The frequency of marker changes in sugarcane plants regenerated from callus culture

This study investigates the frequency of apparent and permanent expression of marker change following two types of tissue culture, conventional callus and direct regeneration cultures, and for two markers it relates this frequency to that following breeding. Each clone was used for only one marker. After conventional callus culture, plants of the sugarcane clone Arundoid B, a clone having a growth habit with shortened internodes and leaves, were freed of this marker at a rate of 1 in 172 plants. Marker remission in a second clone with a leaf blotch was enhanced in the presence of a mutagen. Callus culture alone gave a remission rate of 1/280 plants, while treatment of callus with ethyl methanesulfonate gave a remission rate of 1/42 plants. Of two markers subjected to vegetative and sexual transmission, the first, a leaf marker, was stable in callus culture with no remissions; crossing with non-marker parents produced progeny with 54% lacking the marker. The second, a stalk marker (multibud), showed epigenetic effects during two generations of vegetative propagation; plants lacking the multibud marker produced vegetative progeny in which the marker reappeared. Nine crosses to nonmarker parents produced progeny of which an average of 29% had the marker. The use of stalk chimeras as markers demonstrated that passage through conventional callus or direct regeneration culture resulted in the loss of the donor phenotype in all plants regenerated. Phenotypic variation in plants derived from callus culture appears to arise from several sources; chimeral segregants, epigenetic transients, and mutational variants.     

Structure of equine corticotropin releasing factor

A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. The purified peptide is equipotent with human corticotropin releasing factor in an in vitro bioassay and in a human plasma binding protein assay.     

Blood Types in Relation to Depressions and Schizophrenia: A Preliminary Report

The distribution of the ABO, A₁, D, E, C, and Kell blood types in relation to selected psychiatric diagnoses was studied in over 700 consecutive admissions to a provincial mental hospital. Predicted relationships based upon recent work in other laboratories were supported by consistent trends approaching statistical significance. In addition, blood group O was found to be significantly associated with involutional melancholia.     

Inositol 1,3,4,5-tetrakisphosphate is essential for sustained activation of the Ca2+-dependent K+ current in single internally perfused mouse lacrimal acinar cells

Summary We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca²⁺-activated K⁺ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P₃) (10–100 m) evoked a transient increase in the Ca²⁺-sensitive K⁺ current that was independent of the presence of Ca²⁺ in the external solution. The transient nature of the Ins 1,4,5 P₃ effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5mm 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P₃, as well as with the stable analoguedl-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)₃) (100 m). Ins 1,3,4 P₃ (50 m) had no effect, whereas 50 m Ins 2,4,5 P₃ evoked responses similar to those obtained by 10 m Ins 1,4,5 P₃. A sustained increase in Ca²⁺-dependent K⁺ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P₄) (10 m) was added to the Ins 1,4,5 P₃ (10 m)-containing solution and this effect could be terminated by removal of external Ca²⁺. The effect of Ins 1,3,4,5 P₄ was specifically dependent on the presence of Ins 1,4,5 P₃ as it was not found when 10 m concentrations of Ins 1,3,4 P₃ or Ins 2,4,5 P₃ were used. Ins 2,4,5 P₃ (but not Ins 1,3,4 P₃) at the higher concentration of 50 m did, however, support the Ins 1,3,4,5 P₄-evoked sustained current activation. Ins 1,3,4 P₃ could not evoke sustained responses in combination with Ins 1,4,5 P₃ excluding the possibility that the action of Ins 1,3,4,5 P₄ could be mediated by its breakdown product Ins 1,3,4 P₃. Ins 1,3,4,5 P₄ also evoked a sustained response when added to an Ins 1,4,5 P(S)₃-containing solution. Ins 1,3,4,5,6 P₅ (50 m) did not evoke any effect when administered on top of Ins 1,4,5 P₃. In the absence of external Ca²⁺, addition of Ins 1,3,4,5 P₄ to an Ins 1,4,5 P₃-containing internal solution evoked a second transient K⁺ current activation. Readmitting external Ca²⁺ in the continued presence internally of Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ made the response reappear. We conclude that both Ins 1,4,5 P₃ and Ins 1,3,4,5 P₄ play crucial and specific roles in controlling intracellular Ca²⁺ homeostasis.     

A myo-inositol D-3 hydroxykinase activity in Dictyostelium.

A soluble ATP-dependent enzyme which phosphorylates myo-inositol has been characterized in Dictyostelium. The myo-inositol kinase activity was partially purified from amoebae by chromatography on DEAE-Sepharose and phenyl-Sepharose columns. The product of both the partially purified activity and of a crude cytosolic fraction was myo-inositol 3-phosphate. The partially purified preparations of myo-inositol kinase (a) possessed a Km for myo-inositol of 120 microM (in the presence of 5 mM-ATP) and for ATP of 125 microM (in the presence of 1 microM-myo-inositol), (b) did not recognize allo-, epi-, muco-, neo-, scyllo-, 1 D-chiro or 1 L-chiro-inositol as substrates, (c) were competitively inhibited by three naturally occurring analogues of myo-inositol: 1 L-chiro-inositol (Ki 49.5 +/- 0.7 microM: the structural equivalent of myo-inositol, except that the D-3 hydroxy moiety is axial), D-3-deoxy-myo-inositol [Ki 103 +/- 1 microM: (-)-viburnitol], and sequoyitol (Ki 271 +/- 7 microM; unlike 1 L-chiro-inositol and D-3-deoxy-myo-inositol, this was a substrate for the kinase), and finally (d) were apparently non-competitively inhibited by myo-inositol 3-phosphate. The product of myo-inositol kinase could be detected in intact amoebae and was a substrate for the first in a series of inositol polyphosphate kinases present in Dictyostelium which ultimately yield myo-inositol hexakisphosphate. The activity of myo-inositol D-3-hydroxykinase in Dictyostelium lysates showed evidence of developmental regulation.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

SATELLITE-MONITORED MOVEMENTS AND DIVE BEHAVIOR OF A BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) IN TAMPA BAY, FLORIDA

An adult, female bottlenose dolphin ( Tursiops trucncatus ) was radio tagged and monitored via satellite-based Argos receivers for 25 d from 28 June to 23 July 1990, in Tampa Bay, Florida. A total of 794 transmissions were obtained during 106 satellite passes. A mean of 3.9 (SE = 0.24) locations/day were determined by Service Argos and showed the animal remained in the bay, usually close to the southeastern shore. The dolphin moved at least 581 km at a minimum mean speed of 1.2 (SE = 0.1) km/h. Data from 63, 922 dives were recorded. The animal spent an average of 87.1 (SE = 0.6)% of the time submerged, with a mean dive duration of 25.8 (SE = 0.5) sec. Mean dive duration differed significantly between four periods of the day, as did the mean percent of time spent submerged. During the early morning the animal spent more time at the surface, averaged shorter dives, and was submerged less than other times of day. This is the first study to demonstrate die1 dive cycles in a bottlenose dolphin. Four months after tag loss, the dolphin was photographed with no evidence of necrosis or disfigurement of the dorsal fin. Satellite telemetry was demonstrated as an effective means of documenting the movements and dive behavior of a small inshore cetacean.     

Allylic substitutions in tri-O-acetyl-glycals and related compounds

The reaction of tri-O-acetyl-d-allal or -d-glucal, ethyl 4,6-di-O-acetyl-2,3-dideoxy-α-d)-erythro-hex-2-enopyranoside, or 1,4,6-tri-O-acetyl-α,β-d-erythro-hex-2-enopyranose with sodium azide in acetonitrile under catalysis by boron trifluoride diethyl etherate yields a mixture of 4,6-di-O-acetyl-3-azido-3-deoxy-d-allal and -d-glucal, together with both anomers of 4,6-di-O-acetyl-2,3-dideoxy-d-erythro-hex-2-enopyranosyl azide. The mechanism of these reactions is discussed. 3-Amino-3-deoxy-d-allal and -d-glucal and their derivatives are described.     

Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.