Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Density- and frequency-dependent predation of artificial bird nests

Predators are often expected to vary their relative predation rates according to the frequency of prey types in the environment (frequency-dependent predation). The underlying cause for this must lie in some dependency of absolute predation rates on the density of prey types in the environment (density-dependent predation). However, frequency-dependent predation may either be caused by 'simple' density-dependent predation, in which the absolute predation rate on a given prey type depends purely on the density of that type, or by more complex responses in which absolute rates depend also on the density of other prey types. It is usually difficult to distinguish the underlying cause of frequency-dependent predation, because frequencies tend to change as densities change. Here, we describe the results of an experiment conducted to disentangle these phenomena under two prey richness (low and high) conditions. We used artificial bird nests (placed on shrubs and on saplings) baited with quail eggs placed in natural forests as models of natural bird nests. Our results indicate that both the absolute and relative predation rates on the prey types may vary in complex ways. Predation rates depend on a complex interaction between the prey's own density, other prey density and the diversity of prey in the environment. Neglecting to include, or consider, these complexities into analyses may lead to erroneous conclusions in studies of absolute or relative predation rates.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Glial α1-receptors probably inhibit the high-affinity uptake of noradrenaline into astrocytes in the rat brain in vivo

The effect of ₂-receptor blockage on the extraneuronal turnover of noradrenaline (NA) has been studied in the intact rat brain. Tropolone and yohimbine, along with reserpine or desmethylimipramine, were given 30 min after intracerebroventricular injection of [7-³H]NA, i.e. after the tracer had been stored or inactivated. Tropolone given alone did not change the fractions of³H-activity recovered as [³H]NA from hypothalamus, septum, striatum and pons-medulla, but in the presence of yohimbine improved the [³H]NA recovery in all areas except pons-medulla. The maximum effect was seen in the hypothalamus of reserpine-treated rats. Since the ₂-autoreceptors were blocked, the increased [³H]NA recovery does not reflect a down-regulated neuronal NA turnover. Instead it seems to show that a fraction greater than normal of neuronally released NA had been taken up into astrocytes and remained unmetabolized if catechol-O-methyltransferase was inactive. It is assumed that yohimbine enabled the protective tropolone effect by blocking astrocytic ₂-receptors that otherwise, either by itself or by antagonizing -receptor-induced hyperpolarization or cAMP formation, had impaired parameters that stimulate the high-affinity NA Uptake₁ of astrocytes (e.g. membrane potential, Na⁺, K⁺-ATPase) or control the gap junction permeability in the glial syncytium.     

5-HT,dopamine, norepinephrine,and related metabolites in brain of low alcohol drinking (LAD) rats shift after chronic intra-hippocampal infusion of harman

Harman (1-methyl--carboline) has been shown to induce preference for alcohol in the genetically bred, low alcohol drinking (LAD) rat. This study was undertaken in the LAD rat to determine whether monoamines and their metabolites in different regions of the brain are altered by harman infused chronically into the dorsal hippocampus. For this purpose, a cannula was implanted stereotaxically into the dorsal hippocampus. The cannula was attached to an osmotic minipump implanted subcutaneously within the intrascapular space. The pump was filled with either an artificial cerebrospinal fluid (CSF) vehicle or harman, which was delivered at a rate of 1.0 or 3.0 g/h (i.e., 5.5 or 16.5 nmol/h, respectively) for a period of 14 days. Four days after surgery, a standard preference test for ethyl alcohol was given to the rats over 10 days in which concentrations were increased daily from 3%–30%. The higher concentration of harman infused into the hippocampus elevated the level of serotonin (5-HT), both ipsilateral and contralateral to the hippocampal site of infusion, as well as in the midbrain, frontal cortex, striatum and nucleus accumbens. Similarly, this treatment resulted in a rise in the levels of norepinephrine in the hippocampus and midbrain but aecreases in dopamine levels in the pons. The levels of 5-hydroxyindoleacetic acid (5-HIAA) and: 3,4-dihydroxyphenylacetic acid (DOPAC) were diminished in the pons of rats given 3.0 g/h harman, whereas both concentrations of the -carboline reduced the level of homovanillic acid (HVA) in the frontal cortex. These harman-induced changes in the metabolism of the amines are possibly the result of an inhibition of monoamine oxidase (MAO). When the harman-induced shifts in the neurochemical values were compared to the alcohol intakes of the rats as reported previously, no significant correlation was found. The absence of this concordance suggests that the alterations in the monoamine neurotransmitters produced by harman and the voluntary intake of alcohol induced by this -carboline may not originate from the same systems in the brain.     

Refocusing revisited: An optimized,gradient-enhanced refocused HSQC and its applications in 2D and 3D NMR and in deuterium exchange experiments

Summary 2D ¹⁵N-¹H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T₂ relaxation during the refocusing delays. However, we show here that, when high ¹⁵N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH₂ geminal peaks and their truncation artefacts at a modest cost in sensitivity.     

Infrared spectra of carbon monoxide bound to mitochondria from diverse species and tissues reveal structurally similar cytochrome c oxidase dioxygen reaction sites

Infrared bands for CO bound to mitochondria from bovine and porcine hearts, bovine brain, rat kidney, and blowfly flight muscle and to intact blowfly flight muscle have been measured in the carbon-oxygen stretch region. Each spectrum contains a narrow band near 1963 cm-1 similar to the major band found earlier for the carbonyl cytochrome c oxidase purified from bovine heart. A second band near 1959 cm-1 ascribed to a less stable conformer of the purified oxidase carbonyl is also detected in mitochondria. These spectra support very similar CO (and O2) binding sites among all the oxidases examined whether the enzyme is purified or is still within mitochondria or intact tissue and therefore suggest that the reduced heme A ligand binding site has been highly conserved during evolution.     

Increase in histone acetylation and transitions in histone variants during Friend cell differentiation

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.     

Response of excised embryos of rice (Oryza sativa L.) to X-rays

Summary The response of rice (Oryza sativa L.) embryos to X-rays (M₁ to M₃) was studied. By means of irradiating excised embryos, both chlorophyll and macromutation were successfully induced in three genotypes of rice. However, differential responses in terms of mutation frequency, mutation spectrum and optimal levels of X-rays required for induction of mutation (chlorophyll as well as morphological) were found to exist between cultivars. In Satika and Ashkhata, LD₅₀ values and maximum induced seed sterility are concomitant to optimum level of radiation required for triggering chlorophyll mutation. However, optimum dose for induction of macromutation in Satika and Kerangserang is independent of either LD₅₀ and/or induced seed sterility.Chances of obtaining both dominant and locus specific recessive mutations in the immediate X-ray treated generation (M₁) are large. This indicates the very high degree of effectiveness of the excised embryo irradiation technique with rice.