SDS-PAGE Analysis Of Proteins Research Articles

Extraction and purification of seaweed protein from Ulva sp. - Challenges to overcome

Ulva sp. has gained attention as a promising and sustainable protein source, due to its high essential amino acid content. This study investigated traditional extraction methods using a double screw press followed by acid or heat precipitation. Furthermore, the screw pressing was combined by a novel purification method that combines mild heat treatment with microfiltration for removal of chlorophyll, to produce white Ulva protein. The double screw press followed by acid precipitation or heat denaturation showed low total protein yields, with 14% of the biomass protein extracted, of which half was soluble. The highest total protein yield (5.5%) was achieved with acid precipitation at pH 2, while heat denaturation at 90 °C resulted in a total protein yield of 4.1%. Acid precipitation proved to be effective with ∼ 81% precipitation of solubilized protein compared to ∼ 43% for heat denaturation. While microfiltration effectively retained chlorophyll in the retentate, the recovery of protein in the permeate remained challenging. SDS-PAGE analysis of proteins in different microfiltration-fractions showed that larger protein complexes (>250 kDa) were unable to transmit through the membranes, resulting in a maximum recovery of 24.9% of the feed-protein in the permeate. Further refinement of Ulva fractionation protocols is imperative for improvement.

Genes expression in Penaeus monodon of Bangladesh; challenged with AHPND-causing Vibrio parahaemolyticus

Vibrio parahaemolyticus, the causative agent of Acute hepatopancreatic necrosis disease (AHPND), was discovered in 2013 as a unique isolate that produces toxins and kills penaeid shrimps in devasting nature in Bangladesh and causes severe economic losses. This research aimed to understand the expressions of immune genes in different stages of the host species, Penaeus monodon, against virulence and toxin genes upon being challenged with V. parahaemolyticus. Healthy post-larvae (PL) samples were collected from southwestern of Bangladesh from July 2021 to August 2022. The tryptic soy agar with 1.5% sodium chloride (NaCl) was used to inoculate the cells of V. parahaemolyticus, and the tryptic soy broth (TSB) with 1.5% NaCl was used to transfer the colonies. The spectrophotometry measured bacteria density. PCR, qPCR, SDS-PAGE, and Western blot measured gene expression and survivability after the immersion challenge. The 1 × 105CFU/mL of V. parahaemolyticus was used for 144 h.p.i (hours post-infection) challenge to six stages of post-larvae (PL) of P. monodon (PL20, PL25, PL30, PL35, PL40, and PL45), PL30 and PL35 showed 100% mortality by day 72 (h.p.i.) after exposure that indicated most vulnerable to V. parahaemolyticus. The expression of immune and toxic genes was confirmed by qPCR. The immune genes toll-like receptors (TLR), prophenoloxidase (ProPO), lysozyme (lyso), and penaeidin (PEN) of PL20 and PL25 of P. monodon were expressed robustly up-trends. PL30 and PL35 showed the lowest gene expression at the end of 72 (h.p.i.). At the end of the 144 (h.p.i.) exposure, the immune genes TLR, ProPO, lyso, and PEN expressed highest in PL45 than other post-larvae stages of P. monodon. The toxic genes (pirA, ToxR, ToxA, ToxB, tlh, tdh, and trh) in PL30 and PL35 of P. monodon after exposure of V. parahaemolyticus were expressed highest at the end of the 72 (h.p.i.). The lowest toxic genes expressions were revealed in PL20 and PL45 at the end of the 144 (h.p.i.). The SDS-PAGE analysis of proteins from the bacterium revealed identical protein profiles with toxic genes, and those toxins were further confirmed by Western blot. The 20 kDa, 78 kDa (ToxR), 20 kDa, 25 kDa (ToxA), 25 kDa (ToxB), 20 kDa, 27 kDa, 75 kDa (tdh), and 20 kDa, 27 kDa, 75 kDa, and 78 kDa (trh) proteins were strong responses in Western blot, indicating the crucial involvement of these immune-related genes in the defense and recovery of the first-line defense mechanisms during V. parahaemolyticus infection to shrimp. The all-toxic genes showed a unique homology and those derived from the common ancestor compared with V. parahaemolyticus (NCBI accession no. AP014859.1). All clades were derived with different traits with very low genetic distance, where the overall mean distance was 3.18 and showed a very uniform and homogenous pattern among the lineages. The V. parahaemolyticus infection process in different PL stages in P. monodon revealed novel insights into the immune responses. The responses may lead to the subsequent production of a DNA vaccine, enhancing shrimp health management to minimize the economic losses due to AHPND experiencing an outbreak of early mortality syndrome (EMS) toward sustainable production P. monodon (shrimp).

SDS-PAGE analysis of hormone treated larvae of honeybee Apis cerana indica (F.) (Hymenoptera: Apidae)

SDS-PAGE analysis of protein was carried out in fifth instar larvae of worker and drone caste at 24 hrs and 36 hrs treatment with acetone (control), JH-III and 20-HE. 52kDa to 106 kDa protein bands were observed in both caste of larvae after JH-III, 20-HE and acetone treatment after 24 hrs and 36 hrs. Female specific 170kDa vitelogenin like protein band was observed in larvae of both caste. Caste specific protein band 191kDa and 32kDa was observed in worker larvae and 38kDa protein band was observed in drone larvae. This difference is due to different developmental timing and difference in physiology of both caste.

VARIATIONS OF PROXIMAL COMPOSITION IN PILEA MICROPHYLLA (L.) LIEBM DUE TO DESICCATION AND REHYDRATION IMPACTS

Water deficiency is the most significant abiotic stress factor for land plants. Most plants are unable to survive desiccation to the air dry state. There are however a few species from lower plant groups to flowering plants that tolerate desiccation known as resurrection plants. So, the present investigation was undertaken tostudy the biochemical changes in Pileamicrophylla against desiccation (1, 3, 5 and 7 days) and rehydration (30 min) stress. As an initial part of the study total sugar, protein and proline content were analyzed and showed a gradual decline during the stress periods. The SDS-PAGE analysis of protein indicated thedisapperance of certain bands in the desiccated and rehydrated samples (7D) when compared to the control indicating the denaturation of proteins during stress. Similarly, the appearance of new bands (15kDa on 3D and 20 and 17kDa on the 5D) were noticed suggesting the formation of stress related proteins to tide over desiccation. The amount of free amino acids increased in P. microphylla, in pace with desiccation periods. Constitutive increase in the level of proline (the stress amino acid) accumulation is seen when compared with the control i.e., 6 fold higher than that of the control, after exposed to desiccation for 5 D. Increase in proline level relating to exposure time of desiccation stress suggests its role as osmolyte. Similarly, a reduction in chlorophyll level and an increase in carotenoid were also observed during stress. Decrease of total chlorophyll content was observed with duration of desiccation. The decrease in chlorophyll a and b was recovered during rehydration up to 5 D. Further studies are warranted at molecular level in terms of stress proteins and genes involved in desiccation tolerance in this plant.

MOLECULAR ANALYSIS OF THE SUPPRESSION OF COLLETROTRICHUM FALCATUM BY TRICHODERMA HARZIANUM IN SUGARCANE

Detailed tritrophic interaction studies were conducted under in vitro and in vivo conditions among sugarcane, Colletotrichum falcatum, the red rot pathogen of sugarcane and its antagonist Trichoderma harzianum. Two way interaction under in vitro conditions clearly indicated that T. harzianum was able to inhibit the growth of C. falcatum which was evidenced by degradation of C. falcatum proteins and expression of new proteins by Trichoderma both at intra and extra cellular level. Similar suppression of the pathogen by the antagonist was also observed during three way interaction with sugarcane tissue in vitro and reduction of symptom production in stalk tissue in vivo. At the molecular level, SDS-PAGE analysis of proteins extracted from different samples of three way interaction in vitro revealed that there were differentially expressed proteins (ca. 100-150 kDa) when C. falcatum was inoculated in sugarcane tissue followed by Trichoderma at different intervals, particularly 72 h after pathogen inoculation. Under field conditions, C. falcatum symptoms in the stalk tissue were inhibited by simultaneous inoculation of Trichoderma and C. falcatum. Protein profiles of these samples in SDS-PAGE indicated that the sugarcane proteins (ca. 80-100 kDa) were degraded during the host-pathogen interaction and the same proteins were retained during three way interaction of pathogen along with the antagonist on sugarcane. Furthermore, the growth and proliferation of T. harzianum over C. falcatum inside the stalk tissue was shown by duplex PCR, tissue bioassay, and microscopic observations. All these results confirm that T. harzianum was able to suppress C. falcatum in sugarcane by antagonistic mechanism and expression of newer proteins that could be related to defence / antifungal nature.

SDS-PAGE analysis of urinary proteins in dogs with heartworm disease

The aim of the study was to describe the urinary electrophoretic pattern of dogs with heartworm disease. Urine samples from 15 heartworm-infected and 15 healthy dogs were taken. Urinary specific gravity, urinary protein concentration and the urine protein/creatinine (U P/C) ratio were determined. Urine proteins were fractionated using SDS-PAGE. Results showed statistically significant differences for the U P/C ratio (P 0.05) between groups. Urinary protein SDSPAGE analysis showed eight distinct bands in the urine of heartworm-infected dogs. The presence of proteins exclusively found in the urine of infected dogs suggests renal damage, even in cases of light proteinuria, indicating that SDS-PAGE is a sensitive method for the identification and characterisation of renal proteinuria in dogs with heartworm disease.

Functional and Structural Characterization of PaeM, a Colicin M-like Bacteriocin Produced by Pseudomonas aeruginosa

Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg(2+) ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

Plant growth promoting rhizobacterial (PGPR) bioconsortia mediates induction of defense-related proteins against infection of root rot pathogen in mulberry plants

The mixture of Pseudomonas fluorescens isolate Pf1 and Py15 and Bacillus subtilis isolate 16 (Bs16) was found to protect mulberry plants from root rot disease caused by Macrophomina phaseolina. Induction of defense-related proteins and chemicals by the mixture of Pf1, Py15 and Bs16 against challenge inoculation with M. phaseolina in mulberry were studied. The activity of defense enzymes peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), β-1, 3-glucanase, chitinase and defense chemical total phenols were found to be significantly higher in combined application of biocontrol agents Pf1+Py15+Bs16 followed by Pf1+Bs16, Pf1+Py15 and Py15+Bs16 treated plants when compared to individual treatments and control. The SDS-PAGE analysis of protein in biocontrol agents treated plant showed eight proteins with molecular weight of 14, 23, 30, 35, 45, 50, 60 and 98 kDa. In healthy plants only six proteins (excepting 14 and 98 kDa) appeared in biocontrol agents treated plant two additional new proteins appeared. These results suggest that induction of defense enzymes involved in phenyl propanoid pathway and accumulation of total phenols and PR-proteins might have contributed to restriction of invasion of M. phaseolina in mulberry roots.

Spore stage expression of a vegetative insecticidal gene increase toxicity of Bacillus thuringiensis subsp. aizawai SP41 against Spodoptera exigua

To enhance the toxicity of the Bacillus thuringiensis subsp. aizawai strain SP41 (SP41), the vegetative insecticidal protein (Vip) gene vip3A from SP41 was redirected to the sporulation stage by replacing its native promoter with the strong promoter P19 of the cry11Aa operon. Compared to the wild type, SP41 with PVIP ( vip3A with its native promoter and ter) had the relative expression ratios of 457, 548, and 290 at 8, 14, and 20 h of cultivation, respectively, as measured by quantitative reverse transcription polymerase chain reaction (PCR). SP41 transformed by P19VIP ( vip3A controlled by P19 promoter with vip3A ter) showed higher expressions (23, 2055, 1831) at the same time points. SP41 with P19VIP20 ( vip3A controlled by P19 promoter and containing P20 and operon ter) had the lowest expression levels (3, 11, 9) at any time point. SDS-PAGE analysis of proteins in the culture supernatant of the P19VIP at 8, 14, and 20 h demonstrated a significant increase in Vip3A at the sporulation stage. Using the surface contamination bioassay, the 50% lethal concentration (LC 50) of whole culture of PVIP, P19VIP, and P19VIP20 at 20 and 48 h of cultivation against Spodoptera exigua larvae were (68.3, 21.2, and 60.2 μg cm −2) and (69.8, 41.8, and 74.6 μg cm −2), respectively, compared with 86.6 and 104.4 μg cm −2 for SP41. The results showed that Vip from P19VIP, expressed at spore stage at 20 and 48 h, can increase the toxicity of SP41 for 4.1- and 2.5-fold, respectively.

Change of Proteins in Instant Chinese Noodle by the Fermentation of Lactobacillus plantarum NRIC 0380 Affects the Noodle Quality

The lactic acid fermentation of instant Chinese noodle sheet by Lactobacillus plantarum NRIC 0380 changed the noodle quality as evaluated by texture and sensory tests. This interesting change was induced by only a short 2-h fermentation time, however the noodle quality subsequently degraded with prolongation of the fermentation of up to 24 h. SDS-PAGE analysis of proteins in the noodle sheet indicated no change in proteins extracted from the 2-h fermented noodle sheet compared to non-fermented noodle sheet. In contrast, native-PAGE analysis showed a shift in molecular weight of gluten proteins, with those extracted from the 2-h fermented noodle sheet having a higher molecular weight than those from non-fermented noodle sheet. These results strongly suggested a conformational change of gluten proteins in the noodle sheet caused by the short 2-h fermentation. The 2-h lactic acid fermentation decreased the pH from 8.5 to 7.5, but gluten proteins extracted from noodle sheet made with the addition of lactic acid to adjust the pH to 7.3 did not show this increase in molecular weight. Thus, the change from native state of gluten proteins does not seem to be induced by the presence of the lactic acid itself but by other factor(s) associated with lactic acid fermentation. On the other hand, the reduction of noodle quality by prolonged fermentation for up to 24 h seems to induce degradation of albumin and globulin proteins as revealed by SDS-PAGE analysis and also the change in the native state of gluten proteins as detected by native-PAGE analysis.

Phosphodiesterase Activity of CvfA Is Required for Virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.

Heat Shock-Induced Changes in Lipid and Protein Metabolism in the Endoplasmic Reticulum of Barley Aleurone Layers

Heat shock in barley aleurone layers induces heat shock protein synthesis and suppresses secretory protein synthesis by selectively destabilizing their mRNAs. In addition, the endoplasmic reticulum (ER) membranes upon which secretory protein mRNAs are translated become vesiculated during heat shock, leading to the hypothesis that ER dissociation and targeted mRNA destabilization are linked mechanistically. Supporting this, ER can be heat adapted, and heat-adapted ER has higher levels of fatty acid saturation in membrane phospholipids which do not vesiculate upon heat shock. Secretory protein mRNAs are also more stable in heat-adapted cells. To understand better heat shock-induced changes in ER membranes, we examined ER membrane proteins and enzymes involved in phosphatidylcholine biosynthesis and phospholipid turnover in heat-shocked aleurone cells. Heat shock significantly increased the activity of phospholipases A2 and D, and shortly thereafter significant but gradual increases in choline kinase and phosphocholine glyceride transferase activities and a sharp increase in phosphorylcholine citidyl transferase activity were observed. Only minor changes were observed in SDS-PAGE analyses of proteins from sonicated ER membranes fractionated on continuous sucrose gradients. Overall, heat shock reduced total lipid in ER membranes relative to protein, and in intact, ultracentrifuged aleurone cells examined by light and electron microscopy the ER band appeared to increase in density. The changes in phospholipid metabolism coupled with the suppression of secretory protein synthesis indicate that in addition to inducing a classic heat shock response, high temperature also induces a classic unfolded protein response in the ER of this secretory cell.

SORGHUM VERTICILLIFLORUM: A NATURAL HOST OF SORGHUM YELLOW BANDING VIRUS IN VENEZUELA

Apparently virus- infected Sorghum verticilliflorum (wild sorghum) plants were observed at Maracay, Aragua State. These plants showed yellow speckles, chlorotic streaks and bands, stunting, and light necrosis. Symptomatic plants were collected, and extracts from these plants were mechanically inoculated to a range of plant species. A virus was transmitted at relatively low rate (7%) to wild sorghum and sorghum (S. bicolor) plants. Symptoms appeared 17-24 days after inoculation. Infected wild sorghum plants were sterile or had only a few spikelets. The virus was not transmitted by Rhopalosiphum maidis or Peregrinus maidis, nor was it found in progeny of seed from an infected plant. In agar double immunodiffusion tests forty-two isolates from wild sorghum reacted positively with antiserum to Sorghum yellow banding virus, SrYBV (Garrido et al., 2000). Examination of clarified viral concentrate of some isolates consistently revealed isometric particles 25 nm in diameter. SDS-PAGE analysis of protein extracted from these preparations yielded a single band with a mol. wt. of approximately 29 kDa. Based on these results, the virus is considered to be SrYBV (Garrido et al., 2000; Klaassen and Falk, 1989). Probably, S. verticilliflorum is an important host in the epidemiology of SrYBV in Venezuela, since it is a widely distributed weed in the country. This is the first report of S. verticilliflorum as natural host of SrYBV.

Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro.

Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture system.

AFLP Fingerprint Analysis of Bradyrhizobium Strains Isolated from Faidherbia albida and Aeschynomene Species

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.

Effect of 5-azacytidine and sinefungin on streptomyces development

The effect of two DNA-methyltransferase inhibitors, 5-azacytidine (5azaC) and sinefungin (Sf), on the development of Streptomyces antibioticus ETH7451 (Sa) was studied. Pulse labeling experiments and SDS-PAGE analysis of proteins from cells grown in sporulation synthetic medium showed that both inhibitors affect a limited number of systems. Synthesis of the antibiotic rhodomycin was increased in the presence of 5azaC. 5azaC also stimulated the production of actinorhodin in cultures of S. coelicolor A3(2) grown in minimal medium. The analog did not affect the expression of whiB and whiG, two sporulation genes from S. coelicolor A3(2) whose homologues are present in Sa. Overall results indicated that 5azaC and Sf affect specific events associated with differentiation and secondary metabolism in Streptomyces

Neisseria gonorrhoeae strain MS11 harbouring a mutation in gene aroA is attenuated and immunogenic

An aroA mutant of gonococcal strain MS11 was constructed (JKD298) and compared with the wild-type (JKD288). The requirement of JKD298 for aromatic compounds, typical of an aroA mutant, was demonstrated using defined media. Other than the expected auxotrophy, no further differences could be demonstrated between the parent and the aroA mutant. SDS-PAGE analysis of protein and lipopolysaccharide (LPS) profiles showed no differences between the strains. Bactericidal assays using human and guinea-pig normal sera showed that both strains were serum sensitive and were similarly converted to serum resistance by in vitro sialylation using CMP-NANA. Infectivity experiments in guinea-pig subcutaneous chambers showed considerably reduced virulence of the aroA JKD298, which could only infect chambers at very high doses. Established infections by either strain elicited a strong PMN response in which similar proportions of each strain were seen intracellularly. Infections by JKD298 provoked a strong antibody response as detected by ELISA using whole sonicated gonococci. This is the first demonstration of attenuation of Neisseria gonorrhoeae by introduction of a defined mutation in a metabolic gene.

Solubilization and functional reconstitution of the human placental taurine transporter

The taurine transporter from purified human placental brush-border membranes was solubilized and reconstituted into proteoliposomes in a functional form. Solubilization was done with 2.5% cholate in the presence of 4 M urea. The proteins in the solubilizate were precipitated with 6% poly(ethylene glycol) and the precipitated proteins were reconstituted into proteoliposomes with an asolectin/protein ratio of 10:1. Under these experimental conditions, the taurine transport activity in the proteoliposomes was maximal. SDS-PAGE analysis of proteins, however, revealed that the proteoliposomes still contained a majority of the proteins originally present in the brush-border membranes. Uptake of taurine in the reconstituted proteoliposomes was obligatorily dependent on the presence of Na + as well as Cl −. Substitution of Na + with other monovalent cations such as K + and Li + reduced the taurine transport activity drastically. Similarly, substitution of Cl with other monovalent anions such as SCN −, F −, I and NO 3 − could support the transport activity only to a maximum of 30% of the control activity. In the presence of Cl −, the uptake rate was sigmoidally related to Na + concentration, resulting in a Na +/taurine coupling ratio of 2:1. The apparent dissociation constant for Na + was about 195 mM. In the presence of Na +, the uptake rate was hyperbolically related to Cl − concentration, indicating a Cl −/taurine coupling ratio of 1:1. The apparent dissociation constant for Cl − was about 205 mM. The NaCl-dependent taurine uptake was stimulated by an inside-negative membrane potential, showing that the uptake process was electrogenic. The uptake system was specific for β-amino acids. The affinity of the system for taurine was high with an apparent dissociation constant of 2.7 ± 0.1 μM. It is concluded that the taurine transporter can be dislodged from the placental brush-border membranes and reconstituted in a catalytically active from in proteoliposomes with no significant change in its characteristics.

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.