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Biomedical subjects

H L Smits

Publications and source records attributed to H L Smits.

At least 19 recordsLinked to original sources
PubMed1992

The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11.

Previous results indicated that SV40 small t is essential for SV40-induced transformation of diploid cells but dispensable for the transformation of cells with a deletion on the short arm of chromosome 11 (del-11 cells). From these results we concluded that del-11 cells contain a cellular 'SV40 small t-like' factor, which is able to transactivate the HPV16 long control region (LCR) and to complement SV40 large T in transformation. Since SV40 small t and the regulatory 55 kDa subunit (PR55) of protein phosphatase 2A (PP2A), have been shown to inhibit the enzyme activity of PP2A, the PR55 beta subunit could be the putative 'small t-like' factor. In accordance with this hypothesis, we show that the PR55 beta subunit is highly expressed in del-11 but not in diploid cells and is able to trans-activate the HPV16 LCR in diploid cells. Moreover, inhibition of PP2A by okadaic acid resulted in trans-activation of the HPV16 LCR in diploid cells. Alignment of PR55 and SV40 small t showed a common four amino acid motif DKGG. We present evidence that the integrity of this motif is necessary for the PP2A-mediated ability of SV40 small t to trans-activate the HPV16 LCR.

Amino Acid Sequence↗
PubMed1992

Detection of human papillomavirus types by the polymerase chain reaction and the differentiation between high-risk and low-risk cervical lesions.

By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a statistically highly significant rise in the prevalence of the oncogenic HPV types (16, 18, 31, 33) with increasing severity of cervical intraepithelial neoplasia (CIN I to III), indicating a role for these HPV types in the pathogenesis of cervical cancer. The prevalence of other HPV types decreased significantly with the severity of the lesion, suggesting that these HPV types play a less significant role in this process. These data indicate that HPV typing with PCR may be a valuable tool for distinguishing between high-risk and low-risk cervical lesions. Furthermore, our results suggest that the detection of HPV types by consensus PCR in the cervix of patients with an abnormal smear but without histologically detectable CIN is a useful tool for predicting which of these patients will eventually develop CIN. Finally, a relatively low percentage (3%) of HPV double infections is reported in this study.

Biopsy↗
PubMed1992

Modulation of the human papillomavirus type 16 induced transformation and transcription by deletion of loci on the short arm of human chromosome 11 can be mimicked by SV40 small t.

The human papillomavirus (HPV) type 16 enhancer-promoter has been shown to be active in human fibroblasts with a deletion on the short arm of one chromosome 11 (karyotype 46,del(11)(p11.11p15.1)) but is virtually inactive in diploid human fibroblasts (Smits, Smits, Jebbink, and ter Schegget, 1990b, Virology, 176, 158-165). In diploid human embryonic fibroblasts, activation of the HPV16 enhancer-promoter could be achieved by expression of the SV40 small t. By cotransfecting SV40 small t cDNA together with HPV16 DNA into diploid cells, it was possible to increase the transforming activity of HPV16 by 10- 15-fold. Furthermore, SV40 small t was essential for the SV40 large T-induced morphological transformation of human diploid fibroblasts, whereas SV40 small t was dispensable for transformation of del-11 cells. We propose that, as a result of the deletion of loci on the short arm of chromosome 11 in del-11 cells, functions are expressed that mimic those of SV40 small t in transformation and trans-activation.

Antigens, Polyomavirus Transforming↗
PubMed1992

Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types.

DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.

Base Sequence↗
PubMed1991

Human papillomavirus type 16 transcripts expressed from viral-cellular junctions and full-length viral copies in CaSki cells and in a cervical carcinoma.

We have mapped using the RNA PCR the viral-cellular junctions of HPV16 viral-cellular cotranscripts expressed in CaSki cells and a cervical carcinoma to nt 3728 and 881, respectively. Both junctions were located within the E1-E2 region. Examination of the cellular sequences of the cotranscripts showed the presence of a polyadenylation signal in each of the transcripts. In CaSki cells and in the cervical carcinoma transcripts derived from the full-length early region including the E2 transcript were also detected. Our results suggest that the utilization of a cellular polyadenylation site could be important in the development of cancer by HPV.

Base Sequence↗
PubMed1990

The short arm of chromosome 11 likely is involved in the regulation of the human papillomavirus type 16 early enhancer-promoter and in the suppression of the transforming activity of the viral DNA.

Loci on the short arm of chromosome 11 between 11p11 and 11p15 likely are involved directly or indirectly in the regulation of the HPV-16 enhancer-promoter strength and this may contribute to the control of the level of viral early gene expression. Transient expression assays have shown that the HPV-16 enhancer-promoter functions much stronger in fibroblasts in which this region of chromosome 11 (del-11 cells) is deleted than in normal diploid human embryonic fibroblasts. The differential regulation of the HPV-16 long control region may be due either to the presence or activity of a cellular transcription factor which up-regulates HPV-16 early gene expression in del-11 cells or to a factor which down-regulates expression in diploid cells. Since the HPV-16 enhancer containing fragment (nt 7003 to nt 57) in combination with the SV40 promoter functions equally well in del-11 cells as in diploid cells a target of this factor likely is located in the putative HPV-16 early promoter region. The relative resistance of diploid human embryonic fibroblasts to HPV-16 induced transformation could be explained by the inactivity of the HPV-16 early promoter as these cells could be transformed by HPV-16 DNA constructs in which the early gene expression was driven from a heterologous enhancer-promoter. These results may indicate that loci on the short arm could suppress HPV-16-induced transformation by down-regulating the activity of the viral early promoter.

Cells, Cultured↗
PubMed1990

Uniformity of the splicing pattern of the E6/E7 transcripts in human papillomavirus type 16-transformed human fibroblasts, human cervical premalignant lesions and carcinomas.

We utilized the RNA polymerase chain reaction (PCR) to analyse the transcripts of the E6/E7 open reading frames of human papillomavirus type 16 (HPV-16). Total RNA was isolated from 14 cervical squamous carcinomas, nine cervical intraepithelial neoplasias and from human fibroblasts transformed with different HPV-16 constructs. In all specimens two spliced transcripts were detected. Sequence analysis of the cloned PCR products showed that both transcripts were generated by splicing out an intron in E6, from nucleotides (nt) 226 to 409 in one transcript and from nt 226 to 526 in the other. The major transcript present in all RNA specimens had the smallest intron in E6. The RNA PCR described here is the method of choice for analysing splice and donor sites in tissue specimens where a limited amount of RNA is available. Results obtained with transformed cells revealed no difference in splicing whether HPV-16 was controlled by its homologous promoter or by a heterologous promoter, the Rous sarcoma virus long terminal repeat.

Base Sequence↗
PubMed1989

Keratin 13 expression is linked to squamous differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid.

Rabbit tracheal epithelial (RbTE) cells in primary culture undergo at confluence a multistep program of squamous differentiation. This study examines the expression of keratins in RbTE cells in relation to this differentiation process. During the exponential growth phase RbTE cells are undifferentiated and express three major keratins, K5, K14, and K19, and two minor keratins, K6 and K16. Squamous differentiation is accompanied by increased expression of keratins K6, K16, and K19, and in particular of keratin K13, which reacts specifically with the monoclonal antibody AE8. These changes in keratin synthesis coincide with the commitment to terminal differentiation. Retinoic acid, an inhibitor of the expression of the squamous differentiated phenotype, inhibits the increase in the expression of K6, K16, and K13 and reduces the expression of K5 and K14; however, retinoic acid treatment results in increased levels of keratin K19 and K18. Retinoic acid inhibits the expression of K16 and K13 at concentrations as low as 10(-9)-10(-10) M. At least some of these changes in keratins appear to be related to alterations in the cellular levels of the respective mRNAs. Our results indicate that specific changes in keratin expression, in particular keratin K13, correlate with the onset of squamous differentiation in RbTE cells. Induction of the expression of keratin K13 may function as a marker of squamous differentiation in tracheobronchial epithelial cells.

Animals↗
PubMed1989

Squamous differentiation in normal and transformed rat tracheal epithelial cells.

Morphological observations suggest that rate tracheal epithelial (RTE) cells undergo squamous differentiation when maintained in cell culture. The purpose of the studies presented here was to examine and define differentiation of cultured RTE cells with the help of markers previously shown to be specific for squamous differentiation. Furthermore, we wanted to determine whether neoplastic transformation of these cells causes significant disruption of their differentiation program. Our experiments showed that squamous differentiation occurs in normal primary RTE cell cultures. Epidermal transglutaminase (transglutaminase type I) activity increased approximately 20-fold in RTE cultures as a function of time. Cholesterol sulfate, another marker of squamous differentiation, increased only modestly with time. A significant number of cells formed cross-linked envelopes in cultures growth-arrested at a cell density of approximately 250 cells/mm2. However, no significant changes in keratin expression were detected. Neoplastically transformed RTE cells which exhibit a greatly increased growth capacity expressed the same three markers of squamous differentiation as normal RTE cells. However, transglutaminase type I activity was relatively low. The cross-linked envelope formation was independent of cell density in the transformed cells. Like in normal RTE cultures, cholesterol sulfate accumulation only increased moderately with increasing cell density. The keratin pattern of transformed RTE cell lines was identical to that of normal primary RTE cells. A well-differentiated squamous cell carcinoma derived from one of the neoplastic cell lines expressed in vivo keratin markers typical of keratinization (56 kd acidic keratin and 65-67 kd basic keratins). We draw the following conclusions. (i) The biochemical studies confirm that normal RTE cells undergo squamous differentiation. The pathway of terminal squamous differentiation is cell density dependent. (ii) In transformed RTE cells, growth as well as differentiation are less subject to regulation by cell density than in normal cells. (iii) Transformed RTE cells are differentiation competent; the main abnormality appears to be that in transformed cell populations proliferation and differentiation occur concomitantly.

Animals↗
PubMed1988

Evaluation of different DNA-DNA hybridisation techniques in detection of HPV 16 DNA in cervical smears and biopsies.

The sensitivities of dot blot hybridisation and in situ filter hybridisation for the detection of HPV DNA were compared. Dot blot hybridisation was 10-50 times more sensitive than in situ filter hybridisation in detecting HPV 16 DNA in the cervical cancer cell lines SiHa and CaSki. Cervical smears collected from 51 women with a history of one or more abnormal cervical smears were tested by both hybridisation techniques for the presence of HPV 16 DNA; 11 were positive in the in situ filter hybridisation, 35 in the dot blot hybridisation. Thirty-five cervical biopsies available from this group of 51 women were processed for dot blot hybridisation. In 30 of the 35 cases the results of this hybridisation corresponded with the results of the dot blot hybridisation on the smears.

Biopsy↗
PubMed1988

Induction of anchorage-independent growth of human embryonic fibroblasts with a deletion in the short arm of chromosome 11 by human papillomavirus type 16 DNA.

Human embryonic fibroblasts with a large deletion (11p11.11p15.1) in the short arm of one chromosome 11 (del-11 cells) appeared to be susceptible to transformation by early human papillomavirus type 16 (HPV-16) DNA, whereas diploid human embryonic fibroblasts were not. This difference in susceptibility might be explained by the absence of a tumor suppressor gene located within the deleted part on the short arm of chromosome 11. The presence of abundant viral early-gene transcripts in transformed cells suggests that transformation was induced by an elevated level of an HPV-16 early-gene product(s). The low transcriptional activity of HPV-16 in diploid cells may indicate that cellular genes affect viral transcription. Interruption of the HPV-16 E2 early open reading frame is probably required for high-level HPV-16 early-gene expression driven from the homologous enhancer-promoter region.

Blotting, Southern↗
PubMed1988

Nursing home reimbursement and the allocation of rehabilitation therapy resources.

Most public funding methods for long-term care do not adequately match payment rates with patient need for services. Case-mix payment systems are designed to encourage a more efficient and equitable allocation of limited health care resources. Even nursing home case-mix payment systems, however, do not currently provide the proper incentives to match rehabilitation therapy resources to a patient's needs. We were able to determine by a review of over 8,500 patients in 65 nursing homes that certain diagnoses, partial dependence in activities of daily living (ADLs), clear mental status, and improving medical status are associated with the provision of rehabilitation services to nursing home residents. These patient characteristics are clinically reasonable predictors of the need for therapy and should be considered for use in nursing home case-mix reimbursement systems. Primary payment source also was associated with the provision of rehabilitation services even after taking into account significant patient characteristics. It is unclear how much of the variation in service use across payers is due to differences in patient need as opposed to differences in the financial incentives associated with current payment methods.

Activities of Daily Living↗
PubMed1987

Case mix of public patients in skilled nursing facilities in Connecticut.

The case mix of publicly funded residents in 73 skilled nursing facilities (SNFs) in two Connecticut counties was examined. Data collected in 1980-1981 for utilization review by a professional standards review organization were used for the analysis. The findings indicate that considerable variation exists in case mix across the SNFs. Medicaid per diem rates, which are based on historic costs, have a low and negative correlation with case mix. Case mix indices are significantly higher for rural facilities, with a for-profit type of ownership, and with less than 75% of the bed days devoted to Medicaid residents.

Connecticut↗
PubMed1987

Molecular cloning of gene sequences regulated during squamous differentiation of tracheal epithelial cells and controlled by retinoic acid.

A cDNA library was constructed from polyadenylated RNA present in squamous differentiated rabbit tracheal epithelial cells. Screening of the cDNA library was aimed at identifying RNAs that were abundant in squamous cells and expressed at low levels in undifferentiated cells. Two different recombinants were obtained containing inserts, 0.86 and 0.77 kilobases (kb) in size, that hybridized to mRNAs 1.0 and 1.25 kb in length. These RNAs were present at approximately 50-fold higher levels in squamous cells than in proliferative or confluent retinoic acid-treated cells. The increase in the levels of the 1.0- and 1.25-kb RNAs correlated closely with the onset of squamous differentiation and was not related to induction of terminal cell division. Treatment of rabbit tracheal epithelial cells with transforming growth factor beta, which induces squamous differentiation in these cells, also resulted in elevated levels of the 1.0- and 1.25-kb RNAs. The increased levels of these RNAs in squamous cells appeared to a large extent to be regulated at a posttranscriptional level. Retinoic acid not only inhibited the increase in the levels of the 1.0- and 1.25-kb RNAs but also reversed the expression of these RNAs in squamous cells. These results suggest that retinoic acid affects, directly or indirectly, molecular events that induce alterations in the posttranscriptional processing of the transcripts corresponding to the 1.0- and 1.25-kb RNAs.

Animals↗
PubMed1986

Can Medicare prospective payment survive the ICD-9-CM disease classification system?

The Medicare prospective payment system represents a fundamental change in hospital payment. The diagnosis-related group (DRG) patient classification scheme serves as the modifier of payment for this system. The DRG definitions are, in turn, based on the International Classification of Disease, 9th Revision, Clinical Modification (ICD-9-CM). Deficiencies in the ICD-9-CM coding system directly affect the equity of the Medicare payment system. A review of the ICD-9-CM system identifies three principal problems: the inability of the system to reflect clinically important patient attributes adequately; the use of outcome, rather than approach, to code surgical procedures; and the blurring of clinical specificity by the adoption of certain coding rules. If these deficits in coding specificity are not corrected, it is unlikely that DRGs will adequately distinguish clinically unique types of patients. This inability to differentiate among patients threatens to undermine the equity of Medicare payments. Physicians must become more aware of disease coding and more involved in its development and implementation.

Diagnosis-Related Groups↗
PubMed1978

Identification of mitochondrial gene products by DNA-directed protein synthesis in vitro.

1. A cell-free system, derived from Escherichia coli is highly active in the linked transcription-translation of yeast mtDNA from both wild-type and petite strains. 2. The products of synthesis are short (Mr less than 10 000) hydrophobic polypeptides, which show a high tendency to aggregate in a specific fashion with E. coli and mitochondrial proteins. Aggregation is extremely persistent: alkali, sodium dodecyl sulphate/urea, guanidinium . HCl and carboxymethylation reduce it, but do not eliminate it completely. 3. Nevertheless, results of indirect immunoprecipitation tests suggest that antigenic determinants of cytochrome c oxidase are among the products synthesized. The immunoprecipitation appears specific by criteria including competition experiments and its absence when mtDNA from low complexity petites, retaining only the gene for 21 S rRNA and some flanking sequences, is used to programme protein synthesis. Electrophoretic analysis of material precipitated by anti-cytochrome c oxidase sera reveals four discrete polypeptides with molecular weights of 7400, 6400, 5000 and 4100, which probably represent polypeptide fragments carrying antigenic determinants of cytochrome c oxidase.

Bacterial Proteins↗
PubMed1975

The primary-care practitioner -- specialist or jack-of-all-trades.

Primary-care physicians, too often viewed as low-level generalists, are more appropriately thought of as specialists whose work demands specific skills. These physicians function as managers, advocates, educators, and counselors for their patients while also serving as co-ordinators of other professionals involved in primary care. The basis for education in primary care should be a recognition of the specialized nature of the work that primary-care physicians perform. Faculty members in primary-care programs should themselves be primary-care physicians; these educators deserve the same titles and status given other clinical specialists within the academic community. The setting of standards for primary-care practice and the design of research into areas such as the quality of care are tasks that should be undertaken by, or in collaboration with, physicians who are primary-care specialists.

Counseling↗