Catecholamine innervation of enkephalinergic neurons in guinea pig hypothalamus: demonstration by an in vitro autoradiographic technique combined with a post-embedding immunogold method

To study the relationship between the catecholamine (CA) nerve endings and the enkephalinergic cell bodies in the magnocellular dorsal nucleus (MDN) of guinea pig hypothalamus, double-labeling experiments were performed on the same tissue section at the electron microscopic level. An in vitro autoradiographic (ARG) method for [3H]-norepinephrine (NE) or [3H]-dopamine (DA) was combined with a post-embedding immunogold cytochemical technique for Met-enkephalin (Met-enk) in colchicine-treated animals. Hypothalamic slices (450 micrograms) were perfused with [3H]-NE or [3H]-DA at the fluid-gas interface, then fixed by immersion with glutaraldehyde and osmic acid. Semi-thin sections processed from the thickness of the slices showed adequate penetration of the tracers to all parts of the tissue. Frontal sections permitted visualization of some CA-uptake structures distributed around the cells. At the ultrastructural level, preservation appeared good on about 60% of the thickness of slices, and [3H]-CA structures were easily distinguished. Ultra-thin sections were successively incubated with Met-enk and colloidal gold-labeled antisera, followed by ARG processing. At the electron microscopic level, the good integrity of the tissue made possible visualization of [3H]-CA nerve terminals making synaptic contacts with enkephalinergic perikarya. These results provide morphological evidence for direct catecholaminergic control of enkephalinergic neurons of the MDN.     

Generation of different nucleosome spacing periodicities in vitro. Possible origin of cell type specificity

We have been able to generate ordered nucleosome arrays that span the physiological range of spacing periodicities, using an in vitro system. Our system (a refinement of the procedure previously developed) uses the synthetic polynucleotide poly[d(A-T)], poly[d(A-T)], core histones, purified H1, and polyglutamic acid, a factor that increases nucleohistone solubility and greatly promotes the formation of ordered nucleosome arrays. This system has three useful features, not found in other chromatin assembly systems. First, it allowed us to examine histones from three different cell types/species (sea urchin sperm, chicken erythrocyte, and HeLa) as homologous or heterologous combinations of core and H1 histones. Second, it allowed us to control the average packing density (core histone to polynucleotide weight ratio) of nucleosomes on the polynucleotide; histone H1 is added in a second distinct step in the procedure to induce nucleosome alignment. Third, it permitted us to study nucleosome array formation in the absence of DNA base sequence effects. We show that the value of the spacing periodicity is controlled by the value of the initial average nucleosome packing density. The full range of physiological periodicities appears to be accessible to arrays generated using chicken erythrocyte (or HeLa) core histones in combination with chicken H5. However, chromatin-like structures cannot be assembled for some nucleosome packing densities in reactions involving some histone types, thus limiting the range of periodicities that can be achieved. For example, H1 histone types differ significantly in their ability to recruit disordered nucleosomes into ordered arrays at low packing densities. Sea urchin sperm H1 is more efficient than chicken H5, which is more efficient than H1 from HeLa or chicken erythrocyte. Sea urchin sperm core histones are more efficient in this respect than the other core histone types used. These findings suggest how different repeat lengths arise in different cell types and species, and provide new insights into the problems of nucleosome linker heterogeneity and how different types of chromatin structures could be generated in the same cell.     

Resistance ofSpodoptera frugiperda [Lep.: Noctuidae] to a nuclear polyhedrosis virus in the field and laboratory

Median lethal doses (LD₅₀s) of nuclear polyhedrosis virus (NPV) were determined in neonatal offspring ofSpodoptera frugiperda (J. E. Smith) (Sf) larvae captured in southeastern Louisiana in 1981, 1982, and 1984. These LD₅₀s ranged from 1.8 to 16.3 polyhedral inclusion bodies (PIB)/insect. The LD₅₀s significantly (P<0.05) increased during the season of 1982 but had no pattern in 1981 or 1984. However, the Sf populations increased in heterogeneity of response to the NPV during all 3 years. The LD₅₀ increased from 4.1 to 18.7 PIB/insect in a Sf laboratory colony exposed to the NPV LD₈₀ for 7 generations, whereas in a control colony not exposed to NPV the LD₅₀ was 5.9 PIB/insect after 7 generations.     

Cellular differences in the regeneration of murine skeletal muscle: a quantitative histological study in SJL/J and BALB/c mice

Summary Skeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.     

Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.     

Sensitization of low-dose-rate irradiation by nonlethal hyperthermia

To assess whether hyperthermia could radiosensitize cells irradiated at a low dose rate, Chinese hamster V79 cells were simultaneously heated and irradiated at 0.86 Gy/h. The data showed that heat treatments at 39 and 40 degrees C, which did not induce heat killing alone or high-dose-rate radiosensitization, resulted in enhanced cell killing with low-dose-rate irradiation. The dose-modification factor (ratio of the slopes of the curves for low dose rate and high dose rate) was reduced to 1.8 at 39 degrees C and 1.4 at 40 degrees C, compared to a value of 2.1 at 37 degrees C. These data indicate that nonlethal heat treatments can cause enhanced radiosensitization under low-dose-rate conditions. The implications of these results for interstitial thermoradiotherapy are discussed.     

A review of the USAF/NASA proton bioeffects project: rationale and acute effects.

Ionizing radiation represented one of the important hazards facing the first manned lunar mission. A combined USAF/NASA project was conducted from 1963 through 1969 to estimate the relative biological effectiveness (RBE) of the radiations of space. Approximately 2000 primates (Macaca mulatta) and 5000 mice were irradiated with protons and electromagnetic radiations. The proton energies studied were selected to be representative of the proton spectrum in space. Much of the project was concerned with the use of cyclotrons for proton irradiations and with dosimetry. Biological measurements included clinical findings, physiological changes, hematological changes, histopathology, and mortality. When allowance was made for variation of response as a consequence of depth-dose distribution, the RBE for protons was approximately 1. This was anticipated from earlier theoretical studies and radiation therapy in humans with high-energy charged-particle beams.     

The influence of cell size on marine bacterial motility and energetics

The influence of Brownian motion on marine bacteria was examined. Due to their small size, marine bacteria rotate up to 1,400 degrees in one second. This rapid rotation makes directional swimming difficult or impossible, as a bacterium may point in a particular direction for only a few tens of milliseconds on average. Some directional movement, however, was found to be possible if swimming speed is sufficiently great, over approximately 100 μm sec⁻¹. This led to the testable hypothesis that marine bacteria with radiii less than about 0.75 μm should exceed this speed. The result of the increased speed is that marine bacteria may spend in excess of 10% of their total energy budget on movement. This expenditure is 100 times greater than values for enteric bacteria, and indicates that marine bacteria are likely to be immotile below critical size-specific nutrient concentrations.     

Foundations of vectorial metabolism and osmochemistry

Chemical transformations, like osmotic translocations, are transport processes when looked at in detail. In chemiosmotic systems, the pathways of specific ligand conduction are spatially orientated through osmoenzymes and porters in which the actions of chemical group, electron and solute transfer occur as vectorial (or higher tensorial order) diffusion processes down gradients of total potential energy that represent real spatially-directed fields of force. Thus, it has been possible to describe classical bag-of-enzymes biochemistry as well as membrane biochemistry in terms of transport. But it would not have been possible to explain biological transport in terms of classical transformational biochemistry or chemistry. The recognition of this conceptual asymmetry in favour of transport has seemed to be upsetting to some biochemists and chemists; and they have resisted the shift towards thinking primarily in terms of the vectorial forces and co-linear displacements of ligands in place of their much less informative scalar products that correspond to the conventional scalar energies. Nevertheless, considerable progress has been made in establishing vectorial metabolism and osmochemistry as acceptable biochemical disciplines embracing transport and metabolism, and bioenergetics has been fundamentally transformed as a result.