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—Phytochrome (Pfr)-mediated threshold control of apparent lipoxygenase (LOG) synthesis in the cotyledons of the mustard seedling depends on the presence of the hypocotylar hook. With isolated cotyledons, no suppression by light of apparent LOG synthesis can be detected. As soon as the cotyledons are separated from the hook, the control of apparent LOG synthesis by Pfr is completely lost. The data are consistent with the hypothesis advanced in a previous paper [Oelze-Karow, H. and H. Mohr (1973) Photochem. Photobiol.18, 319–330] that apparent LOG synthesis in the cotyledons is controlled by phytochrome located in the hypocotylar hook. The data support the concept [De Greef, J. A. and R. Caubergs (1972) Arch. Int. Physiol. Biochim.80, 959–960] that a very precise and highly ordered biophysical recognition and transmission system of light signals exists in plants.

Isolated rabbit kidneys were perfused at 37 ° C with a cell-free solution containing 0.0, 1.4, 2.1, and 2.8 m DMSO for 60 min. Electron microscopic examination of cortical structures revealed relatively high sensitivity of proximal tubular cells to DMSO-induced alterations, which included cytoplasmic clarification and disruption of microvilli. Glomeruli exhibited proliferation of rough endoplasmic reticulum, but were otherwise well preserved after perfusion with and without DMSO. Vascular endothelial cells were also intact after perfusion with DMSO, suggesting that DMSO-induced resistance changes are not necessarily associated with degeneration of capillary cells. Perfusion without DMSO resulted in good preservation of all cortical structures. These studies suggest that ultrastructural effects of DMSO must be considered in assessment of damage done in whole kidneys during freeze-preservation studies.

We investigate thes inclusive pion spectra in the electroproduction reaction ep → eπ±+ anything in a coincidence experiment at DESY. Data are presented from the kinematical region 0.1 < |q2| < 0.8 GeV2 and 3.15 < W < 2.8 GeV as a function of the transverse momentum squared pT2, the Feynman variable x2, and the azimuth angle φ. The charge ratio π+π− is given in terms of the variables q2 and x. The inclusive spectra are compared with results from photoproduction.

Isolated rabbit kidneys were perfused at normothermia and at low flow rates with perfusates of various compositions. K+- and Mg2+-rich perfusates appeared to ameliorate loss of renal function encountered with salt solutions of extracellular composition (Tyrode's). A vascular mechanism for the initial vasoconstriction observed during renal perfusion is suggested, and addition of isoxsuprine, a beta-adrenergic agonist, blocked the initial vasoconstriction. Normothermic, low-flow perfusion is able to maintain functional viability of the rabbit kidney for periods up to 90 min, after which some deterioration of function is noted. Na and K reabsorption, creatinine clearance, oxygen consumption, and urine acidification are, furthermore, observable in the isolated, normothermically perfused rabbit kidney at low flows. Normothermic perfusion for periods up to 90 min is a suitable means of administering cryoprotectant drugs in preparing whole kidneys for freezing studies.

In a previous paper (HOCK, KuHNERT and MOHR, 1965) we have shown that fat degradation in the cotyledons of the mustard seedling (Sinapis alba L). is under the control of active phytochrome (=P730), at least to a certain extent. One could imagine that P730 might exert this control over fat consumption through isocitritase (E C 4. 1. 3. 1). In the present paper we demonstrate that P730 does not have any influence on the activity of isocitritase in the mustard seedling, although the enzyme activity does show a strong increase and a following decline during the time of our experimentation (Fig. 8, 9). - Isocitritase could be assayed in a satisfactory manner in the raw extract only after the adsorption of phenolic compounds on "Divergan" (Fig. 1, 3). Furthermore the biological unit of the "seedling" turned out to be the only reliable system of reference for the enzyme activity determined in the extract. Puromycin strongly inhibits isocitritase synthesis whereas Actinomycin D is hardly effective (Fig. 10). It has been concluded that there might exist a relatively stable m-RNA which has been formed during the early stage of germination. Regulation of enzyme synthesis might occur on the level of translation. The evidence for this conclusion is not convincing, however; other interpretations are not excluded.

Electroproduction of π°-mesons on protons at backward angles was investigated by measuring the scattered electron and the recoiling proton in coincidence. The differential cross section was determined as a function of W, q2, θπoucms and u respectively, in the following kinematical region: W = √(πo + p′)2 = 1.5 − 2.1 GeV, |q2| = |(e − e′)2| = 0.3 − 1.0 GeV2/c2, 100° ⩽ θπocms ⩽ 180°, − 60° ⩽ Φπo ⩽ + 60°.

Freezing an organ and thawing it to a state of viable function is an extremely complex problem with innumerable sources of damage to the tissues. This study has evaluated the toxicity of these solute moderators since if they are toxic, the freezing procedure is doomed to failure in spite of control of all other factors. Glycerol and dimethyl sulfoxide (DMSO) in varying concentrations and saturated chloroform solution were perfused through isolated rat hearts at temperatures above 25 °C and below 15 °C. Chloroform exhibited no toxic effect. Both 10% glycerol and 20% DMSO are toxic if introduced into the heart at room temperature or above, although DMSO is the better tolerated.

The electroproduction of a π-meson and of a Δ(1236) nucleon resonance on hydrogen, ep → eπΔ (1236), was investigated in the two charge states π−Δ++ and π+Δ0 by measuring the scattered lepton and the produced π-meson in coincidence. The differential cross sections as funcions of W, q2, t−tmin and oπq were determine in the following kinematical region: w = (π + δ)2 = 2.0 − 3.0 GeV , |q2| = |(e−e′)2 | = 0.15 − 0.8 GeV2/c2 , |t − tmin| = 0-0.5GeV2/c2 with t = (p − δ)2 , φπq = 0 − 360° .

The electroproduction of single π+ mesons on hydrogen, ep → e′π+n, has been investigated by measuring coincidences between the scattered electron and the produced π+ meson. The differential cross sections as functions of so, q2, t and φπq were determined in the following kinematical region: so = (π+ + n)2 = 4.0 − 6.0 GeV2; |q2| = |(e − e′)2| = 0.1 − 0.9 GeV2/c2; |t| = |(p − n)2| = |tmin| − 0.15 GeV2/c2; φπq = 0 − 2π; (φπq = angle between polarizatin plane (e, e′) and production plane (π+, n); e, e′, π+, p, n = four-momenta of primary and scattered electron, π+ meson, target proton and recoil neutron).

Summary When hearts from rats were frozen with 15% dimethyl sulfoxide (DMSO) to −20°C and thawed, 80% resumed mechanical activity when the DMSO was washed out. Hearts treated similarly, except that the DMSO was 30%, failed to recover in all cases 8 except one. Hearts treated with 30% DMSO did not freeze. None of the hearts from animals pretreated for 4 days before the experiment with 2.0 mg per day of cortisone acetate and then cooled to −20°C with 30% DMSO resumed mechanical activity after warming. However, the cortisone did reduce edema formation in the hearts to 17% of that formed in hearts from untreated rats. The results demonstrate that 30% DMSO is toxic in rat heart preparations, in contrast to previous reports of the lack of toxicity of similar concentrations in other tissues.

Isolated rat hearts were perfused with balanced salt solution (BSS), then with BSS containing DMSO in one of several concentrations (0.14, 0.70, 1.41, 2.11, or 2.82 m ) at +30 °C, sealed in a metal cannister, and cooled slowly (1 °C/min) to a core temperature of −17 °C (total cooling time (TCT) = 37 min), thawed rapidly and reperfused with BSS. Groups of protected (0.70 m DMSO) or nonprotected hearts were cooled to −10 °C; of these, some were thawed immediately upon reaching −10 °C (TCT = 30 min), others were maintained at −10 °C for an additional 7 min and thawed (TCT = 37 min). Contractile activity was recorded during prefreeze and postthaw perfusion periods. Hearts which exhibited spontaneous A-V function after thawing were considered to be survivors. No hearts treated with 0.14 or 2.82 m DMSO survived thawing from −17 °C, but approximately half of all hearts treated with 0.70, 1.41 or 2.11 m DMSO and thawed from −17 °C survived. Survivors had diminished contractile strengths and heart rates, and gave no or negative inotropic responses to ouabain, a cardiotonic drug. Electron microscope study of all hearts revealed subcellular damage which differed little between surviving and nonsurviving hearts in each of these three groups. Cellular and subcellular membranes as well as nuclear fine structure were damaged, but the severity and extent of such injury was markedly less than that seen in thawed hearts treated with 0.14 or 2.82 m DMSO or no DMSO at all. DMSO largely prevented rupture of outer nuclear and mitochondrial membranes, but mitochondrial swelling was evident. Sarcomere alteration was minimal and usually limited to lateral compression by adjacent swollen mitochondria. Some regions of the myocardium contained generally disrupted cells, but many cells showed remarkable ultrastructural preservation. The relative ratio of “damaged” to “undamaged” cells was higher in surviving hearts than in nonsurvivors. All hearts treated with 0.70 m DMSO and thawed immediately from −10 °C survived; only half survived when DMSO was omitted. Seventythree percent of DMSO-treated hearts held at −10 °C for 7 min survived; none survived this brief storage interval when DMSO was not used. Protected hearts thawed immediately from −10 °C showed minor ultrastructural changes, whereas prolonging duration of cooling or omitting DMSO caused increased ultrastructural alteration qualitatively similar to those seen in hearts cooled to −17 °C. These results suggest that physical parameters such as water flux, crystal growth and cell volume are predominately responsible for freeze damage rather than chemical injury, and that percent cell water converted to ice and solute concentration per se are not directly related to ultimate cardiac cryoinjury.

The temporal pattern of control by phytochrome of the enzyme lipoxygenase (EC 1.13.1.13) in the mustard seedling (Sinapis alba L.) is described. The biosynthetic system which produces lipoxygenase does not respond to phytochrome (Pfr) up to 33.25 h after sowing (25°C). By this time the control by Pfr comes suddenly and fully into play. At 48 h after sowing the system escapes from the control by Pfr. Neither the beginning nor the end of the period of control is influenced by phytochrome. Interpretation of the data requires a double action control mechanism. It is argued that such a mechanism is necessarily a characteristic of development in multicellular organism.

The toxicity of four cryoprotective agents (glycerol, dimethylsulfoxide, dextran, magnesium ion) as a function of time was assessed at 30° on isolated rabbit atria. Glycerol, 2·0 M, in Ringer solution killed all atria in less than 30 min. Atria tolerated 2·1 M dimethylsulphoxide in Ringer for 1 hr, 0·7 M glycerol in Ringer for 4 hr, 6% (w/v) dextran in saline for 6 hr and 6·6 times 10−2 M magnesium chloride in Ringer for 8 hr. After washing out the cryoprotective agents the tissue was examined histologically and for its response to isoprenaline, MJ-1999 [4,2-(isopropylamino-10-hydroxyethyl)methanesulphonilide, a β-blocking agent] and isoprenaline and ouabain. These tests were the more sensitive indicators of functional integrity.

— Apparent synthesis* of the enzyme lipoxygenase in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (Pfr ground state)† through a threshold (all-or-none) mechanism. This response was used to determine physiologically the photostationary states, Λ that is, the [Pfr]/[Ptot] ratios established by different wavelengths in the red and far-red range of the spectrum, including the standard red and far-red sources used in this laboratory (Mohr, 1966). Under the premises (for which justification has been given on previous occasions) that the [Pfr]/[Ptot] ratio for standard red light is 0.8, and that the decay of Pfr is a first-order process with a half-life of 45 min, the [Pfr]/[Ptot] ratios determined physiologically by means of the lipoxygenase response agree with the [Pfr]/[Ptot] ratios determined spectrophotometrically by Hartmann and Spruit (cf. Fig. 9 in Hanke et al., 1969) in hypocotyl hooks of mustard seedlings. In the hook the fr, that is, the [Pfr]/[Ptot] ratio for standard far-red, is found to be 0.023. In the cotyledons, this ratio is several times higher (Schafer et al., 1972). The conclusion that apparent lipoxygenase synthesis in the cotyledons is controlled by phytochrome located in the hook has been substantiated by further spectrophotometric (Schafer et al., 1973) and physiological experiments (H. Oelze-Karow and H. Mohr, in preparation). The minimum steepness of the threshold was determined. An increase of the Pfr level from 118 (relative units) to 130 leads to an instantaneous and total suppression of apparent lipoxygenase synthesis; a corresponding decrease from 130 (relative units) to 118 leads to an immediate resumption of apparent LOG synthesis at full speed. It is concluded that an explanation of the experimental facts requires a cooperative effect on the level of Pfr, a high degree of synchrony on the cellular and organismic level and rapid communication between the hypocotyl hook and the cotyledons. *The term ‘apparent synthesis’ is used operationally in the present paper to denote any increase of enzyme activity, although de novo synthesis of lipoxygenase has not so far been rigorously demonstrated. The usual inhibitor experiments (cf. Oelze-Karow et al., 1970) have led to the conclusion that intact RNA and protein synthesis is required for an increase of lipoxygenase activity.

Hearts isolated from adult rats were perfused with one of several concentrations of DMSO (0.00, 0.14, 0.70, 1.41, 2.11, 2.82 m ) in a balanced salt solution (BSS), and then with DMSO-free BSS. During perfusion cardiac contractions were recorded. After perfusion the ventricular muscle was studied by electron microscopy. At all concentrations of DMSO studied there was a significant negative chronotropism, proportional to the concentration, and completely reversible by DMSO washout. DMSO concentrations of 0.14 and 0.70 m caused a positive inotropic effect which is reversible by drug washout. DMSO concentrations greater than 0.70 m caused a negative inotropic effect which was completely reversible only at 1.41 m . The ultrastructural effects of DMSO in concentrations up to 1.41 m were minimal. At higher concentrations one observes marked alteration or destruction of membrane-limited organelles: mitochondria, sarcoplasmic reticulum, transverse tubules, and nuclei. Although the myofilaments were contorted by swollen mitochondria, the ultrastructural integrity of the myofilaments was apparently unaltered, even at the highest concentration of DMSO studied.

The electroproduction of single π + − mesons on hydrogen, e + p → e′ + π + + n, has been measured up to values of | q 2 | ⩽ 0.9 GeV 2 /c 2 . The differential cross sections have been determined for small momentum transfer to the nucleon over the full azimuthal angle O π q .

The electroproduction of a π+ meson and of the Δ0(1236) nucleon resonance on hydrogen, ep→e'π+Δ0(1236), was investigated by measuring the scattered electron and the produced π+ meson in coincidence. The differential cross sections as functions of so, q2, t−tmin or θπq+, and φπq were determined in the following kinematical region: so = (π+ + Δo)2 = 4.4−6.3 GeV2, |q2| = |(e−e')2| = 0.2−0.8 GeV2/c2, |t−tmin| = 0−0.1 GeV2/c2, θπq∗ = 0o–20o, φπq = 0−360o.

Isolated rat hearts were perfused with balanced salt solution (BSS) for 20 min, sealed in a metal cannister, and cooled in a −20 °C acetone bath at a rate of 1 °C/min to one of four subzero core temperatures (−10, −12, −17, or −20 °C). Upon attainment of the desired temperature the hearts were rapidly thawed (40–50 ° C/min) and reperfused with BSS for an additional 20 min. Approximately half of the hearts cooled to −10 or −12 °C resumed spontaneous contractile activity after thawing. One of 16 hearts survived cooling to −17 °C, while no heart survived cooling to −20 °C. Nonfrozen controls gave a positive inotropic response to a standard test dose of ouabain; none of the thawed survivors did. The electron microscopic appearance of survivors of freezing to −10 or −12 °C showed little alteration of normal cardiac ultrastructure. Nonsurviving hearts from all groups demonstrated marked cryoinjury, primarily to membranes of the mitochondria, nuclei, sarcotubular system, and the sarcolemmal membrane. Thaw rigor, an irreversible postthaw contracture of muscle, was often the culmination of cryoinjury to the contractile apparatus and was characterized by the appearance of irregular dense sarcomeres which lacked the banding patterns typical of normal striated muscle; normal myofilament geometry was also found to be altered.

The inhibition of hypocotyl lengthening in intact mustard seedlings is controlled by two photosensitive systems which can be experimentally separated. 1. Kinetics of the growth response in the dark after red and far-red irradiation (Figs. 1, 2) demonstrate the operation of Pfr in the ground state via a threshold mechanism similar to the regulation of lipoxygenase synthesis in the mustard cotyledons (c. f. ref. [27]). This threshold mechanism determines the duration of the growth inhibition (Δt) following irradiation (Fig. 1, 2). Δt is dependent on the relative Pfr concentration at the beginning of the dark period and on the half life of Pfr destruction, but it is independent of the quantum flux density of far-red light (Fig. 4). The effect of 5 min red light on Δt can be fully reversed by 5 min far-red light (Fig. 3). The data reveal a quantitative relationship between the relative Pfr concentration and the photomorphogenetic response, Δt (Fig. 6). This relationship may explain in principle the logarithmic correlation between the percentage of phytochrome converted to Pfr by an initial irradiation and the subsequent response which has also been reported in the literature. 2. In continuous far-red light the velocity constant of the steady state growth is controlled by a “high intensity reaction” which shows the usual logarithmic dependence on quantum flux density (Fig. 4, 5), but no phytochrome destruction is apparent (Fig. 2). According to Hartmann (ref.[11–13]) this “high intensity reaction” can also be attributed to Pfr, which in this case acts through some excited state, P fr * .

Summary Whole isolated rat hearts were frozen at −20°C for 20 min using one of three cryoprotective agents: 15% DMSO in Ringer's solution, 6% high molecular weight dextran in saline, or 1.3% MgCl 2 in Ringer's solution. Also rat, guinea pig, rabbit, and dog hearts were frozen under similar conditions using 5% MgSO 4 as a cryoprotective agent. A series of rat hearts were frozen in Ringer's solution alone. Less than 10% of the hearts frozen in Ringer's solution resumed spontaneous atrial and ventricular contraction after thawing, while more than 85% of the hearts in each of the experimental groups met this criterion of survival. Typical pharmacological responses were elicited from thawed rat hearts receiving either DMSO, dextran, or MgCl 2 cryoprotection when tested with isoproterenol, MJ-1999 (a beta blocking agent), and ouabain. Magnesium ions seem to be potentially useful as cryoprotective agents. The mechanism of action of the agents used in this study is thought to be stabilization of macromolecules during freezing and prevention of bound water extraction leading to denaturation.

Summary A theory, based on alterations of intra- and extracellular water is presented to explain the mechanisms of cellular damage by freezing and the effects of cryoprotective agents. The concepts are based on recent developments in knowledge of the physical chemistry of water and on histological studies of frozen tissues. Bound water in the form of lattices seems to be essential to cell integrity, especially protein structure and function. The cellular morphological changes during freezing and the physical events of crystallization suggest that cellular membranes are preserved during slow freezing because of increased stability of the lattice structures. Death in freezing seems to occur primarily as a result of extraction of bound water from vital cellular structures. The extracted water, incorporated into growing ice crystals, leaves proteins dehydrated and denatured. During rapid freezing, intracellular ice formation consumes not only the free water but also portions of the bound water, thereby weakening the lattice structures. Rapid freezing can both physically derange the membranes and promote their denaturation with subsequent breakdown. If the lattice structures are left intact during freezing, intracellular ice formation does not result in death. Agents protecting cells during freezing probably act by inducing additional lattice or clathrate formation, either intracellularly or extracellularly. Much of the free water of the cellular environment is thus structuralized, being available for dissolved electrolytes but reducing the amount available for hexagonal ice formation.

Photophosphorylation and oxygen evolution were measured in 8-day-old dark-grown bean leaves (Phaseolus vulgaris) after various times of greening in far red light and in white light. The sequence of development was the same for both greening regimes, but the processes were much more rapid in white light. The capacity for photophosphorylation, as assayed by the firefly luciferase assay, appeared after 12 hours in far red light. At this stage and for times up to 24 hours, photophosphorylation was not inhibited by 10(-5)m 3-(3,4-dichlorophenyl)-1,1-dimethylurea. At 24 hours, the capacity for oxygen evolution appeared and photophosphorylation became partially inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations which inhibited oxygen evolution. In white light photophosphorylation appeared after 15 minutes, and oxygen evolution at one hour. Photophosphorylation became partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea when oxygen evolution appeared. Carbonylcyanide m-chlorophenyl-hydrazone inhibited photophosphorylation and photosynthesis at low concentrations, 10(-5)m, with immature leaves, but the leaves developed resistance to carbonylcyanide m-chlorophenyl-hydrazone as they greened.

The present study was conducted to determine the physiological and ultrastructural effects of hyperbaria on the mammalian myocardium. Isolated rat hearts were perfused in vitro for 20 minutes on a Langendorff apparatus, chilled to +5°C and subjected to hydrostatic pressures of 10,000 or 15,000 psi for periods of time of 10–40 minutes. After warming the hearts were reperfused for an additional 30 minutes. Contractile function was recorded during both perfusion periods and declines in contractile strength after high pressure were calculated. A control group of hearts was kept refrigerated at atmospheric pressure for 45 minutes between perfusion periods. Hearts could tolerate 10,000 psi for up to 20 minutes and 15,000 psi for 10 minutes. Alteration of atrial ultrastructure was found in all groups studied but was most prominent in hearts exposed to 10,000 psi for more than 20 minutes and in all hearts subjected to 15,000 psi regardless of time. Disruption of myocardial fine structure resembled that known to result from ischemia. Mitochondria were severely altered as evidenced by swelling, loss of cristae, and ruptures of the mitochondrial envelope. Specific atrial granules were depleted, edema was prominent in the perinuclear and interfibrillar regions, at the lateral cell junctions, and at the interealated dises whose interspaces were significantly widened; sarcotubular elements were also swollen. Nuclear changes included aggregation of chromatin, increased size of the perinuclear cisternae, and rarely, rupture of the outer nuclear membranes. Contractile elements were apparently passively distorted as a result of mitochondrial swelling and generalized interfibrillar edema. Control hearts not subjected to high pressure also showed marked disruption of fine structure, suggesting that ischemic injury played an important role in producing lesions in all hearts.