Sites of lymph follicle formation in the draining popliteal lymph nodes of mice locally injected with antigenic and mitogenic substances

Our previous studies showed that some antigenic and mitogenic substances, when locally injected into mice, efficiently produced new lymph follicles outside pre-existing follicles in draining lymph nodes, whereas others had virtually no effect. In the present experiments, young adult male mice were injected with several antigens and mitogens in the rear footpad, and the number and development sites of newly produced lymph follicles in the draining popliteal nodes were studied using serial sections of the nodes obtained between 5 and 21 days after injection. In the unstimulated state, each popliteal node contained a limited number of lymph follicles which mostly lay in a portion of the peripheral cortex overlaying the deep cortex (this portion is referred to as the PCOU), whereas a portion of the peripheral cortex extending beyond the deep cortex (referred to as the PCBU) was underdeveloped with only occasional follicles. Mice treated with soluble PHA or fluid tetanus toxoid developed germinal centers in association with existing follicles but failed to produce new follicles. The PCBU of the draining nodes remained underdeveloped, and the number and distribution pattern of lymph follicles within a draining node were comparable to those in the control node. Animals treated with LPS (50 micrograms), Con A, alum-precipitated PHA or alum-precipitated tetanus toxoid produced significantly large numbers of new follicles outside pre-existing follicles in the draining nodes, the new follicles produced in the PCBU being generally more numerous than those in the PCOU. In these draining nodes, the peripheral cortex, comprising a number of follicles, was found to overlie the deep cortex and extend beyond the deep cortex towards the hilar region. In animals given a less effective stimulant, such as ferritin or a smaller dose of LPS (10 micrograms), the draining nodes produced a relatively small number of new follicles, most of which were formed in the PCBU. The present results indicate that in the mouse popliteal node, the PCBU is morphologically underdeveloped under normal conditions, but develops lymph follicles in response to exogenous stimuli more readily than the PCOU, and that substances efficient in inducing follicle formation can be regarded as capable of stimulating the development of the peripheral cortex.     

Chymotrypsin-like enzymes are involved in sperm penetration through the vitelline coat of Ciona intestinalis egg

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

Immunocytochemical detection of Ca2+-dependent subspecies of protein kinase C in mouse embryos before and during compaction

Summary To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca²⁺-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.     

Glycosphingolipids in cultured lens epithelial cells from dog and rhesus monkey

Vertebrate lens tissues contain several species of acidic andneutral glycosphingolipids in relatively high amounts. However,the epithelia with capsule from dog and rhesus monkey lenseshad a simpler composition and lower content of glycosphingolipidsthan whole lenses. Gangliosides and neutral glycosphingolipidsin monolayer cultures of lens epithelial cells were also differentfrom those in whole lenses. Although -galactosyl (Gal1-3Ga1-R)or Lewis^x (Galß1-4[Fuc1-3]GlcNAc-R) epitopes werefound in glycosphingolipids from whole lenses, they were notdetected in those from monolayer cultures of dog and rhesusmonkey lens cells. In addition, significant changes in ganglio-seriesgangliosides were induced in monolayer cultures of both cells,where GM3 and GD3 were predominant. Immunofluorescence studyrevealed a characteristic distribution of cell surface gangliosidesin confluent monolayers. These findings suggest that glycosphingolipidsynthesis in lens epithelia is intrinsically different fromthat in cortical and nuclear fibres, and that the expressionof Lewis^x and -galactosyl epitopes in glycosphingolipids appearsto be associated with the differentiation of epithelial cellsto fibres. gangliosides glycosphingolipids lens epithelial cells Lewis^x rhesus monkey.     

Chloroplast DNA variation inDesmodium subgenusPodocarpium (Leguminosae): Infrageneric phylogeny and infraspecific variations

Chloroplast DNA (cpDNA) restriction site variation was examined in five species ofDesmodium subgenusPodocarpium (Leguminosae; Papilionoideae; Desmodieae). Twenty four phylogenetically informative cpDNA mutations were scored. The cladistic analysis of characters based on the 24 mutations resulted in the most parsimonious tree which supports the monophyly of the subgenus.Desmodium elegans of subgenusDollinera was the sister group of subgenusPodocarpium in this tree. The groupings obtained from the cpDNA characters were consistent with the present infrageneric classification system for the subgenus except for the infraspecific taxa ofD. podocarpum. Three groups withinD. podocarpum, which were incongruent with the infraspecific classification of the species, were distinguished by a total of four site mutations. The first group consisted of subsp.podocarpum, subsp.fallax, and subsp.oxyphyllum var.oxyphyllum; the second subsp.oxyphyllum var.oxyphyllum; and the last subsp.oxyphyllum var.oxyphyllum and var.mandshuricum.     

The taxonomy and distribution ofEuchresta japonica (Leguminosae)

The taxonomic position ofEuchresta trifoliolata is discussed, and it is concluded that it is identical withE. japonica. Consequently,E. japonica can be considered to be discontinuously distributed between SW. Japan and S. China and represents a unique pattern of distribution, which has not been known for the flora of Japan and China.E. japonica is also considered to represent a relic distribution pattern.     

A compound possessing antitumor and interferon-inducing activities derived from the common antigen (OEP) of Pseudomonas aeruginosa.

OEP, a component consisting mainly of protein with small amounts of lipids and sugars, has been isolated from the autolysate of Pseudomonas aeruginosa and purified by physicochemical methods. It possesses remarkable biological properties, showing antitumor and interferon-inducing activities. As regards the antitumor activity of the sample, the ED50 value against ascites sarcoma-180 was 1 microgram/kg mouse/day, and its interferon-inducing activity amounted to 15 units at a concentration of 0.01 microgram/ml. Both activities increased after protease digestion, reaching about ten times those of the sample which had not undergone digestion. The protease-treated OEP contained 17% protein, 14.5% total sugars, 31% lipids, 12.5% hexosamine, 3.8% KDO, and 2.7% phosphorus. Neutral sugars consisted of 12.4% rhamnose, 2.7% mannose, 66.9% glucose, and other unidentified material. Total lipids derived from OEP consisted of 65% loosely-bound and 35% covalently-bound lipids; the former contained C14:10, C16:0, C16:1, C18:0, and C15:1 acids and the latter, beta-OH C10:0, C12:0, alpha-OH C12:0, beta-OH C12:0, C16:0, and C16:1 acids. The antitumor and interferon-inducing activities of OEP remained after the removal of loosely-bound lipids from OEP.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.