Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 m in diameter) and less electron-dense than the D-granules (less than 0.6 m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.     

Effects of NO2 and O3 Alone or in Combination on Kidney Bean Plants (Phaseolus vulgaris L.): Growth, Partitioning of Assimilates and Root Activities

Ten-day old kidney bean plants (Phaseolus vulgaris L. cv. Shin-edogawa)were exposed to 2.0 and 4–0 parts 10^–6 NO2, and0.1, 0.2, and 0.4 parts 10^–6 O3 alone or in combinationfor 2, 4, and 7 d. The effects of these air pollutants wereexamined with respect to the growth, partitioning of assimilates,nitrogen uptake, soluble sugar content, and root respiration. Decreased dry matter production was significant with all treatmentsexcept 2.0 parts 10^–6 NO₂ and 0.1 parts 10^–6 O₃.Exposure to mixtures of the gases produced more severe suppressionof growth than exposure to the single gases. Root/shoot ratiowas significantly lowered at 7 d by the gas treatments otherthan 2.0 parts 10^–6 NO₂ and 0.1 parts 10^–6 O₃. Thetotal nitrogen content of plants was increased by all treatments;the higher percent of nitrogen found with O₃ exposure will resultfrom the growth retardation which increases the concentrationof nitrogen in the plants because the absorption of nitrogenby roots was unaffected. The combination of O₃ with NO₂ significantlydecreased the assimilation of NO₂ by the plants. The concentration of soluble sugars in roots was decreased bythe gas treatments. There was a strong positive correlationbetween soluble sugar content and dry weight of the roots harvestedat 7 d. Root respiration was relatively unchanged until 5 dand then decreased significantly at 7 d by 2.0 parts 10^–6NO₂ and 0–2 parts 10^–6 O₃. Retarded growth of theroots and the decreased root respiration may be due to diminishedtranslocation of sugars from leaves to roots caused by exposureto air pollutants. The uptake of soil nitrogen was not closelyrelated with root respiration in the case of O₃ exposure. Key words: NO₂, O₃, Phaseolus vulgaris, Growth, Sugars, Root respiration     

The relation between calcium and cell wall in growing rice leaf

The percentage of pectic substances in the cell wall of riceleaf decreased with the ageing of the leaf but other componentsin the cell wall changed little during leaf growth. Cell wallcomponents were not affected by a nutritional deficiency ofcalcium. At the beginning of the growth of rice leaf, calciumin the cell wall existed only in the pectic substance fractionboth in the calcium sufficient and deficient leaves. However,in the cell wall of the mature leaf, a considerable amount ofcalcium was found in the lignin fraction. The amount of calciumin this form was larger in calcium sufficient leaves than indeficient leaves. Calcium seems to occur in two forms in the cell wall, combinedwith pectic substances and with ligneous substances. This assumptionwas further supported by calcium distribution in enzymaticallydegradated fractions of the cell wall. Calcium seems to be combined with pectic substances by a strongerchemical bond than with the ligneous substances. ¹Present address: Tohoku Agricultural Experiment Station, Omagari,Akita.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

The Presence of Tyrosinase and Related Proteins in Human Epidermis and Their Relationship to Melanin Type

The present study was carried out to investigate the abundance of tyrosinase and related proteins (TRP-1 and TRP-2) in human epidermis and their relationship to melanin type. Positive immunocytochemical staining was seen for all three proteins in epidermal melanocytes. For each protein the numbers of positively stained melanocytes were similar in all subjects studied irrespective of skin type. Following 5 daily suberythemal doses of UVB the melanocytes were larger, more dendritic, and increased in number. With TRP-1 and TRP-2 the increase in number in response to UVB was unrelated to skin type and, hence, with melanin type but with tyrosinase there was a much greater increase in skin types III and IV than in skin type I and II. The enhanced numbers of tyrosinase-positive melanocytes were accompanied by increased staining intensity, suggesting a greater expression of tyrosinase in the melanocytes from skin types III and IV compared with skin types I and II. This increase in tyrosinase could be related to the greater levels of eumelanin found in skin types III and IV, and this is in keeping with the view that higher levels of tyrosinase are associated with the production of eumelanin than phaeomelanin.     

Isolation of a new cDNA clone encoding an Rh polypeptide associated with the Rh blood group system

We searched for a human chromosome that would restore the cholesterol metabolism in 3T3 cell lines (SPM-3T3) derived from homozygous sphingomyelinosis mice (spm/spm). Mouse A9 cells containing a single copy of pSV2neo-tagged chromosomes 9, 11, or 18 derived from normal human fibroblasts served as donor cells for transfer of human chromosomes. Purified A9 microcells were fused with SPM-3T3 cells, and the microcell hybrids were selected in medium containing G418 antibiotics. The microcell hybrids that contained human chromosomes 9, 11, or 18 in a majority of cells were examined. The accumulation of intracellular cholesterol in the microcell hybrids containing a chromosome 18 decreased markedly, whereas in the microcell hybrids containing either chromosomes 9 or 11 it was similar to that in SPM3T3 cells. The SPM-3T3 cells with an intact chromosome 18 were further passaged and subcloned. Clones which again accumulated intracellular cholesterol had concurrently lost the introduced chromosome 18. The abnormal accumulation was associated with a decrement in the esterification of exogenous cholesterol. These findings suggest that the gene responsible for the abnormal cholesterol metabolism in the spm/spm mice can be restored by a hu man chromosome 18. The gene was tentatively mapped on 18pter18p11.3 or 18q21.3qter that was lost during subcloning, thereby resulting in reaccumulation of the intracellular cholesterol.     

Antimelanoma Activity of Chloroquine,an Antimalarial Agent With High Affinity for Melanin

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in up-take of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin     

The occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation

A previous electron microscopic study of the cat testis revealed that spermatids derived from the same spermatogonium are joined together by intercellular bridges. The present paper records the observation of similar connections between spermatocytes and between spermatids in Hydra, fruit-fly, opossum, pigeon, rat, hamster, guinea pig, rabbit, monkey, and man. In view of these findings, it is considered likely that a syncytial relationship within groups of developing male germ cells is of general occurrence and is probably responsible for their synchronous differentiation. When clusters of spermatids, freshly isolated from the germinal epithelium are observed by phase contrast microscopy, the constrictions between the cellular units of the syncytium disappear and the whole group coalesces into a spherical multinucleate mass. The significance of this observation in relation to the occurrence of abnormal spermatozoa in semen and the prevalence of multinucleate giant cells in pathological testes is discussed. In the ectoderm of Hydra, the clusters of cnidoblasts that arise from proliferation of interstitial cells are also connected by intercellular bridges. The development of nematocysts within these groups of conjoined cells is precisely synchronized. Both in the testis of vertebrates and the ectoderm of Hydra, a syncytium results from incomplete cytokinesis in the proliferation of relatively undifferentiated cells. The intercellular bridges between daughter cells are formed when the cleavage furrow encounters the spindle remnant and is arrested by it. The subsequent dissolution of the spindle filaments establishes free communication between the cells. The discovery of intercellular bridges in the two unrelated tissues discussed here suggests that a similar syncytial relationship may be found elsewhere in nature where groups of cells of common origin differentiate synchronously.     

gamma-Butyrobetaine as a specific antagonist for carnitine in the development of the early chick embryo

The effect of γ-butyrobetaine alone and with the addition of carnitine on the development of the early excised chick embryo has been studied. γ-Butyrobetaine in appropriate amounts exerts an inhibitory effect which can be relieved or annulled by the inclusion of appropriate amounts of carnitine. This has been interpreted as a metabolite-antimetabolite relationship, in which the normal metabolite, carnitine, is antagonized by the structurally closely related γ-butyrobetaine, and is regarded as evidence of an important role of carnitine in the metabolism of the developing chick embryo.