Multidrug-resistant tuberculosis in children: A practical update on epidemiology, diagnosis, treatment and prevention.

Pediatric multidrug-resistant tuberculosis (MDR-TB) remains a significant global problem, and there are numerous barriers preventing children with MDR-TB from being identified, confirmed with microbiologic tests, and treated with a safe, practical, and effective regimen. However, several recent advances in diagnostics and treatment regimens have the promise to improve outcomes for children with MDR-TB. We introduce this review with two cases that exemplify both the challenges in management of MDR-TB in children, but also the potential to achieve a positive outcome. More than 30,000 cases of MDR-TB per year are believed to occur in children but less than 5% are confirmed microbiologically, contributing to poorer outcomes and excess mortality. Rapid molecular-based testing that provides information on rifampin susceptibility is increasingly globally available and recommended for all children suspected of TB disease--but remains limited by challenges obtaining appropriate samples and the paucibacillary nature of most pediatric TB. More complex assays allowing better characterization of drug-resistant isolates are emerging. For children diagnosed with MDR-TB, treatment regimens have traditionally been long and utilize multiple drugs associated with significant side effects, particularly injectable agents. Several new or repurposed drugs including bedaquiline, delamanid, clofazimine and linezolid now allow most treatment regimens to be shorter and all-oral. Yet data to support short, all-oral, novel regimens for young children containing pretomanid remain insufficient at present, and there is a compelling need to conduct pediatric trials of promising therapeutics and MDR-TB treatment regimens.

SARS-CoV-2 antibody prevalence by industry, workplace characteristics, and workplace infection prevention and control measures, North Carolina, 2021 to 2022.

Background: The COVID-19 pandemic has disproportionately affected workers in certain industries and occupations, and the workplace can be a high risk setting for SARS-CoV-2 transmission. In this study, we measured SARS-CoV-2 antibody prevalence and identified work-related risk factors in a population primarily working at industrial livestock operations. Methods: We used a multiplex salivary SARS-CoV-2 IgG antibody assay to determine infection-induced antibody prevalence among 236 adult (≥18 years) North Carolina residents between February 2021 and August 2022. We used the National Institute for Occupational Safety and Health Industry and Occupation Computerized Coding System (NIOCCS) to classify employed participants' industry and compared infection-induced IgG prevalence by participant industry and with the North Carolina general population. We also combined antibody results with reported SARS-CoV-2 molecular test positivity and vaccination history to identify evidence of prior infection. We used logistic regression to estimate odds ratios of prior infection by potential work-related risk factors, adjusting for industry and date. Results: Most participants (55%) were infection-induced IgG positive, including 71% of animal slaughtering and processing industry workers, which is 1.5 to 4.3 times higher compared to the North Carolina general population, as well as higher than molecularly-confirmed cases and the only other serology study we identified of animal slaughtering and processing workers. Considering questionnaire results in addition to antibodies, the proportion of participants with evidence of prior infection increased slightly, to 61%, including 75% of animal slaughtering and processing workers. Participants with more than 1000 compared to 10 or fewer coworkers at their jobsite had higher odds of prior infection (adjusted odds ratio [aOR] 4.5, 95% confidence interval [CI] 1.0 to 21.0). Conclusions: This study contributes evidence of the severe and disproportionate impacts of COVID-19 on animal processing and essential workers and workers in large congregate settings. We also demonstrate the utility of combining non-invasive biomarker and questionnaire data for the study of workplace exposures.

SARS-CoV-2 Antibody Prevalence among Industrial Livestock Operation Workers and Nearby Community Residents, North Carolina, 2021 to 2022.

Industrial livestock operations (ILOs), particularly processing facilities, emerged as centers of coronavirus disease 2019 (COVID-19) outbreaks in spring 2020. Confirmed cases of COVID-19 underestimate true prevalence. To investigate the prevalence of antibodies against SARS-CoV-2, we enrolled 279 participants in North Carolina from February 2021 to July 2022: 90 from households with at least one ILO worker (ILO), 97 from high-ILO intensity areas (ILO neighbors [ILON]), and 92 from metropolitan areas (metro). More metro (55.4%) compared to ILO (51.6%) and ILON participants (48.4%) completed the COVID-19 primary vaccination series; the median completion date was more than 4 months later for ILO compared to ILON and metro participants, although neither difference was statistically significant. Participants provided a saliva swab we analyzed for SARS-CoV-2 IgG using a multiplex immunoassay. The prevalence of infection-induced IgG (positive for nucleocapsid and receptor binding domain) was higher among ILO (63%) than ILON (42.9%) and metro (48.7%) participants (prevalence ratio [PR], 1.38; 95% confidence interval [CI], 1.06 to 1.80; reference category ILON and metro combined). The prevalence of infection-induced IgG was also higher among ILO participants than among an Atlanta health care worker cohort (PR, 2.45; 95% CI, 1.80 to 3.33) and a general population cohort in North Carolina (PRs, 6.37 to 10.67). The infection-induced IgG prevalence increased over the study period. Participants reporting not masking in public in the past 2 weeks had higher infection-induced IgG prevalence (78.6%) than participants reporting masking (49.3%) (PR, 1.59; 95% CI, 1.19 to 2.13). Lower education, more people per bedroom, Hispanic/Latino ethnicity, and more contact with people outside the home were also associated with higher infection-induced IgG prevalence. IMPORTANCE Few studies have measured COVID-19 seroprevalence in North Carolina, especially among rural, Black, and Hispanic/Latino communities that have been heavily affected. Antibody results show high rates of COVID-19 among industrial livestock operation workers and their household members. Antibody results add to evidence of health disparities related to COVID-19 by socioeconomic status and ethnicity. Associations between masking and physical distancing with antibody results also add to evidence of the effectiveness of these prevention strategies. Delays in the timing of receipt of COVID-19 vaccination reinforce the importance of dismantling vaccination barriers, especially for industrial livestock operation workers and their household members.

Microbial diversity and nitrogen-metabolizing gene abundance in backyard food waste composting systems.

AIMS: The microbial diversity of backyard compost piles is poorly understood compared to large-scale, highly regulated composting systems. The purpose of this study was the identification of the microbial community composition and associated change over time among three different backyard composting styles. METHODS AND RESULTS: Food waste was composted in a household backyard compost bin, a small-scale aerated windrow or a semi-aerated static pile. Samples were obtained from each sequential phase of the composting process for 16s rRNA sequencing and relationships between temperature, moisture and microbial communities were examined. The Bacilli dominated in the early phases of composting then transitioned to Proteobacteria in the later stages. Different bacterial species increased and decreased dramatically in different composting systems and at different phases of the composting process. We performed qPCR to quantify gene abundance of nirS to profile the nitrogen-metabolizing bacteria present in each composting system. Gene abundance of nirS varied with temperature, but peaked during the cooling phase in the aerated windrow. CONCLUSIONS: Although the phases of decomposition were not as distinct as large-scale regulated piles, the microbial diversity mirrored the appropriate phases. Interestingly, different backyard composting styles were marked by the predominance of certain bacterial species. In particular, nitrogen-metabolizing bacterial communities peaked in the later stages of decomposition. SIGNIFICANCE AND IMPACT OF THE STUDY: A profile of the compost microbiome yields important clues about how differences in backyard food waste composting systems influence bacterial species that may facilitate or hinder nitrogen metabolism.

Drivers of professional mobility in the Northern Territory: dental professionals.

INTRODUCTION: Attracting and retaining an efficient allied health workforce is a challenge faced by communities in Australia and overseas. High rates of staff turnover in the professional workforce diverts resources away from core business and results in the loss of valuable skills and knowledge. Understanding what attracts professionals to a particular place, and why they leave, is important for developing effective strategies to manage turnover and maximise workforce productivity. The Northern Territory (NT) faces particular workforce challenges, in part because of its geographic location and unusual demography. Do these factors require the development of a tailored approach to recruitment and retention? This article reports on a study undertaken to examine the motivations for coming to, staying in and leaving the NT for dental professionals, and the implications of results on workforce management practices. METHODS: In 2006, dentists, dental specialists, dental therapists and dental hygienists who were working or had worked in the NT, Australia, in the recent past were surveyed to collect demographic and workforce data and to establish the relative importance of social and work-related factors influencing their migration decisions. Multivariate logistic regression models were generated to describe the demographic characteristics of dental professionals who stayed in the NT for more than 5 years and to analyse why dental professionals left. The analyses, based on a 42% response rate, explained 60-80% of the variation in responses. RESULTS: Generally dental professionals who had stayed for more than 5 years were older, had invested in the purchase of homes and were more involved in social and cultural activities. Those who moved to the NT as a result of financial incentives or who had strong expectations that working in the NT would be an exciting, novel experience tended to stay for no more than 5 years, often leaving because they found the work environment too stressful. In contrast, those who stayed longer came because they had existing social networks and were familiar with the NT environment, staying primarily because they have enjoyed the NT lifestyle, particularly the sense of community and the opportunities available through living in smaller centres. CONCLUSION: There are benefits in actively engaging newly recruited professionals and their families in social networks. Work related stress and departure was associated with administrative deficiencies within the management system. Despite the NT's unusual demographic profile, the factors influencing recruitment and retention are not markedly different from those reported elsewhere.

A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells.

The utilization of adult stem cells in tissue engineering is a promising solution to the problem of tissue or organ shortage. Adult bone marrow derived mesenchymal stem cells (MSCs) are undifferentiated, multipotential cells which are capable of giving rise to chondrocytes when maintained in a three-dimensional culture and treated with members of the transforming growth factor-beta (TGF-beta) family of growth factors. In this study, we fabricated a nanofibrous scaffold (NFS) made of a synthetic biodegradable polymer, poly(-caprolactone) (PCL), and examined its ability to support in vitro chondrogenesis of MSCs. The electrospun PCL porous scaffold was constructed of uniform, randomly oriented nanofibers with a diameter of 700 nm, and structural integrity of this scaffold was maintained over a 21-day culture period. MSCs cultured in NFSs in the presence of TGF-beta1 differentiated to a chondrocytic phenotype, as evidenced by chondrocyte-specific gene expression and synthesis of cartilage-associated extracellular matrix (ECM) proteins. The level of chondrogenesis observed in MSCs seeded within NFSs was comparable to that observed for MSCs maintained as cell aggregates or pellets, a widely used culture protocol for studying chondrogenesis of MSCs in vitro. Due to the physical nature and improved mechanical properties of NFSs, particularly in comparison to cell pellets, the findings reported here suggest that the PCL NFS is a practical carrier for MSC transplantation, and represents a candidate scaffold for cell-based tissue engineering approaches to cartilage repair.

Filgrastim support during combination chemotherapy using cisplatin, doxorubicin, and cyclophosphamide to treat advanced or recurrent endometrial cancer: a clinical study and literature review.

In a private practice setting, 16 patients with advanced or recurrent endometrial carcinoma received cisplatinum 50 mg/m2, doxorubicin 50 mg/m2, and cyclophosphamide 750 mg/m2 every three weeks. Growth factor support using filgrastim was initiated on the first cycle of therapy and each subsequent cycle. Sixteen patients were entered into the study with 13 being evaluable. No patient had previously received chemotherapy. The overall response rate was 54% with two complete responses (15%) and five partial responses (38%). Stable disease was seen in 46% of patients. Progression-free survival was observed to be a median of 8.5 months for a complete response, a median of 8.5 months for a partial response and a median of 7 months for stable disease. Fifteen percent of the patients and 3% of all chemotherapy cycles had febrile neutropenic events. There were no deaths due to myelotoxicity. Only one patient required a dose reduction due to neutropenia. Four of the 13 patients required dose reductions due to previous nadir thrombocytopenia. Grade 4 granulocytopenia occurred in 28% of treatment cycles and grade 3 granulocytopenia occurred in 12% of treatment cycles. The use of filgrastim (G-CSF) allowed patients to stay on therapy for an average of seven treatments. Neutropenia was not the dose-limiting toxicity from this dose-intense regimen.

Estrogenic endocrine disruptive components interfere with calcium handling and differentiation of human trophoblast cells.

During development, calcium (Ca) is actively transported by placental trophoblasts to meet fetal nutritional and the skeletal mineralization needs. Maternal exposure to estrogenic pesticides, such as 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and methoxychlor (MTC), has been shown to result in reproductive disorders and/or abnormal fetal development. In this study, we have examined the effects of exposure of trophoblastic cells to MTC and DTT, in comparison to 17beta-estradiol (E2) and diethylstilbestrol (DES), to test the hypothesis that cellular Ca handling is a target for these endocrine disruptive components. Treatment with DDT, MTC, DES, or E2 increased cellular Ca uptake, and the expression of trophoblast-specific human Ca binding protein (HCaBP) was down-regulated by both MTC and DDT. Treatment with MTC, DDT, and DES inhibited cell proliferation, induced apoptosis, and suppressed expression of several trophoblast differentiation marker genes. These effects were reversed by overexpression of metallothionein IIa, a gene highly responsive to cadmium and other metals. These results strongly suggest that trophoblast Ca handling functions are endocrinally modulated, and that their alteration by candidate endocrine disruptors, such as MTC and DDT, constitutes a possible pathway of the harmful effects of these components on fetal development.

Effect of cartilage oligomeric matrix protein on mesenchymal chondrogenesis in vitro.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) mutations have been identified as responsible for two arthritic disorders, multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). However, the function of COMP in chondrogenic differentiation is largely unknown. Our investigation focuses on analyzing the function of normal COMP protein in cartilage biology. METHODS AND RESULTS: To explore the function of COMP we make use of an in vitro model system for chondrogenesis, consisting of murine C3H10T1/2 mesenchymal cells maintained as a high-density micromass culture and stimulated with bone morphogenetic protein 2 (BMP-2). Under these culture conditions, C3H10T1/2 cells undergo active chondrogenesis in a manner analogous to that of embryonic limb mesenchymal cells, and have been shown to serve as a valid model system to investigate the mechanisms regulating mesenchymal chondrogenesis. Our results indicate that ectopic COMP expression enhances several early aspects of chondrogenesis induced by BMP-2 in this system, indicating that COMP functions in part to positively regulate chondrogenesis. Additionally, COMP has inhibitory effects on proliferation of cells in monolayer. However, at later times in micromass culture, ectopic COMP expression in the presence of BMP-2 causes an increase in apoptosis, with an accompanying reduction in cell numbers in the micromass culture. However, the remaining cells retain their chondrogenic phenotype. CONCLUSIONS: These data suggest that COMP and BMP-2 signaling converge to regulate the fate of these cells in vitro by affecting both early and late stages of chondrogenesis.

The murine COMP (cartilage oligomeric matrix protein) promoter contains a potent transcriptional repressor region.

OBJECTIVE: A subgroup of patients with pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) have been found to harbor mutations within the cartilage oligomeric matrix protein (COMP) gene. These two diseases are autosomal dominant disorders that are characterized by an early onset of osteoarthritis (OA). The COMP gene is expressed primarily in chondrocytes in articular cartilage as well as in tendon and ligament. Therefore, control over tissue specific COMP expression may be an important aspect in cartilage biology. To begin an analysis of the regulation of COMP expression, we have cloned, sequenced and characterized the entire genomic clone for mouse COMP that includes the COMP promoter. METHODS AND RESULTS: The COMP coding region spans 19 exons over approximately 8.4 kb of DNA. The arrangement and size of the exons have a remarkable similarity to those of the human COMP genomic sequence, indicating a significant degree of genomic conservation. Analysis of a 453 basepair region of the putative COMP promoter reveals two strong transcriptional repressor elements located between position -356 and -304 and between -251 and -180, relative to the start site for transcription. These repressor elements down-regulate transcription from the promoter in a broad spectrum of cell lines. Removal of the repressor DNA sequence from the COMP promoter leads to significant enhancement in transcriptional activity, indicating that this region acts in a dominant manner to transcriptional activators located more proximal to the start site of transcription. This region also represses transcription when linked to a heterologous promoter. CONCLUSIONS: This repressor region probably down-regulates transcription from the COMP promoter in vivo. It may help to repress transcription of COMP in non-cartilaginous tissues and/or may aid in the expression of COMP to the appropriate level in tissues such as cartilage, tendon and ligament.

Laminated multi-segment rib graft in anterior column reconstruction.

Rib harvested during thoracotomy can be effectively used for anterior column reconstruction. An innovative technique is described to convert multiple individual rib segments into a robust single anterior column reconstruction graft using cortical screws.

Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro.

OBJECTIVE: This study aims to apply gene expression profiling technology to gain insight into the molecular regulation of mesenchymal chondrogenesis. METHODS: The experimental system consists of micromass cultures of C3H10T1/2 cells, a murine multipotential embryonic cell line, treated with the chondroinductive growth factor, bone morphogenetic factor-2 (BMP-2). In this system, chondrogenic differentiation characterized by both morphological changes and cartilage matrix gene expression has been shown to be completely dependent upon BMP-2 treatment and the high cell plating density of micromass cultures. To identify candidate genes that may have key functional roles in chondrogenesis, we have applied subtractive hybridization to isolate genes whose expression is significantly up- or down-regulated during chondrogenesis. RNA was isolated from micromass cultures treated with BMP-2 for 24 h and analysed for representational differences by means of a subtractive hybridization screening method. RESULTS: Sixteen different genes were identified whose expression was up-regulated between two- and 12-fold by B,P-2, and twelve different genes were identified whose expression was down-regulated between two- and seven-fold by BMP-2. CONCLUSIONS: The potential of this screening methodology to identify new BMP-2 regulated genes is suggested by the fact that a majority of the identified genes are indeed novel. Identification and characterization of these genes should provide insight as to how chondrogenesis is regulated and also should provide important new markers for the study of osteoarthritis.

Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2.

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.

A dose-escalating study of weekly bolus topotecan in previously treated ovarian cancer patients.

OBJECTIVE: Topotecan is an established topoisomerase I inhibitor for the treatment of relapsed ovarian cancer. Myelotoxicity and suboptimal patient convenience associated with daily topotecan, however, have prompted investigators to explore alternate regimens, including a weekly regimen of topotecan. The objective of this study was to determine the maximum tolerated dose (MTD) of topotecan given as a weekly bolus in previously treated ovarian cancer patients. METHODS: Second- and third-line ovarian cancer patients with measurable disease or elevated cancer antigen 125 received weekly bolus topotecan intravenously starting at 1.5 mg/m(2). Topotecan was escalated in dose increments of 0.5 mg/m(2) every 21 days as tolerability allowed. Dose-limiting toxicity was defined as grade 3/4 neutropenia or thrombocytopenia. RESULTS: Thirty-two of 35 patients were evaluable for safety and tolerability. No notable toxicity was observed with weekly topotecan doses < 4 mg/m(2). Additionally, there was an absence of dose-limiting myelotoxicity and thrombocytopenia with weekly topotecan. The MTD of weekly topotecan without the use of granulocyte colony-stimulating factor support was 4 mg/m(2), with grade 2 anemia, chronic fatigue, and grade 2 gastrointestinal toxicity limiting further dose escalation. Weekly topotecan also demonstrated antitumor activity at doses >2 mg/m(2). CONCLUSIONS: The establishment of a well-tolerated, weekly regimen of topotecan (4 mg/m(2), with a maximum recommended dose of 6 mg/m(2)) provides the basis for further investigation in phase II studies of single-agent and combination regimens in previously treated ovarian cancer patients.

A transcriptionally inactive E2F-1 targets the MDM family of proteins for proteolytic degradation.

E2F-1-activated transcription promotes cell cycle progression and apoptosis. These functions are regulated by several factors including the E2F-1-binding protein MDM2 and the retinoblastoma protein pRb. Using a yeast two-hybrid screen we have identified the MDM2-related protein, MDMX, as an E2F-1-binding protein. In these studies we find that coexpression of MDMX with E2F-1 results in degradation of the MDMX protein. Although this proteolytic degradation can be blocked by the protease inhibitors bafilomycin A(1), N-acetyl-Leu-Leu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism. Using an E2F-1 deletion mutant (E2F-1(180-437)) we show that E2F-1-targeted degradation of MDMX does not require the E2F-1 DNA binding domain and therefore is independent of E2F-1-driven transcription. We also find that this transcriptionally inactive E2F-1 mutant is capable of degrading the MDMX-related protein MDM2 and the MDMX isoform MDMX-S. Mapping of the E2F-1 C terminus reveals that neither a previously characterized C-terminal MDM2 binding domain nor the pRb binding domain on E2F-1 is required for MDMX and MDM2 degradation.

Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability.

Inhibition of eosinophil apoptosis by exposure to interleukin-5 (IL-5) is associated with the development of tissue eosinophilia and may contribute to the inflammation characteristic of asthma. Analysis of the signaling events associated with this process has been hampered by the inability to efficiently manipulate eosinophils by the introduction of active or inhibitory effector molecules. Evidence is provided, using a dominant-negative N17 H-Ras protein (dn-H-Ras) and MEK inhibitor U0126, that activation of the Ras-Raf-MEK-ERK pathway plays a determining role in the prolongation of eosinophil survival by IL-5. For these studies, a small region of the human immunodeficiency virus Tat protein, a protein transduction domain known to enter mammalian cells efficiently, was fused to the N-terminus of dn-H-Ras. The Tat-dn-H-Ras protein generated from this construct transduced isolated human blood eosinophils at more than 95% efficiency. When Tat-dn-H-Ras-transduced eosinophils were treated with IL-5, they exhibited a time- and dosage-dependent reduction in extracellular regulated kinase 1 and 2 activation and an inhibition of p90 Rsk1 phosphorylation and IL-5-mediated eosinophil survival in vitro. In contrast, Tat-dn-H-Ras did not inhibit CD11b up-regulation or STAT5 tyrosine phosphorylation. These data demonstrate that Tat dominant-negative protein transduction can serve as an important and novel tool in studying primary myeloid cell signal transduction in primary leukocytes and can implicate the Ras-Raf-MEK-ERK pathway in IL-5-initiated eosinophil survival.

Leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation.

Encephalomyocarditis virus (EMCV) is the prototype member of the cardiovirus genus of picornaviruses. For cardioviruses and the related aphthoviruses, the first protein segment translated from the plus-strand RNA genome is the Leader protein. The aphthovirus Leader (173-201 amino acids) is an autocatalytic papain-like protease that cleaves translation factor eIF-4G to shut off cap-dependent host protein synthesis during infection. The less characterized cardioviral Leader is a shorter protein (67-76 amino acids) and does not contain recognizable proteolytic motifs. Instead, these Leaders have sequences consistent with N-terminal zinc-binding motifs, centrally located tyrosine kinase phosphorylation sites, and C-terminal, acid-rich domains. Deletion mutations, removing the zinc motif, the acid domain, or both domains, were engineered into EMCV cDNAs. In all cases, the mutations gave rise to viable viruses, but the plaque phenotypes in HeLa cells were significantly smaller than for wild-type virus. RNA transcripts containing the Leader deletions had reduced capacity to direct protein synthesis in cell-free extracts and the products with deletions in the acid-rich domains were less effective substrates at the L/P1 site, for viral proteinase 3Cpro. Recombinant EMCV Leader (rL) was expressed in bacteria and purified to homogeneity. This protein bound zinc stoichiometrically, whereas protein with a deletion in the zinc motif was inactive. Polyclonal mouse sera, raised against rL, immunoprecipitated Leader-containing precursors from infected HeLa cell extracts, but did not detect significant pools of the mature Leader. However, additional reactions with antiphosphotyrosine antibodies show that the mature Leader, but not its precursors, is phosphorylated during viral infection. The data suggest the natural Leader may play a role in regulation of viral genome translation, perhaps through a triggering phosphorylation event.

The ZF87/MAZ transcription factor functions as a growth suppressor in fibroblasts.

ZF87/MAZ is a zinc finger transcription factor that activates expression of tissue-specific genes and represses expression of the c-myc proto-oncogene. Infection of NIH3T3 fibroblasts with a retrovirus expressing ZF87/MAZ leads to a significant reduction in G418-resistant colonies, compared to cells infected with a retroviral control. Further, only a small fraction of the G418-resistant colonies express ZF87/MAZ. When the ZF87/MAZ-expressing colonies are expanded, they demonstrate a slow growth phenotype, a delayed transit through G1 phase and a decrease in endogenous c-myc gene expression and cyclin A and cyclin E protein levels. Consistent with a partial G1 arrest, the ZF87/MAZ-expressing cells show a reduced sensitivity to the S phase specific chemotherapeutic agent camptothecin. These data indicate that ZF87/MAZ is a growth suppressor protein in nontransformed cells, in part, by affecting the levels of key cell cycle regulatory proteins.

Molecular cloning and expression patterns of mouse cartilage oligomeric matrix protein gene.

OBJECTIVE: To develop transgenic mice harboring mutations in the COMP gene as animal models for pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), autosomal dominant disorders characterized by early onset osteoarthritis and epiphyseal abnormalities. As a first step in generating a mouse model for COMP mutations, we have cloned the cDNA of mouse COMP and examined its tissue expression pattern. DESIGN: Total mRNA was isolated from the skeletal tissues of newborn C57BL/6j mice and used as a template for oligo(dT) first-strand cDNA synthesis. The cDNA was used for PCR amplification of COMP using three oligonucleotide primer pairs designed from the published rat COMP cDNA sequence. Nested PCR was used to complete the sequence between the amplified fragments. The entire cDNA was sequenced and the expression pattern of the corresponding transcripts examined by Northern hybridizations. RESULTS: A full-length COMP cDNA was isolated. Analysis showed that the entire translated region of the mouse COMP gene is 2268 bp and the derived amino acid sequence shows 90% homology to human COMP. Of eight adult mouse non-cartilage tissues tested, COMP expression was detected only in testis.

Regulation of scleraxis function by interaction with the bHLH protein E47.

Scleraxis is a basic helix-loop-helix (bHLH) protein whose function has been postulated to be preconfigurative of sclerotomal mesenchymal patterning during early embryonic development by regulating expression of differentiation-specific genes, particularly those involved in chondrogenesis. To gain understanding of the molecular action of scleraxis we test the hypothesis that it heterodimerizes with another bHLH protein to activate gene expression. Transient coexpression of scleraxis and E47, a candidate bHLH protein, showed that scleraxis dimerizes with E47 in vivo and that this complex binds to a classic E-box DNA sequence better than either factor alone. Further, when expressed together, scleraxis and E47 synergistically enhanced transcription from a promoter containing multiple E-box binding sites.