Selection of Optimal Host Strain for Molecular Pathogenesis Studies on <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>

Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.     

Reliability of an electrophoretic and image processing analysis of human skeletal muscle taken from m. vastus lateralis

The relative content of myosin heavy chain (MHC) isoforms IIb, IIa and I in human skeletal muscle taken from the m. vastus lateralis of 30 healthy male subjects was analysed using mini-gel electrophoresis. Repeated electrophoretic gels utilizing the same methods were produced for all subjects and the determination of MHC protein bands was performed using a digital scanner and National Institutes of Health (NIH) Image software and laser densitometry. A comparison between the NIH Image processing technique and laser densitometry revealed differences of 6.47%, 6.35% and 6.84% between these measurement techniques for MHC-IIb, -IIa and -I isoforms, respectively. The percentage technical error of measurement (TEM%) between electrophoretic gels was shown to be 19.1%, 17.8% and 14.2%, with regard to percentage of occurrence of MHC-IIb, -IIa and -I isoforms respectively. The variation in electrophoretic gel analyses was shown to be 5.7%, 7.3% and 5.5%, with regard to the percentage of MHC-IIb, -IIa and -I isoforms respectively. Intra-class correlations comparing NIH Image and laser densitometry produced r values in the range 0.38–0.63. Comparisons between and within gel analyses produced r values in the range 0.59–0.94 and 0.93–0.98, respectively. Analyses of variance revealed no significant differences (P < 0.05) between analysis techniques, between␣gels or within gels for the measurement of MHC-IIb, -IIa and -I isoforms. The inter-gel error between fibre subgroups was moderate for the two type-II MHC populations and less for type-I MHC; the intra-individual error in the measuring technique used for classifying the MHC-IIb, -IIa and -I protein bands was small. The results obtained in this investigation showed consistent trends which may reflect a false classification of the type-II MHC populations for the inter-gel and intra-individual analyses. The NIH Image software and digitizing process was shown to be a valid and reliable method for distinguishing between MHC protein bands of human skeletal tissue as separated by mini-gel electrophoretic techniques. Accepted: 6 January 1997     

Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae

The name Enterobacter kobei sp. nov. is proposed for a group of organisms referred to as NIH Group 21 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, motile rods conforming to the definition of the family Enterobacteriaceae. The DNA relatedness of 23 strains of NIH Group 21 to the representative proposed as the type strain of this species averaged 82% at 70°C, whereas the relatedness to other species within the family Enterobacteriaceae was less than 42%. Because the phenotypic resemblance to Enterobacter cloacae is very close and the DNA relatedness (12–42%) is closer to species of the genus Enterobacter than to other species of the family, the members of NIH Group 21 were placed in the genus Enterobacter. Close phenotypic and genetic relationships were also found between NIH Group 21 and a member of a group of organisms referred to as Enteric Group 69 at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. It is suggested that the latter could be regarded as a subspecific rank of E. kobei, though this is subject to study of further strains. The majority of strains of E. kobei were isolated from clinical specimens. A culture of the type strain (NIH 1485-79) has been deposited in the Japan Collection of Microorganisms as JCM 8580. Received: 22 March 1996 / Accepted: 19 April 1996     

Inhibition of PID1/NYGGF4/PCLI1 gene expression highlights its role in the early events of the cell cycle in NIH3T3 fibroblasts

Abstract

The PID1/NYGGF4/PCLI1 gene encodes for a protein with a phosphotyrosine-binding domain, which interacts with the lipoprotein receptor-related protein 1. Previous work by us and others suggested a function of the gene in cell proliferation of NIH3T3 fibroblasts and 3T3-L1 pre-adipocytes. The molecular characterization of PCLI1 protein, ectopically expressed in NIH3T3 fibroblasts, revealed two phosphorylation sites at Ser154 and Ser165. In order to clarify the functions of this gene, we analyzed the effects of its downregulation on cellular proliferation and cell cycle progression in NIH3T3 cell cultures. Downregulation of PID1/NYGGF4/PCLI1 mRNA levels by short hairpin RNAs (shRNAs) elicited decreased proliferation rate in mammalian cell lines; cell cycle analysis of serum-starved, synchronized NIH3T3 fibroblasts showed an increased accumulation of shRNA-interfered cells in the G1 phase. Decreased levels of FOS and MYC mRNAs were accordingly associated with these events. The molecular scenario emerging from our data suggests that PID1/NYGGF4/PCLI1 controls cellular proliferation and cell cycle progression in NIH3T3 cells.     

Common swine leucocyte antigen (SLA) haplotypes in NIH and Sinclair miniature swine have similar effects on the expression of an inherited melanoma

Mixed lymphocyte culture and serological typing of NIH and Sinclair miniature swine indicate that the two herds share a common SLA haplotype. NIH haplotype a (International Haplotype H10) appears identical to Sinclair haplotype B, which has significant effects on the penetrance of Sinclair swine cutaneous malignant melanoma (SSCM). Offspring of crosses between melanoma-bearing Sinclair swine homozygous for the B haplotype and non-melanoma NIH aa swine have tumour incidence identical to Sinclair melanoma BB× Sinclair non-melanoma BB offspring. Our results provide further support for the involvement of the swine leucocyte antigen (SLA) complex in the inheritance of SSCM, and identify a new source of non-tumour animals that have all of the genes for SSCM except those at the tumour-initiator locus.     

Enterobacter cowaniisp. nov., a New Species of the Family Enterobacteriaceae

The name Enterobacter cowanii sp. nov. is proposed for a group of organisms referred to as NIH Group 42. Members of this species are Gram-negative, motile rods conforming to the definition of the family Enterobacteriaceae. The DNA relatedness of nine strains of NIH Group 42 to the proposed type strain of this species averaged 85% at 70°C, whereas the relatedness to other species within the family Enterobacteriaceae was less than 38%. Because the DNA relatedness (5–38%) is closer to species of the genus Enterobacter than to other species of the family, the members of NIH Group 42 were placed in the genus Enterobacter. The majority of strains of E. cowanii were isolated from clinical specimens. A culture of the type strain (888-76) has been deposited in the Japan Collection of Microorganisms as JCM 10956. Received: 22 June 2000 / Accepted: 26 June 2000     

Transformation of NIH/3T3 to Anchorage Independence by H-Ras Is Accompanied by Loss of Suppressor Activity

Despite their familiar sensitivity to transformation by dominant-acting ras oncogenes, NIH/3T3 cells carry a ras suppressor. When tested by cell fusion they were able to suppress the anchorage-independent phenotype of both mouse and human cells transformed by activated H-ras or N-ras. This suppression occurred without a decrease in expression of the activated ras oncogene. Ras-transformed NIH/3T3 clones cured of their oncogene by benzamide treatment reverted to a non-transformed phenotype, but had lost the ability to suppress other ras transformants, indicating that their initial transformation was accompanied by suppressor loss. In hamster cells an active ras oncogene increased the rate of chromosome segregation by >100-fold. These results suggest that in vitro transformation of NIH/3T3 cells by ras may be more similar to multistep in vivo tumor development than previously suspected, involving not only expression of an active oncogene but also loss of a suppressor activity, perhaps induced by the clastogenic oncogene.     

Transcriptional analysis of the titin cap gene

Mutations in titin cap (Tcap), also known as telethonin, cause limb-girdle muscular dystrophy type 2G (LGMD2G). Tcap is one of the titin interacting Z-disc proteins involved in the regulation and development of normal sarcomeric structure. Given the essential role of Tcap in establishing and maintaining normal skeletal muscle architecture, we were interested in determining the regulatory elements required for expression of this gene in myoblasts. We have defined a highly conserved 421 bp promoter proximal promoter fragment that contains two E boxes and multiple putative Mef2 binding sequences. This promoter can be activated by MyoD and myogenin in NIH3T3 fibroblast cells, and maintains the differentiated cell-specific expression pattern of the endogenous Tcap in C2C12 cells. We find that while both E boxes are required for full activation by MyoD or myogenin in NIH3T3 cells, the promoter proximal E box has a greater contribution to activation of this promoter in C2C12 cells and to activation by MyoD in NIH3T3 cells. Together, the data suggest an important role for MyoD in activating Tcap expression through the promoter proximal E box. We also show that myogenin is required for normal expression in vivo and physically binds to the Tcap promoter during embryogenesis.     

NIH ImageJ and Slice-O-Matic computed tomography imaging software to quantify soft tissue

Objective: To compare reliability and limits of agreement of soft tissue cross‐sectional areas obtained using Slice‐O‐Matic and NIH ImageJ medical imaging software packages. Research Methods and Procedures: Abdominal and midthigh images were obtained using single‐slice computed tomography. Two trained investigators analyzed each computed tomography image in duplicate. Adipose tissue and skeletal muscle cross‐sectional areas (centimeters squared) were calculated using standard Hounsfield unit ranges (adipose tissue: ?190 to ?30 and skeletal muscle: ?29 to 150). Regions of interest included abdominal total area, total fat area, subcutaneous fat area, visceral fat area (AVF), and right and left thigh total area, fat area, and skeletal muscle area. Results: For all images, intra‐investigator coefficients of variation ranged from 0.2% to 3.4% and from 0.4% to 5.6% and inter‐investigator coefficients of variation ranged from 0.9% to 4.8% and 0.2% to 2.6% for Slice‐O‐Matic and NIH ImageJ, respectively, with intra‐ and inter‐investigator coefficients of reliability of R² = 0.99. Mean AVF values for investigators A and B ranged from 168 to 170 cm² using Slice‐O‐Matic and NIH ImageJ. Bland‐Altman analyses revealed that Slice‐O‐Matic and NIH ImageJ results were comparable. The mean differences (95% confidence intervals) between the AVF cross‐sectional areas obtained using the Slice‐O‐Matic and NIH ImageJ medical imaging software were +2.5 cm² (?5.7, +10.8 cm²) or +1.4% (?3.4%, +6.4%). Discussion: These findings show that both the Slice‐O‐Matic and NIH ImageJ medical imaging software systems provide reliable measurements of adipose tissue and skeletal muscle cross‐sectional areas.     

Effect of the pSV2-neo Plasmid on NIH 3T3 Cell Motion Detected Electrically

Advantage was taken of DNA transfection techniques to investigate the effect of the pSV2-neo plasmid and its derivatives on recipient NIH 3T3 cell motion. Cell spreading and motion were followed by a newly developed electrical method to monitor cell morphology, referred to as electric cell-substrate impedance sensing. Using this method, we found that the eukaryotic--prokaryotic shuttle vector pSV2-neo had a strong effect on the recipient NIH 3T3 cell spreading and cell motion. However, two new neo plasmids, pSK-neo and pSP-neo, which were constructed by modifying the pSV2-neo plasmid, did not have a significant effect on the recipient cell activities. The results suggest that there may be some sequences in pSV2-neo which affect recipient cell behavior.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Defective autophagy in multidrug resistant cells may lead to growth inhibition by BH3‐mimetic gossypol

The clinical efficacy of many chemotherapeutic agents has been reduced due to the development of drug resistance. In this article, we aimed to validate gossypol, a natural BH3 mimetic found in cottonseeds, as a potential therapeutic to overcome multidrug resistance (MDR). Gossypol was found to retain its efficacy in v‐Ha‐ras‐transformed NIH 3T3 cells that overexpressed P‐glycoprotein (Ras‐NIH 3T3/Mdr), which was similar to the efficacy observed in their parental counterparts (Ras‐NIH 3T3). A rhodamine assay revealed that the alteration of MDR activity did not contribute to the cytotoxic effect of gossypol. Gossypol caused a G₂/M arrest by the induction of p21^Cip1 and the down‐regulation of p27^Kip1 expression in Ras‐NIH 3T3 cells, whereas no significant G₂/M arrest was exhibited in Ras‐NIH 3T3/Mdr cells. Surprisingly, a 48‐h treatment with gossypol induced apoptotic cell death in Ras‐NIH 3T3 cells; however, gossypol induced both apoptosis and necrosis in Ras‐NIH 3T3/Mdr cells, as determined with flow cytometry analysis. More notably, gossypol preferentially induced autophagy in Ras‐NIH 3T3 cells but not in Ras‐NIH 3T3/Mdr cells. Coimmunoprecipitation and flow cytometric analysis revealed that gossypol‐induced autophagy is independent of the dissociation of Beclin 1 from Bcl‐2 in Ras‐NIH 3T3 cells. Taken together, these results suggest that the antiproliferative activity of gossypol appears to be due to cell‐cycle arrest at the G₂/M phase, with the induction of apoptosis in Ras‐NIH 3T3 cells. In addition, defective autophagy might contribute to apoptotic and necrotic cell death in response to gossypol in Ras‐NIH 3T3/Mdr cells. J. Cell. Physiol. 228: 1496–1505, 2013. © 2012 Wiley Periodicals, Inc.     

Cyclin T1 overexpression induces malignant transformation and tumor growth

Human PTEFb is a protein kinase composed by CDK9 and Cyclin T that controls the elongation phase of RNA Pol II. This complex also affects the activation and differentiation program of lymphoid cells. In this study we found that several head and neck tumor cell lines overexpress PTEFb. We also established that Cyclin T1 is able to induce transformation in vitro, as we determined by foci and colony formation assays. Nu/nu mice s.c. injected with stable transfected Cyclin T1 cells (NIH 3T3 Cyclin T1) developed tumors faster than animals injected with control cells (NIH 3T3 b-gal). In vitro, NIH 3T3 Cyclin T1 cells show increased proliferation and CDK4-Rb phosphorylation. Even more, silencing E2F1 expression (shRNA E2F1) in NIH 3T3 cells resulted in a dramatic inhibition of Cyclin T1-induced foci. All these data demonstrate for the first time the Cyclin T1 oncogenic function and suggest a role for this protein in controlling cell cycle probably via Rb/E2F1 pathway.     

CD36 mediates binding of soluble thrombospondin-1 but not cell adhesion and haptotaxis on immobilized thrombospondin-1

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [¹²⁵I]-TSP1 and [¹²⁵I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats,^18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd.     

Tutorial on large deviations for the binomial distribution

We present, in an easy to use form, the large deviation theory of the binomial distribution: how to approximate the probability ofk or more successes inn independent trials, each with success probabilityp, when the specified fraction of successes,a≡k/n, satisfies 0<p<a<1. Supported by NIH grant GM 36230 and NSF grant DMS 8601986. Supported by NIH grant GM 36230 and a grant from the System Development Foundation.     

RNA Interference-Mediated Inhibition of Wild-Type Torsin A Expression Increases Apoptosis Caused by Oxidative Stress in Cultured Cells

To assess RNAi mediated inhibition of the expression of wt-DYT1 on H₂O₂-induced toxicity in NIH 3T3 cells and primary cortical neurons. To detect the function of wild-type Torsin A and the effect of SiRNA on the wt-DYT1 gene. The shRNA expression vector was constructed by ligating annealed complementary shRNA oligonucleotides into the down-stream of the human U6 promoter (PU6) of the RNAi-ready pSIREN-Shuttle vector. Then, the pSIREN-Shuttle-DYT1-shRNA cassette was ligated to Adeno-X Viral DNA to construct the recombinant adenoviral vector pAd-DYT1-shRNA. Cultured cerebral cortical neurons and NIH 3T3 cells were transfected with pAd-DYT1-shRNA and pSIREN-Shuttle-DYT1-shRNA. We evaluated NIH 3T3 cells and neurons in the presence of oxidative stress using a TUNEL assay under different conditions. The knockdown efficacy of the DYT1 was confirmed by real-time RT-PCR and Western Blot analysis. After exposure to H₂O_2, the quantity of NIH 3T3 cells transfected with pSIREN-Shuttle-DYT1-shRNA, which stained positively in the TUNEL assay, was significantly higher than the cells transfected with pSIREN-Shuttle-negative control-shRNA. (44.85 ± 1.81% vs. 8.98 ± 2.73%, t = 26.168). There were significantly more apoptotic neurons infected with pAd-DYT1-shRNA (45.63 ± 7.53%) than neurons infected with pAd-X-negative control-shRNA (17.33 ± 2.43%) (t = 9.816). The observed silencing of wild-type Torsin A expression by DYT1-shRNA was sequence-specific. RNAi-mediated inhibition of the expression of wild-type Torsin A increases apoptosis caused by oxidative stress. It is reasonable to consider that wild-type Torsin A has the capacity to protect cortical neurons against oxidative stress, and in the development of DYT1-delta GAG-dystonia the neuroprotective function of wild-type Torsin A may be compromised.