Daten von 1979 bis 1991 zusammengefaßt) sowie die Geschlechterverhältnisse der Einzeljahre wichen nicht signifikant von einem ausgeglichenen Geschlechterverhältnis ab. Eine statistische Überprüfung aller zum sekundären Geschlechterverhältnis beim Schwarzspecht vorliegenden Literaturangaben ergab ebenfalls in keinem Fall einen statistisch signifikanten Unterschied zwischen den ermittelten Geschlechterverhältnissen und einem Verhältnis von 1:1. Die mehrmals in der Literatur vertretene Meinung, bei Schwarzspechtnestlingen sei ein -Überschuß festzustellen, ist somit nicht länger haltbar.

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Is there a surplus of males in Black Woodpecker nestlings (Dryocopus martius)?

Summary A population of Black Woodpeckers was studied in an area of about 200 km² in southern Germany from 1978 up to 1991. Once a year the nesting trees were visited to record the age and number of young and to ring the nestlings. In 1979 the sex of seven birds (two broods) was determined additionally (crown feathers showing red from tenth day in , bluish-black in — only nape is red). From 1980 onwards the young of all broods censused were sexed (445 birds; 140 broods; age of the birds: 14 to 20 days). In contrast to several studies which had pointed to a -biased secondary sex ratio in the Black Woodpecker, but corresponding to findings in various bird species sexually monomorphic in size, neither the overall sex ratio (0,92 :1 ; pooled data from 1979 to 1991) nor the ratios of single years showed any significant deviations from 1:1. Likewise, we did not find any differences between the sex ratios of Black Woodpecker nestlings reported in the literature and a balanced ratio. For these reasons it is not tenable any longer to speak about a surplus of in Black Woodpecker nestlings.

     

Die Feinstruktur der Zirbeldrüse normaler,trächtiger und experimentell beeinflußter Meerschweinchen

Zusammenfassung In der Meerschweinchenzirbeldrüse lassen sich elektronenmikroskopisch helle und dunkle Pinealzellen sowie einzelne Gliazellen nachweisen. In den bei weitem überwiegenden hellen Pinealzellen zeichnet sich ein Teil der vesicle-crowned rodlets (VCR) durch lokale Auftreibungen aus. Von VCR deutlich abzugrenzen sind die vesicle-crowned balls (VCB). Erstmalig beschrieben wird das Vorkommen von sog. Zylindern, die als Vorstufen von VCB aufgefaßt werden. In den relativ seltenen dunklen Pinealzellen, die sich durch chromatinreiche Kerne und elektronendichtes Zytoplasma auszeichnen, sind Vesikel, VCR, VCB und Zylinder seltener als in hellen Pinealzellen. Die reichlich vorhandenen marklosen Nervenfasern finden sich vor allem in perivasculären Räumen, seltener im Parenchym. Synapsen zwischen Nerven und Pinealzellen wurden nicht beobachtet. In den Zirbeldrüsen trächtiger Meerschweinchen zeichnen sich in der 2. Hälfte der Tragzeit die hellen Pinealzellen durch stärkere Lappung der Kerne, gehäuftes Auftreten von laktiven Zonen, Vermehrung von Mitochondrien, glattem ER, agranulären Vesikeln, VCR, VCB und Zylindern aus. Die dunklen Pinealzellen nehmen während der Tragzeit an Zahl zu. Post partum bilden sich diese Veränderungen innerhalb einer Woche zurück. Längerer Aufenthalt der Tiere in Dunkelheit führt zu einer Aktivierung der hellen Pinealzellen mit auffallender Vermehrung der VCR und zu einer Zunahme der dunklen Zellen. Unter Dauerbelichtung kommt es in den hellen Zellen zu einer Abnahme fast aller Zellorganellen und zu einer starken Vermehrung der VCR, die nach 70 Tagen auch Formveränderungen aufweisen. Nach Reserpinbehandlung beobachtet man eine Verminderung und degenerative Veränderungen der VCR. Es wird diskutiert, daß die VCR als prae- bzw. postsynaptische Strukturen der Erregungsübertragung von Nerven zu Pinealzellen bzw. von Pinealzellen untereinander dienen könnten.

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The fine structure of the pineal gland of normal, pregnant and experimentally affected guinea-pigs

Summary By means of electron microscopy light and dark pinealocytes can be distinguished in the guinea-pig pineal gland. Glial cells are rare. In the light pinealocyte. the most frequent cell type, some vesicle-crowned rodlets (VCR) show circumscribed thickenings. From these structures vesicle-crowned balls (VCB) have to be clearly distinguished. Furthermore cylinders occur, which, it is suggested, are precursors of VCB. Dark pinealocytes characterized by chromatin-rich nuclei and electron-dense cytoplasm are rare and contain fewer vesicles, VCR, VCB and cylinders than light pinealocytes. Numerous non-myelinated nerve fibres are situated within perivascular spaces, a few also in the parenchyma. Synapses between nerve fibres and pinealocytes were not observed. In the pineal gland of pregnant guinea-pigs the following changes can be observed in the second half of gestation. The light cells show many nuclear indentations and an increase of active zones, mitochondria, smooth ER, agranular vesicles, VCR, VCB, and cylinders respectively. The dark cells increase in number. After birth these changes reverse to normal within one week. Constant darkness leads to an activation of the light cells accompanied by an increase of the VCR and to an increase in number of the dark cells. Under constant illumination the light cells show a decrease of their organelles and a strong increase of the VCR. After 70 days the VCR also show a change in shape. Following reserpine treatment the VCR decrease in number and show signs of degeneration. It is discussed that the VCR function as pre- or postsynaptic structures and that they are involved either in transmitting impulses from nerve fibres to pinealocytes or from one pinealocyte to the other.

Untersuchung unter Leitung von Univ.-Doz. Dr. L. Vollrath.     

Synthesis of a new neoglycopeptide for inhibition of lactose permease. Dedicated to Professor Fritz J?hnig, who inspired this work

A new neoglycopeptide was synthesized and tested for its capability to bind to lactose permease of Escherichia coli and to inhibit the transport of lactose. The free 5- carboxypentyl-1-thio--D-galactopyranoside or the protected 2,3,4,6-tetra-O-acetyl-5-carboxypentyl-1-thio--D- galactopyranoside was linked to the N-terminal -amino group of the resin bound heptapeptide H-Phe-Phe-Gly-Gly-Gly-Gly-Ala-OH by different activation methods. Upon cleavage from the resin, deacetylation and purification, a neoglycopeptide which showed a significant inhibition of lactose permease was obtained.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

Electrophoretic analysis of the high-molecular-weight glutenin subunits of Triticum monococcum,T. urartu,and the A genome of bread wheat (T. aestivum)

Summary The high molecular weight (HMW) subunit composition of glutenin was analysed by sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE) in the A genome of 497 diploid wheats and in 851 landraces of bread wheat. The material comprised 209 accessions of wild Triticum monococcum ssp. boeoticum from Greece, Turkey, Lebanon, Armenia, Iraq, and Iran; 132 accessions of the primitive domesticate T. monococcum ssp. monococcum from many different germplasm collections; one accession of free-threshing T. monococcum ssp. sinskajae; 155 accessions of wild T. urartu from Lebanon, Turkey, Armenia, Iraq, and Iran; and landraces of T. aestivum, mainly from the Mediterranean area and countries bordering on the Himalayan Mountains. Four novel HMW glutenin sub-units were discovered in the landraces of bread wheat, and the alleles that control them were designated Glu-Ald through Glu-Alg, respectively. The HMW subunits of T. monococcum ssp. boeoticum have a major, x subunit of slow mobility and several, less prominent, y subunits of greater mobility, all of which fall within the mobility range of HMW subunits reported for bread wheat. In T. monococcum ssp. monococcum the range of the banding patterns for HMW subunits was similar to that of ssp. boeoticum. However, two accessions, while containing y subunits were null for x subunits. The single accession of Triticum monococcum ssp. sinskajae had a banding pattern similar to that of most ssp. boeoticum and ssp. monococcum accessions. The HMW subunit banding patterns of T. urartu accessions were distinct from those of T. monococcum. All of them contained one major x and most contained one major y subunit. In the other accessions a y subunit was not expressed. The active genes for y subunits, if transferred to bread wheat, may be useful in improving bread-making quality.     

The evolution of the type I interferon gene family in mammals

A phylogenetic analysis of mammalian type I interferon (IFN) genes showed: (1) that the three main subfamilies of these genes in mammals (IFN-, IFN-, and IFN-) diverged after the divergence of birds and mammals but before radiation of the eutherian orders and (2) that IFN- diverged first. Although apparent cases of interlocus recombination among mouse IFN- genes were identified, the hypothesis that coding regions of IFN- genes have been homogenized within species by interlocus recombination was not supported. Flanking regions as well as coding regions of IFN- were more similar within human and mouse than between these species; and reconstruction of the pattern of nucleotide substitution in IFN- coding regions of four mammalian species by the maximum parsimony method suggested that parallel substitutions have occurred far more frequently between species than within species. Therefore, it seems likely that IFN- genes have duplicated independently within different eutherian orders. In general, type I IFN genes are subject to purifying selection, which in the case of IFN- and IFN- is strongest in the putative receptor-binding domains. However, analysis of the pattern of nucleotide substitution among IFN- genes suggested that positive Darwinian selection may have acted in some cases to diversify members of this subfamily at the amino acid level.     

Effect of “fasting” and different concentrations of glucose on α-linolenic acid metabolism in HTC cells. Correlation with the ultrastructural study

Summary HTC cells are able to convert -linolenic acid into higher homologs by desaturating and elongating reactions. When the cells were cultured in a Krebs Ringer bicarbonate solution (fasted cells) a decrease in both biosynthetic reactions took place. Refeeding the cells with Swim's 77 medium without glucose enhanced the biosynthesis of polyunsaturated fatty acids from -linolenic acid family, but when glucose was added to the medium, -linolenic acid was preferably elongated rather than converted into eicose-pentaenoic acid.The ultrastructural study revealed HTC cells with a simple cytoplasmic organization, showing little evidence of their origin from hepatocytes. The cells cultured in a complete medium appeared well preserved and were similar to those fasted for 12 hours and refed for another 12 hours using Swim's 77 medium without serum. The amount of glucose in the medium plays a role in preserving the cell structure. This effect does not occur if glucose is added in the absence of aminoacids and vitamins.