DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

To investigate the response of cells to one type of DNA damage — namely DNA crosslinks — cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 μg/ml plus 5 kJ/m² or 1.0 μg/ml plus 1–2.5 kJ/m²)_halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [³H]thymidine into cellular DNA immediately after the treatment. Then, after 8 h of incubation, the incorporation of [³H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [³]Hthymidine were correlated with the halt and resumption in the cell-cycle process.Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.     

The development of the female accessory gland in the insect Rhodnius prolixus

The specialized cell types and two distinct regions of the adult Rhodnius prolixus cement gland develop from a simple pseudostratified epithelial tube during the 20–22 days of the fifth stadium. Feeding initiates the first phase, proliferation. Cells round up and divide tangentially to the lumen. Following the proliferation phase, differentiative mitoses occur and differentiation, resulting in secretory units (consisting of a ductule, gland cell and cuticular lining), ensues in the distal region. Ductule morphogenesis occurs without pseudocilia, thus differing from other insect glands. The complex changes in cell shape and interaction occur during development of the secretory unit. The secretory cell and end-apparatus develop from a double cell unit at the base of elongating ductules. The inner cell produces a complex end-apparatus of epicuticle that mirrors the microvillar pattern and then it degenerates. The ductules are lined by cuticulin and inner epicuticle while the central gland lumen has a layer of endocuticle as well. The epithelium of the proximal region remains simple producing the thick corrugated cuticle characteristic of the adult secretory duct. The mesodermal covering forms a thick longitudinal striated muscle layer that adheres to the epithelium via desmosomes.     

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.     

Comparison between the in vitro activities of in vivo-induced hapten-specific suppressor cells and supernatants of a hapten-specific suppressor hybridoma

A trinitrophenyl (TNP)-specific suppressor hybridoma was obtained by fusing hapten-binding spleen cells (SC) of BALB/c mice 1 week after intravenous (iv) injection of TNP-modified syngeneic lymphocytes with the AKR lymphoma BW5147. The suppressive activity of supernatants from one clone (TNP-44) was compared with that of in vivo-induced TNP-specific suppressor cells. Both the TNP-specific suppressor cells (TsTNP) and the TNP-44 were hapten binding and hapten specific. They suppressed the functional activity of TNP-haptenized T as well as B cells. TNP-44 supernatant also inhibited the proliferation of TNP-modified cells. Using native target cells, both TNP-44 supernatant and the in vivo-induced suppressor cells suppressed the anti-TNP B-cell response to TNP-bound T-dependent soluble or cellular antigens, but not to TNP-lipopolysaccharide (LPS). Furthermore, the function of TNP-specific helper T cells (THTNP) was impaired in the presence of TSTNP or supernatant from TNP-44. From these observations it was concluded that both the TSTNP and a TNP-specific factor derived from a suppressor hybridoma function via an antigen bridge at the TH or at the TH-dependent B-cell subset.     

Fusion of neurohypophyseal membranes in vitro

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca²⁺ in micromolar concentrations. Mg²⁺ and Sr²⁺ were ineffective. Mg²⁺ inhibited Ca²⁺-induced fusion.In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca²⁺. Mg²⁺ and Sr²⁺ were ineffective in replacing Ca²⁺ as trigger for fusion or vasopressin release.Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosome were exposed to cold.     

The histology and fine structure of the olfactory organ of the squid Lolliguncula brevis Blainville.

The olfactory organ of the squid has a thick, pseudostratified epithelium containing five morphological types of ciliated receptors. In the simplest receptors the cilia originate separately in the distal pole of the cell. All other receptors have some type of cilia filled cavity, varying from a simple pocket of cilia at the surface to a completely closed vesicle filled with cilia in cells deep in the epithelium. The receptors are compared to cells in the rhinophore of Nautilus and the olfactory organs of coleoid cephalopods. Possible functions of the olfactory organ, based on its morphology, are discussed.     

Mutagenesis in multinucleate cells: the effects of N-methyl-N'-nitro-N-nitrosoguanidine on Phycomyces spores

Multinucleate cells, such as the spores of the fungus Phycomyces, are unsuitable for the isolation of recessive mutants. Nuclear killing by N-methyl-N'-nitro-N-nitrosoguanidine (henceforth nitrosoguanidine) eliminates all but one of the nuclei in some of the cells and allows the expression of recessive mutations. Even in the best conditions, only about 35% of the survivors have a single functional nucleus. Functionally uninucleate cells can be positively selected. This involves the exposure to nitrosoguanidine of the spores of a heterokaryon and selection for a recessive marker present in a small fraction of its nuclei. The optimal conditions for nitrosoguanidine mutagenesis in Phycomyces differ from those for bacteria and yeast. Buffer composition and pH are less important than in other organisms. Survival is an exponential function and mutation induction a linear function of the dose of the mutagen (concentration X time). Spore germination leads to an immediate increase in the number of gene copies per cell, thus further hindering the expression of recessive mutations; dominant mutations are then nearly always isolated in heterokaryotic form.     

Opioid-specific recognition sites of the mu- and the delta-type in rat striatum after lesions with kainic acid

The binding of 3H-dihydromorphine (3H-DHM) and of 3H-D-ala2, D-leu5-enkephalin (3H-DADL), which are regarded as relatively selective ligands for mu- or delta-type opioid receptors, respectively, was estimated in total particulate fraction of the striatum of rats in vitro, either in tissue of rats after striatal chemolesions with kainic acid or in control rats (not operated or saline injected into the striatum). Kainate lesions reduced the Bmax values of 3H-DHM by about 78 - 88% depending on the method of calculation, and of 3H-DADL by greater than 90%. Furthermore they lowered the Kd-values, suggesting an increase in affinity. The results are discussed with regard to recent hypotheses on the structure and function of opioid receptors.     

Absence of hydrocortisone from cytoplasmic hormone-protein complexes formed in vivo after administration of biologically active doses of [3H]hydrocortisone

After administration of [³H]hydrocortisone to adrenalectomized rats, hormone-protein complexes were isolated from liver cytosol by DEAE-cellulose chromatography. After application of biologically active and inactive doses of hydrocortisone five binding components were detected eluting at the same salt concentrations as the hormone-protein complexes observed after incubation of cytosol with [³H]hydrocortisone in vitro. The isolated hormone-protein fractions were acidified and extracted with ethylacetate and the steroids were analyzed by thin-layer chromatography. No significant amount of hydrocortisone could be detected in any of the complexes formed in vivo 5–60 min after administration of biologically active doses of hydrocortisone. 3ξ,11β,17α,20ξ, 21-Pentahydroxypregnane, steroidal carboxy acids, glucuronides and a very polar conjugate of hydrocortisone were found in the different fractions. After an in vivo dose of hydrocortisone of about 1/5000th of the minimal dose required for enzyme induction, hydrocortisone could be found in all cytoplasmic hormone-protein complexes formed. In contrast to the cytoplasmic hormone-protein complexes, hydrocortisone could be readily demonstrated in nuclei isolated after the administration of biologically active doses of hormone, although acid metabolites were found to represent the main part of the radioactive compounds present in the nuclei. These acid metabolites were located in ronide on the basis of its chromatographic behavior. The biological significance of this conjugate of hydrocortisone as well as that of the extremely polar conjugate found in fraction DE-3 cannot be understood on the basis of the published data pertaining to biological functions and metabolism of glucocorticosteroids.Our finding that no ‘classical’ glucocorticosteroid receptor can be detected in rat liver cytosol raises again the question of the way in which hydrocortisone and its active metabolites enter the nucleus. On the basis of the published data, the possibility cannot be ruled out that glucocorticosteroids are transported via the endoplasmic reticulum. A transport by this way has been inferred for the uptake of sodium and inulin by liver nuclei [40–42].     

Metabolic activation of selected aromatic amines and polycyclic hydrocarbons by isolated subcellular fractions of Drosophila melanogaster

The applicability of microsomal preparations from Drosophilamelanogaster as the metabolic factor in the Salmonella mutagenicity assay with strains TA98 and TA100 was evaluated. Isolated cellular fractions (S27) from PB-pretreated flies activated N-acetyl-2-aminofluorene (2-AAF), N-hydroxy-N-aceyl-2-aminofluorene (N-OH-AAF), benzo[a]pyrene (BP), 9,10-dimethylanthracene (DA) and 2 -naphythylamine (NA)_into mutagenic metabolites, 7,-12-Dimethylbenz[a]-anthracene (DMBA) was ineffective under the conditions of the test.This study was performed in an effort to determine optimal conditions for activating, by Drosophila enzymes, aromatic amines and polycyclic hydrocarbons, with 2-AAF and BP as model mutagens. The following alterations improved the sensitivity of this combined Salmonella/Drosophila assay. (1) Incubation of the plates at 25°C for 1 night instead of permanent exposure at 37°C. (2) Isolation of S27 fractions instead of the conventional S9, because 9000 × g was not sufficient tio spin down Drosphila mitochondria.     

Stimulation of polypeptide synthesis by spermidine at the level of initiation in rabbit reticulocyte and wheat germ cell-free systems

It is shown that the stimulation of eukaryotic polypeptide synthesis by spermidine is due to the stimulation at the level of initiation by following reasons. The incorporation of formylmethionine into polypeptides was stimulated by spermidine at the same degree to the incorporation of leucine into polypeptides. Fluorography of the polypeptides formed showed that the number of chains of individual protein synthesized was larger when spermidine was added. The formation of the complex of Met-tRNA_f, globin mRNA and 40-S ribosomal subunits was stimulated by spermidine.     

Relationship between induction of aryl hydrocarbon hydroxylase and de novo synthesis of cytochrome P-448 (P1-450) in mice

Phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzpyrene, 3-methylcholanthrene (3-MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were administered i.p. for 1 or 3 days to genetically “responsive” (C57BL/6J) and genetically “non-responsive” (DBA/2J) mice. 3-MC or benzpyrene stimulated aryl hydrocarbon hydroxylase (AHH) activity in C57BL/6J (B6) mice but not in DBA/2J (D2) mice. TCDD induced AHH activity in both B6 and D2 mice. Time-course studies showed that in the first 12 h after a single injection of 3-MC to B6 mice there was no shift in the reduced cytochrome P-450-CO complex absorption spectra from 450 to 448 nm, although AHH activity increased 4–5 times over (above) that of the control group. The relationship between induction of AHH activity by polycyclic hydrocarbons in B6 mice and the concomitant synthesis of cytochrome P-448 is discussed.     

The reduction kinetics of chlorophyll a1 as an indicator for proton uptake between the light reactions in chloroplasts

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll a₁ (P-700) after far-red preilluination have been studied with high time resolution in spinach chloroplasts.

1. 1. The kinetics of chlorophyll a₁ exhibits a pronounced lag phase of 2–3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II.

2. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction.

3. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values.

4. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll a₁. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules.

5. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylakoid membrane.

6. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll a₁ reduction, is estimated as > 10¹¹ M⁻¹ · s⁻¹. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution.

Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

Effects of hormones (calcitonin, GIP) and pharmacological antagonists (ranitidine and famotidine) on isolated rat parietal cells

The rationale for the present study was to compare calcitonin and gastric inhibitory polypeptide (GIP) versus two histamine H2 receptor antagonists with respect to their potency of inhibiting parietal cell functions. Adenylate cyclase activity and acid production ([14C]aminopyrine uptake) of isolated rat parietal cells were stimulated by histamine. At 10(-7) and 10(-6) mol/l, calcitonin and GIP reduced the response to histamine by 10-20% following noncompetitive kinetics. Ranitidine and famotidine (MK 208) inhibited the response to histamine by about 50% at 10(-7)-10(-6) mol/l, and at 10(-5) mol/l abolished the histamine effect. On a molar basis famotidine turned out to be 6 times more potent than ranitidine. Both antagonists revealed competitive kinetics. Our data suggest direct inhibition of the parietal cells by the tested compounds which were shown to interfere at the adenylate cyclase cAMP system or at the histamine H2 receptor. However, compared to the histamine H2 receptor antagonists, hormonal inhibition is less pronounced and mediated by a different mechanism.     

Properties of reticulum cell sarcomas in SJL/J mice: II. Fate of labeled tumor cells in normal and irradiated syngeneic mice

Transplantable reticulum cell sarcoma (RCS) cells were labeled with ³H-uridine or ³H-thymidine in vitro and injected intravenously into normal and irradiated syngeneic SJL/J mice. RCS cells exhibited typical B cell migration characteristics in peripheral lymphoid organs in both normal and irradiated recipients, localizing in follicles in a pattern resembling that of labeled normal bone marrow cells. However, over the first 72 hr after transfer, RCS cells diluted their label much less in irradiated than in normal recipients, reflecting their inability to proliferate in the irradiated hosts. The presence of unlabeled tumor cells did not significantly affect the distribution of labeled normal bone marrow or lymph node cells in the recipients. Thus, RCS fails to grow in irradiated recipients in spite of undisturbed homing characteristics and in the absence of any evidence of cytotoxic influences from the host.     

The influence of phospholipid polar groups on gramicidin channels

The influence of well-defined changes in the polar part of phospholipid molecules on the properties of black lipid membranes was studied using a series of phospholipids with identical hydrocarbon chains, but systematically changed polar groups. The hydrocarbon tails of the lipids under study were composed of 1,2-dipentadecylmethylidene glycerol. The polar parts differed in the degree of $\text{N-}\text{methylation}$ and comprised phosphocholine, $\text{-N,N-}\text{dimethylethanolamine}$, $\text{-N-}\text{methylethanolamine}$ and ethanolamine. Stable black lipid membranes could be formed with the solvents octane, decane, dodecane, tetradecane and hexadecane. The properties of gramicidin-induced single ionic channels changed systematically in membranes from the phosphatidylcholine to the phosphatidylethanolamine analogue, as indicated by an increase in the amplitude A of the unit conductance step and a decrease in the average channel life-time or duration τ. The series of τ-values was opposite to that expected from hydrocarbon thickness (specific capacitance). It is suggested that the surface tension γ is a relevant parameter for the prediction of τ-values.     

Trans-bilayer pseudocontact shifts produced by lanthanide ions in the 1H and 31P-nmr spectra of phospholipid vesicular membranes and their temperature variation

1. Shifts in the ¹H and ³¹P-nmr signals originating from the outer and inner phosphorylcholine head-groups and from the lipid acyl chains are observed when phosphatidylcholine vesicles are treated with increasing extravesicular concentrations of the lanthanides Eu³⁺, Pr³⁺, Yb³⁺, and Dy³⁺. 2. The addition of KNCS to increase the binding of the lanthanide ions to the outer head-groups is used to demonstrate that the intravesicular group shifts are not caused by bulk susceptibility effects. 3. The magnitude and direction of the observed shifts in the ¹H-nmr spectrum are shown to be consistent with (a) pseudocontact interaction of the paramagnetic lanthanide ions with the outer phospholipid head-groups, (b) current views of the conformation of the phosphatidylcholine head-group in the presence of lanthanides, and (c) a conservation of magnetic field within the vesicles due to their spherical nature. 4. Variation of the shifts with temperature are compared for egg phosphatidylcholine and dipalmitoyl phosphatidylcholine. The temperature variation in shifts is also used to study phase transitions in each monolayer and phase separations in mixed lipid systems.     

Characterization of experimental phenylketonuria augmentation of hyperphenylalaninemia with α-methylphenylalanine and p-chlorophenylalanine

Phenylalanine in conjuction with p-chlorophenylalanine or α-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterous effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing α-methylphenylalalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to expereimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, γ-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in t he brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age results obtained with animal models and the clinical findings in untreated phenylketonuric patients.