Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

Characterization of lignin in situ by photoacoustic spectroscopy

Photoacoustic spectroscopy is a recently developed nondestructive analytical technique that provides ultraviolet, visible, and infrared absorption spectra from intensely light scattering, solid, and/or optically opaque materials not suitable for conventional spectrophotometric analysis. In wood and other lignocellulosics, the principal ultraviolet absorption bands, in the absence of photosynthetic pigments, arise from the aromatic lignin component of the cell walls. Photoacoustic spectra of extracted lignin fragments (milled wood lignin) and synthetic lignin-like polymers contain a single major absorption band at 280 nanometers with an absorption tail extending beyond 400 nanometers. Photoacoustic spectra of pine, maple, and oak lignin in situ contain a broad primary absorption band at 300 nanometers and a longer wavelength shoulder around 370 nanometers. Wheat lignin in situ, on the other hand, exhibits two principle absorption peaks, at 280 nanometers and 320 nanometers. The presence of absorption bands at wavelengths greater than 300 nanometers in intact lignin could result from (a) interacting, nonconjugated chromophores, or (b) the presence of more highly conjugated structural components formed as the result of oxidation of the polymer. Evidence for the latter comes from the observation that, on the outer surface of senescent, field-dried wheat culms (stems), new absorption bands in the 350 to 400 nanometer region predominate. These new bands are less apparent on the outer surface of presenescent wheat culms and are virtually absent on the inner surface of either senescent or presenescent culms, suggesting that the appearance of longer wavelength absorption bands in senescent wheat is the result of accumulated photochemical modifications of the ligin polymer. These studies also demonstrate photoacoustic spectroscopy to be an important new tool for the investigation of insoluble plant components.     

Membrane Development in the Cyanobacterium, Anacystis nidulans, during Recovery from Iron Starvation

Deprivation of iron from the growth medium results in physiological as well as structural changes in the unicellular cyanobacterium Anacystis nidulans R2. Important among these changes are alterations in the composition and function of the photosynthetic membranes. Room-temperature absorption spectra of iron-starved cyanobacterial cells show a chlorophyll absorption peak at 672 nanometers, 7 nanometers blue-shifted from its normal position at 679 nanometers. Iron-starved cells have decreased amounts of chlorophyll and phycobilins. Their fluorescence spectra (77K) have one prominent chlorophyll emission peak at 684 nanometers as compared to three peaks at 687, 696, and 717 nanometers from normal cells. Chlorophyll-protein analysis of iron-deprived cells indicated the absence of high molecular weight bands. Addition of iron to iron-starved cells induced a restoration process in which new components were initially synthesized and integrated into preexisting membranes; at later times, new membranes were assembled and cell division commenced. Synthesis of chlorophyll and phycocyanins started almost immediately after the addition of iron. The absorption peak slowly returned to its normal wavelength within 24 to 28 hours. The fluorescence emission spectrum at 77K changed over a period of 14 to 24 hours during which the 696- and 717-nanometer peaks grew to their normal levels, and the 684 nanometer peak moved to 687 nanometers and its relative intensity decreased to its normal level. Analysis of chlorophyll-protein complexes on polyacrylamide gels showed that high molecular weight chlorophyll-protein bands were formed during this time, and that low molecular weight bands (related to photosystem II) disappeared. The origin of the fluorescence emission at 687 and 696 nanometers is discussed in relation to the specific chlorophyll-protein complexes formed during iron reconstitution.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

Spectral Irradiance and Distribution of Pigments in a Highly Layered Marine Microbial Mat

The spectral irradiance from 400 to 1,100 nm was measured with depth in the intertidal sand mats at Great Sippewissett Salt Marsh, Mass. These mats contained at least four distinct layers, composed of cyanobacteria, purple sulfur bacteria containing bacteriochlorophyll a (Bchl a), purple sulfur bacteria containing Bchl b, and green sulfur bacteria. Spectral irradiance was measured directly by layering sections of mat on a cosine receptor. Irradiance was also approximated by using a calibrated fiber-optic tip. With the tip, irradiance measurements could be obtained at depth intervals less than 250 μm. The irradiance spectra were correlated qualitatively and quantitatively with the distribution of the diverse chlorophyll pigments in this mat and were compared with spectra recorded in plain sand lacking pigmented phototrophs. We found that the shorter wavelengths (400 to 550 nm) were strongly attenuated in the top 2 mm of the mat. The longer wavelengths (red and near infrared) penetrated to much greater depths, where they were attenuated by Bchl a, b, and c-containing anoxygenic phototrophic bacteria. The specific attenuation bands in the irradiance spectra correlated with the specific in vivo absorption bands of the Bchl-protein complexes in the bacteria. We concluded that the pigments in the phototrophs had a profound affect on the light environment within the mat. It seems likely that the diverse Bchl-protein complexes found in the anoxygenic phototrophs evolved in dense mat environments as a result of competition for light.     

Action spectra for nitrate and nitrite assimilation in blue-green algae

Action spectra for the assimilation of nitrate and nitrite have been obtained for several blue-green algae (cyanobacteria) with different accessory pigment composition. The action spectra for both nitrate and nitrite utilization by nitrate-grown Anacystis nidulans L-1402-1 cells exhibited a clear peak at about 620 nanometers, corresponding to photosystem II (PSII) C-phycocyanin absorption, the contribution of chlorophyll a (Chl a) being barely detectable. The action spectrum for nitrate reduction by a nitrite reductase mutant of A. nidulans R2 was very similar. All these action spectra resemble the fluorescence excitation spectrum of cell suspensions of the microalgae monitored at 685 nanometers—the fluorescence band of Chl a in PSII. In contrast, the action spectrum for nitrite utilization by nitrogen-starved A. nidulans cells, which are depleted of C-phycocyanin, showed a maximum near 680 nanometers, attributable to Chl a absorption. The action spectrum for nitrite utilization by Calothrix sp. PCC 7601 cells, which contain both C-phycoerythrin and C-phycocyanin as PSII accessory pigments, presented a plateau in the region from 550 to 630 nanometers. In this case, there was also a clear parallelism between the action spectrum and the fluorescence excitation spectrum, which showed two overlapped peaks with maxima at 562 and 633 nanometers. The correlation observed between the action spectra for both nitrate and nitrite assimilation and the light-harvesting pigment content of the blue-green algae studied strongly suggests that phycobiliproteins perform a direct and active role in these photosynthetic processes.     

Photosynthetic Enhancement in the Diatom Phaeodactylum tricornutum

Enhancement phenomena in photosynthesis of the diatom Phaeodactylum tricornutum Lewin were studied by means of a Haxo oxygen electrode and 2 monochromatic light beams. It was necessary to correct for a minor non-linearity in rate oxygen evolution vs. intensity similar to that reported for Chlorella. Action spectra on complementary backgrounds and derived enhancement spectra were compared to in vivo absorption spectra in identifying the character of pigment systems 1 and 2. Fucoxanthin, chlorophyll c, and chlorophyll a-670 are clearly assignable to pigment system 2 which absorbs in excess at wavelengths 400 to 678 mμ. Chlorophyll a-1 (and a portion of the fucoxanthin) are assignable to pigment system 1 which absorbs in excess at wavelengths 678 to 750 mμ. Enhancement values were generally lower than those observed in Chlorella or Anacystis and lead to conclusion that diatom pigmentation provides an effective light harvesting apparatus.     

Analysis of absorption spectra changes induced by temperature lowering on phycobilisomes,thylakoids and chlorophyll-protein complexes

Using fourth derivative analysis, differences between room and low temperature absorption spectra were studied. The positions of most absorption bands of the water-soluble, accessory pigment complex, the phycobilisome, remained unchanged after cooling. The stability of the wavelength positions of chlorophyll a forms in vivo as a function of temperature (Gulyaev, B.A. and Litvin, F.F. (1967) Biofizika 12, 845–854) was generally confirmed. The wavelength positions of all chlorophyll a forms in the P-700 chlorophyll a protein complex were unchanged when the preparations were cooled to ?196°C. Likewise, with other chlorophyll-containing materials: the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex and the thylakoids of higher plants, algae, and cyanobacteria, the wavelength positions of most chlorophyll a forms were stable upon cooling. An exception was a 680 nm chlorophyll a band which was generally split at low temperature into two bands with the materials investigated. An interpretation of the multiplicity of chlorophyll spectral forms and the spectral changes induced by cooling for these forms is given using exciton theory and the energy-coupling variation of chlorophyll a molecules.     

A new form of chlorophyll C involved in light-harvesting

A new form of chlorophyll c has been isolated from the pyrmnesiophyte Pavlova gyrans Butcher. This pigment is spectrally similar to chlorophyll c₂, but all the absorption maxima (454, 583, and 630 nm in diethyl ether) are shifted 4 to 6 nanometers to longer wavelengths. The new pigment can be separated from other chlorophyll c-type pigments by reversed-phase high performance liquid chromatography and thin layer chromatography. Both chlorophylls c₁ and c₂ are found with the new chlorophyll c pigment in P. gyrans, and it has also been detected in the chrysophyte Synura petersenii Korsh. The light-harvesting function of the new chlorophyll c pigment is indicated by its presence along with chlorophyll c₁ and c₂ in a light-harvesting pigment-protein complex isolated from P. gyrans in which chlorophyll c pigments efficiently transfer absorbed light energy to chlorophyll a.     

Water-soluble chlorophyll protein of Brassica oleracea var. Botrys (cauliflower)

A water-soluble chlorophyll protein was prepared from Brassica oleracea var. Botrys (cauliflower) and purified by (NH₄)₂SO₄ fractionation and by chromatography on a DEAE-cellulose column. The chlorophyll protein contained chlorophylls a and b in the ratio 6:1, and no carotenoids. The molecular weight, determined by means of gel filtration on Sephadex G-100, was 78000. The chlorophyll protein showed absorption peaks at 273, 340, 384, 420, 438, 465, 628, 674 and 700 nm. Since the three bands at 384, 420 and 438 nm all have approximately the same height, the spectrum is different from that of chlorophyll a in organic solvents. The fluorescence of the chlorophyll protein showed a peak at 683 nm, with shoulders at 706 and 745 nm at room temperature, and peaks at 685, 706 and 744 nm at the temperature of liquid N₂. An apo-protein was prepared by removing the chlorophylls with 2-butanone and purified by precipitation with (NH₄)₂SO₄. The apo-protein thus prepared had an absorption band at 273 nm but none at longer wavelengths. The apo-protein could be combined with chlorophylls, forming a chlorophyll protein which had spectral characteristics similar to those of the original.     

Indépendance des formes spectrales de chlorophylle a et des holochromes chlorophylliens

Independence of special forms of chlorophyll a and chlorophyll holochromesZea mays L. seedlings were cultivated for 10 days with submission to 4 s illumination periods interspersed with dark periods varying in length from 30 min to 6 h depending on the lot analyzed. The results show that, for the case in which the dark periods were shorter than 1 h, the relative proportions of different spectroscopic chlorophyll forms (maxima at 662, 670, 677.5, and 684 nm) were constant. For longer durations of darkness between illuminations, the relative proportion of the form Ca₆₇₀ increases, while that of Ca₆₈₄ diminishes with the length of darkness; to a lesser extent, the relative proportion of Ca₆₆₂ increases and a form Ca₆₉₂ disappears. A scheme is proposed to explain the evolution of the relative proportions of the different spectral forms.The different chlorophyll holochromes present in the chloroplasts were also analysed. If the dark period was longer than 1 h, chlorophyll was associated with peptide chains of molecular weights 21 000 and 29 000. If the dark period was shorter than 1 h chlorophyll was associated with four peptide chains of molecular weights 21 000, 25 000, 29 000 and 70 000.The results taken together demonstrate that a given spectral chlorophyll a form cannot be associated with a definite chlorophyll holochrome.     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

In vivo chlorophyll forms in higher plants and algae sensitive to treatment with lutein

Lamella preparations of spinach, Chlorella, Phaeodactylum, Anabatnaand Porphyra were treated with a hydrophobic reagent, lutein,and the absorption and fluorescence spectra in the red regionbefore and after treatment were compared for changes causedby the treatment. Absorption spectra of all these preparationsunderwent the same spectral change, transformation of a bandat 684 nm into a band at 666 nm. The longer the maximum wavelengthof the red peak, the greater was the fractional absorbance decreaseat 684 nm. The content of C684 (the chlorophyll form responsiblefor the 684 nm band) in the lamellae was estimated from thefractional decreases as being progressively higher in the orderof Phaeodactylum, Porphyra, Anabatna, Chlorella and spinach.The fluorescence spectra at liquid nitrogen temperature beforetreatment showed two bands. The longer wavelength band was transformedby the treatment into a shorter wavelength band(s), as describedbelow, according to the maximum wavelengths: spinach, F735F695(or F686); Chlorella, F715F700 (or F686); Phaeodactylum, anunidentified componentF690; Anabaena, F732F685 (or F695); Porphyra,F726F683. These chlorophyll forms with fluorescence maxima between715 and 735 nm were, therefore, designated C684 based on absorptionspectrophotometry, and are considered to play a role in photosystemII. (Received August 15, 1972; )     

Partial analysis of the bands seen in circular dichroism spectra of green and blue-green algal cells and thylakoids

A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.     

Greening of etiolated bean leaves in far red light

Eight-day-old dark-grown bean leaves were greened by prolonged irradiation with far red light. Growth, chlorophyll content, oxygen-evolving capacity, photophosphorylation capacity, chloroplast structure (by electron microscopy), and in vivo forms of chlorophyll (by low temperature absorption and derivative spectroscopy on intact leaves) were followed during the greening process. Chlorophyll a accumulated slowly but continuously during the 7 days of the experiment (each day consisted of 12 hours of far red light and 12 hours of darkness). Chlorophyll b was not detected until the 5th day. The capacity for oxygen evolution and photophosphorylation began at about the 2nd day. Electron microscopy showed little formation of grana during the 7 days but rather unfused stacks of primary thylakoids. The thylakoids would fuse to give grana if the leaves were placed subsequently in white light. The low temperature spectroscopy of intact leaves showed that the chlorophyll a was differentiated into three forms with absorption maxima near 670, 677, and 683 nanometers at −196 C during the first few hours and that these forms accumulated throughout the greening process. Small amounts of two longer wavelength forms with maxima near 690 and 698 nanometers appeared at about the same time as photosynthetic activity.     

Energy Transfer from the Phycobilisomes to Photosystem II Reaction Centers in Wild Type Cyanidium caldarium

Nonsaturating light at 600 or 436 nanometers was used to excite specifically phycocyanin or chlorophyll a, respectively, both of which participate in light capture in photosystem II of Cyanidium caldarium. The ratio of absorption of light by phycocyanin to chlorophyll in photosystem II in this organism is >20 at 600 nanometers and ≤0.2 at 436 nanometers.     

Light- and MgCl2-dependent characteristics of four chlorophyll-protein complexes isolated from the marine dinoflagellate,Glenodinium sp.

Chlorophyll-protein complexes previously isolated from low-light (80 μE·m⁻²·s⁻¹) log cultures of the marine dinoflagellate, Glenodinium sp., were further characterized. SDS solubilization in combination with polyacrylamide gel electrophoresis in the presence of Deriphat 160-C resolved four discrete chlorophyll-protein bands. In order to elucidate the functional role of Glenodinium sp., room-temperature absorption and fluorescence spectra, protein composition, and pigment molar ratios were obtained for each complex. Results indicated that complex I was analogous to the green plant Photosystem I complex and was also associated with light-harvesting chlorophyll c₂. Complex II was highly enriched in chlorophyll c₂, devoid of peridinin, and demonstrated energy transfer from chlorophyll c to chlorophyll a within the complex, indicating the presence of a light-harvesting component. Based on peridinin: chlorophyll a ratios and fluorescence excitation spectra analyses for complexes III and IV, it was concluded that these complexes contained functional peridinin-chlorophyll a-protein complexes. Changing the ionic environment during isolation of the complexes, or altering the growth irradiance of Glenodinium sp. cultures, resulted in a significant alteration of distribution of chlorophyll a among the chlorophyll-protein complexes.     

Treatment of the thylakoid membrane with surfactants : assessment of effectiveness using the chlorophyll a absorption spectrum

Treatment of higher plant (Nicotiana tabacum L. var. Samsun) chloroplast thylakoid membranes with surfactants results in a shift of the chlorophyll a absorption maximum in the red spectral region from its in vivo value of 678.5 nanometers to shorter wavelengths. The magnitude of this shift is correlated with membrane disruption, and is not necessarily due to the release of pigment from pigment-protein complexes present in the membrane. Membrane disruption has been measured by the amount of pigment in the supernatant fraction after centrifugation of surfactant treated membranes. For an equivalent amount of disruption, the extent of the blue-shift is influenced by the ionic nature of the surfactant: anionic surfactants cause small shifts, cationic surfactants cause the largest (~10 nanometers) shifts, and nonionic surfactants produce intermediate shifts. The wavelength of maximum absorbance of chlorophyll a in the red region is a convenient criterion for assessing the potential utility of different surfactants for studies on the structure, composition and function of higher plant thylakoid membranes.     

Studies on the absorption spectra of chlorophyll a in aqueous dispersions of lipids from the photosynthetic membranes

The absorption spectra of chlorophyll a were studied in aqueousdispersions of four major lipid components present in the thylakoidmembranes. Chlorophyll a in aqueous dispersions of uncharged galactolipidsrevealed two absorption bands, at 670 and 745 nm, when the molecularratio of chlorophyll to lipid was higher than 0.2. The latterband may be due to the formation of microcrystals of chlorophylla. Chlorophyll a in aqueous dispersions of negatively chargedlipids revealed a single absorption band at 670 nm. However,chlorophyll a was decomposed during measurement in these lipiddispersions. The absorption spectra of chlorophyll a in aqueous dispersionsof mixture of galactolipid and charged lipid were apparentlysimilar to those of chlorophyll a in the charged lipid dispersion.Chlorophyll a, however, was not decomposed in these aqueousdispersions of lipid mixtures. It is concluded that the presence of both galactolipid and chargedlipid are necessary to reconstruct the state of chlorophylla dissolved in the lipid phase in the thylakoid membranes. The red absorption band of chlorophyll a in the reconstructedsystem composed of chlorophyll a, charged and uncharged lipids,appeared at 670 nm with a half bandwidth of 22 nm. Analysisof the absorption spectrum in the fourth derivative and thecurve-fitting methods indicated that the red band was composedmainly of a single band with a peak at 670–671 nm. ¹ Present address: Department of Biology, College of GeneralEducation, University of Tokyo, Komaba, Meguro-ku, Tokyo 153,Japan. (Received October 13, 1977; )