PHOTOSYNTHETIC PHYSIOLOGY OF PROROCENTRUM MARIAE-LEBOURIAE (DINOPHYCEAE) DURING ITS SUBPYCNOCLINE TRANSPORT IN CHESAPEAKE BAY1

This paper presents results of field studies on the estuarine dinoflagellate Prorocentrum mariae-lebouriae (Parke & Ballantine) Faust in Chesapeake Bay. We tested the hypothesis that the photosynthetic physiology of Prorocentrum shows adaptive responses to low-light during a lengthy subpycnocline transport in estuarine circulation. Prorocentrum underwent a seasonal, northward trnasport between February and June, 1984 and 1985. Low cell densities occurred in the seaward part of the estuary during winter and early-spring, subpycnocline populations progressed up-estuary in the ensuring 2–3 months, and dense surface populations developed in the mesohaline portion of the estuary thereafter. We sampled Prorocentrum from surface and subpycnocline waters and measured photosynthesis-light (P-I) relations with in situ incubations. The photophysiology of Prorocentrum collected below the pycnoline differed from that of cells in the surface mixed layer in that photosynthetic efficiency, α-cell^?1, was higher, photosynthetic capacity, P_max-cell^?1 was ·4 times greater for subpycnocline (≦ 10m) samples than for those from the surface mixed layer (≧ 6m). Comparison of in situ photosynthetic properties to those generated in laboratory studies showed that values of α·cell^?1 for both surface and subpycnocline samples were in the range found for cultures in low-light. Concentrations of Chls a, c and peridinin·cell^?1 and molar pigment ratios peridinin: Chl a and Chl a: Chl c were not significantly different for the surface and subpycnocline samples, nor were C · cell^?1 or C : Chl a. Chloroplast and starch volume fractions and the number of thylakoids were the same for samples collected at different depths, and there was no evidence of cytoplasmic vacuolization in any field samples. These morphometric data for cells from natural populations of Prorocentrum most closely resembled data for laboratory cultures grown at or near 2.6E·m-^?2·4d^?1. A lower growth irradiance of 0.3E·m^?2·d^?1 produced indications of stress in cultures, including starch depletion and vacuolization, that were never observed in natural populations. Based on the combination of these findings, we conclude that Prorocentrum is adapted to low-light both in the surface mixed layer and beneath the pycnocline, although certain photophysiological characteristics distinguish these two groups of samples.     

High diversity of alpha-globin haplotypes in a Senegalese population, including many previously unreported variants.

RFLP haplotypes at the alpha-globin gene complex have been examined in 190 individuals from the Niokolo Mandenka population of Senegal: haplotypes were assigned unambiguously for 210 chromosomes. The Mandenka share with other African populations a sample size-independent haplotype diversity that is much greater than that in any non-African population: the number of haplotypes observed in the Mandenka is typically twice that seen in the non-African populations sampled to date. Of these haplotypes, 17.3% had not been observed in any previous surveys, and a further 19.1% have previously been reported only in African populations. The haplotype distribution shows clear differences between African and non-African peoples, but this is on the basis of population-specific haplotypes combined with haplotypes common to all. The relationship of the newly reported haplotypes to those previously recorded suggests that several mutation processes, particularly recombination as homologous exchange or gene conversion, have been involved in their production. A computer program based on the expectation-maximization (EM) algorithm was used to obtain maximum-likelihood estimates of haplotype frequencies for the entire data set: good concordance between the unambiguous and EM-derived sets was seen for the overall haplotype frequencies. Some of the low-frequency haplotypes reported by the estimation algorithm differ greatly, in structure, from those haplotypes known to be present in human populations, and they may not represent haplotypes actually present in the sample.     

Conformational changes induced in bovine lens alpha-crystallin by carbamylation. Relevance to cataract.

Carbamylation of lens proteins may contribute to cataractogenesis in certain medical conditions where blood urea is elevated for prolonged periods. This paper reports on the effects of carbamylation on the physicochemical properties of one of the major lens structural proteins, alpha-crystallin. In particular it is shown that carbamylation alters the tertiary and secondary structure of the protein, leading to an increased reactivity of protein thiols, resulting in interchain disulphide bonding.     

Isolation and hydroxylysine glycoside content of some cyanogen bromide-cleaved fragments of collagen from bovine corneal stroma.

1. Guinea-pig low-density lipoproteins were isolated by ultracentrifugation and iodinated either by the IC1 method or by the chloramine-T procedure. 2. The efficiency of labelling by both methods was essentially the same. 3. When the two products were compared by ultracentrifugation, gel chromatography and immunodiffusion analysis, no significant difference in their properties was detected. 4. When they were compared by gradient-gel electrophoresis, aggregates were found in the product of the IC1 method, but not in the lipoprotein iodinated by the chloramine-T process. 5. Both products were metabolized by the guinea pig with essentially the same fractional catabolic rate. 6. The fractional catabolic rate of lipoprotein iodinated by the chloramine-T method was not significantly different from that of lipoprotein biologically labelled in the protein moiety with [75Se]selenomethionine. 7. It is concluded that the products of both methods of iodination are almost equally acceptable, provided that the optimum conditions for the chloramine-T reaction are carefully established.     

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells.

Phagocytic processing of heat-killed Listeria monocytogenes by peritoneal macrophages resulted in degradation of these bacteria in phagolysosomal compartments and processing of bacterial antigens for presentation to T cells by class II MHC molecules. Within 20 min of uptake by macrophages, Listeria peptide antigens were expressed on surface class II MHC molecules, capable of stimulating Listeria-specific T cells. Within this period, degradation of labeled bacteria to acid-soluble low molecular weight catabolites also commenced. Immunoelectron microscopy was used to evaluate the compartments involved in this processing. Upon uptake of the bacteria, phagosomes containing Listeria fused rapidly with both lysosomes and endosomes. Class II MHC molecules were present in a tubulo-vesicular lysosome compartment, which appeared to fuse with phagosomes, as well as in the resulting phagolysosomes containing internalized Listeria; these compartments were all positive for Lamp 1 and cathepsin D and lacked 46-kD mannose-6-phosphate receptors. In addition, class II MHC and Lamp 1 were co-localized in vesicles of the trans Golgi reticulum, where they were segregated from 46-kD mannose-6-phosphate receptors. Vesicles containing both Listeria-derived components and class II MHC molecules were also observed; some of these may represent vesicles recycling from phagolysosomes, potentially bearing processed immunogenic peptides complexed with class II MHC. These results support a central role for lysosomes and phagolysosomes in the processing of bacterial antigens for presentation to T cells. Tubulo-vesicular lysosomes appear to represent an important convergence of endocytic, phagocytic and biosynthetic pathways, where antigens may be processed to allow binding to class II MHC molecules and recycling to the cell surface.     

Pearson syndrome and mitochondrial encephalomyopathy in a patient with a deletion of mtDNA.

A patient is described who has features of Pearson syndrome and who presented in the neonatal period with a hypoplastic anemia. He later developed hepatic, renal, and exocrine pancreatic dysfunction. At the age of 5 years he developed visual impairment, tremor, ataxia, proximal muscle weakness, external ophthalmoplegia, and a pigmentary retinopathy (Kearns-Sayre syndrome). Muscle biopsy confirmed the diagnosis of mitochondrial myopathy. Analysis of mtDNA from leukocytes and muscle showed mtDNA heteroplasmy in both tissues, with one population of mtDNA deleted by 4.9 kb. The deleted region was bridged by a 13-nucleotide sequence occurring as a direct repeat in normal mtDNA. Both Pearson syndrome and Kearns-Sayre syndrome have been noted to be associated with deletions of mtDNA; they have not previously been described in the same patient. These observations indicate that the two disorders have the same molecular basis; the different phenotypes are probably determined by the initial proportion of deleted mtDNAs and modified by selection against them in different tissues.     

1H NMR studies on bovine cyclophilin: preliminary structural characterization of its complex with cyclosporin A

Cyclophilin (163 residues, Mr 17737), a peptidyl prolyl cis-trans isomerase, is a cytosolic protein that specifically binds the potent immunosuppressant cyclosporin A (CsA). The native form of the major bovine thymus isoform has been analyzed by 2D NMR methods, COSY, HOHAHA, and NOESY, in aqueous media. The 156 main-chain amides in CyP yield 126 observable NH/alpha CH couplings (81%, Gly pairs counted as 1). Following exhaustive D2O exchange, 44 amide resonances remain visible. Further analysis of the NH/NH, NH/alpha CH, and alpha CH/alpha CH regions of the COSY and NOESY data sets indicates that the residual amides in D2O form a coherent hydrophobic domain which yields 2D NMR features suggestive of a beta-sheet. Many (43/126) of the amide resonances have been classified according to amino acid type. In the aromatic region of the spectra, the assignment of the ring spin systems is nearly complete (12/15 Phe, 2/2 Tyr, 1/1 Trp, and 3/4 His). This has successfully lead to the complete assignment of all of their beta CH's, main-chain alpha CH resonances, and many of the backbone amide resonances (8/12 Phe, 2/2 Tyr, 1/1 Trp, and 2/3 His). In other regions of the spectrum, the side-chain and main-chain resonances for 10/23 Gly, 9/9 Ala, 5/11 Thr, 5/9 Val, and 1/6 Leu have been completely assigned. The drug-free cyclophilin and CsA-bound cyclophilin form two discrete protein structures that are in slow exchange on the NMR time scale. Comparison of the fingerprint regions from the COSY spectra obtained from the two forms of the protein reveals a minimum of 16 cross-peaks which are clearly shifted upon complexation. In fact, on the basis of chemical shift changes observed in assigned side-chain and main-chain resonances, only a relatively few of the amino acid residues identified to date are perturbed by complex formation. These include 3 Phe (8, 12, and 14) and the Trp in the aromatic region and 2 Ala (7 and 8) in the Ala/Thr region. In the upfield-shifted methyl region, an assigned Leu and Val spin system and a spin system labeled X10 (an Ile or Leu) are affected by complex formation. In addition, a new aliphatic spin system, labeled X11, which shows a close spatial relationship to the perturbed Phe12, is observed in this region of the spectrum. In summary, the regions of the protein altered by complex formation can be divided into two categories: a hydrophobic and a H2O-accessible domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

A new mitochondrial disease associated with mitochondrial DNA heteroplasmy.

A variable combination of developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal neurogenic muscle weakness, and sensory neuropathy occurred in four members of a family and was maternally transmitted. There was no histochemical evidence of mitochondrial myopathy. Blood and muscle from the patients contained two populations of mitochondrial DNA, one of which had a previously unreported restriction site for AvaI. Sequence analysis showed that this was due to a point mutation at nucleotide 8993, resulting in an amino acid change from a highly conserved leucine to arginine in subunit 6 of mitochondrial H(+)-ATPase. There was some correlation between clinical severity and the amount of mutant mitochondrial DNA in the patients; this was present in only small quantities in the blood of healthy elderly relatives in the same maternal line.