Identification of a rhodopsin photoreceptor in Euglena gracilis.

Visual pigments are a class of receptor proteins that absorb light and trigger sensory signals. Retinal-containing proteins are used in nature as photoreceptors mainly in animals vision. Mammalian rhodopsin is the best studied example of a light sensor which couples photon absorption to a cascade of biochemical reactions amplifying the input signal. A surprising discovery was to find rhodopsin also in Archaebacteria and in unicellular eukaryotes. On the basis of absorption microspectroscopic measurements and of inhibition experiments on pigment biosynthetic pathways, we have recently suggested that a rhodopsin could be the functional receptor of the visual process in Euglena gracilis, a flagellate which can use light directly to promote photosynthetic reactions, or as an incident flux of information to adjust its swimming orientation. We here report purification and identification of all-trans-retinal by column chromatography, HPLC and GC-MS in E. gracilis; these findings indicate with absolute certainty that rhodopsin is the photoreceptor molecule of this microorganism.

The photoreceptor protein of Euglena gracilis.

We isolated the photoactive protein Erh, isolated from the photoreceptor of the unicellular photosynthetic flagellate Euglena gracilis. It is a 27 kDa protein with a photocycle resembling that of sensory rhodopsin, but with at least one stable intermediate. We recorded the absorption spectrum of the parent form of this protein both under native form and in the presence of hydroxylamine and sodium borohydride, and the fluorescence spectra of both the parent and intermediate forms. We suggest that Erh is a rhodopsin-like protein and propose a simple photocycle. This protein shows optical bistability, without thermal deactivation.

Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use.

The biocompatibility of two cyanoacrylate surgical glues (Glubran and Glubran 2), supplied by General Enterprise Marketing, Viareggio, Lucca, Italy, was tested through cytotoxicity and blood compatibility tests and the evaluation of antimicrobial activity. Cytotoxicity and blood compatibility tests were performed on the polymerized glues. Using the neutral red uptake test, the extracts from Glubran and Glubran 2 after polymerization were non-toxic to L929 cells only when diluted 1: 10 with culture medium. Glubran and Glubran 2 induced a significant decrease of activated partial thromboplastin time (APTT), which is favourable with regard to the desired haemostasis. The APTT shortening determines a haemostatic effect and therefore contribute to the tissue adhesion induced by the glues. Otherwise, no significant variation of prothrombin activity, fibrinogen, platelet number, total and differential leukocyte count was induced by the glues, which, in addition, did not show haemolytic effect. There was no difference between Glubran and Glubran 2 regarding haemocompatibility. The antimicrobial ability of the unpolymerized glues was tested onto Bacillus subtilis var. niger for 3 weeks: neither Glubran nor Glubran 2 were found effective in this respect. In conclusion, we can assume that cytotoxicity was severe with the undiluted glues, but was acceptable when glues were diluted. On the contrary, blood compatibility was acceptable for the intended use of the glues. No difference was found between Glubran and Glubran 2 after polymerization.

Effects of hydroxylamine, digitonin and triton X-100 on photoreceptor (paraflagellar swelling) and photoreception of Euglena gracilis.

We present experiments that test the effects of agents commonly used in visual pigment investigations, namely hydroxylamine (NH2OH), digitonin and triton X-100, on the photoreceptor and photoreception of Euglena. Hydroxylamine reacts with free and opsin-bound retinal, in aqueous solution, to form stable oximes, whereas digitonin and triton X-100 are the most common extractants of rhodopsin. Since previous data indicate that the chromophore present in Euglena photoreceptor is retinal, we investigated the influence of these chemicals on this organelle. The effects of these agents were studied by means of phase contrast, fluorescence and transmission electron microscopy and photobehaviour experiments. Hydroxylamine inhibited the formation of the Euglena photoreceptor. Photoaccumulation experiments on hydroxylamine-treated cells showed that they are unable to perceive light. Digitonin solubilized the crystalline structure of the photoreceptor, whereas the triton effect was limited to the membranous structures of the cell, leaving the photoreceptor unimpaired.

A complex photoreceptive structure in the cyanobacterium Leptolyngbya sp.

Among the terrestrial epilithic cyanobacteria isolated from Roman hypogea at extremely low light intensity, a non-heterocystous strain, belonging to the genus Leptolyngbya, showed a marked photobehavior. These red cyanobacteria possess an orange spot at the tip of the apical cell. Micro-spectrophotometric analysis of this tip showed an absorption spectrum with two bands, centered at 456 and 504 nm, respectively. Experiments on photo-orientation impairment of these cells, and micro-spectrophotometric analysis of the tip of impaired trichomes showed that a rhodopsin-like protein might be present in this structure. All these data could support the hypothesis of the presence of a complex photoreceptive system in this prokaryote.

Ultrastructure of the pellicle of Euglena gracilis.

Deep-etching technique was used to investigate the organization of the pellicle complex of Euglena gracilis. The interpretation of the images was further supported by SEM and TEM investigations. Our results mainly validate data obtained by previous freeze-fracture studies on the E and P faces of the outer cortical membrane. At the level of the ridges, the outer E fracture face is highly organized in a regular striated pattern, whereas the P inner face shows a particulate structure. However, our images reveal that this particulate organization of the P face is not limited to the ridges, but it is displayed also by the grooves. Moreover, this face shows two distinct layers, a particulate layer facing the cytoplasm and a striated layer facing the E face; these layers represent different true fracture levels of the same P face.

Microorganism track reconstruction: an image processing approach.

This paper presents an automatic system for the analysis of microorganism behaviour. The movements of free swimming microorganisms are videotaped by means of a television camera mounted on a microscope. The analysis is performed off-line by digitizing the video signal through the use of an image processor unit. Microorganism tracks are reconstructed spatially and chronologically by means of image processing techniques. From these tracks cell movement parameters are obtained. The results of our experiment in testing photoinduced movements follow.

Identification of cellular and subcellular features by means of digital microscopy.

This paper presents two examples of the application of quantitative digital microscopy to two different fields of biological analysis: DNA content determination in microstructures and the study of the behaviour of microorganisms. In both cases the application of segmentation and labelling procedures was found to be essential in order to obtain the desired results, because it made it possible to overcome the measurement limits of traditional instrumentation and to obviate drawbacks normally present when analysis is placed in the hands of the human operator.

Chitosan as flocculant for concentrating Euglena gracilis cultures.

The practical criteria for the usefulness of an algal separation process for laboratory routine being effectiveness and time consumption, we tested the feasibility of a flocculation procedure to harvest large volumes of Euglena gracilis in culture. This procedure turned out to be a technically viable system which avoided tedious centrifugation and preserved E. gracilis flagellar apparatus integrity. E. gracilis cultures were treated with chitosan, a by-product derived from chitin from the exoskeleton of crustaceans. Since this polymer carries a positive charge, it functions as a polycationic coagulating agent by adsorbing onto particles in suspension and by bridging together into agglomerates, or flocs. A 96-98% reduction of suspended cells in cultures with 200 mg/l of chitosan, at pH 7.5, was obtained.

In vivo photocycle of the Euglena gracilis photoreceptor.

We present the light-induced photocycle of the paraflagellar swelling of Euglena gracilis. The kinetics of this process was reconstructed by sampling its fluorescence emission and switching the excitation light from 365 nm to 436 nm. Stable intermediates in the photocycle were manifested. The measured millisecond resolution kinetics best fits a Michaelis-Menten equation. The data provide strong evidence that the paraflagellar swelling, a three-dimensional natural crystal of a light-detecting protein, is the true Euglena photoreceptor.

Ultrastructure of the apical zone of Euglena gracilis: photoreceptors and motor apparatus.

Euglena is an organism that every student of biology has observed; its morphology has been a subject of interest since the early microscopic literature for its enigmatic role of "plant-like" or "animal-like" organism. Therefore, this review has no pretensions to absolute novelty, but, like a journey to the centre of the earth, will attempt to arouse the reader's curiosity by taking him inside the cell Euglena, through the canal opening into the reservoir chamber. In light of the most recent knowledge, though much remains to be clarified, the aim is to provide information from ultramicroscopical studies on the apical zone of Euglena and possible functional meanings of the structures present therein. The survey of these structures is carried on as a study in correlation: TEM of cells after various treatments is correlated with SEM of cells fixed by means of different techniques. Notes on locomotion and other features of cytological and biological interest are added to assist with the comprehension of this microorganism.

First identification and localization of a visual pigment in Hydra (Cnidaria, Hydrozoa).

The cnidarian Hydra does not possess identified photoreceptive structures or specialized cells for light detection; nevertheless, it shows a marked photosensitivity. So far no evidence has been previously reported about the localization of the proteins involved in the photoresponse. We used polyclonal antibodies and immunofluorescence microscopy on whole-mount Hydra to identify a putative rhodopsin-like protein. Our results show an immunoreactivity in the ectodermal layer of Hydra, which corresponds in position to the nervous epidermal sensory cells. These data provide the first identification of a rhodopsin-like protein in a phylogenetically old invertebrate and give a new insight into the Hydra photoreceptive response.

Presence of HIV-1 proviral sequences in saliva of infected individuals in relation with clinical stage and CD4+ lymphocyte count.

We detected by PCR (Polymerase Chain Reaction) HIV-1 proviral sequences in saliva cells of 89 HIV+ subjects at different stages of disease. Twenty negative individuals not at risk of HIV infection were considered as controls. The amplification of DNA was performed using the primers of genomic env region SK68 and SK69. The presence of HIV-1 in DNA was found in 16/89 (18.0%) saliva samples from HIV-1 subjects but in none of the 20 saliva samples from healthy subjects. No statistically significant difference in the presence of HIV-1 proviral sequences in saliva was observed when comparing patients receiving AZT or no treatment and patients at different stages of infection. Conversely, a statistically significant difference among subjects with CD4+ cell counts > 400/cmm and those with CD4+ cell counts from 200 to 400/cmm, was found stratifying the subjects according to their CD4+ cell counts.

Partial nucleotide sequencing of six subtype 2c hepatitis C viruses detected in Italy.

The great majority of 121 hepatitis C virus (HCV) isolates obtained from 117 Italian patients with community-acquired infection could readily be typed by genotype-specific PCR. Subtype 1b was dominant (74 isolates); subtypes 2b, 2a, and 1a followed, with 19, 14, and 8 isolates, respectively. The six isolates that remained untyped by this method were classified as subtype 2c on the basis of sequence analysis of PCR amplicors obtained from the core and NS5 genes. These findings indicate that HCV subtype 2c has a relatively high prevalence in Italy. Sequencing the core region from positions 160 to 259 is sufficient to distinguish subtype 2c from other known HCV genotypes.

[HIV-1 proviral DNA sequences in the saliva of patients with HIV infection]. / Sequenze provirali di HIV-1 nella saliva di soggetti con infezione da HIV.

In order to understand the significance of presence of HIV-1 in saliva, we searched for by PCR HIV-1 proviral sequences in the saliva cells of 49 HIV-1 infected patients. Seven out 49 specimens resulted positive, 4 of which were from patients with PGL, 1 with ARC and 2 with AIDS. Four patients had a CD4+ lymphocyte counts < 200/cmm and in 3 patients the CD4+ lymphocyte count ranged from 200 to 400/cmm. Two patients were treated with AZT, 1 with DDI and 4 had no antiretroviral treatment. In conclusion, although HIV-1 proviral sequences have been found in saliva of HIV-1 infected patients, a larger group of patients should be investigated to define more precisely the role of HIV-1 in saliva.

Lymphotropic virus infection of peripheral blood mononuclear cells in B-cell non-Hodgkin's lymphoma.

Some lymphotropic viruses such as Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) have been proposed as causative agents of B cell non-Hodgkin's lymphoma (NHL). More recently, the presence of hepatitis C virus (HCV), which is both a hepatotropic and lymphotropic virus, has been reported in one third of B cell NHL patients. The aim of this study was to investigate in a series of B cell NHL the prevalence of three lymphotropic viruses, i.e. EBV, HHV-6 and HCV, in peripheral blood mononuclear cells (PBMC). Eighteen unselected B cell NHL patients (10 men, 8 women; mean age 62 +/- 12 years, range 31-77 years; mean disease duration 1.8 +/- 1.4 years) and 40 age- and sex-matched healthy controls were included in the study. In all cases, an acquired-immunodeficiency-syndrome-related lymphoma was excluded. By means of the polymerase chain reaction technique, EBV DNA, HHV-6 DNA and HCV RNA were detected in PBMC. HCV genomic sequences were significantly more frequent in PBMC of NHL patients than in controls (33 vs. 2.5%; p < 0.01); on the other hand, in the same two groups EBV DNA (39 vs. 60%; p = not significant) and HHV-6 DNA (22 vs. 32%; p = not significant) were present in a comparable percentage of individuals in the same two groups. The infection of PBMC by HCV alone was present in the majority (5 of 6) of HCV-positive NHL. These data support the implication of HCV infection in a statistically significant number of B cell NHL, whereas a possible co-operation between HCV and other well-known lymphotropic viruses seems to be excluded.

Human herpesvirus-6 reactivation in a longitudinal study of two HIV-1 infected patients.

After primary infection, human herpesvirus-6 (HHV-6) persists in latent form and can be reactivated in immunocompromised subjects. A longitudinal study of HHV-6 infection was carried out in two HIV-1 seropositive patients to provide in vivo evidence of HHV-6 reactivation. Concomitant with a significant rise of anti-HHV-6 IgG detected by IFA, a transient increase of HHV-6 viral load was shown in PBLs by PCR. During HHV-6 reactivation it was also identified either cell-free HHV-6 by PCR in plasma or IgM antibody titers. HHV-6 reactivation was followed by a temporary decrease in CD4+ count and by a progressive dramatic loss of CD4+ during the following 18 months. HHV-6 strain characterization by PCR demonstrated that first patient (MM) initially showed the B variant, followed by reactivation and persistence of the A variant, while in the second (SG) only the A variant was detected. The evidence of HHV-6 reactivation suggests its involvement in immunologic damage underlying the disease.

Quantitation of HCV viraemia by branched DNA signal amplification in patients treated with alpha-interferon--a longitudinal study.

Using bDNA, the plasma viral load trend of HCV-infected patients undergoing IFN therapy was analyzed. Nine patients were enrolled, each assigned to one of three groups, based on IFN response as determined by ALT and AST level trend. HCV was genotyped using DEIA. Each patient's clinical stage was determined by liver biopsy analysis. In nonresponding patients elevated viral loads and biochemical parameters were observed. These values were not influenced by IFN treatment. In relapsed patients the cessation of IFN treatment increased viral load; this was associated with a rise in ALT and AST values. In responders ALT and AST levels remained normal; viral load was low. Patients with elevated HCV viral load showed a worsening in their liver histology during the follow-up period. These results confirm that plasma viral load is a good marker of biochemical change and disease progression.