The differentiation and maintenance of Th17 cells require a unique cytokine milieu and activation of lineage-specific transcription factors. This process appears to be antagonized by the transcription factor T-bet, which controls the differentiation of Th1 cells. Considering that T-bet-deficient (T-bet−/−) mice are largely devoid of natural killer (NK) cells due to a defect in the terminal maturation of these cells, and because NK cells can influence the differentiation of T helper cells, we investigated whether the absence of NK cells in T-bet-deficient mice contributes to the augmentation of autoreactive Th17 cell responses. We show that the loss of T-bet renders the transcription factors Rorc and STAT3 highly responsive to activation by stimuli provided by NK cells. Furthermore, reconstitution of T-bet−/− mice with wild-type NK cells inhibited the development of autoreactive Th17 cells through NK cell-derived production of IFN-γ. These results identify NK cells as critical regulators in the development of autoreactive Th17 cells and Th17-mediated pathology.

Purpose

To investigate the feasibility of bypassing occluded segments of retinal venous main vessels in isolated, arterially perfused caprine eyes via the closed-sky vitrectomy approach using keratoprosthesis.

Methods

Isolated caprine eyes were used in this study. For each eye, the retinal vessel was perfused by Krebs solution via ophthalmic artery, and pars plana vitrectomy was performed using temporary keratoprosthesis. All retinal micro-vascular maneuvers were performed in a closed-sky eyeball. The main retinal vein was blocked by endodiathermy at the site of the vessel's first branching. Two openings, several millimeters apart, were created by vascular punctures in both the main vein and its branch vein wall straddling the induced occluded segment. Catheterization was achieved using a flexible polyimide tube, with each end inserted into the vessel wall opening. A sealed connection between the vessel and the tube was obtained by endodiathermy. Bypass of the occluded retinal vein segment was thus achieved, and the patency of this vascular bypass was confirmed by intravascular staining.

Results

Puncturing, catheterization, and endodiathermy were viable by closed-sky approach using keratoprosthesis. Bypassing of the occluded retinal main vein segment was accomplished with the combination of these maneuvers. Good results were obtained in 23 of 38 (60%) caprine eyes.

Conclusions

This study demonstrated that bypassing the occluded segment of retinal main vein can be successfully performed in a closed-sky eyeball model of isolated, arterially perfused caprine eye. This early work indicated that the more advanced retinal vascular bypass surgery in in vivo eye may be feasible in the future.

Purpose

To investigate the effects of Merogel coverage on ostial patency in endonasal endoscopic dacryocystorhinostomy (EES-DCR) for primary chronic dacryocystitis (PCD).

Methods

In all, 260 patients with unilateral PCD were randomized into two groups: the Merogel group and the control group. All patients underwent EES-DCR. The Merogel group received Merogel covering the wound 1–2 mm around the ostium and the control group received no treatment. Patients were followed up for 9 months. The mucosal epithelialization of the wound, the proliferation of fibrosis tissue, and the success rate of ostial patency were compared.

Results

Our study included 112 patients in the Merogel group and 115 patients in the control group. At the 2-week review, intact mucosal epithelium lined the ostia in 96 Merogel patients compared with 80 control patients (ITT analysis: χ 2=4.502, P=0.034). At the 9-month review, scars were present in 18 patients in the Merogel group compared with 39 patients in the control group (ITT analysis: χ 2=9.909, P=0.002, ITT analysis). No differences were observed in the granulation formation between the two groups. The success rate of ostial patency reached 94.6% (106/112) in the Merogel group compared with 80% (92/115) in the control group (ITT analysis: χ 2=4.151, P=0.042).

Conclusion

Merogel coverage may enhance the success rate of EES-DCR for PCD by promoting mucosal epithelial healing and preventing excessive scarring.

Recent researches have shed light on the biological importance of microRNAs (miRNAs) in colorectal cancer (CRC) genesis, progression and response to treatments. The potential utility of miRNAs in the preclinical stage have been explored and investigated. In this review, we explored the literature and reviewed the cutting edge progress in the discovery of noninvasive plasma and faecal miRNAs for CRC early diagnosis, as well as their measurability and predictability. We also discussed the utility of miRNAs as novel prognostic and predictive markers, and their association with CRC clinical phenotypes including recurrence, metastasis and therapeutic outcomes. Finally, we summarised miRNA-related single-nucleotide polymorphisms and their potential influence on sporadic CRC susceptibility and therapeutic response. In conclusion, the use of miRNAs as biomarker for CRC is still in its infancy and need further characterisation and evaluation.

Recently our group identified in murine bone marrow (BM) and human cord blood (CB), a rare population of Very Small Embryonic-Like (VSEL) stem cells. We hypothesize that these cells are deposited during embryonic development in BM as a mobile pool of circulating pluripotent stem cells (PSC) that play a pivotal role in postnatal tissue turnover both of non-hematopoietic and hematopoietic tissues. During in vitro co-cultures with murine myoblastic C2C12 cells, VSELs form spheres that contain primitive stem cells. Cells isolated from these spheres may give rise to cells from all three germ layers when plated in tissue specific media. The number of murine VSELs and their ability to form spheres decreases with the age and is reduced in short-living murine strains. Thus, developmental deposition of VSELs in adult tissues may potentially play an underappreciated role in regulating the rejuvenation of senescent organs. We envision that the regenerative potential of these cells could be harnessed to decelerate aging processes.

Neuro-axonal degeneration occurs progressively from the onset of multiple sclerosis and is thought to be a significant cause of increasing clinical disability. Several histopathological studies of multiple sclerosis and experimental autoimmune encephalomyelitis have shown that the accumulation of sodium in axons can promote reverse action of the sodium/calcium exchanger that, in turn, leads to a lethal overload in intra-axonal calcium. We hypothesized that sodium magnetic resonance imaging would provide an indicator of cellular and metabolic integrity and ion homeostasis in patients with multiple sclerosis. Using a three-dimensional radial gradient-echo sequence with short echo time, we performed sodium magnetic resonance imaging at 3 T in 17 patients with relapsing–remitting multiple sclerosis and in 13 normal subjects. The absolute total tissue sodium concentration was measured in lesions and in several areas of normal-appearing white and grey matter in patients, and corresponding areas of white and grey matter in controls. A mixed model analysis of covariance was performed to compare regional tissue sodium concentration levels in patients and controls. Spearman correlations were used to determine the association of regional tissue sodium concentration levels in T2- and T1-weighted lesions with measures of normalized whole brain and grey and white matter volumes, and with expanded disability status scale scores. In patients, tissue sodium concentration levels were found to be elevated in acute and chronic lesions compared to areas of normal-appearing white matter (P < 0.0001). The tissue sodium concentration levels in areas of normal-appearing white matter were significantly higher than those in corresponding white matter regions in healthy controls (P < 0.0001). The tissue sodium concentration value averaged over lesions and over regions of normal-appearing white and grey matter was positively associated with T2-weighted (P ≤ 0.001 for all) and T1-weighted (P ≤ 0.006 for all) lesion volumes. In patients, only the tissue sodium concentration value averaged over regions of normal-appearing grey matter was negatively associated with the normalized grey matter volume (P = 0.0009). Finally, the expanded disability status scale score showed a mild, positive association with the mean tissue sodium concentration value in chronic lesions (P = 0.002), in regions of normal-appearing white matter (P = 0.004) and normal-appearing grey matter (P = 0.002). This study shows the feasibility of using in vivo sodium magnetic resonance imaging at 3 T in patients with multiple sclerosis. Our findings suggest that the abnormal values of the tissue sodium concentration in patients with relapsing–remitting multiple sclerosis might reflect changes in cellular composition of the lesions and/or changes in cellular and metabolic integrity. Sodium magnetic resonance imaging has the potential to provide insight into the pathophysiological mechanisms of tissue injury when correlation with histopathology becomes available.

During gated intensity-modulated radiotherapy (IMRT) treatment for patients with inoperable non-small cell lung cancer (NSCLC), the end-expiration (EE) phase of respiratory is more stable, whereas end-inspiration (EI) spares more normal lung tissue. This study compared the relative plan quality based on dosimetric and biological indices of the planning target volume (PTV) and organs at risk (OARs) between EI and EE in gated IMRT. 16 Stage I NSCLC patients, who were scanned by four-dimensional CT, were recruited and re-planned. An IMRT plan of a prescription dose of 60 Gy per respiratory phase was computed using the iPlan treatment planning system. The heart, spinal cord, both lungs and PTV were outlined. The tumour control probability for the PTV and normal tissue complication probability for all OARs in the EE and EI phases were nearly the same; only the normal tissue complication probability of the heart in EE was slightly lower. Conversely, the conformation number of the PTV, V20 of the left lung, V30 of both lungs, Dmax of the heart and spinal cord, V10 of the heart and D5% of the spinal cord were better in EE, whereas Dmean of the PTV, V20 of the right lung and maximum doses of both lungs were better in EI. No differences reached statistical significance (p<0.05) except Dmax of the spinal cord (p _ 0.033). Overall, there was no expected clinical impact between EI and EE in the study. However, based on the practicality factor, EI is recommended for patients who can perform breath-hold; otherwise, EE is recommended.

We report a rational synthesis of maghemite (γ-Fe2O3) short-nanotubes (SNTs) by a convenient hydrothermal method and subsequent annealing process. The structure, shape, and magnetic properties of the SNTs were investigated. Room-temperature and low-temperature magnetic measurements show that the as-fabricated γ-Fe2O3 SNTs are ferromagnetic, and its coercivity is nonzero when the temperature above blocking temperature (TB). The hysteresis loop was operated to show that the magnetic properties of γ-Fe2O3 SNTs are strongly influenced by the morphology of the crystal. The unique magnetic behaviors were interpreted by the competition of the demagnetization energy of quasi-one-dimensional nanostructures and the magnetocrystalline anisotropy energy of particles in SNTs.

We report a rational synthesis of maghemite (γ-Fe2O3) short-nanotubes (SNTs) by a convenient hydrothermal method and subsequent annealing process. The structure, shape, and magnetic properties of the SNTs were investigated. Room-temperature and low-temperature magnetic measurements show that the as-fabricated γ-Fe2O3 SNTs are ferromagnetic, and its coercivity is nonzero when the temperature above blocking temperature (TB). The hysteresis loop was operated to show that the magnetic properties of γ-Fe2O3 SNTs are strongly influenced by the morphology of the crystal. The unique magnetic behaviors were interpreted by the competition of the demagnetization energy of quasi-one-dimensional nanostructures and the magnetocrystalline anisotropy energy of particles in SNTs.

Background

Surgical closure of patent ductus arteriosus (PDA) with severe pulmonary arterial hypertension in adults carries higher risk than in children.

Objectives

To investigate the application of self‐expandable occluders for transcatheter closure of PDA associated with severe pulmonary arterial hypertension in adults, and the assessment of immediate and short‐term results.

Methods

29 adult patients (6 men, 23 women) underwent attempted transcatheter closure of PDA at a mean (standard deviation (SD)) age of 31.1 (11.4) years (range 18–58 years) and a mean (SD) weight of 54.1 (7.1) kg (range 42–71 kg). On the basis of haemodynamic and clinical data obtained before and after trial occlusion, the final duct occlusion was determined and carried out. Radiographs of the chest, electrocardiograms and echocardiograms were used for follow‐up evaluation of the treatment within 1 day, 1 month and 3–6 months after successful closure.

Results

20 of the 29 patients had successful occlusion (group 1), and 9 patients failed (named group 2). In group 1, in which occlusion was successful, mean (SD) pulmonary arterial pressures decreased markedly after trial occlusion: 78 (19.3) mm Hg (range 50–125 mm Hg) before occlusion and 41 (13.8) mm Hg (range 23–77 mm Hg) after occlusion. Systemic arterial oxygen saturation was found to be >90% in 19 patients and <90% in the remaining patient before inhalation of oxygen, and >95% during inhalation of oxygen or after occlusion in all 20 patients. In group 2, the occlusion was not successful, because in two patients the device was not available; another two patients showed worsening of symptoms. The other five patients showed increased pulmonary arterial pressures after trial closure; their mean (SD) pulmonary arterial pressures increased by 10.3 (6) mm Hg (4–16 mm Hg) after trial occlusion, and systemic arterial oxygen saturation was 85.5% (2.6%) (range 82.6–88%) before inhalation of oxygen and 94.7% (1.7%) (range 90.7–99.1%) during inhalation of oxygen. In group 1, the dimensions of the left atrium, left ventricle and pulmonary artery increased considerably in 3–6‐months of follow‐up compared with those of preocclusion.

Conclusions

Transcatheter closure is an effective treatment for adults with PDA associated with reversible severe pulmonary arterial hypertension. Further research is needed for the evaluation of long‐term results.

Ni nanocrystals (NCs) were embedded in BaTiO3 epitaxial films using the laser molecular beam epitaxy. The processes involving the self-organization of Ni NCs and the epitaxial growth of BaTiO3 were discussed. With the in situ monitoring of reflection high-energy electron diffraction, the nanocomposite films were engineered controllably by the fine alternation of the self-organization of Ni NCs and the epitaxial growth of BaTiO3. The transmission electron microscopy and the X-ray diffraction characterization confirmed that the composite film consists of the Ni NCs layers alternating with the (001)/(100)-oriented epitaxial BaTiO3 separation layers.

The mechanisms mediating the establishment of totipotency during the egg-to-embryo transition in mammals remain poorly understood. The cytoplasmic lattices (CPLs), a structure unique to mammalian oocytes and preimplantation embryos, have long been predicted to function as a storage form for the maternal contribution of ribosomes and mRNA to the early embryo. Here, we demonstrate that oocyte- and embryo-restricted peptidylarginine deiminase 6 (PADI6) is required for incorporation of ribosomal components into the oocyte CPLs. Next, we show that the abundance and localization of ribosomal components is dramatically affected in PADI6-/- two-cell embryos and that de novo protein synthesis is also disregulated in these embryos. Finally, we demonstrate that embryonic genome activation (EGA) is defective in PADI6 -/- two-cell embryos. These results suggest that, in mammals, ribosomal components are stored in the oocyte CPLs and are required for protein translation during early development.

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent fatal cancers in the world. Despite advances in early diagnosis and improvements in surgical techniques, the survival of patients with HCC even after resection is poor because of the high incidence of recurrences. Therefore, the identification of prognostic factors may be helpful in the development of new treatment protocols.

Aims: To investigate HER-2/neu status in HCC by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), and to explore the possibility of using trastuzumab in the treatment of HCC.

Methods: Eight hundred and sixty eight surgical samples from patients with primary HCC were examined for their HER-2/neu status. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the PathVysion HER-2 DNA probe kit. The correlations between HER-2/neu overexpression and clinicopathological characteristics were analysed statistically.

Results: HER-2/neu overexpression was detected in 21 (2.42%) of the 868 primary HCCs. Only one specimen showed HER-2/neu gene amplification by FISH. No significant associations were found between HER-2/neu overexpression and the clinicopathological parameters.

Conclusions: There is a low frequency of HER-2/neu overexpression/amplification in HCC. There appears to be no role for HER-2/neu as a prognostic marker and no benefit of anti-HER-2/neu trastuzumab treatment in patients with HCC.

The slit genes have recently been found to encode proteins with a conserved chemorepulsive activity for axons in invertebrates and vertebrates. We have determined the expression pattern of a slit gene in Xenopus embryos. In the neural tube, slit is expressed at the ventral and dorsal midlines, and the motor neurons. slit is also expressed in a changing pattern in the retina. The full-length Xenopus Slit protein is secreted extracellularly, whereas its receptor Roundabout can not be secreted. Using a myc-tagged secreted Slit protein, we confirmed the binding of Slit to Roundabout expressed on the cell surface.

These results confirm Slit–Roundabout interactions and the biochemical properties of Slit and Roundabout proteins, and further support the idea that Slit may guide axon projections in multiple regions of the embryo.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.

Exposure of Staphylococcus aureus to cell wall inhibitors induces massive overexpression of a number of genes, provided that the VraSR two-component sensory regulatory system is intact. Inactivation of vraS blocks this transcriptional response and also causes a drastic reduction in the levels of resistance to beta-lactam antibiotics and vancomycin. We used an experimental system in which the essential cell wall synthesis gene of S. aureus, pbpB, was put under the control of an isopropyl-β-d-thiogalactopyranoside-inducible promoter in order to induce reversible perturbations in cell wall synthesis without the use of any cell wall-active inhibitor. Changes in the level of transcription of pbpB were rapidly followed by parallel changes in the vraSR signal, and the abundance of the pbpB transcript was precisely mirrored by the abundance of the transcripts of vraSR and some additional genes that belong to the VraSR regulon. Beta-lactam resistance in S. aureus appears to involve a complex stress response in which VraSR performs the critical role of a sentinel system capable of sensing the perturbation of cell wall synthesis and allowing mobilization of genes that are essential for the generation of a highly resistant phenotype. One of the sites in cell wall synthesis “sensed” by the VraSR system appears to be a step catalyzed by PBP 2.

Pregnancy-related lumbopelvic pain has puzzled medicine for a long time. The present systematic review focuses on terminology, clinical presentation, and prevalence. Numerous terms are used, as if they indicated one and the same entity. We propose “pregnancy-related pelvic girdle pain (PPP)”, and “pregnancy-related low back pain (PLBP)”, present evidence that the two add up to “lumbopelvic pain”, and show that they are distinct entities (although underlying mechanisms may be similar). Average pain intensity during pregnancy is 50 mm on a visual analogue scale; postpartum, pain is less. During pregnancy, serious pain occurs in about 25%, and severe disability in about 8% of patients. After pregnancy, problems are serious in about 7%. The mechanisms behind disabilities remain unclear, and constitute an important research priority. Changes in muscle activity, unusual perceptions of the leg when moving it, and altered motor coordination were observed but remain poorly understood. Published prevalence for PPP and/or PLBP varies widely. Quantitative analysis was used to explain the differences. Overall, about 45% of all pregnant women and 25% of all women postpartum suffer from PPP and/or PLBP. These values decrease by about 20% if one excludes mild complaints. Strenuous work, previous low back pain, and previous PPP and/or PLBP are risk factors, and the inclusion/exclusion of high-risk subgroups influences prevalence. Of all patients, about one-half have PPP, one-third PLBP, and one-sixth both conditions combined. Overall, the literature reveals that PPP deserves serious attention from the clinical and research communities, at all times and in all countries.

An unusual case of retroperitoneal ectopic pregnancy is reported. The patient, a 34 year old nulligravida, underwent IVF/ET following bilateral salpingectomy. A small, degenerating, intrauterine gestational sac suggested failing intrauterine pregnancy. There was no intraperitoneal free fluid. On Day 41 after ET, the patient was hospitalized because of acute epigastric pains. A pseudocyst of the head of pancreas was demonstrated by CT scan. A day later, exploratory laparotomy, because of a precipitous drop in the hemoglobin, revealed a massive retroperitoneal hematoma and an embryo in the gestational sac attached to the head of pancreas and major blood vessels. The patient did well following surgery. The mechanisms of retroperitoneal embryo migration are discussed and literature is reviewed.

The diversity of cellular and tissue functions within organs requires that local communication circuits control distinct populations of cells. Recently, we reported that cardiac myocytes regulate the expression of both von Willebrand factor (vWF) and a transgene with elements of the vWF promoter in a subpopulation of cardiac microvascular endothelial cells (J. Cell Biol. 138:1117). The present study explores this communication. Histological examination of the cardiac microvasculature revealed colocalization of the vWF transgene with the PDGF alpha-receptor. Transcript analysis demonstrated that in vitro cardiac microvascular endothelial cells constitutively express PDGF-A, but not B. Cardiac myocytes induced endothelial expression of PDGF-B, resulting in PDGF-AB. Protein measurement and transcript analysis revealed that PDGF-AB, but not PDGF-AA, induced endothelial expression of vWF and its transgene. Antibody neutralization of PDGF-AB blocked the myocyte-mediated induction. Immunostaining demonstrated that vWF induction is confined to PDGF alpha-receptor-positive endothelial cells. Similar experiments revealed that the PDGF-AB/alpha-receptor communication also induces expression of vascular endothelial growth factor and Flk-1, critical components of angiogenesis. The existence of this communication pathway was confirmed in vivo. Injection of PDGF-AB neutralizing antibody into the amniotic fluid surrounding murine embryos extinguished expression of the transgene. In summary, these studies suggest that environmental induction of PDGF-AB/alpha-receptor interaction is central to the regulation of cardiac microvascular endothelial cell hemostatic and angiogenic activity.

Brief ischemic episodes confer marked protection against myocardial stunning 1-3 d later (late preconditioning [PC] against stunning). The mechanism of this powerful protective effect is poorly understood. Although protein kinase C (PKC) has been implicated in PC against infarction, it is unknown whether it triggers late PC against stunning. In addition, the entire PKC hypothesis of ischemic PC remains controversial, possibly because the effects of PKC inhibitors on PC protection have not been correlated with their effects on PKC activity and/or translocation in vivo. Thus, conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In the control group (group I, n = 7), the recovery of systolic wall thickening after the six O/R cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg before the first O on day 1 (group II, n = 10) abrogated the late PC effect against stunning, whereas a 10-fold lower dose (0.5 mg/kg; group III, n = 7) did not. Administration of 5 mg/kg of chelerythrine 10 min after the sixth reperfusion on day 1 (group IV, n = 6) failed to block late PC against stunning. When rabbits were given 5 mg/kg of chelerythrine in the absence of O/R (group V, n = 5), the severity of myocardial stunning 24 h later was not modified. Pretreatment with phorbol 12-myristate 13-acetate (4 microg/kg) on day 1 without ischemia (group VI, n = 11) induced late PC against stunning on day 2 and the magnitude of this effect was equivalent to that observed after ischemic PC. In vehicle-treated rabbits (group VIII, n = 5), the six O/R cycles caused translocation of PKC isoforms epsilon and eta from the cytosolic to the particulate fraction without significant changes in total PKC activity, in the subcellular distribution of total PKC activity, or in the subcellular distribution of the alpha, beta1, beta2, gamma, delta, zeta, iota, lambda, and mu isoforms. The higher dose of chelerythrine (5 mg/kg; group X, n = 5) prevented the translocation of both PKC epsilon and eta induced by ischemic PC, whereas the lower dose (0.5 mg/kg; group XI, n = 5) prevented the translocation of PKC eta but not that of epsilon, indicating that the activation of epsilon is necessary for late PC to occur whereas that of eta is not. To our knowledge, this is the first demonstration that a PKC inhibitor actually prevents the translocation of PKC induced by ischemic PC in vivo, and that this inhibition of PKC translocation results in loss of PC protection. Taken together, the results demonstrate that the mechanism of late PC against myocardial stunning in conscious rabbits involves a PKC-mediated signaling pathway, and implicate epsilon as the specific PKC isoform responsible for the development of this cardioprotective phenomenon.

Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaerobic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H(inf2)-CO(inf2)), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobacterium formicicum.

In an earlier study on minus-strand DNA synthesis catalyzed by murine leukemia virus reverse transcriptase, we described a prominent pause site near the polypurine tract (J. Guo, W. Wu, Z. Y. Yuan, K. Post, R. J. Crouch, and J. G . Levin, Biochemistry 34:5018-5029, 1995). We now report that pausing at this site is due to a stem-loop structure in the RNA template, formed by interaction of a number of bases in the polypurine tract, including the six G's, and a 3' sequence which includes four C's. Addition of human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein to reverse transcriptase reactions reduces pausing by approximately 8- to 10-fold and stimulates synthesis of full-length DNA. Thus, NC functions as an accessory protein during elongation of minus-strand DNA and increases the efficiency of DNA synthesis, in this case, by apparently destabilizing a region of secondary structure in the template. Since NC is associated with genomic RNA in the viral core and is likely to be part of a viral replication complex, these results suggest that NC may also promote efficient DNA synthesis during virus replication. Mutational analysis indicates that the features of HIV-1 NC which are important for reduction of pausing include the basic amino acids flanking the first zinc finger, the zinc fingers, and the cysteine and aromatic amino acids within the fingers. These findings suggest that reverse transcription might be targeted by drugs which inactivate the zinc fingers of HIV-1 NC.

An endospore-forming, butyrate-degrading bacterium (strain BH) was grown on butyrate in monoxenic coculture with a methanogen. The culture formed dense aggregates when Methanobacterium formicicum was the methanogenic partner, but the culture was turbid when Methanospirillum hungatei was the partner. In contrast, a propionate-degrading, lemon-shaped bacterium (strain PT) did not form aggregates with Methanobacterium formicicum unless an acetate-degrading Methanosaeta sp. was also included in the culture. Fatty acid-degrading methanogenic granules were formed in a laboratory-scale upflow reactor at 35(deg)C fed with a medium containing a mixture of acetate, propionate, and butyrate by using defined cultures of Methanobacterium formicicum T1N, Methanosaeta sp. strain M7, Methanosarcina mazei T18, propionate-degrading strain PT, and butyrate-degrading strain BH. The maximum substrate conversion rates of these granules for acetate, propionate, and butyrate were 43, 9, and 17 mmol/g (dry weight)/day, respectively. The average size of the granules was about 1 mm. Electron microscopic observation of the granules revealed that the cells of Methanobacterium formicicum, Methanosaeta sp., butyrate-degrading, and propionate-degrading bacteria were dispersed in the granules. Methanosarcina mazei existed inside the granules as aggregates of its own cells, which were associated with the bulk of the granules. The interaction of different species in aggregate formation and granule formation is discussed in relation to polymer formation of the cell surface.

We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The resulting lacZalpha peptide fusion protein remained functional. Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation. With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis. Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations. DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis. RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum. The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites. Results indicate that recombination is another source of mutations during viral replication.