Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.     

iNOS-Dependent Increase in Colonic Mucus Thickness in DSS-Colitic Rats

Aim

To investigate colonic mucus thickness in vivo in health and during experimental inflammatory bowel disease.

Methods

Colitis was induced with 5% DSS in drinking water for 8 days prior to experiment, when the descending colonic mucosa of anesthetized rats was studied using intravital microscopy. Mucus thickness was measured with micropipettes attached to a micromanipulator. To assess the contributions of NOS and prostaglandins in the regulation of colonic mucus thickness, the non-selective NOS-inhibitor L-NNA (10 mg/kg bolus followed by 3 mg/kg/h), the selective iNOS-inhibitor L-NIL (10 mg/kg bolus followed by 3 mg/kg/h) and the non-selective COX-inhibitor diclofenac (5 mg/kg) were administered intravenously prior to experiment. To further investigate the role of iNOS in the regulation of colonic mucus thickness, iNOS −/− mice were used.

Results

Colitic rats had a thicker firmly adherent mucus layer following 8 days of DSS treatment than untreated rats (88±2 µm vs 76±1 µm). During induction of colitis, the thickness of the colonic mucus layer initially decreased but was from day 3 significantly thicker than in untreated rats. Diclofenac reduced the mucus thickness similarly in colitic and untreated rats (−16±5 µm vs −14±2 µm). While L-NNA had no effect on colonic mucus thickness in DSS or untreated controls (+3±2 µm vs +3±1 µm), L-NIL reduced the mucus thickness significantly more in colitic rats than in controls (−33±4 µm vs −10±3 µm). The importance of iNOS in regulating the colonic mucus thickness was confirmed in iNOS−/− mice, which had thinner colonic mucus than wild-type mice (35±3 µm vs 50±2 µm, respectively). Furthermore, immunohistochemistry revealed increased levels of iNOS in the colonic surface epithelium following DSS treatment.

Conclusion

Both prostaglandins and nitric oxide regulate basal colonic mucus thickness. During onset of colitis, the thickness of the mucus layer is initially reduced followed by an iNOS mediated increase.     

In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.     

Evidence that ecdysteroids and methyl farnesoate control allometric growth and differentiation in a crustacean.

Allometric, or disproportionate, growth of body parts is a basic problem in morphogenesis. Male spider crabs, Libinia emarginata, have several forms or morphotypes. During the terminal molt, the propodus enlarges disproportionately, exceeding the carapace length by as much as 35%. Even though shorter clawed males are reproductive, the large-clawed males become primary reproductives. We stimulated penultimate stage males to molt by eyestalk ablation, which removes molt inhibiting hormone (MIH) and mandibular organ inhibiting hormone (MIOH), and measured ecdysones by radioimmunoassay and methyl farnesoate (MF) in hemolymph by high-performance liquid chromatography (HPLC) using an internal standard. Eyestalk ablation accelerated molting and increased ecdysteroids to peak at 150 ng/ml before the molt. In control animals the ecdysteroids peaked at 90 ng/ml 3 days before the molt, with MF remaining less than 0.5 ng/ml. These became large males with large allometric claws. In contrast, the ablated ones, with increased MF (1 to 1.5 ng/ml), increased carapace size, but retained shorter non-allometric claws, with length shorter than the carapace. The results are consistent with experiments that we have performed with MF administration (Abdu et al., Biol. Bull., Woods Hole, MA 195 (1998) 112; Laufer et al., Gen. Comp. Endocrinol. 111 (1998) 113; Laufer et al., in: IV Symp. Aquaculture in Central America: Focusing on Shrimp and Tilapia, (1997a), p. 161; Laufer et al., Invert. Reprod. Dev. 31 (1997b) 63) which led to the interpretation that ecdysteroids and low MF concentrations promote allometric growth, while ecdysones with relatively higher concentrations of MF inhibited allometric growth. These results indicate and support the conclusion that MF and ecdysteroids determine the control of morphogenesis in allometric growth of Crustacea.     

Methyl farnesoate binding proteins in tissues of the spider crab, Libinia emarginata

Methyl farnesoate (MF) binding proteins (MFBPs) were found in the ovaries, testes, accessory glands, and hemolymph of the spider crab Libinia emarginata, by photoaffinity labeling the tissues in vitro with tritiated farnesyl diazomethyl ketone ([3H]-FDK). Specificity was demonstrated by competitive displacement of [3H]-FDK with MF. SDS-PAGE followed by fluorography revealed several labeled proteins in the hemolymph and testes with molecular masses ranging from 29 to 116 kDa, and two in the ovary that were 97 and 70 kDa. Tissues from reproductive animals bound twice as much label per gram weight compared to those that were from non-reproductive crabs.     

Monoclonal antibodies recognizing nitrite oxidoreductase of Nitrobacter hamburgensis, N. winogradskyi, and N. vulgaris.

Three monoclonal antibodies (MAbs) against nitrite oxidoreductase (NOR) of Nitrobacter hamburgensis were produced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of the purified enzyme showed that the MAbs named Hyb 153.1 and Hyb 153.3 both recognized a protein with a molecular mass of 64,000 Da, while Hyb 153.2 recognized a protein with a molecular mass of 115,000 Da. The molecular masses of these proteins are in the same range as those of the proteins of the alpha (115,000-Da) or beta (65,000-Da) subunit of the NOR. By using the antibodies, the amount of NOR was shown to be dependent on the growth conditions. The highest level of NOR was observed in N. hamburgensis when cells were growing mixotrophically. Analysis of whole-cell extracts of N. hamburgensis, N. winogradskyi, and N. vulgaris indicated serological homology of the NORs from these species of the genus Nitrobacter. The immunological analysis enables detection of the key enzyme of the genus Nitrobacter.     

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.     

Response of Biomass Development,Essential Oil,and Composition of Plectranthus amboinicus (Lour.) Spreng. to Irrigation Frequency and Harvest Time

A greenhouse experiment was conducted to study the effects of four irrigation intervals (4, 8, 12, and 16 days) and six harvests (2, 4, 6, 8, 10, and 12 months after transplanting) on biomass, essential oil content, and composition of Plectranthus amboinicus (Lour .) Spreng . Fresh weight and essential oil yield decreased with increasing irrigation interval; whereas, essential oil content was stimulated by water stress and increased as the irrigation interval increased. Fresh weight of Plectranthus amboinicus irrigated every 4 days peaked when harvested at 6 months, but essential oil content peaked when irrigated every 16 days and harvested at 2 months after transplantation. On the other hand, essential oil yield peaked when irrigated every 8 days and harvested at 6 months. Thymol, p‐cymene, γ‐terpinene, and β‐caryophyllene were the major compounds, and they peaked at different irrigation intervals and harvest times. This study showed biomass, essential oil content, and yield as well as the major and minor constituents of Plectranthus amboinicus are influenced by irrigation interval and the timing of harvest.     

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence

Lactobacillus reuteri is a symbiont that inhabits the gastrointestinal (GI) tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable in vivo and in vitro detection of L. reuteri and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc) and red fluorescent protein mCherry in L. reuteri strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM) and conventional plate count (PC), and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an in vivo imaging system (IVIS), which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1×10¹⁰ CFU and luminescence signals at doses ranging from 1×10⁵ to 1×10¹⁰ CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an in vitro experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI) of L. reuteri and may provide useful tools for future studies of in vivo and in vitro interactions between the bacteria and the host.