Intracellular trafficking of HIV-1 Gag via Syntaxin 6-positive compartments/vesicles: Involvement in tumor necrosis factor secretion.

HIV-1 Gag protein is synthesized in the cytosol and is transported to the plasma membrane, where viral particle assembly and budding occur. Endosomes are alternative sites of Gag accumulation. However, the intracellular transport pathways and carriers for Gag have not been clarified. We show here that Syntaxin6 (Syx6), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane fusion in post-Golgi networks, is a molecule responsible for Gag trafficking and also for tumor necrosis factor-α (TNFα) secretion and that Gag and TNFα are cotransported via Syx6-positive compartments/vesicles. Confocal and live-cell imaging revealed that Gag colocalized and cotrafficked with Syx6, a fraction of which localizes in early and recycling endosomes. Syx6 knockdown reduced HIV-1 particle production, with Gag distributed diffusely throughout the cytoplasm. Coimmunoprecipitation and pulldown show that Gag binds to Syx6, but not its SNARE partners or their assembly complexes, suggesting that Gag preferentially binds free Syx6. The Gag matrix domain and the Syx6 SNARE domain are responsible for the interaction and cotrafficking. In immune cells, Syx6 knockdown/knockout similarly impaired HIV-1 production. Interestingly, HIV-1 infection facilitated TNFα secretion, and this enhancement did not occur in Syx6-depleted cells. Confocal and live-cell imaging revealed that TNFα and Gag partially colocalized and were cotransported via Syx6-positive compartments/vesicles. Biochemical analyses indicate that TNFα directly binds the C-terminal domain of Syx6. Altogether, our data provide evidence that both Gag and TNFα make use of Syx6-mediated trafficking machinery and suggest that Gag expression does not inhibit but rather facilitates TNFα secretion in HIV-1 infection.

Non-contrast magnetic resonance angiography for systemic artery evaluation in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis; systemic arteries other than the coronary arteries should therefore also be evaluated. This study investigated the feasibility of evaluating coronary aneurysms, systemic artery aneurysms (SAAs), and cerebrovascular diseases in patients with KD using non-contrast magnetic resonance angiography (NC-MRA). METHODS: Coronary artery protocols, including coronary magnetic resonance angiography (MRA) and vessel wall imaging, were performed in 57 examinations of 28 patients. Systemic artery protocol, including SAA scans and head MRA, along with coronary artery protocol, were performed in 42 examinations of 42 patients. The image quality of the SAAs was evaluated on a 4-point scale. Examination time and sedation dosage were compared between the protocols. The presence of SAAs and cerebrovascular disease was also evaluated. RESULTS: The image quality score of SAAs was 4 (interquartile range [IQR]: 4-4) for the aorta, 4 (IQR: 3-4) for the subclavian artery, 4 (IQR: 3-4) for the renal artery, and 3 (IQR: 3-4) for the iliac artery. No differences were found between examination time (47.0 [IQR: 43.0-61.0] min vs. 51.0 [IQR: 45.0-60.0] min, p = 0.48) and sedative dose (4.63 [IQR: 3.93-5.79] mg/kg vs. 4.21 [IQR: 3.56-5.71] mg/kg, p = 0.37) between the protocols. Systemic artery protocol detected SAAs in three patients (7.1%), and cerebrovascular disease was not detected. CONCLUSIONS: Evaluating the coronary and systemic arteries in patients with KD using NC-MRA on a single examination was possible without compromising examination time or sedation dose. The systemic artery protocol was useful in finding SAAs.

Effect of renal tubular damage on non-cancer mortality in the general Japanese population living in cadmium non-polluted areas.

This study aimed to clarify the cause-effect relationship between renal tubular damage and non-cancer mortality in the general Japanese population. We conducted a 19-year cohort study including 1110 men and 1,03 women who lived in three cadmium-non-polluted areas in 1993 or 1994. Mortality risk ratios based on urinary ß2-microglobulin (ß2MG) and N-acetyl-ß-glucosaminidase (NAG) concentrations were estimated for specific non-cancer diseases using the Fine and Gray competing risks regression model. In men, continuous urinary NAG (+1 µg/g cre) concentrations were significantly correlated with increased mortality caused by diseases of the respiratory system (hazard ratio (HR): 1.09, 95% confidence interval (CI): 1.03-1.15). Urinary ß2MG (+100 µg/g cre) concentrations were significantly correlated with increased mortalities caused by kidney and urinary tract diseases (HR: 1.01, 95% CI: 1.00-1.03), renal diseases (HR: 1.01, 95% CI: 1.00-1.03), renal failure (HR: 1.02, 95% CI: 1.00-1.03), and external causes of mortality (HR: 1.01, 95% CI: 1.00-1.02). In women, urinary NAG (+1 µg/g cre) concentrations were significantly associated with increased mortality caused by ischemic heart diseases (HR: 1.02, 95% CI: 1.00-1.04) and kidney and urinary tract diseases (HR: 1.01, 95% CI: 1.00-1.04). Urinary ß2MG (+100 µg/g cre) concentrations were significantly correlated with increased mortality caused by cardiovascular diseases (HR: 1.01, 95%CI: 1.00-1.02), ischemic heart diseases (HR: 1.01, 95%CI: 1.00-1.02), and kidney and urinary tract diseases (HR: 1.02, 95% CI: 1.01-1.03). The present study indicates that renal tubular damage was significantly related to several non-cancer disease causes of mortality in Japan's general population living in cadmium-non-polluted areas.

A Proteomic Analysis of Detergent-Resistant Membranes in HIV Virological Synapse: The Involvement of Vimentin in CD4 Polarization.

The cell-cell contact between HIV-1-infected and uninfected cells forms a virological synapse (VS) to allow for efficient HIV-1 transmission. Not only are HIV-1 components polarized and accumulate at cell-cell interfaces, but viral receptors and lipid raft markers are also. To better understand the nature of the HIV-1 VS, detergent-resistant membrane (DRM) fractions were isolated from an infected-uninfected cell coculture and compared to those from non-coculture samples using 2D fluorescence difference gel electrophoresis. Mass spectrometry revealed that ATP-related enzymes (ATP synthase subunit and vacuolar-type proton ATPase), protein translation factors (eukaryotic initiation factor 4A and mitochondrial elongation factor Tu), protein quality-control-related factors (protein disulfide isomerase A3 and 26S protease regulatory subunit), charged multivesicular body protein 4B, and vimentin were recruited to the VS. Membrane flotation centrifugation of the DRM fractions and confocal microscopy confirmed these findings. We further explored how vimentin contributes to the HIV-1 VS and found that vimentin supports HIV-1 transmission through the recruitment of CD4 to the cell-cell interface. Since many of the molecules identified in this study have previously been suggested to be involved in HIV-1 infection, we suggest that a 2D difference gel analysis of DRM-associated proteins may reveal the molecules that play crucial roles in HIV-1 cell-cell transmission.

Correlation between Quality of Life under Treatment and Current Life Satisfaction among Cancer Survivors Aged 75 Years and Older Receiving Outpatient Chemotherapy in Ishikawa Prefecture, Japan.

Life satisfaction is increasingly important for older cancer survivors as the global population ages and the life expectancy 29 of cancer survivors increases. This study sought to identify factors associated with physical symptoms, quality of life under treatment, and current life satisfaction in cancer survivors aged 75 years and older receiving outpatient chemotherapy. Information about treatment for cancer survivors was collected from electronic medical records, and interviews were conducted to assess life satisfaction under treatment. Participants were older cancer survivors in Ishikawa, Japan. Of the participants, 80% lived on the Noto Peninsula. The average linear distance traveled for treatment was 40.7 km. The factors associated with patients' dissatisfaction with their current lives included general malaise (odds ratio: 9.61; 95% confidence interval: 1.28-72.22) and being less happy now than when they were younger (odds ratio: 10.559; 95% confidence interval: 1.50-74.24). In outpatient cancer treatment for survivors aged 75 years and older, support should consider the distance to the hospital. As in past studies, general malaise was shown to have a negative impact on the lives of cancer survivors aged 75 years or older. Support providers should pay attention to patients' general malaise when providing support.

Pitfall for systemic artery aneurysms evaluation using electrocardiogram-gated subtracted three-dimensional fast spin echo sequence of magnetic resonance imaging in patients with Kawasaki disease.

Kawasaki disease (KD) is described as a syndrome that causes both coronary and systemic artery aneurysms (SAAs). This report describes the pitfall for SAAs' evaluation when using electrocardiogram (ECG)-gated subtracted three-dimensional fast spin echo (3D FSE) sequence of magnetic resonance imaging in KD patients. A 12-year-old male was diagnosed with KD at 3 months of age. We acquired ECG-gated 3D FSE images in the diastole and systole phases with coronal sections. Subtraction was then performed from diastolic phase imaging to systolic phase imaging. A 15.5 mm right axillary artery aneurysm and an 8.0 mm left axillary artery aneurysm were identified with ECG-gated 3D FSE in the diastolic phase. However, we observed signal loss in the right axillary artery aneurysm when subtraction was performed to selectively detect arteries; further, the brachial artery was poorly detected. ECG-gated subtracted 3D FSE sequence of magnetic resonance imaging can compromise the image quality of both aneurysm and peripheral artery images when detecting SAAs.

Renal tubular dysfunction and cancer mortality in the Japanese general population living in cadmium-non-contaminated areas.

The relationship between cadmium exposure, exposure-related renal tubular dysfunction, and mortality have been reported, mainly in the residents of Cd-contaminated areas in Japan. The aim of this study was to establish the cause-effect relationship between renal tubular dysfunction and cancer mortality in the general population in non-contaminated areas. A 19-year cohort study was conducted in 1110 men and 1703 women in 1993 or 1994, who lived in three cadmium-non-contaminated areas. Mortality risk ratios of urinary ß2-microglobulin (ß2MG) and N-acetyl-ß-glucosaminidase (NAG) for all malignant neoplasms and specific cancers were estimated using the Fine and Gray competing risks regression model. Significant hazard ratios (HRs) for liver and pancreas cancer were observed for NAG (liver: HR corresponding to an increase of 1 IU/g cr, 1.10, 95%CI, 1.02-1.19, pancreas: HR, 1.10, 95%CI, 1.02-1.19) in men. In women, a negative HR was observed for NAG (lung cancer: HR 0.80, 95% CI, 0.67-0.96) and for ß2MG (all malignant neoplasms: HR, 0.97, 95% CI, 0.93-1.00). The present study indicated that renal tubular dysfunction was significantly related to mortality in the general population of cadmium-non-contaminated areas in Japan.

Work-related experiences of people living with young-onset dementia in Japan.

Dementia that occurs before age 65 years is defined as young-onset dementia (YOD). YOD develops during the prime of a person's working life and has a major impact on not only work but life in general. Therefore, Japan is promoting measures to support work and social participation, taking into account the characteristics of people living with YOD. Given the rarity of YOD, few studies have examined the difficulties faced by people living with YOD during this life stage or the kinds of support they require. We believe that studying these difficulties and support requirements will contribute to prolonging the careers of people living with YOD and to providing them support during social, emotional and economic crises. This qualitative study aims to clarify the experiences of working-age Japanese people living with YOD to examine, from their perspective, possibilities for employment support. For this study, people living with YOD who were currently working were recruited by snowball sampling. This study was conducted in Japan between September 2018 and February 2019. A semi-structured interview was conducted, and the contents were transcribed and analysed using Colaizzi's qualitative methodology. Four categories were derived: (a) crisis from continuing to work, (b) seeking support, (c) overcoming challenges to work and (d) reaffirming a sense of purpose by resuming work and social participation. Participants were able to continue working and socialising by letting others know about their illness and seeking support. In the process of reaffirming a sense of purpose by resuming work and social participation, participants continued working or transitioned to socially active lives after leaving their job thanks to the support of work colleagues and medical and healthcare/welfare professionals who understand YOD. The results indicate a need for awareness raising in the workplace to promote understanding of such professional support and its significance.

Identification of the 5'-Terminal Packaging Signal of the H1N1 Influenza A Virus Neuraminidase Segment at Single-Nucleotide Resolution.

The genome of the influenza A virus is an eight-segmented negative-strand RNA (vRNA). Progeny vRNAs replicated in the nucleus selectively assemble into a single set of eight different segments, probably in the cytoplasm, and are packaged into progeny virions at the cell membrane. In these processes, a region of approximately 100 nucleotides at both ends of each segment is thought to function as a selective assembly/packaging signal; however, the details of the mechanism, such as the required sequences, are still unknown. In this study, we focused on the 5'-terminus of the sixth neuraminidase gene segment vRNA (Seg.6) to identify the essential sequence for selective packaging. The 5'-terminal region of the A/Puerto Rico/8/34 strain Seg.6 was divided into seven regions of 15 nucleotides each from A to G, and mutations were introduced into each region by complementary base substitutions or synonymous codon substitutions. Mutant viruses were generated and compared for infectious titers, and the relative ratios of the eight segments packaged into virions were measured. We also ascertained whether mutant vRNA was eliminated by competitive packaging with wild-type vRNA. Mutations in the A-C regions reduced infectious titers and eliminated mutant vRNAs by competition with wild-type vRNA. Even under non-competitive conditions, the packaging efficiency of the A or B region mutant Seg.6 was reduced. Next, we designed an artificial vRNA with a 50-nucleotide duplication at the 5'-terminal region. Using this, a virus library was created by randomly replacing each region, which became an untranslated region (UTR), with complementary bases. After selecting proliferative viruses from the library, nine wild-type nucleotides in the A and B regions were identified as essential bases, and we found that these bases were highly conserved in Seg.6 vRNAs encoding the N1 subtype neuraminidase. From these results, we conclude that the identified bases function as the 5'-terminal packaging signal for the N1 subtype Seg.6 vRNA.

The Relationship between the Urinary Cadmium Concentration and Cause-Specific Mortality in Subjects without Severe Renal Damage: A 35-Year Follow-Up Study in a Cadmium-Polluted Area of Japan.

We evaluated the association between urinary cadmium concentration (uCd, µg/g Cr) and risk of cause-specific mortality according to urinary ß2-microglobulin (MG) concentration. Participants were 1383 male and 1700 female inhabitants of the Cd-polluted Kakehashi River basin. The uCd and ß2-MG were evaluated in a survey in 1981-1982, where those participants were followed-up over 35 years later. Among the participants with a urinary ß2-MG < 1000, the hazard ratios (HRs) (95% confidence interval) for mortality were significantly higher in those with a uCd of ≥10.0 compared with <5.0 for cardiovascular disease [HR 1.92 (1.08-3.40) for men, 1.71 (1.07-2.71) for women], pneumonia or influenza [2.10 (1.10-4.00) for men, 2.22 (1.17-4.19) for women], and digestive diseases [for men; 3.81 (1.49-9.74)]. The uCd was significantly associated with mortality from heart failure in women and digestive diseases in men, after adjustment for other causes of death using the Fine and Gray competing risk regression model. For participants with a urinary ß2-MG of ≥1000, no significant association was observed between uCd and any major cause of death. In the absence of kidney damage, Cd may increase the risk of death from cardiovascular disease, pneumonia, and digestive diseases.

Efficient Transendothelial Migration of Latently HIV-1-Infected Cells.

A small fraction of HIV-1-infected T cells forms populations of latently infected cells when they are a naive T-cell subset or in transit to a resting memory state. Latently HIV-1-infected cells reside in lymphoid tissues and serve as viral reservoirs. However, whether they systemically recirculate in the body and re-enter the lymphoid nodes are unknown. Here, we employed two in-vitro cell coculture systems mimicking the lymphatic endothelium in lymph nodes and investigated the homing potential, specifically the transendothelial migration (TEM), of two latently HIV-1-infected cell lines (J1.1 and ACH-2). In trans-well coculture systems, J1.1 and ACH-2 showed higher TEM efficiencies than their parental uninfected and acutely infected cells. The efficiency of TEM was enhanced by the presence of stromal cells, such as HS-5 and fibroblastic reticular cells. In an in-vitro reconstituted, three-dimensional coculture system in which stromal cells are embedded in collagen matrices, J1.1 showed slightly higher TEM efficiency in the presence of HS-5. In accordance with these phenotypes, latently infected cells adhered to the endothelial cells more efficiently than uninfected cells. Together, our study showed that latently HIV-1-infected cells enhanced cell adhesion and TEM abilities, suggesting their potential for efficient homing to lymph nodes.

The Association between Anthropometric Indices of Obesity and Chronic Kidney Disease in Middle-aged Japanese Men and Women: A Cohort Study.

Objective This study investigated associations between three indices of obesity-the body mass index (BMI), waist circumference (WC), and waist-to-height ratio (WHtR)-and the incidence of chronic kidney disease (CKD). Methods The employees of a company in Japan (1,725 men, 1,186 women; aged 35-55 years) had BMI, WC, and WHtR measured in health examinations. The incidence of CKD was determined at annual medical examinations over a six-year period. The hazard ratios for CKD were calculated using proportional hazard models, and the χ2 statistic was used to compare the strengths of the associations. Results The mean BMI (kg/m2), WC (cm), and WHtR were 23.6, 84.3, and 0.49 for men and 22.3, 79.7, and 0.50 for women, respectively. The incidence of CKD (/1,000 person-years) was 18.1 for men and 8.4 for women. In men, positive linear associations were observed between the BMI, WC, and WHtR and the risk of CKD, even after adjusting for the presence of metabolic abnormalities (p for trend <0.001, 0.012, and 0.023, respectively). In women, a linear association was observed only between the WHtR and CKD, not the BMI or WC (p for trend =0.042, 0.057, and 0.186). The χ2 statistics were the highest for the BMI in both men and women. Conclusion The BMI, WC, and WHtR were linearly associated with the risk of CKD independently of metabolic abnormalities in men, while the associations were weaker or not significant in women. The BMI was the most strongly associated with the incidence of CKD in both men and women.

Evaluating for systemic artery aneurysms using noncontrast magnetic resonance angiography in patients with Kawasaki disease: A report of two cases.

Kawasaki disease (KD) involves coronary aneurysms and can infrequently cause systemic artery aneurysms (SAAs). Therefore, patients with KD should be evaluated for both coronary and systemic arterial aneurysms. This report describes 2 cases of SAA evaluated using the diastolic phase image of electrocardiogram-gated three-dimensional fast spin echo during noncontrast magnetic resonance angiography. The first case was a 1-year-old male who diagnosed with KD at 2 months of age. Multiple right axillary artery aneurysms measuring 6.0 mm and 2.5 mm and left axillary artery aneurysms measuring 12.0 mm, 4.0 mm, and 3.0 mm were observed by scanning for 94 seconds. The second case was a 13-year-old male who diagnosed with KD at 4 months of age, with a 7.0-mm right axillary artery aneurysm observed by scanning for 101 seconds. Electrocardiogram-gated three-dimensional fast spin echo in the diastolic phase can help evaluate SAA in patients with KD and does not require a prolonged scanning time or contrast medium.

The relationship between cadmium exposure and renal volume in inhabitants of a cadmium-polluted area of Japan.

This follow-up study was conducted over 30 years in a cadmium-polluted area of Japan. Urinary cadmium (U-Cd) concentration decreased by nearly half from 1986 to 2008 in men and women. However, it increased from 2008 to 2014 and maintained similar levels in 2016. Because renal atrophy may induce an increase in U-Cd, kidney volumes were determined using magnetic resonance imaging (MRI) scans in 2018. Based on the MRI results, we divided the participants into two groups, namely the normal group (n = 6, three men and three women) and the lesion group (n = 6, three men and three women). The level of urinary N-acetyl-ß-d-glucosaminidase/creatinine (U-NAG/Cr) in the lesion group was significantly higher than in the normal group. The level of serum alkaline phosphatase (Al-P) was positively associated with U-Cd. Age and renal cortex volumes showed significantly negative associations. However, U-Cd and renal cortex and kidney volumes showed no significant associations. These results suggest that U-NAG and serum Al-P were sensitive biomarkers to reflect renal tubular dysfunction and bone damage caused by cadmium poisoning. Individuals chronically exposed to Cd should be observed carefully, due to the increased effect of aging on renal cortex volumes.

Environmental cadmium exposure and noncancer mortality in a general Japanese population in cadmium nonpolluted regions.

The objective of this study was to assess the effect of environmental cadmium exposure according to urinary cadmium concentration (U-Cd) on noncancer mortality in a general Japanese population. We conducted a longitudinal study for 19 years in 2804 inhabitants (1107 men and 1697 women) in some cadmium nonpolluted regions in Japan. The participants were classified into quartiles based on U-Cd (µg/g cre) adjusted for urinary creatinine. Hazard ratio (HR) and 95% confidence interval (95% CI) for continuous U-Cd or the quartiles of U-Cd were calculated for noncancer mortality. By applying a Fine and Gray competing risk model, continuous U-Cd (+1 µg/g cre) showed significant HR for cardiocerebrovascular diseases (HR 1.05, 95% CI: 1.00-1.11), cerebrovascular diseases (HR 1.08, 95% CI: 1.01-1.16), and cerebral infarction (HR 1.11, 95% CI: 1.04-1.20) in men. However, notable significant HR for continuous and quartered U-Cd were not observed in women. In this study, U-Cd was associated with increased cardiocerebrovascular mortality in a general Japanese population, suggesting that environmental cadmium exposure is detrimental to the life prognosis in cadmium nonpolluted regions in Japan.

Relationship between urinary ß2 -microglobulin concentration and mortality in a cadmium-polluted area in Japan: A 35-year follow-up study.

The relationship between urinary ß2 -microglobulin (ß2 -MG) and the risk of all-cause mortality and cause-specific mortality in a cadmium (Cd)-polluted area was investigated in 3139 inhabitants (1404 men and 1735 women) of the Kakehashi River basin in Japan at 35-year follow-up. The subjects had been participants in the 1981-1982 health impact survey that assessed Cd-induced renal dysfunction, as measured by the urinary ß2 -MG concentration. Hazard ratios were calculated to assess the risk of all-cause and cause-specific mortality according to the urinary ß2 -MG concentrations. Risk ratios (RRs) were assessed using the Fine and Gray regression model to account for competing risks of cause-specific mortality. The mortality rate was significantly higher in participants with urinary ß2 -MG concentrations >1000 µg/g creatinine (Cr) for men and >300 µg/g Cr for women. In the proportional hazard model, higher urinary ß2 -MG concentrations were associated with higher risks of circulatory disease, digestive system diseases, and kidney and urinary tract diseases in men and women, and with senility for women. However, when competing risk was accounted for, the RRs were significantly higher only for kidney and urinary tract diseases in men and women (RR for each increment of 1000 µg/g Cr [95% confidence interval]: 1.02 [1.00-1.04] for men, and 1.01 [1.00-1.02] for women). The long-term prognosis of participants with renal tubular dysfunction was poor, most likely due to kidney and renal tract diseases.

Frequency of consumption of balanced meals, bodyweight gain and incident risk of glucose intolerance in Japanese men and women: A cohort study.

AIMS/INTRODUCTION: This cohort study assessed the risk for bodyweight gain and development of glucose intolerance based on the frequency of consumption of balanced meals including grain, fish or meat and vegetables. MATERIALS AND METHODS: The participants (8,573 men, 3,327 women) were employees of a company in Japan. A self-administered questionnaire was used to evaluate the frequency of balanced meal consumption. Bodyweight changes and the incidence of glucose intolerance (glycated hemoglobin >6.0%) during the 3-year follow-up period were determined through annual health examinations. RESULTS: The mean bodyweight change over a period of 3 years was 0.78 kg for men and 0.84 kg for women. A lower frequency of balanced meals was associated with a higher bodyweight gain for men (P for trend = 0.004), but not for women. During the study, 464 men and 115 women developed glucose intolerance. Overall, the frequency of balanced meals was not associated with the risk of glucose intolerance in either sex. However, the interaction between the frequency of balanced meals and degree of obesity had a significant effect on the incidence of glucose intolerance in men (P = 0.005), with less frequent consumption of balanced meals being associated with a higher risk for glucose intolerance among men with a BMI ≥25.0 kg/m2 (P for trend = 0.007). CONCLUSIONS: A higher frequency of balanced meals, including grain, fish or meat and vegetable dishes - important components of healthy Japanese food - was associated with a lower risk of glucose intolerance in obese men, but not in non-obese men and women.

Identification and functional analysis of glutamine transporter in Streptococcus mutans.

BACKGROUND: Streptococcus mutans, a biofilm-forming bacterium, possesses several transporters that function as import/export molecules. Among them, the PII protein family is composed of members that regulate glutamine synthesis in bacterial species. OBJECTIVE: In this study, we characterized the function of the glutamine transporter in S. mutans MT8148. METHODS: The SMU.732 gene, corresponding to glnP in S. mutans, is homologous to the glutamine transporter gene in Bacillus subtilis. We constructed a glnP-inactivated mutant strain (GEMR) and a complement strain (comp-GEMR) and evaluated their biological functions. RESULTS: Growth of GEMR was similar in the presence and absence of glutamine, whereas the growth rates of MT8148 and comp-GEMR were significantly lower in the presence of glutamine as compared to its absence. Furthermore, biofilms formed by MT8148 and comp-GEMR were significantly thicker than that formed by GEMR, while the GEMR strain showed a significantly lower survival rate in an acidic environment than the other strains. Addition of n-phenyl-2-naphthylamine, used to label of the membrane, led to increased fluorescence intensity of MT8148 and GEMR, albeit that was significantly lower in the latter. CONCLUSIONS: These results suggest that glnP is associated with glutamine transport in S. mutans, especially the import of glutamine involved in biofilm formation.

Cell-Based Influenza A/H1N1pdm09 Vaccine Viruses Containing Chimeric Hemagglutinin with Improved Membrane Fusion Ability.

The H1N1 influenza pandemic vaccine has been developed from the A/California/07/09 (Cal) virus and the well-known high-yield A/Puerto Rico/8/34 (PR8) virus by classical reassortment and reverse genetics (RG) in eggs. Previous studies have suggested that Cal-derived chimeric hemagglutinin (HA) and neuraminidase (NA) improve virus yields. However, the cell-based vaccine of the H1N1 pandemic virus has been less investigated. RG viruses that contained Cal-derived chimeric HA and NA could be rescued in Madin-Darby canine kidney cells that expressed α2,6-sialyltransferase (MDCK-SIAT1). The viral growth kinetics and chimeric HA and NA properties were analyzed. We attempted to generate various RG viruses that contained Cal-derived chimeric HA and NA, but half of them could not be rescued in MDCK-SIAT1 cells. When both the 3'- and 5'-terminal regions of Cal HA viral RNA were replaced with the corresponding regions of PR8 HA, the RG viruses were rescued. Our results were largely consistent with those of previous studies, in which the N- and C-terminal chimeric HA slightly improved virus yield. Importantly, the chimeric HA, compared to Cal HA, showed cell fusion ability at a broader pH range, likely due to amino acid substitutions in the transmembrane region of HA. The rescued RG virus with high virus yield harbored the chimeric HA capable of cell fusion at a broader range of pH.

HIV Reactivation in Latently Infected Cells With Virological Synapse-Like Cell Contact.

HIV reactivation from latency is induced by cytokines but also by cell contact with other cells. To better understand this, J1.1 cells, a latent HIV-1-infected Jurkat derivative, were cocultured with its parental Jurkat. J1.1 cells became p17MA-positive and produced a high level of HIV p24CA antigen, only when they were cocultured with stimulated Jurkat with cell-to-cell contact. In contrast, very little p24CA was produced when they were cocultured without cell contact. Similar results were obtained when latent ACH-2 and its parental A3.01 cells were cocultured. Confocal microscopy revealed that not only HIV-1 p17MA and gp120Env but also LFA-1, CD81, CD59, and TCR CD3 accumulated at the cell contact site, suggesting formation of the virological synapse-like structure. LFA-1-ICAM-1 interaction was involved in the cell-to-cell contact. When J1.1 was cocultured with TCR-deficient Jurkat, the p17MA-positive rate was significantly lower, although the cell-to-cell contact was not impaired. Quantitative proteomics identified 54 membrane molecules, one of which was MHC class I, that accumulated at the cell contact site. Reactivation from latency was also influenced by the presence of stromal cells. Our study indicated that latent HIV-1 in J1.1/ACH-2 cells was efficiently reactivated by cell-to-cell contact with stimulated parental cells, accompanying the virological synapse-like structure.