Liquid-phase mega-electron-volt ultrafast electron diffraction.

The conversion of light into usable chemical and mechanical energy is pivotal to several biological and chemical processes, many of which occur in solution. To understand the structure-function relationships mediating these processes, a technique with high spatial and temporal resolutions is required. Here, we report on the design and commissioning of a liquid-phase mega-electron-volt (MeV) ultrafast electron diffraction instrument for the study of structural dynamics in solution. Limitations posed by the shallow penetration depth of electrons and the resulting information loss due to multiple scattering and the technical challenge of delivering liquids to vacuum were overcome through the use of MeV electrons and a gas-accelerated thin liquid sheet jet. To demonstrate the capabilities of this instrument, the structure of water and its network were resolved up to the 3 rd hydration shell with a spatial resolution of 0.6 Å; preliminary time-resolved experiments demonstrated a temporal resolution of 200 fs.

Femtosecond gas-phase mega-electron-volt ultrafast electron diffraction.

The development of ultrafast gas electron diffraction with nonrelativistic electrons has enabled the determination of molecular structures with atomic spatial resolution. It has, however, been challenging to break the picosecond temporal resolution barrier and achieve the goal that has long been envisioned-making space- and-time resolved molecular movies of chemical reaction in the gas-phase. Recently, an ultrafast electron diffraction (UED) apparatus using mega-electron-volt (MeV) electrons was developed at the SLAC National Accelerator Laboratory for imaging ultrafast structural dynamics of molecules in the gas phase. The SLAC gas-phase MeV UED has achieved 65 fs root mean square temporal resolution, 0.63 Å spatial resolution, and 0.22 Å-1 reciprocal-space resolution. Such high spatial-temporal resolution has enabled the capturing of real-time molecular movies of fundamental photochemical mechanisms, such as chemical bond breaking, ring opening, and a nuclear wave packet crossing a conical intersection. In this paper, the design that enables the high spatial-temporal resolution of the SLAC gas phase MeV UED is presented. The compact design of the differential pump section of the SLAC gas phase MeV UED realized five orders-of-magnitude vacuum isolation between the electron source and gas sample chamber. The spatial resolution, temporal resolution, and long-term stability of the apparatus are systematically characterized.

Abalone collagens: immunological properties and seasonal changes of their mRNA levels.

The antisera were raised against pepsin-solubilized abalone collagen and its corresponding gelatin. The reactivity against abalone collagen was higher with the anti-collagen than anti-gelatin antiserum. The two antisera recognized all type I collagens from various vertebrates, whereas these had no reactivity against vertebrate type III and type V collagens. Furthermore, both antisera reacted with only alpha 2(I) chains from chicken, rat, and calf. The strong reactivity was observed against the two antisera in the case of invertebrate and protochordate collagens, especially for turban shell collagen. The seasonal changes of collagen mRNA levels were examined in relation to those of collagen content. Haliotis discus collagens (Hdcols) 1 alpha and 2 alpha coding for abalone collagen pro alpha-chains showed quite similar patterns. The highest mRNA levels in adductor and foot muscles for the two collagens were observed in December and January, in good agreement with the increase of collagen content. The mRNA levels decreased in July and August when collagen content decreased. These results indicate that collagen transcription levels are closely related to collagen contents.

Carnosine stimulates vimentin expression in cultured rat fibroblasts.

Two-dimensional electrophoretic gel profiles were compared between rat 3Y1 fibroblasts cultured in the presence and absence of 30 mM L-carnosine (beta-alanyl-L-histidine) for one week without any replenishment of medium. While a number of cellular proteins changed their expression levels by the addition of carnosine, we identified one of the most prominently varied proteins as vimentin. Immunoblot analysis with anti-vimentin antibody demonstrated that the vimentin levels increased about 2-fold after one-week culture in the presence of carnosine. We also confirmed that the increase of vimentin expression was dependent on the concentration of carnosine added to the medium. Moreover, when cultured cells were stained with anti-vimentin antibody and observed by light microscopy, most cells grown in the presence of carnosine were found to have markedly developed vimentin filaments. The increase of vimentin expression was also observed by adding with carnosine related dipeptides, N-acetylcarnosine and anserine.

The occurrence of two types of collagen proalpha-chain in the abalone Haliotis discus muscle.

Acid-soluble collagens were prepared from connective tissues in the abalone Haliotis discus foot and adductor muscles with limited proteolysis using pepsin. Collagen preparation solubilized with 1% pepsin contained two types of alpha-chains which were different in their N-terminal amino acid sequences. Accordingly, two types of full-length cDNAs coding for collagen proalpha-chains were isolated from the foot muscle of the same animal and these proteins were named Hdcols (Haliotis discus collagens) 1alpha and 2alpha. The two N-terminal amino acid sequences of the abalone pepsin-solubilized collagen preparation corresponded to either of the two sequences deduced from the cDNA clones. In addition, several tryptic peptides prepared from the pepsin-solubilized collagen and fractionated by HPLC showed N-terminal amino acid sequences identical to those deduced from the two cDNA clones. Hdcols 1alpha and 2alpha consisted of 1378 and 1439 amino acids, respectively, showing the primary structure typical to those of fibril-forming collagens. The N-terminal propeptides of the two collagen proalpha-chains contained cysteine-rich globular domains. It is of note that Hdcol 1alpha completely lacked a short Gly-X-Y triplet repeat sequence in its propeptide. An unusual structure such as this has never before been reported for any fibril-forming collagen. The main triple-helical domains for both chains consisted of 1014 amino acids, where a supposed glycine residue in the triplet at the 598th position from the N-terminus was replaced by alanine in Hdcol 1alpha and by serine in Hdcol 2alpha. Both proalpha-chains of abalone collagens contained six cysteine residues in the carboxyl-terminal propeptide, lacking two cysteine residues usually found in vertebrate collagens. Northern blot analysis demonstrated that the mRNA levels of Hdcols 1alpha and 2alpha in various tissues including muscles were similar to each other.

[A case of spontaneous spinal epidural hematoma with spontaneous remission before surgery].

A 61-year-old man was referred to our department with spontaneously remitting sensorimotor disturbance and bowel and bladder dysfunctions. He had no specific previous history and neither received any drug nor suffered from hypertension. On onset, complete flaccid paralysis of lower extremities, almost complete sensory disturbance of all modalities below the level of Th12, and bowel and bladder dysfunctions were observed. MRI and CT scan revealed an epidural hematoma in the posterior region of the spinal cord at Th11 level. Afterwards he continued to improve gradually both clinically and radiologically. Four days subsequent to onset, on admission to our department, he had slight sensorimotor disturbance, and bowel and bladder dysfunctions still existed. MRI demonstrated a shrinking and flattened hematoma. We performed laminectomy and evacuation of the hematoma. Grossly and histologically, no underlying lesion was revealed. Postoperative course was not eventful. He was discharged without residual deficits. In our case, surgery accomplished three goals: definitive diagnosis, secure decompression and prevention of recurrence. We believe, the possibility of spontaneous resolution of spontaneous spinal epidural hematomas (SSEDHs) with spontaneous remission may be high, but prompt surgical evacuation should be the treatment of choice for such cases, even if no underlying lesion is revealed by MRI, except in cases where operative morbidity and mortality rate is high and in cases with no neurological deficits other than pain. A review of the literature indicated that not all SSEDHs with spontaneous remission resolved spontaneously and completely.

Trachoma therapy with topical tetracycline and oral erythromycin: a comparative trial.

Because topical antibiotic treatment has had a limited effect in previous controlled trials against trachoma, treatment with oral erythromycin was compared with topical tetracycline in 6-8-year-old children in southern Tunisia who had potentially blinding active trachoma. A total of 169 children were divided into two groups that were carefully matched for age, sex, locality, and intensity of disease. Oral erythromycin ethyl succinate in a paediatric dosage form was administered to one group and topical 1% tetracycline ointment to the other group, twice daily, six days a week for three weeks. The two treatments were equivalent in effectiveness and resulted in a substantial decrease in disease intensity and a marked reduction in chlamydial infection detected in conjunctival smears. To maintain blood levels of antibiotics known to be effective in the treatment of chlamydial infections with a dosage schedule possible in a trachoma control programme, one of the long-acting tetracyclines (doxycycline or minocycline) might be considered. Such systemic chemotherapy should be limited to selective treatment of individuals who can be adequately monitored.

Intermittent trachoma chemotherapy: a controlled trial of topical tetracycline or erythromycin.

In communities with endemic blinding trachoma, mass (or "blanket") treatment with a topically applied tetracycline derivative is a standard control measure. The widely used "intermittent" treatment schedule consists of the twice daily application of antibiotic ointment for five consecutive days once a month for six months. In this study, the efficacy of "intermittent" treatment was evaluated for the treatment of severe and moderate intensity trachoma in children in southern Tunisia. Tetracycline or erythromycin ointments (specific antichlamydial drugs) were compared with 5% boric acid ointment (a simple antiseptic) given by the intermittent schedule during the winter and spring. There was a statistically significant degree of improvement at only one examination, four weeks after the full course of treatment had been completed. When re-examined five months later there were no differences in intensity in the three groups. The limited effect of topical chemotherapy might be attributable to several causes, among which could be inadequate drug levels, inadequate treatment periods, reinfection from non-treated children in the community, and auto-infection from extraocular sites (e.g., respiratory tract) in the same child. The possible value of short-term (two weeks) systemic antimicrobial therapy as an additional strategy to prevent blindness of children with potentially blinding active trachoma is discussed.

Antibiotic susceptibility of Chlamydia trachomatis.

The antibiotic susceptibility of Chlamydia trachomatis isolates was determined in a tissue culture system. Representatives of all currently recognized serotypes of trachoma-inclusion conjunctivitis agents were tested. Tetracycline and erythromycin yielded similar results, with 1.0 mug/ml preventing chlamydial replication. Rifampin was the most active antibiotic, with 0.25 mug/ml completely suppressing inclusion formation of all strains. Fifty percent end points were usually achieved at one-fourth to one-eighth the 100% suppression level. Penicillin was not as effective, and the assays were often irregular. Antibiotic susceptibility of these chlamydiae was essentially the same, regardless of serotype, anatomic site infected, geographic origin, or antibiotic use in the community.

Resistance to reinfection with a chlamydial agent (guinea pig inclusion conjunctivitis agent).

Although most chlamydial infections are chronic or recurrent, infection of the guinea pig's eye with guinea pig inclusion conjunctivitis (GPIC) agent induces a marked resistance to reinfection. To characterize this resistance to GPIC agent, we compared the disease and infection in previously infected guinea pigs with that in animals infected for the first time. In animals experiencing primary infection, even the lowest dose (10 egg-lethal doses [ELD50]) produced the disease and chlamydial inclusions in conjunctival smears, but the incubation period became progressively shorter with the highest inocula (10(4) and 10(5) ELD50). In animals with previous infection only these two highest inocula produced disease and infection, but the disease was short-lived, and replication of the agent was severely limited. The mechanism of this resistance may be due to secretory antibody in the tears, cellular immunity, or other local factors.

Cytology as a guide to the presence of chlamydial inclusions in Giemsa-stained conjunctival smears in severe endemic trachoma.

Microscopical examination of 927 Giemsa-stained conjunctival smears from children with chronic trachoma in southern Tunisia showed 93 (10 per cent.) with typical trachoma (chlamydial) inclusions in epithelial cells. The accompanying cytological features were a useful indicator for inclusions. Inclusions were found only in slides with numerous polymorphonuclear neutrophils (PMNs) and separation of the epithelial cells. When these two features alone were present, 3 per cent. of the smears were inclusion-positive; when many lymphocytes were present also, 25 per cent. were inclusion positive; when other cytological features (plasma cells, macrophages, blastoid, and stem cells) were present as well, 70 per cent. of the smears were inclusion-positive. The occurrence of these sets of cytological features can be a useful guide for selecting smears for intensive examination for chlamydial inclusions. Immunofluorescent (FA) staining and Giemsa staining of 527 pairs of matched smears detected trachoma agent in 67 (13 per cent.); in thirty by both methods, in thirteen by Giemsa staining alone, and in 24 by FA alone. The examination of Giemsa-stained smears for chlamydial inclusions is a useful technique for the diagnosis of trachoma or inclusion conjunctivitis by laboratories that do not have the specialized facilities for the identification of these chlamydial infections by the technically more complex procedures of immunofluorescent staining or isolation in embryonated eggs or tissue cultures.

Comparison of media for the isolation of haemophilus species from cases of seasonal conjunctivitis associated with severe endemic trachoma.

Media for isolation of Haemophilus sp. from the conjuctiva were compared in an oasis in southern Tunisia where severe trachoma and seasonal epidemic purulent conjunctivitis are common. Of 89 children tested, IsoVitaleX-supplemented chocolate agar yielded Haemophilus in 87%, plain chocolate agar in 75%, sheep blood agar with a stab of Staphylococcus epidermidis in 74%, and Fildes medium in 58%. Since other microbial pathogens are easily identified in the modified blood agar, it was the most useful single medium.