Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication

In C. elegans, genome-wide screens have identified just five essential centriole-duplication factors: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 [1-8]. These proteins are widely believed to comprise a conserved core duplication module [3, 9-14]. In worm embryos, SPD-2 is the most upstream component of this module, and it is also essential for pericentriolar material (PCM) recruitment to the centrioles [1, 4, 15, 16]. Here, we show that Drosophila Spd-2 (DSpd-2) is a component of both the centrioles and the PCM and has a role in recruiting PCM to the centrioles. DSpd-2 appears not, however, to be essential for centriole duplication in somatic cells. Moreover, PCM recruitment in DSpd-2 mutant somatic cells is only partially compromised, and mitosis appears unperturbed. In contrast, DSpd-2 is essential for proper PCM recruitment to the fertilizing sperm centriole, and hence for microtubule nucleation and pronuclear fusion. DSpd-2 therefore appears to have a particularly important role in recruiting PCM to the sperm centriole. We speculate that the SPD-2 family of proteins might only be absolutely essential for the recruitment of centriole duplication factors and PCM to the centriole(s) that enter the egg with the fertilizing sperm.     

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton $\text{T}{}_1$ NMR and obtained a value of 4.0 · 10^?3 cm · s^?1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.     

The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis

The fidelity of protein synthesis depends on the rate constants for the reaction of ribosomes with ternary complexes of elongation factor Tu (EF-Tu), GTP, and aminoacyl (aa)-tRNA. By measuring the rate constants for the reaction of poly(U)-programmed ribosomes with a binary complex of elongation factor (EF-Tu) and GTP we have shown that two of the key rate constants in the former reaction are determined exclusively by ribosome-EF-Tu interactions and are not affected by the aa-tRNA. These are the rate constant for GTP hydrolysis, which plays an important role in the fidelity of ternary complex selection by the ribosome, and the rate constant for EF-Tu.GDP dissociation from the ribosome, which plays an equally important role in subsequent proofreading of the aa-tRNA. We conclude that the fidelities of ternary complex selection and proofreading are fundamentally dependent on ribosome-EF-Tu interactions. These interactions determine the absolute value of the rate constants for GTP hydrolysis and EF-Tu.GDP dissociation. The ribosome then uses these rate constants as internal standards to measure, respectively, the rate constants for ternary complex and aa-tRNA dissociation from the ribosome. These rates, in turn, are highly dependent on whether the ternary complex and aa-tRNA are cognate or near-cognate to the codon being translated.     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

Effect of steroid hormones on endotoxin-mediated cartilage degradation

Summary Cartilage degradation is a characteristic feature of various types of human arthritis, notably rheumatoid arthritis and osteoarthritis. The influence of glucocorticoid and other steroid hormones on cartilage proteoglycan breakdown was examined in a model system in which breakdown is readily quantified by the release of proteoglycan from cultured bovine nasal cartilage discs. Endotoxin (bacterial lipopolysaccharides) treatment enhanced the depletion of cartilage proteoglycan by 2–3 fold. This was inhibited in a concentration-dependent manner by hydrocortisone (10^–9 to 10^–5M) or other glucocorticoid hormones (dexamethasone, prednisolone, cortisone). Inhibition required the continued presence of the steroid. Removal of hydrocortisone (3 × 10^–7M) after 4 days from endotoxin-treated cultures resulted in the rapid restoration of an endotoxin response, so that proteoglycan release approached maximum levels during a second 4-day culture period. Other C-21 steroid hormones (progesterone, aldosterone) were also inhibitory at 10^–5M, but testosterone and -estradiol showed little influence on endotoxin action. Proteoglycan products of smaller average mol wt (Sepharose CL-2B chromatography), consistent with core protein cleavages, were released from endotoxin-treated cartilage. Cleavage was unaffected by -estradiol, partially blocked by aldosterone and largely prevented by hydrocortisone administration.     

In situ hybridization and immunocytochemistry in serial sections of rabbit skeletal muscle to detect myosin expression

We performed in situ hybridization of myosin heavy-chain (MHC) mRNA on rabbit muscle using a biotin-labeled complementary RNA probe. An 1107-nucleotide fragment from an alpha-cardiac MHC cDNA was used to transcribe an RNA probe 97% similar to slow-twitch and 75% similar to fast-twitch sequences. Serial sections were used to identify slow-twitch fibers in medial gastrocnemius, soleus, and tibialis anterior by immunofluorescence of slow MHC and oxidative capacity by histochemistry. Slow-twitch fibers hybridized by the RNA probe stained heavily after detection with streptavidin-alkaline phosphatase (89% dark and 11% medium density). Fast-oxidative fibers stained intermediately (26% dark, 58% medium, and 16% light) and fast-glycolytic fibers stained lightly (12% medium and 88% light). Biotin-labeled probe and enzymatic detection allowed greater resolution of the subcellular location of the MHC mRNA, a distinct advantage over isotope labeling and autoradiography. A non-uniform distribution of MHC mRNA was recognized within an adult skeletal muscle fiber. High concentrations of MHC mRNA were found under the sarcolemma and between the myofibrils, suggesting the existence of a distribution mechanism. The combination of in situ hybridization and immunocytochemistry allows rapid subcellular localization of both MHC mRNA and its translated protein.     

Effects of environmental conditions on the fixation and transfer of nitrogen from alfalfa to associated timothy

Nitrogen fixation (NF) by alfalfa and nitrogen transfer (NT) from alfalfa to associated timothy was studied under different environmental conditions in controlled growth chambers, using the¹⁵N dilution technique. Evidence was obtained of NT from alfalfa to the associated timothy. Conditions that favored NF by alfalfa resulted in an increase in its NT. Of 3 different temperature regimes (25/20, 16/14, and 12/9°C day/night), 16–25/14–20°C was the best range for NF by alfalfa and resulted in the greatest NT. High light intensity (550 uE.m⁻².sec⁻¹) and long days (16–20 h) also caused increased NF by alfalfa and benefitting timothy more than in a regime of low light intensity (by shading 50% or 75%) or short days (12/12 or 16/8 h day/night). When the inoculated (Rhizobium meliloti) root systems of plants were kept free from other microorganisms (axenic condition) to minimize possible decomposition of dead tissues, lower NT from alfalfa was observed, especially at later cuts, compared to non-axenic plants. This suggests that both direct excretion and decomposition of dead alfalfa tissues are sources of N benefit from alfalfa to associated timothy. Contribution no 1065 of the Plant Research Centre.     

Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3

Silver nitrate effectively promoted shoot regeneration in wheat (Triticum aestivum L.) callus cultures derived from immature embryos. This effect could be observed in both weakly and strongly regenerating cultivars, and in using material from both field and greenhouse grown plants. The role of silver ions as an inhibitor of ethylene action was supported by a reversal of the inhibitory effects of 2,4-D and ethylene on morphogenesis in wheat callus cultures.Enhancement of shoot regeneration by silver nitrate was also observed in callus cultures of non-regenerating or weakly regenerating mutants of Nicotiana plumbaginifolia Viv. derived from cell cultures.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Ethrel 2-chlorethylphosphonic acid - NAA 1-naphthaleneacetic acid - NR^– nitrate reductase deficient     

黄地老虎核型多角体病毒的一些特性

黄地老虎核型多角体病毒(Agrotis segetum Nulear Polyhedrosis Virus简称AsNPV)的国内分离株(AsNPV^C),多角体呈六边形,大小1.7—2.6μm,为多粒包埋类型.每个病毒束内有2—7个核衣壳,大小约52nm×308nm.感染烟青虫(Heliothis assttlta)后分离到的多角体(As-HaNPV)其形状不规则,大小0.7—2.6μm,亦为多粒包埋类型.核衣壳2—6个不等,大小约40nm×300nm.EcoR1和HindⅢ限制性内切酶电泳图谱分析表明,AsNPV^CDNA和As-HaNPV DNA的EcoRI、HindIII酶切图谱一致,两者与HaNPV DNA的EcoRI,HindⅢ酶切图谱存在明显差异,AsNPV^C DNA的EcoRI酶切图谱共有15个片段,分子量在12.74×10⁶—1.18×10⁶道尔顿之间,总分子量约88.6×10⁶道尔顿,相当于134.25kbp.HaNPV DNA的EcoRI酶切图谱共有19个片段,分子量在13.89×10⁶—1.10×10⁶道尔顿之间,总分子量约93.86×10⁶道尔顿,相当于142.25kbp.AsNPV对黄地老虎2龄和4龄幼虫以及对烟青虫4龄幼虫的LD_{50分别为:1.4×10⁵pIB、7.4×10⁴PIB和2.61×10⁴PIB.}