The incorporation of acetate,stearate and d(?)-β-hydroxybutyrate into milk fat by the isolated perfused mammary gland of the goat

1. Mammary glands of lactating goats were perfused for 12.5-15hr. with heparinized whole blood and infused with a substrate mixture of glucose, acetate and amino acids (and sometimes chylomicra) containing either [1-(14)C]acetate, d(-)-beta-hydroxy[1-(14)C]butyrate or [U-(14)C]stearate. 2. There was a substantial net uptake of acetate by the glands and transfer of radioactivity into milk fat. Acetate was extensively utilized for the synthesis of milk fatty acids of chain length up to C(14) and to a smaller extent for the synthesis of palmitate. 3. There was a small and variable net uptake of stearate and beta-hydroxybutyrate and negligible oxidation of these substrates. However, tissue uptake was demonstrated by a substantial fall in specific radioactivity across the glands and an extensive transfer of radioactivity into milk fatty acids. 4. With beta-hydroxybutyrate the labelling of milk fat was very similar to that with acetate, but the distribution of radioactivity suggested a cleavage into C(2) fragments of about 40%. 5. Labelled stearate gave rise to highly labelled stearate and oleate in the milk fat. Small amounts of radioactivity were detected in stearate of plasma triglycerides and oleate of plasma free fatty acids. 6. In experiments where there was a decline in milk-fat secretion late in perfusion, the milk fatty acids showed a marked decline in the proportion of stearate and oleate and a rise in the proportion of myristate and palmitate. This did not occur in experiments where milk-fat secretion was maintained at a higher level. 7. The present results confirm that there is a large pool of long-chain fatty acids in mammary tissue that can act as an endogenous source of these substrates.     

溴氰菊酯对鼠脑蛋白质磷酸化作用的影响

以小鼠大脑碎片与[γ-~(32)P]ATP一起保温,观察到溴氰菊酯对蛋白1—3磷酸化的刺激作用和对4、5磷酸化的抑制作用,表明溴氰菊酯对大脑蛋白质磷酸化产生了影响。从鼠脑分离了C、D、S三个组分,分别进行的蛋白质磷酸化试验结果表明,C、D组分可能是重要的磷酸化部位。 蛋白1、2、3的磷酸化明显地受到溴氰菊酯的刺激,这三个蛋白质可能是“蛋白Ⅲb”的几种形式。溴氰菊酯对“蛋白Ⅲb”磷酸化的刺激,可能会影响神经末梢的神经激素释放,从而影响到与其相关的某些神经功能。     

Increased regional vascular albumin permeation in the rat during anaphylaxis

The changes in vascular albumin permeation induced by systemic anaphylaxis were studied simultaneously in 21 different tissues of the same animal. Before Ag challenge sensitized rats were injected i.v. with 125I-albumin (test tracer), 51Cr-RBC (vascular space marker) and 57Co-EDTA (extravascular space marker). The index of vascular permeation used was the tissue to blood isotope ratio (tbir), which was obtained by dividing the ratio of 125I/51Cr counts in each tissue by the ratio of the same isotopes in the arterial blood sample. After Ag challenge, the increase in the tbir varied considerably among the different tissues. The most pronounced increase was noted in the lymph node (ninefold) followed by the aorta and mesentery (six- to sevenfold) and the various parts of the gastrointestinal tract (four- to sixfold). In the skin less than skeletal muscle less than lung less than liver and eye two- to fourfold increases occurred. Relatively minor increases in albumin permeation (less than twofold) were observed in the brain less than kidney less than heart and less than spleen. The testis was the only organ in which no significant change occurred. For some of the tissues there was also an increase in the tbir for 57Co/51Cr (an index of the extracellular fluid space) suggesting edema formation. The highest increase was noted in the aorta (fourfold). Minor increases occurred in the atrium of the heart, stomach, duodenum, and lymph nodes. There was also a 36% increase in hematocrit. Therefore, systemic anaphylaxis caused extensive extravasation of albumin and hemoconcentration.     

The MgATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme.

An ATP-like affinity labeling reagent, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), was used to probe the MgATP-binding site of smooth muscle myosin light chain kinase from chicken gizzard (smMLCK) and its calmodulin (CaM) complex. Native smMLCK has an absolute requirement for the binding of the calcium complex of CaM for expression of its catalytic activity. FSBA reacted with smMLCK-CaM and with the CaM-free, inactive enzyme as well. Both reactions were dependent on time and FSBA concentration. Reaction was accompanied by the incorporation of covalently bound [14C]FSBA into smMLCK protein at a molar ratio of approximately 1:1 in each case. p-(Fluorosulfonyl)benzoic acid, an analogue of FSBA lacking the adenosine targeting group, did not react at a significant rate with either form of smMLCK. Reaction of CaM-free and CaM-bound smMLCK with FSBA displayed saturation kinetics. The first-order rate constants for the conversion of the reversible, noncovalent enzyme-FSBA complex to form the irreversibly inhibited, covalently modified enzyme were similar for both smMLCK and smMLCK-CaM, 0.15 and 0.07 min-1, respectively. The concentrations of FSBA yielding the half-maximal rate of inactivation, KI, were essentially identical--0.65 and 0.64 mM, respectively--for smMLCK and smMLCK-CaM. MgATP, but not MgGTP or a substrate peptide, potently inhibited reaction with FSBA. Inhibition by MgATP was competitive. The measured inhibitory constant for MgATP was essentially the same--33 versus 34 microM--for both smMLCK and smMLCK-CaM. It therefore is concluded that the MgATP-binding site on smMLCK remains accessible and recognizable as such when the enzyme becomes inactivated upon dissociation of CaM.     

Sequence-dependent distortions induced in DNA by monofunctional platinum(II) binding.

The effects on thermal stability and conformation of DNA produced by the monofunctional adducts of chlorodiethylenetriamineplatinum(II) chloride ([Pt(dien)Cl]Cl) have been investigated. Oligodeoxyribonucleotide duplexes of varying lengths (9-20 base pairs) and of varying central trinucleotide sequences were prepared and characterized that contained site-specific and unique N(7)-guanine adducts. Included are adducts at the sequences of d(AGC), d(AGT), d(CGA), d(TGA), d(TGC), and d(TGT). All these monofunctional adducts decrease the melting temperature (Tm) of the duplexes. This destabilization effect exhibits a sequence-dependent variability. The highest lowering of Tm is observed for the modified duplexes containing the central sequence of pyrimidine-guanine-pyrimidine. The destabilization effect is reduced with decreasing concentrations of Na+. Polarography, circular dichroism, phenanthroline-copper, and chemical probes reveal conformational distortions spreading over several base pairs around the adduct. The effects of monofunctional platinum(II) adducts on conformational distortions in DNA exhibit a sequence-dependent variability similar to those on thermal stability of DNA. The influence of the monofunctional adduct formed by cis-diamminemonoaquamonochloroplatinum(II) on the stability of the oligonucleotide duplex has been also studied. This lesion decreases thermal stability of DNA in the same way as does the adduct of [Pt(dien)Cl]Cl.     

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.