Identification of a novel two-partner secretion system from<Emphasis Type="Italic"> Burkholderia pseudomallei</Emphasis>

Two adjacent genes, bpaA and bpaB, whose products display significant similarity to a number of two-partner secretion (TPS) systems have been identified in Burkholderia pseudomallei strain 08, but are absent from the closely related avirulent species B. thailandensis. They possess a number of sequence features characteristic of TPS systems, including the presence of an NPNGI motif in a region of BpaA which strongly resembles a TPS secretion domain. BpaA is a very large protein (~530 kDa) and contains three repeats, each 600–800-amino acids long. Putative membrane-spanning regions in BpaB were identified through alignment with TpsB family members, and this also revealed an N-terminal extension not found in other TpsB proteins. The bpaA gene was found to be absent from the majority of B. pseudomallei strains. It appears that bpaAB are located within a putative genomic island that is inserted in close proximity to a methionine tRNA^CAT-encoding gene. Expression of BpaA was undetectable in cells grown in laboratory media. However, owing to the similarity of BpaA to known adhesin molecules, a potential role of BpaA in virulence was investigated in cell culture and in an animal model, but no evidence for such a role was found in these test systems.Communicated by W. Goebel     

Protein expression pattern of P-glycoprotein along the gastrointestinal tract of the Yucatan micropig

The purpose of this study is to characterize the distribution pattern of P-gp protein levels along the entire GI tract in the Yucatan micropig, which is being developed as a model for human drug bioavailability. Small and large intestines were freshly obtained and divided into about 37 segments and 10 segments, respectively (ca., 1 foot/segment). Epithelial cells from the small intestine were obtained by an elution method; whereas, a scraping method was applied to the large intestine. Total cellular protein was isolated from the epithelial cells. Western blot analysis using P-gp antibody showed that the amount of P-gp protein increased distally from the duodenum to the ileum over approximately a 10-fold range. P-gp protein in the large intestine was present at a higher level in the central portion, but the absolute amount was much less than what was found in the small intestine.     

Human embryonal carcinoma tumor antigen,Gp200/GCTM-2, is podocalyxin

We previously characterized a peanut agglutinin-binding tumor antigen, gp200, a surface membrane glycoprotein expressed on human embryonal carcinoma, a malignant stem cell of testicular tumors. Gp200 is remarkably similar to another embryonal carcinoma antigen, GCTM-2, a cell differentiation marker that is also detected in blood of testis cancer patients, yet neither molecular identity is known. We now report the identity of gp200 as podocalyxin. Protein sequence results of gp200 peptides match with podocalyxin sequence. Furthermore, two distinct monoclonal antibodies, specific for podocalyxin, react positively with gp200. Therefore, gp200 is a testicular tumor form of podocalyxin, a surface membrane glycoprotein that was originally discovered as a scaffolding extracellular matrix protein of kidney podocyte cells. Podocalyxin is also expressed on subsets of hematopoietic cells where it has a putative function as a cell adhesion protein. This is the first report of podocalyxin expression on malignant cells.     

Activation of Apical K<Superscript>+</Superscript> Conductances by Muscarinic Receptor Stimulation in Rat Distal Colon: Fast and Slow Components

In the epithelium of rat distal colon the acetylcholine analogue carbachol induces a transient increase of short-circuit current (I_sc) via stimulation of cellular K⁺ conductances. Inhibition of the turnover of inositol-1,4,5-trisphosphate (IP₃) by LiCl significantly reduced both the amplitude and the duration of this response. When the apical membrane was permeabilized with nystatin, LiCl nearly abolished the carbachol-induced activation of basolateral K⁺ conductances. In contrast, in epithelia, in which the basolateral membrane was bypassed by a basolateral depolarization, carbachol induced a biphasic increase in the K⁺ current across the apical membrane consisting of an early component carried by charybdotoxin- and tetraethylammonium-sensitive K⁺ channels followed by a sustained plateau carried by channels insensitive against these blockers. Only the latter was sensitive against LiCl or inhibition of protein kinases. In contrast, the stimulation of the early apical K⁺ conductance by carbachol proved to be resistant against inhibition of phospholipase C or protein kinases. However, apical dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, or a Ca²⁺-free mucosal buffer solution significantly reduced the early component of the carbachol-induced apical K⁺ current. The presence of an apically localized Na⁺/Ca²⁺-exchanger was proven immunohistochemically. Taken together these experiments reveal divergent regulatory mechanisms for the stimulation of apical Ca²⁺-dependent K⁺ channels in this secretory epithelium, part of them being activated by an inflow of Ca²⁺ across the apical membrane.

Glucocorticoids exacerbate the deleterious effects of gp120 in hippocampal and cortical explants

Glucocorticoids (GCs), the adrenal steroids secreted during stress, can compromise the ability of hippocampal neurons to survive numerous necrotic insults. We have previously observed that GCs worsen the deleterious effects of gp120, the glycoprotein of the acquired immune deficiency syndrome virus, which can indirectly damage neurons and which is thought to play a role in the neuropathological features of human immunodeficiency virus infection. Specifically, GCs augment gp120-induced calcium mobilization, ATP depletion, decline in mitochondrial potential, and neurotoxicity in fetal monolayer cultures from a number of brain regions. In the present report, we demonstrate a similar gp120/GC synergy in adult hippocampal and cortical explants. We generated explants from rats that were either adrenalectomized, adrenally intact, or intact and treated with corticosterone to produce levels seen in response to major stressors. Metabolic rates in explants were then indirectly assessed with silicon microphysiometry, and cytosolic calcium concentrations were assessed with fura-2 fluorescent microscopy. We observed that basal levels of GCs tonically augment the disruptive effects of gp120 on metabolism in the CA1 cell field of the hippocampus and in the cortex. Moreover, raising GC concentrations into the stress range exacerbated the ability of gp120 to mobilize cytosolic calcium in a number of hippocampal cell fields. Finally, we observed that the synthetic GC prednisone had similarly exacerbating effects on gp120. Thus, GCs can worsen the deleterious effects of gp120 in a system that is more physiologically relevant than the fetal monolayer culture and in a region-specific manner.     

Early Development of the Secretory Cavity of Peltate Glands in Humulus lupulus L. (Cannabaceae)

Early development of the secretory cavity of chemically fixed peltate glands in Humulus lupulus L. showed secretions with different densities, light, gray and dark, in the cytoplasm of disc cells and in the periplasmic space adjacent to the developing secretory cavity. Secretions were detected in the disc cell wall and subsequently in the developing secretory cavity under the subcuticular wall of the sheath. Light and gray secretions in the cavity possessed a membrane-like surface feature. Secretions were in contact with the irregular inner surface of the cuticle. Secretions contributed to the thickening of the cuticle, whereas the membrane-like surface feature contributed to a network of Cannabis striae distributed throughout the cuticle. This study supports an early development and organization of the secretory cavity in H. lupulus, parallel to those in Cannabis, and may represent common features for lipophilic glands in angiosperms.     

Procathepsin L self-association as a mechanism for selective secretion

The lysosomal cysteine pro-protease procathepsin L was enriched in dense vesicles detectable when microsomes prepared from wild-type or transformed mouse fibroblasts were resolved on sucrose gradients. These dense vesicles did not comigrate with proteins characteristic of the endoplasmic reticulum, Golgi, endosomes or lysosomes. When gradient fraction vesicles were lysed at acidic pH in the presence of excess mannose 6-phosphate to prevent binding to mannose phosphate receptors, the majority of the procathepsin L was associated with the membrane, not the soluble, fraction. Immunogold labeling of procathepsin L in thin sections of cells or gradient fractions, using antibodies directed against the propeptide to avoid detection of the mature enzyme in dense lysosomes, revealed that the proenzyme was concentrated in dense cores localized in small vesicles near the plasma membrane and in multivesicular bodies. Consistent with the density of the gradient fraction and the electron density of the cores, yeast two-hybrid assays indicated the proenzyme could bind itself but could not interact with the aspartic proprotease procathepsin D. The data suggest that in mouse fibroblasts procathepsin L may self-associate into aggregates, initiating the formation of dense vesicles that could mediate the selective secretion of procathepsin L independent of mannose phosphate receptors.     

The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [³⁵S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus. Received: 3 July 1999 / Accepted: 21 August 1999     

Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers

Spider dragline silk is a remarkable fiber made of unique proteins—spidroins—secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (>65.8%) and titer (554.7 mg L^–1) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m⁻³. The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials.