Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Screening of thermostable leucine and alanine dehydrogenases in thermophilicBacillus strains

Summary Screening of leucine and alanine dehydrogenases in thermophilicBacillus strains was carried out to develop their utilization for industrial and analytical catalysts. Out of the 28 thermophilic strains tested, four strains,Bacillus sp. DSM 405, 730 and 1521, andB. sphaericus DSM 462, abundantly produce both the enzymes. Both the enzyme activities in these thermophiles are enhanced by addition of the substrates to a polypeptone medium.     

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5 and 3 ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) _n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) _n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.Paper 3082 of the Forest Research Laboratory, Oregon State University     

Molecular phylogeny of Dipterocarpaceae in Southeast Asia using RFLP of PCR-amplified chloroplast genes

Dipterocarpaceae is the dominant family of Southeast Asia's climax tropical rain forest region, and it contains the region's most important commercial timber species. A molecular phylogeny of the Dipterocarpaceae subfamily Dipterocapoideae was constructed using restriction fragment length polymorphisms of polymerase chain reaction-amplified specific genes in chloroplast DNA. A total of 141 site changes were detected among ten genera and 30 species in 11 different genes: rbcL, psbA, psbD, rpoB, rpoC, petB, atpH, 16S, psaA, petA and trnK. Phylogenetic trees constructed by Wanger parsimony and neighbor-joining methods, using Upuna as the outgroup, displayed five monophytelic groups that included Upuna: HopeaShorea-Parashorea-Neobalanocarpus; Dryobalanops; Dipterocarpus; Anisoptera-Vatica-Cotylelobium; and Upuna. The phylogenetic trees clearly separate species with two different base chromosome numbers: the first group is x=7, and the other is x=11. The x=7 group is thought to be in a synapomorphic character state. Parashorea lucida is a sister to most Shorea species. Neobalanocarpus heimii and Hopea from a clade of a sister to two Shorea species, and Cotylelobium and Vatica are closely related species. Our conclusions agree with a phylogeny derived from wood anatomy data analysis, and with Symington's and Ashton's taxonomic classifications.The raw data of the PCR-RFLP analysis can be obtained from the authors     

Comparative biochemical studies of carotenoids in sea urchins—III. Relationship between developmental mode and carotenoids in the Australian echinoids Heliocidaris erythrogramma and H. tuberculata and a comparison with Japanese species

The sea urchin Heliocidaris tuberculata is typical of most echinoids in having a small egg and a feeding larva, while H. erythrogramma has a large egg and modified development through a non-feeding larvae. The carotenoids in the gonads of these two species were investigated from the comparative biochemical points of view. The carotenoid content of the buoyant eggs of H. erythrogramma was approximately 60 times that of the negatively-buoyant eggs of H. tuberculata. With respect to cytoplasmic volume, however, the carotenoid concentration in the eggs of H. tuberculata was approximately twice that in the eggs of H. erythrogramma. In both species β-echinenone was the principal carotenoid found and their carotenoid patterns were similar. It is very interesting from a functional point of view that carotenoid levels per cytoplasmic volume are conserved across most of the species we have examined irrespective of phylogeny and egg size. In light of this result we suggest that carotenoids may play an important role in developing stage in all echinoids including indirect and direct developers.     

REPRODUCTIVE ISOLATION BY HOST SPECIFICITY IN A PAIR OF PHYTOPHAGOUS LADYBIRD BEETLES

Crossing experiments and food-choice tests show that two sympatric species of phytophagous ladybird beetles, Epilachna niponica and E. yasutomii, are reproductively isolated by host-plant specificity. Adult beetles selected their natural hosts when given choices, though some accepted the host of the other species when no choice was offered. In each species, survival of larvae to the second instar was significantly higher on their own host plant. No evidence for sexual isolation, gametic isolation, hybrid inviability, or reduced hybrid fertility was detected. Reproductive isolation by host specificity is an important prerequisite for certain models of sympatric speciation. Although the present example supports the plausibility of such models, an allopatric origin of host-plant specificity cannot be discounted.     

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.     

Insulin release and metabolism of calcium, adenine and adenosine 3',5'-cyclic monophosphate.

In order to assess further the mechanisms involved in insulin release, we prelabeled rat pancreatic islets of Langerhans by incubating either 45Ca or [2-3H]adenine. When prelabeled islets were perfused with a glucose-free medium (the experiment with 45Ca) and a medium containing 2.8 mM glucose (the experiment with [2-3H]adenine) respectively, a constant rate of efflux of the radioactivity was established by 30 min in each case. D-Glucose at 16.7 mM concentration elicited a rapid efflux of 45Ca and [2-3H]adenine derivatives ([3H]Ad) within 4 to 6 min after commencing the step-wise stimulation by glucose, concomitantly with insulin release. However, L-glucose and D-galactose littel stimulated both 45Ca and [3H]Ad release. Lanthanum chloride caused a burst peak of 45Ca release in the absence of glucose. A rapid efflux of 45Ca was caused by beta-D-glucose and D-glyceraldehyde to much lesser extent than by alpha-D-glucose. The slowly rising concentration of glucose at 0.1 mM/min of gradient level failed to elicit any rapid efflux of 45Ca or [3H]Ad, although insulin release occurred in accordance with an increase in glucose concentration. Even when the gradient of glucose concentration was raised to 0.7 mM/min, glucose failed to stimulate an efflux of [3H]Ad but the subsequent stimulation by 16.7 mM glucose caused a rapid efflux of [3H]Ad concomitantly with the release of insulin. No rapid efflux of 45Ca was observed under a slow-rise glucose stimulation until the gradient level of the glucose concentration was raised to 6.7 mM. Analysis of distribution of the radioactive adenine derivatives after incubation showed that the adenosine fraction had the highest radioactivity in the medium followed by the ATP, adenine and cAMP fraction in that order, and the ATP fraction had the highest radioactivity in the islet. The ratio of radioactivity in the cAMP fraction in the medium to the total count was the highest among all. On the basis of these results, it was suggested that the discharge of [3H]Ad and 45Ca might occur with the alteration of the membrane permeability induced by a rapid change of the glucose concentration, and that their discharge might perhaps link to the glucoreceptor mechanism directly controlling insulin release.     

Insulin release and eflux of [32P]Phosphate from islets of rat Langerhans treated with iodoacetic acid and the anomers of D-glucose.

To clarify the insulin-releasing mechanism, we studied insulin release and the efflux of [32P]phosphate by glucose at 0.1 mM/min of gradient level or at 16.7 mM, and other metabolism in islets of rat Langerhans. When treated with 1 mM iodoacetic acid (IAA) plus the anomers of D-glucose at 2.8 mM for 6 min at 37 degrees C, islets elicited insulin at half the control rate under the step-wise stimulation by glucose and at the same rate as the control under the slow-rise stimulation by glucose. Using islets treated with IAA plus the alpha anomer at 16.7 mM, the step-wise stimulation secreted insulin at half a rate of the control and the slow-rise stimulation at the rate lower than the control, which was not significantly different from the control rate. Treatment with IAA plus the beta anomer at 16.7 mM inhibited insulin release under both types of stimulations by glucose. The step-wise stimulation caused the same rapid efflux of [32P]phosphate from IAA-treated islets as from the control islets, except for islets treated with IAA plus the beta anomer at 16.7 mM. The rate of glucose utilization in islets was inhibited by all IAA-treatments to the same extent, being merely half the control rate. Treatments with IAA plus the anomers at 16.7 mM significantly reduced the formation of [3H]-cAMP and the activity of protein phosphokinase in islets, while in the presence of the anomers at 2.8 mM IAA produced no significant effect. Neither IAA-treatments altered the uptake of 45Ca and the ATP content in islets. The uptake of [14C]IAA was significantly enhanced by the presence of the beta anomer at 16.7 mM to two times the control level. On the basis of these results, we suggested that the B cell might contain both glucoreceptors and rate-sensors of glucose controlling insulin release and the former might be less sensitive to IAA as compared with the latter.