Fatal meconium aspiration in spite of appropriate perinatal airway management: pulmonary and placental evidence of prenatal disease

The cytoskeletal components of hamster oocytes, zygotes, and spontaneously activated parthogenotes were examined after immunocytochemical labeling. Microtubules were found only in the anastral, tangentially arranged second meiotic spindle of unfertilized oocytes. Taxol treatment of unfertilized oocytes greatly augmented astral microtubules in both the metaphase II spindle and the cortex. Disruption of the meiotic spindle microtubules with nocodazole resulted in cortical chromosomal scattering. During hamster sperm incorporation and pronuclear formation, no sperm aster was detected in association with the male DNA. Instead, a large overlapping array of microtubules assembled in the cortex. By mitosis, this interphase array disassembled and an anastral metaphase spindle formed. Microtubule and chromatin configurations were also imaged in hamster oocytes injected with human sperm. Astral microtubules were absent from the sperm centrosome. The implications of these results are discussed in relation to the hamster oocyte penetration assay, a test commonly used by in vitro fertilization clinics to demonstrate the fertilizing ability of human sperm. We conclude that since hamsters and humans follow different methods of centrosome inheritance, maternal and paternal, respectively, the hamster may be an inappropriate model for exploring microtubule and centrosomal defects in humans or for assaying postinsemination forms of human male fertility defects.     

Mutations can cause large changes in the conformation of a denatured protein

Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WTSNase delta) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936-944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748-752]. Because of these and other properties of the SNase delta polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNase delta may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNase delta, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNase delta), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNase delta exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNase delta were all of the m--type (m-substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNase delta that were more extended and coil-like than WT SNase delta were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNase delta, as well as both the m+ and m-substituted versions of SNase delta, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

The survival and growth of ovine afferent lymph dendritic cells in culture depends on tumour necrosis factor-alpha and is enhanced by granulocyte-macrophage colony-stimulating factor but inhibited by interferon-gamma

An in vitro culture system is described which allows an analysis of the signals responsible for the survival, growth and functional maturation of afferent lymph dendritic cells (ALDC), a subpopulation of migrating dermal dendritic cells involved in antigen carriage and presentation to T-cells. Purified ALDC survived and grew for up to 30 days in lymph node conditioned medium and survived 14 days in recombinant ovine (rov) TNF-alpha whereas none were detected after 24 h in rov GM-CSF, rov IFN-gamma or rh M-CSF. However, when rov GM-CSF was added to cultures along with rov TNF-alpha, increased numbers of ALDC compared with input numbers (growth) were recorded on Days 14 and 21. In contrast, when 50-200 units ml-1 of rov IFN-gamma were added to cultures of ALDC along with TNF-alpha or rov TNF-alpha plus rov GM-CSF, cell survival and growth was inhibited. Antibody blocking studies confirmed the cytokine specificity of these effects. ALDC cultured in rov TNF-alpha or rov TNF-alpha plus rov GM-CSF retained MHC Class-II and ov CD-1 antigen expression and accessory function for autologous ov CD-4 T-cell proliferation, although at reduced levels compared with freshly isolated cells. Neither fresh nor cultured ALDC expressed coagulation factor XIIIa.