Drosophila wee1 has an essential role in the nuclear divisions of early embryogenesis.

In Drosophila, the maternally expressed mei-41 and grp genes are required for successful execution of the nuclear division cycles of early embryogenesis. In fission yeast, genes encoding similar kinases (rad3 and chk1, respectively) are components of a cell cycle checkpoint that delays mitosis by inhibitory phosphorylation of Cdk1. We have identified mutations in a gene encoding a Cdk1 inhibitory kinase, Drosophila wee1 (Dwee1). Like mei-41 and grp, Dwee1 is zygotically dispensable but is required maternally for completing the embryonic nuclear cycles. The arrest phenotype of Dwee1 mutants, as well as genetic interactions between Dwee1, grp, and mei-41 mutations, suggest that Dwee1 is functioning in the same regulatory pathway as these genes. These findings imply that inhibitory phosphorylation of Cdk1 by Dwee1 is required for proper regulation of the early syncytial cycles of embryogenesis.

Major DQ8-restricted T-cell epitopes for human GAD65 mapped using human CD4, DQA1*0301, DQB1*0302 transgenic IA(null) NOD mice.

The 65KD isoform of GAD is considered to be a major target autoantigen in many humans with autoimmune prediabetes or diabetes. The major histocompatibility complex class II allele DQA1*0301, DQB1*0302, which encodes HLA-DQ8, confers susceptibility to type 1 diabetes and occurs in up to 80% of affected individuals. To map T-cell epitopes for GAD65 restricted to the diabetes-associated DQ8 heterodimer, we generated transgenic NOD mice expressing HLA-DQ8 and human CD4 while having the mouse class II gene (IA(beta)) deleted. These mice were immunized with full-length purified recombinant GAD65, and the fine specificity of T-cell responses was mapped by examining recall responses of bulk splenocytes to an overlapping set of 20-mer peptides encompassing the entire GAD65 protein. Four different peptides (P121-140, P201-220, P231-250, and P471-490) gave significant T-cell recall responses. P201-220 and P231-250 have been shown previously to bind DQ8, whereas the other two peptides had been classified as nonbinders. Interestingly, the peptide giving the greatest response (P201-220) encompasses residues 206-220 of GAD65, a region that has been shown to be a dominant T-cell epitope in wild-type IA(g7) NOD mice. Overlap in this T-cell epitope likely reflects structural similarities between DQ8 and IA(g7). The fine specificity of antibody responses in the GAD65-immunized mice was also examined by testing the antisera by enzyme-linked immunosorbent assay (ELISA) against the same overlapping set of peptides. The two dominant B-cell epitopes were P361-380 and P381-400; P121-140 and P471-490 appeared to correspond to both B- and T-cell epitopes. Although the NOD human CD4, DQ8, IA(null) transgenic mice generated in these studies do not develop autoimmune diabetes either spontaneously or after cyclophosphamide treatment, they can be used to map DQ8-restricted T-cell epitopes for a variety of human islet autoantigens. They can also be used to test T-cell-specific reagents, such as fluorescently labeled DQ8 tetramers containing GAD65 peptides or other beta-cell peptides, which we believe will be useful in analyzing human immune responses in diabetic and prediabetic patients.

Transcription factor Sp1 is expressed by three different developmentally regulated messenger ribonucleic acids in mouse spermatogenic cells.

Gene expression during spermatogenesis is highly cell- and stage-specific and involves the complex interplay of multiple developmentally regulated transcription factors. Recent evidence suggests that the DNA-binding protein Sp1 functions as an important trans-activator during cell development and differentiation. In the present study, the developmental expression of Sp1 was characterized during mouse spermatogenesis. Three distinct Sp1 transcripts were detected in mouse spermatogenic cells, each with a distinct developmental pattern; an 8.2-kilobase (kb) messenger RNA (mRNA) identical in size to the somatic mRNA expressed in spermatogonial cells, a larger mRNA approximately 8.8 kb in size present in meiotic cells, and a 2.4 kb mRNA in meiotic and postmeiotic germ cells. The 8.8- and 2.4-kb Sp1 transcripts were not observed in somatic cells and, thus, are male germ cell specific. Northern, ribonuclease protection, and RT-PCR assays revealed that the 2.4-kb Sp1 transcript is truncated in both the 5'- and 3'-untranslated regions relative to the somatic mRNA and lacks a short segment of the N-terminal coding region. Polysome analysis further indicated that these germ cell-specific Sp1 mRNAs are translated, albeit with a lower efficiency than the somatic transcript. Consistent with these results, spermatogenic cells were shown to contain approximately 9-fold lower concentrations of Sp1 proteins that are approximately the same size as the somatic form. Of particular interest, the apparent affinity of Sp1 DNA-binding activity in nuclear extracts from mouse germ cells was 5-fold greater than that in extracts from mouse somatic tissues. This may reflect the existence of mechanisms within mouse spermatogenic cells that compensate for the lower nuclear concentrations of Sp1 protein. These results suggest that cell- and stage-specific regulation of Sp1 gene expression and activity may be an important component of the mouse spermatogenic cell developmental program.

Validity of simple approach to scoring and standard setting for standardized-patient cases in an examination of clinical competence.

The results are disappointing, providing little support for the validity of the case-passing decisions based on this simple approach to scoring and standard setting. The case-passing decisions predicted what the case author intended for about only 73% or 74% of the students on average and, with agreement expected by chance removed, predicted what the case author intended for about only 25% of the students. Even with the use of the optimal pass/fail cutoffs and the dropping of students with ambiguous borderline global ratings, the case-passing decisions failed to agree with the case authors' global ratings for 15% to 30% of the students. The findings might be dismissed as simply due to low reliabilities of passing decisions and global ratings based on a single case. Although this concern would apply to intercase reliabilities, which would be subject to case specificity, the appropriate reliabilities here would seem to be intracase (i.e., intrarater), which should be fairly high (if they could be computed). Nevertheless, it seems reasonable to expect much better agreement between results of case scoring and of standard setting developed by the case author and the case author's global ratings of performance on that case, given that the case author might recall the checklist, assign a weight to each item, and so forth. Also, case-passing decisions would possibly agree more with global ratings of live or videotaped performances than with ratings of written summaries of performance; however, that question remains a challenge for further research. In conclusion, the study provides only weak evidence, at best, for the validity of the scoring and standard setting commonly used with SP assessment. The results do not undermine claims about the realism of the SP approach, however, nor do they call into question the standardization afforded by this method of assessing clinical competence. The results do raise serious concerns about this simple approach to scoring and standard setting for SP-based assessments and suggest that we should focus more on the observation and evaluation of actual student performance on SP cases in the development of valid scoring and standard setting.

Clinical and laboratory features of acute sulfur dioxide inhalation poisoning: two-year follow-up.

We present clinical and laboratory results (including nuclear imaging) obtained over a period of two years in two nonsmoking miners who were exposed to high concentrations of sulfur dioxide (SO2) after a mine explosion. Within 3 wk of the accident, both miners had evidence of severe airways obstruction, hypoxemia, markedly reduced exercise tolerance, ventilation-perfusion mismatch, and evidence of active inflammation as documented by positive gallium lung scan. Serial ventilation-perfusion scans over the first 12 months showed progressive improvement without returning to normal. After the initial recovery, there has been no significant change over the subsequent two years postinjury. Pulmonary function and exercise tests also displayed a similar pattern of initial improvement. We conclude that (1) acute exposure to high concentrations of SO2 results in severe airways obstruction, (2) pulmonary function abnormalities are partially reversible, and (3) most of the improvement occurs within 12 months after the initial injury.

Bile salts and bioelectric properties of rat jejunum.

Addition of conjugated bile salts increased transmural- and transserosal-potential differences of sheets of rat jejunum. Removal of Cl- from buffer solutions minimized the bile-sale induced bioelectric changes. Bile-salt induced doubling of tissue resistance was not explained by an observed increase in net Cl- serosa leads to mucosa flux. Electrical effects were unrelated to concentration and were observed only when bile-salt solutions perfused the jejunal mucosa. The molecular events associated with bile-salt interactions with the plasma membrane affecting ion fluxes may relate to their unique effects on sterol absorption.

Surfactants and bioelectric properties of rat jejunum.

The effects of surfactants on bioelectric properties of rat jejunum were determined. Tween 80 (nonionic) and sodium dodecylsulfate (anionic) increased transmural potential differences 20-34% over values in Krebs-Ringer bicarbonate buffer. Short-circuit currents increased 66-112% and net tissue resistance decreased 19-30%. The cationic surfactant cetrimide decreased transmural potential 23%; short-circuit current decreased 32%, and resistance increased 22%. When sulfate replaced chloride in buffer, surfactant effects were minimized or reversed suggesting a role of Cl- flux in the bioelectric effects. Cationic surfactant effects on current and resistance were in the same direction as, but of greater and lesser magnitude, respectively, than alterations observed with bile salts. The current increase was of greater magnitude and resistance decrease less marked. Surfactant molecules may interact specifically with membranes in a more complex manner than simple "detergency".