Yeast Glucan in the Cyst Wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae , which consists of an outer dense layer of mannan, a middle lucent layer of β−1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall P. carinii , as well as the cell wall of S. cerevisiae , can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains β-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is β,3-gIucan laminari-pentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae . These observations indicate that a major component of the cyst wall of P. carinii is β-1,3-glucan.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Light limitation of phytoplankton development in an oligotrophic lake - Loch Ness, Scotland

• 1 The underwater light climate in Loch Ness is described in terms of mixing depth (Z_m) and depth of the euphoric zone (Z_eu). During periods of complete mixing, Z_m equates with the mean depth of the loch (132 m), but even during summer stratification the morphometry of the loch and the strong prevailing winds produce a deep thermocline and an epilimnetic mixed layer of about 30 m or greater. Hence, throughout the year the quotient Z_m/Z_eu is exceptionally high and the underwater light climate particularly unfavourable for phytoplankton production and growth.
• 2 Phytoplankton biomass expressed as chlorophyll a is very low in Loch Ness, with a late summer maximum of less than 1.5 mg chlorophyll a m^-3 in the upper 30 m of the water column. This low biomass and the resulting very low photosynthetic carbon fixation within the water column are evidence that a severe restraint is imposed on the rate at which phytoplankton can grow in the loch.
• 3 The chlorophyll a content per unit of phytoplankton biovolume and the maximum, light-saturated specific rate of photosynthesis are both parameters which might be influenced by the light climate under which the phytoplankton have grown. However, values obtained from Loch Ness for both chlorophyll a content (mean 0.0045 mg mm^-3) and maximum photosynthetic rate (1–4 mg C mg Chla^-1 h^-1) are within the range reported from other lakes.
• 4 Laboratory bioassays with the natural phytoplankton community from Loch Ness on two occasions in late summer when the light climate in the loch is at its most favourable, suggest that even then limitation of phytoplankton growth is finely balanced between light and phosphorus limitation. Hence, for most of the year, when the light climate is less favourable, phytoplankton growth will be light limited.
• 5 Quotients relating mean annual algal biomass as chlorophyll a (c. 0.5 mg Chla m^-3) and the probable annual specific areal loading of total phosphorus (0.4–1.7 g TP m^-2 yr^-1) suggest that the efficiency with which phytoplankton is produced in Loch Ness per unit of TP loading is extremely low when compared with values from other Scottish lochs for which such an index has been calculated. This apparent inefficiency can be attributed to suppression of photosynthetic productivity in the water column due to the unfavourable underwater light climate.
• 6 These several independent sources of evidence lead to the conclusion that phytoplankton development in Loch Ness is constrained by light rather than by nutrients. Loch Ness thus appears to provide an exception to the generally accepted paradigm that phytoplankton development in lakes of an oligotrophic character is constrained by nutrient availability.

     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Novel changes in the plasma membrane and cortical cytoplasm during wound-induced contraction in a giant-celled green alga

Summary Changes in the plasma membrane surface and in the cortical cytoplasm during wound healing in giant green algal cells ofErnodesmis verticillata (Kützing) Brgesen were followed using scanning and transmission electron microscopy. Microvillus-like structures that contain cytoplasmic and cytoskeletal constituents were observed emanating from the surface of the plasma membrane at the retracting/cut end of wounded cells. These delicate structures seem to be remnants of cell wall-plasmalemma connections that draw out the plasma membrane and cortical components from the contracting cytoplasm as it pulls away from the cell wall. Most of these connections break during wound healing and, when contraction stops, the microvillus-like protrusions become progressively shorter. In cells treated with a calmodulin antagonist (W-7), a number of distinctive bodies accumulate that are of unknown composition, are oblong in shape, and have a diameter slightly smaller than the protoplasmic protrusions. Ultrastructural and other data indicate that these bodies result from retrieved constituents of the plasma-membrane protrusions, as they do not accumulate in unwounded drugtreated cells or in cells treated in W-5. These findings suggest that the protoplasmic protrusions accumulate membrane and cytoplasmic components that are retrieved and recycled during wound healing inErnodesmis by a novel mechanism. The combined plasma membrane surfaces of the microvillus-like protrusions may help to account for the drastic decrease in surface area that occurs during wound healing.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy - W-7 N-[6-aminohexyl]-5-chloro-1-naph-thalenesulfonamide - W-5 N-[6-aminohexyl]-1-naphthalenesulfonamide     

The realized food niches of three species of stream-dwelling triclads (Turbellaria)

SUMMARY. 1. The diet of Phagocata vitta, Crenobia alpina and Polycelis felina in a Welsh stream was examined, using a serologicat technique, at monthly intervals over 18 months.
2. For each triclad species, dietary composition changed little with time or increasing body size, and overall feeding intensity was greatest in summer and least in winter.
3. Alt species fed most heavily on oligochaetes, chironomids and Gammarus. Phagocata vitta fed lightly on Plecoptera, Trichoptera and Ephemeroptera whilst the other two triclad species fed substantially on the first two of these prey categories.
4. Food niche breadth indices indicated a wide diet for all species with some specialization in some months. Electivity indices showed positive electivity for oligochaetes and chironomids by all species and for Plecoptera by C. alpina and P. felina .
5. Diets overlapped and none had a food refuge: therefore the potential for interspecific competition for food exists.     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.