How significant is a cervical smear showing glandular dyskaryosis?

OBJECTIVE: To evaluate the incidence, outcome and predictive value of cytology showing glandular dyskaryosis. PARTICIPANTS: Fifty-seven women with a smear diagnosis of glandular dyskaryosis registered between January 1997 and December 2001. SETTING: Colposcopy and cytopathology units in a large district general hospital. RESULTS: Sixty smears in 57 women showing glandular dyskaryosis were identified from a cohort of 135,120 smears, giving an incidence of 0.05%. Hospital records were available for 50 women. Final diagnosis included 13 cases of cervical glandular intraepithelial neoplasia (CGIN), 4 microinvasive cervical adenocarcinomas, 2 undifferentiated tumours, 1 microinvasive squamous carcinoma, 21 cases of CIN and 13 cases of endometrial pathology (8 endometrial cancers). Twelve women had coexistent squamous and glandular disease. Forty-five out of 50 women had significant pathology (positive predictive value 90%). Colposcopy was seen to be of limited value in assessment of smears showing glandular dyskaryosis. Only 1 out of 13 glandular lesions was diagnosed by colposcopy. CONCLUSION: Smears showing glandular dyskaryosis are associated with significant pathology in 90% of cases and malignancy in 32% of cases. Hence, women with a smear showing glandular dyskaryosis should be referred urgently to a colposcopy clinic and flagged up as suspected cancer. Glandular dyskaryosis should be included in the national referral criteria for suspected gynaecological cancer.

Temperature-sensitive lesions in the capsid proteins of the rotavirus mutants tsF and tsG that affect virion assembly.

The SA11 rotavirus mutants tsF and tsG contain temperature sensitive (ts) lesions in the capsid proteins VP2 and VP6, respectively, that interfere with their ability to assemble. To understand the nature of their lesions, full-length cDNAs of tsF gene 2 and tsG gene 6 were prepared from viral mRNA by reverse transcription and polymerase chain reaction. Comparative sequence analysis indicated that the ts phenotype of tsF VP2 is due to an Ala-->Asp substitution at position 387. The mutation falls outside of those regions of VP2 previously suggested to be of functional significance and therefore points to a previously unidentified site in VP2 that is important for the assembly of viral cores. Comparative sequence analysis showed that tsG VP6 contains two mutant amino acids, i.e., Thr-10 and His-13, and therefore one or both of these mutations are responsible for the ts phenotype of the mutant VP6. In the case of other group A and group C VP6 sequences, these residues are Ser and Asp, respectively. Characterization of tsG-infected cells by indirect immunofluorescence staining showed that while viroplasmic inclusions are formed at the nonpermissive temperature, the mutant VP6 accumulates in these structures only at the permissive temperature. While influencing intracellular accumulation, the Thr-10-->Ser and His-13-->Asp mutations in tsG VP6 are probably not directly involved in the interaction of VP6 with VP2, as VP6 deletion mutants lacking residues 10 and 13 retain the ability to bind VP2 in vitro. Analysis of VP6 failed to confirm previous reports that the protein was myristylated and thus excludes the possibility that this cotranslational modification is temperature-dependent for tsG VP6. Together, these data suggest that the amino terminus of VP6 plays an essential role in virus assembly in vivo, perhaps by being necessary for the movement of the protein to viroplasmic inclusions, the site of core and single-shelled particle formation.

Comparative analysis of the rotavirus NS53 gene: conservation of basic and cysteine-rich regions in the protein and possible stem-loop structures in the RNA.

NS53, the product of rotavirus gene 5, is an RNA-binding protein that contains a cysteine-rich region and is a component of early replication intermediates. To gain information about the structure of NS53 and its RNA, we determined the nucleotide sequence of gene 5 for the human viruses Wa (serotype 1) and DS1 (2) and the simian virus SA11 (3) (Patton strain) and compared them and their deduced amino acid sequences to those reported for the bovine viruses UK (6) and RF (6), SA11 (3) (Both strain), the human virus Rohivg803, and the group C porcine virus PRV. The results showed that gene 5 for human, simian, and bovine strains have lengths of 1564-1567, 1611, and 1579-1581 nucleotides (nt) and encode proteins of 486, 495, and 491 amino acids, respectively. Comparison of the protein sequences for NS53 among different serotypes showed that they are extremely divergent with many sharing amino acid homologies of only 36-38%. Even NS53 from viruses isolated from the same species possessed relatively poor homology, e.g., DS1 versus Wa was 68%. The first 150 amino acids of NS53 exhibited a greater degree of conservation than the rest of the protein. Near the amino terminus, NS53 contains three basic regions and a cysteine-rich domain, suggesting that this area is responsible for the RNA-binding activity of the protein. Present in the cysteine-rich domain of all group A and C viruses was the motif C-X2-C-X8-C-X2-C-X3-H-X-C-X2-C-X5-C. Although this motif may form one or two zinc fingers, the fact that it is highly conserved indicates that it plays a critical role in the function of protein. Comparison of the nucleotide sequences for gene 5 showed that the entire 5'-noncoding region and the first 24 nt of the NS53 ORF are conserved. RNA-folding predictions suggest that this region of the NS53 mRNA can interact with itself, producing a stem-loop structure similar to that found near the 5'-terminus of the NS35 mRNA. Thus, such structures may be common to all rotavirus mRNAs, perhaps functioning as signals for packaging of RNAs into replication intermediates or regulating mRNA translation.

Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4.

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.

Nucleotide and amino acid sequence analysis of the rotavirus nonstructural RNA-binding protein NS35.

NS35, a basic protein encoded by gene 8 of SA11 rotavirus, possesses RNA-binding activity and is essential for genome replication. To identify conserved regions in the NS35 gene and its protein product, we determined the nucleotide sequences of the NS35 gene for the mammalian and avian rotaviruses Wa, DS1, SA11 (Patton and Ramig strains), NCDV, and Ty-1 and compared them and their deduced amino acid sequences to those reported for SA11 (Both strain), OSU, and UK. The results indicated that the NS35 genes of the mammalian rotaviruses are 1058-1059 bases in length and encode proteins of 317 amino acids that exhibit high levels of sequence conservation (> or = 83%). The NS35 gene of the turkey rotavirus Ty-1 differed from those of the mammalian rotaviruses with respect to size of the predicted protein (315 amino acids) and of the gene (1042 bases). NS35 of Ty-1 exhibited a relatively low degree of amino acid homology (52-57%) with NS35 of the mammalian viruses. Phylogenetic analysis of the NS35 gene indicated that avian (TY-1) and mammalian rotaviruses are distantly related. Comparison of the predicted sequences of NS35 showed that all possessed a conserved basic domain of 37 amino acids at residues 205-241 that may serve as the RNA-binding domain. Electrophoretic examination showed that NS35 contains a disulfide bond probably located in the amino-terminal half of the protein. Comparison of NS35 genes at the nucleotide level revealed two regions of extensive conservation, (i) a 75-base (b) sequence that includes the 35-base 5'-noncoding region and the first 30 bases of the open reading frame for NS35, and (ii) a 28-b sequence in the 3'-noncoding region of the gene. Secondary structure predictions for the NS35 mRNA suggest that the 75-base sequence can fold to produce a stem double-loop structure. Such a structure may serve as a packaging signal for the assortment of NS35 mRNA into replicase particles.

Rotavirus RNA replication: VP2, but not VP6, is necessary for viral replicase activity.

Temperature-sensitive mutants of simian rotavirus SA11 were previously developed and organized into 10 of a possible 11 recombination groups on the basis of genome reassortment studies. Two of these mutants, tsF and tsG, map to genes encoding VP2 (segment 2) and VP6 (segment 6), respectively. To gain insight into the role of these proteins in genome replication, MA104 cells were infected with tsF or tsG and then maintained at permissive temperature (31 degrees C) until 9 h postinfection, when some cells were shifted to nonpermissive temperature (39 degrees C). Subviral particles (SVPs) were recovered from the infected cells at 10.5 and 12 h postinfection and assayed for associated replicase activity in a cell-free system shown previously to support rotavirus genome replication in vitro. The results showed that the level of replicase activity associated with tsF SVPs from cells shifted to nonpermissive temperature was ca. 20-fold less than that associated with tsF SVPs from cells maintained at permissive temperature. In contrast, the level of replicase activity associated with tsG SVPs from cells maintained at nonpermissive temperature was only slightly less (twofold or less) than that associated with tsG SVPs from cells maintained at permissive temperature. Analysis of the structure of replicase particles from tsG-infected cells shifted to nonpermissive temperature showed that they were similar in size and density to virion-derived core particles and contained the major core protein VP2 but lacked the major inner shell protein VP6. Taken together, these data indicate that VP2, but not VP6, is an essential component of enzymatically active replicase particles.

The community as preclinical classroom: experience with a first-year clerkship.

The required first-year clerkship in family and community medicine at the University of Massachusetts Medical School is a major curricular innovation which has implications for physician education. Students have lived in communities and worked in health service settings with field sponsors, under full-time faculty guidance, in all regions of Massachusetts. This has been one answer to the school's mandate to address the physician maldistribution problem. The goals and objectives and the teaching methods used to implement the program are described. These lessons were drawn from the program experience: community medicine clerkships belong in the first year of the curriculum; full-time medical school faculty working with the field sponsor promotes an optimal learning environment; long-term evaluation remains an important consideration. The first eight years of experience with the clerkship have demonstrated its value, and it should be considered for inclusion in the curriculum of other medical schools.

The preceptorship, an integral unit of the curriculum in community and family medicine.

A curriculum in community and family medicine at the University of Massachusetts Medical School, planned to include a primary care preceptorship as an integral unit, is described. The curriculum has been planned to allow for repeated and increasing exposure of medical students during their undergraduate years to a variety of health care settings within the state. An extensive program of courses is required for all students and consists of a three-week field clerkship in the first year, a field-oriented epidemiology course in the second year, and a six-week field clerkship in the third or fourth year. A preceptorship elective is available to all medical students after they have completed their first two years. Field visits by community and family medicine faculty to the preceptorship site provide overall guidance, facilitate the implementation of objectives, and provide opportunities to strengthen bonds between the practicing physicians of the Commonwealth of Massachusetts and the state medical school. The results to date are discussed.