The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins.

The isolation, characterization, and expression of a full-length cDNA encoding the human T cell glycoprotein CD6 is described. COS cells transfected with the CD6 clone express a 90-kD protein that reacts with all available anti-CD6 monoclonal antibodies. RNA blot hybridization analysis indicates that CD6 transcripts are predominantly restricted to cells in the T lineage. The predicted CD6 sequence is 468 amino acids long, with the typical features of a type I integral membrane protein. The cytoplasmic domain of CD6 contains two serine residues, one or both of which are substrates for phosphorylation during T cell activation. The extracellular domain of CD6 is significantly related to the extracellular domain of the human and mouse T cell antigen CD5, the cysteine-rich domain of the bovine and mouse type I macrophage scavenger receptor, the extracellular domain of the sea urchin spermatozoa protein that crosslinks the egg peptide speract, the mammalian complement factor 1, and the human lung tumor antigen L3. These molecules, therefore, constitute a new gene superfamily that is well conserved across species boundaries.

The use of millipore filters in ultrastructural studies of cell cultures and viruses.

A technique is described for embedding tissue culture cells that have been adsorbed or grown on Millipore filters. The acetone used during the embedding process rendered the filters transparent so that specific areas or cells could be chosen with the aid of the light microscope. Lymphoblastoid cells processed on the filters possessed well-defined plasma membranes and microvilli which were rarely present in cells from parallel cultures that were prepared by pelleting in the centrifuge. Fibroblast cells grown on filters retained their elongated appearance, in contrast to the rounded cells in pelleted preparations. Millipore filters were also used as a means of embedding virus pellets for sectioning. Preparations containing as few as 4 x 10(8) virus particles were suitable for study by the filter technique. Crude tissue-culture harvests of vaccinia virus and purified preparations of Rauscher murine leukemia and adeno-satellite viruses were successfully examined.

Antibody to herpes simplex virus type 2-induced nonstructural proteins in women with cervical cancer and in control groups.

Sera obtained from 15 patients with cervical cancer, 10 patients with breast cancer, and 15 control women, individually matched with the cervical cancer patients, were examined for antibodies to early proteins synthesized in herpes simplex virus type 2 (HSV-2)-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. The relative reactivity to a major early nonstructural protein (VP134) was used to compare these selected sera. The results obtained suggest that cervical cancer patients possess sera with a higher reactivity to VP134 than breast cancer patients or matched healthy women,and that serum reactivity is independent of the level of neutralizing antibodies to HSV-2.

Studies on herpes simplex virus and cancer.

Virus-induced polypeptides of cells infected by herpes simplex virus (HSV) types 1 and 2 were investigated by analysis on polyacrylamide gels and by determination of their antigenicity. Some polypeptides, VP154 and VP134, had immunological reactivity common to both virus types, while others (VP175 and VP123) were type specific. Only the glycosylated polypeptides were able to induce neutralizing antibody. The expression of viral genetic information was studied in newborn mice infected with wild-type and ts mutant viruses; some mutants had become attenuated and had lost pathogenicity for newborn mice while others had not. From induction experiments in HSV=transformed hamster cells, it appears that detection of enhanced replication of ts mutants in human cancer cells would be an indication of resident HSV genetic information. Sera obtained from cancer patients were examined for antibodies to early proteins synthesized in HSV-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. Cervical cancer patients had sera with a higher reactivity to early nonstructural polypeptides than to breast cancer patients or to matched healthy women. In contrast to the results with early polypeptides, little difference was detectable between the matched sera in their reactivity with a major capsid polypeptide, which is synthesized late in the infectious cycle.

Experimental infection of squirrel and marmoset monkeys with attenuated Herpesvirus saimiri.

Herpesvirus saimiri (HVS) was propagated in vero cells for 3 passages at 39 degrees and cloned 3 times at 34 degrees. This virus was inoculated into cotton-topped marmoset and squirrel monkeys; all inoculated monkeys became infected as HVS was reisolated after their circulating lymphocytes were cultured with vero cells and measurable levels of antiviral antibodies developed that were measured by immunofluorescence and/or neutralization tests. None of the inoculated monkeys developed any signs of overt disease and all inoculated monkeys have survived 9 to 14 months postinoculation. The attenuated virus appears to be genetically stable as virus isolated from an infected marmoset was passed 3 times in vitro and then inoculated into other marmosets, which became infected and remained clinically well. Marmosets latently infected with attenuated HVS were not protected when challenged with a large dose (770 plaque-forming units) of oncogenic HVS, although these marmosets survived about 3 times longer than did inoculated control marmosets.

Structure and sequence of the calmodulin gene from Neurospora crassa.

cDNA and genomic clones of Neurospora calmodulin were obtained by PCR. Characterization revealed an open reading frame encoding a predicted protein of 149 amino acids, showing 85% identity to the human calmodulin protein sequence. Comparison of the cDNA and genomic sequence reveals the position of five introns, organized differently than is found in calmodulin genes from higher eukaryotes.

The Drosophila stubarista phenotype is associated with a dosage effect of the putative ribosome-associated protein D-p40 on spineless.

We describe the molecular characterization of the Drosophila melanogaster gene stubarista (sta) that encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. In addition, we have analyzed the basis of the sta1 visible phenotype which consists of shortened antennae and bristles. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. We provide genetic evidence that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. We demonstrate that sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein.

A new enhancer of position-effect variegation in Drosophila melanogaster encodes a putative RNA helicase that binds chromosomes and is regulated by the cell cycle.

In Drosophila melanogaster, position-effect variegation of the white gene has been a useful phenomenon by which to study chromosome structure and the genes that modify it. We have identified a new enhancer of variegation locus, Dmrnahel (hel). Deletion of mutation of hel enhances white variegation, and this can be reversed by a transformed copy of hel+. In the presence of two endogenous copies, the transformed hel+ behaves as a suppressor of variegation. hel is an essential gene and functions both maternally and zygotically. The HEL protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potential HEL homologues have been found in mammals, yeast and worms. HEL protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. We propose that the HEL protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that HEL is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis.

Mouse submandibular gland mucin: embryo-specific mRNA and protein species.

Mouse submandibular salivary gland (SMG) mucin is the primary histodifferentiation product of submandibular epithelia. We demonstrate marked differences between embryonic, neonatal, and adult SMG mucin mRNA and protein by Northern and Western blot analyses: E17 and 1-day-old neonates exhibit two unique mucin transcripts (1.20 and 0.85 kb) which are approximately 19% greater or smaller in size than the single (1.01 kb) adult transcript. Two embryonic protein isoforms (Mr approximately 110 and 152 kDa) are immunodetected compared to a single adult protein (Mr approximately 136 kDa), with the larger (approximately 152 kDa) embryonic isoform persisting in neonatal glands. Mucin transcripts are localized to the branching epithelia in E14 and older SMGs, with increased hybridization signal being seen in terminal bud and proacinar epithelial cells with age; a significant 26% increase in transcript levels is detected by RNase protection assay between E14 and E19. By contrast, submandibular mucin protein is not immunodetected until E17, being primarily immunolocalized to terminal bud and proacinar epithelial cell membranes. Our data clearly shows that substantial qualitative differences exist between embryonic and adult SMG mucin mRNA and protein.

Developmental expression of survivin during embryonic submandibular salivary gland development.

BACKGROUND: The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival. RESULTS: We investigated the developmental expression of survivin in Pseudoglandular (approximately E14), Canalicular (approximately E15) and Terminal Bud (approximately E17) Stage SMGs. We report a significant 26% increase in transcript levels between the Canalicular and Terminal Bud Stages. Immunohistochemical studies demonstrate nuclear-localized survivin protein in epithelial cells bounding forming lumina in Canalicular and Terminal Bud Stage SMGs. CONCLUSIONS: Survivin is known to be a pro-survival and anti-apoptotic factor. Given that survivin translocation into the nucleus is required for the induction of entry into the cell cycle and the inhibition of apoptosis, our demonstration of nuclear-localized survivin protein in presumptive ductal and proacinar lumen-bounding cells suggests that survivin may be a key mediator of embryonic SMG epithelial cell survival.

The functional genomic response of developing embryonic submandibular glands to NF-kappa B inhibition.

BACKGROUND: The proper balance between epithelial cell proliferation, quiescence, and apoptosis during development is mediated by the specific temporal and spatial appearance of transcription factors, growth factors, cytokines, caspases, etc. Since our prior studies suggest the importance of transcription factor NF-kappaB during embryonic submandibular salivary gland (SMG) development, we attempted to delineate the emergent dynamics of a cognate signaling network by studying the molecular patterns and phenotypic outcomes of interrupted NF-kappaB signaling in embryonic SMG explants. RESULTS: SN50-mediated inhibition of NF-kappaB nuclear translocation in E15 SMG explants cultured for 2 days results in a highly significant increase in apoptosis and decrease in cell proliferation. Probabilistic Neural Network (PNN) analyses of transcriptomic and proteomic assays identify specific transcripts and proteins with altered expression that best discriminate control from SN50-treated SMGs. These include PCNA, GR, BMP1, BMP3b, Chk1, Caspase 6, E2F1, c-Raf, ERK1/2 and JNK-1, as well as several others of lesser importance. Increased expression of signaling pathway components is not necessarily probative of pathway activity; however, as confirmation we found a significant increase in activated (phosphorylated/cleaved) ERK 1/2, Caspase 3, and PARP in SN50-treated explants. This increased activity of proapoptotic (caspase3/PARP) and compensatory antiapoptotic (ERK1/2) pathways is consistent with the dramatic cell death seen in SN50-treated SMGs. CONCLUSIONS: Our morphological and functional genomic analyses indicate that the primary and secondary effects of NF-kappaB-mediated transcription are critical to embryonic SMG developmental homeostasis. Relative to understanding complex genetic networks and organogenesis, our results illustrate the importance of evaluating the gene, protein, and activated protein expression of multiple components from multiple pathways within broad functional categories.

Sensory perception in Parkinson disease.

OBJECTIVE: To determine whether there is a complex sensory disturbance that may be contributing to the motor deficit in patients with Parkinson disease. DESIGN: Comparison of performance by patients and healthy, age- and sex-matched subjects in tests of various sensory functions. SETTING: The Center for Human Performance and Testing at a university hospital and research center. PARTICIPANTS: Ten subjects with Parkinson disease and 10 control subjects matched for age and sex. MAIN OUTCOME MEASURE: Performance on 4 subjects of the Sensory Integration and Praxis Test: finger identification, graphesthesia, localization of tactile stimuli, and kinesthesia. RESULTS: Data were analyzed using paired t tests for ratio data and the paired Wilcoxon test for ordinal data. Patients with Parkinson disease performed significantly worse (P = .001) than the control patients on the test of kinesthesia. There were no significant differences between the 2 groups on the other subtests. CONCLUSIONS: Without visual guidance, patients with Parkinson disease had more difficulty in perceiving the extent of a movement made to a target away from the body, a task requiring reliance on proprioceptive feedback. Parkinsonian patients had no more difficulty than controls in making movements to a target on the surface of the body when they could use tactile sensations. Movement difficulties in patients with Parkinson disease may relate in part to a decrease in proprioception. Activities that enhance kinesthetic awareness may be an important adjunct to the treatment of these patients.

Effect of pallidotomy on postural control and motor function in Parkinson disease.

OBJECTIVE: To investigate the effects of pallidotomy on postural reactions and other motor parkinsonian deficits. DESIGN: Comparison of performance by patients before and after pallidotomy on tests of balance and function. SETTING: A Parkinson disease Center of Excellence and Center for Human Performance Testing at a university hospital and research center. PARTICIPANTS: Twenty-nine patients with Parkinson disease undergoing pallidotomy. MAIN OUTCOME MEASURES: Performance results on the United Parkinson's Disease Rating Scale (UPDRS), activities of daily living and motor subscales (parts II and III). and posturography (sensory organization test), which were collected before and 3 and 6 months after surgery with patients in the practically defined off state (medication withheld for at least 12 hours). RESULTS: Data were analyzed with a paired Wilcoxon and Spearman correlation. There was a significant improvement in mean +/- SD UPDRS motor subscale score after pallidotomy (before surgery, 52.43+/-13.46; after surgery, 43.93+/-15.15; z= 3.63; P=.003). There were no significant changes in the UPDRS activities of daily living subscale or average stability scores when the group was examined as a whole. However, examination of individual data revealed that 9 (56%) of 16 patients who could stand independently before surgery showed improvement in either the number of falls or the average stability score. No patient who was unable to stand independently before surgery was able to stand independently after it. CONCLUSION: Pallidotomy helped improve overall motor function in patients with parkinsonism and, for some patients, also improved postural stability.

Bone remodeling in W/Wv mast cell deficient mice.

Strong experimental evidence exists for a relationship between mast cells and bone disease, but the role of mast cells in the regulation of bone remodeling is unknown. In order to address this question, mast cell deficient mice (W/Wv) were paired with their mast cell sufficient (+/+) littermates and evaluated for differences in response to an induced cycle of bone remodeling. This was achieved using a tooth egression protocol, in which a synchronous cycle of bone remodeling was induced in the mandibular buccal alveolar periosteum by extraction of the opposing dentition. Quantitative histomorphometric changes during the activation, resorption, reversal, and formation phases of bone remodeling were documented using standard techniques. Most cell deficient mutants exhibited the following defects in response to an induced cycle of bone remodeling: (a) the onset of the remodeling cycle was delayed by a prolonged activation phase, (b) the duration and extent of the active formation phase was decreased, and (c) the amount of new bone matrix synthesized was diminished while mineralization rates were found to be normal. These results suggest that mast cells and their mediators provide a paracrine mechanism which influences the recruitment of osteoclast and osteoblast progenitors and their participation in bone remodeling. Nonetheless, since bone remodeling occurs in mast cell deficient mice, albeit less efficiently, this mechanism is most likely one of several redundant mechanisms that provide for adequate skeletal homeostasis.

Multiwell chamber chemotaxis assays: improved experimental design and data analysis.

The chemotaxis assay using the Boyden transfilter technique has become widely used in recent years for assessing migratory responses of a wide variety of cell types. In the study reported here we examined the migratory responses of mouse peritoneal macrophages using a multiwell chamber. The experiments were designed to analyze the components of variance in the assay method, to optimize the experimental design, and to develop objective statistical criteria for choosing among experiments with disparate results. Cell counts were obtained with the aid of an image analyzer coupled to a light microscope. Microcomputer software was developed to drive the image analyzer, collect data and conduct statistical analyses. Nested analysis of variance (ANOVA, either 2- or 3-level) was employed to partition the components of variance and F-tests were used to determine their significance. Significant sources of experimental error were identified both within and among wells and were attributed mostly to variability in the chamber/filter assembly and counting procedure. Statistical analyses demonstrated that there was significant variation among assays conducted in different multiwell chambers on the same day, among assays where the same agent was tested on different days in the same chamber, and among replicate counts of the same assay. The following recommendations were made: use ANOVA to distinguish differences due to biological effects from those due to experimental error, design experiments so that all relevant comparisons are included in the same chamber and the same assay, avoid pooling data from different assays unless ANOVA treatment variances are comparable, and when replicate assays yield disparate results choose the assay with the lowest percentage of variation due to experimental error.

Structure-based design and synthesis of substituted 2-butanols as nonpeptidic inhibitors of HIV protease: secondary amide series.

The design, synthesis, and crystallographic analysis of protein-inhibitor complexes is described for a novel series of nonpeptidic HIV protease (HIV Pr)inhibitors. Beginning with a cocrystal structure of a Phe-Pro peptidomimetic bound to the HIV Pr, design was initiated that resulted in the substituted 2-butanol compound 8 as the lead compound (Ki = 24.5 microM, racemic mixture). Modifications on the initial compound were then made on the basis of its cocrystal structure with HIV Pr and inhibition data, resulting in compounds with enhanced potency against the enzyme (compound 18, Ki = 0.48 microM). These inhibitors were found to bind to the enzyme essentially as predicted on the basis of the original design hypothesis. Stereospecific synthesis of individual enantiomers confirmed the prediction of a binding preference for the S alcohol stereochemistry. Modest antiviral activity was demonstrated for several of the more potent HIV Pr inhibitors in a HIV-1 infected CEM-SS cell line.

Bis tertiary amide inhibitors of the HIV-1 protease generated via protein structure-based iterative design.

A series of potent nonpeptide inhibitors of the HIV protease have been identified. Using the structure of compound 3 bound to the HIV protease, bis tertiary amide inhibitor 9 was designed and prepared. Compound 9 was found to be about 17 times more potent than 3, and the structure of the protein-ligand complex of 9 revealed the inhibitor binds in an inverted binding mode relative to 3. Examination of the protein-ligand complex of 9 suggested several modifications in the P1 and P1' pockets. Through these modifications it was possible to improve the activity of the inhibitors another 100-fold, highlighting the utility of crystallographic feedback in inhibitor design. These compounds were found to have good antiviral activity in cell culture, were selective for the HIV protease, and were orally available in three animal models.

The effects of long-term fetal constraint in vitro on the cranial base and other skeletal components.

Recently it was suggested that some cases of premature closure of the sagittal suture in humans may be due to fetal head constraint. Using various modifications of the procedure for long-term shell-less cultivation of chick embryos, we sought to test the hypothesis that fetal head constraint may result in abnormalities of the cranial base and other skeletal structures of the head. Our experiments with various sized containers demonstrate that fetal constraint is associated with both deformation and malformation of the craniofacial skeleton, including the cranial base, squamosal, columella (stapes), and mandible. The severity of both deformation and malformation appears to be a function of the degree of fetal constraint. If, as some suggest, abnormality of the cranial base is the primary anomaly in craniosynostosis, then our results tend to support the fetal constraint hypothesis as one explanation of simple craniostenosis.

Neural tube and neural crest: a new view with time-lapse high-definition photomicroscopy.

The dynamic process of neural tube formation and neural crest migration in live, unstained cultured avian embryos at Hamburger-Hamilton (H.H.) stages 8-11 was investigated by time-lapse cinematography using a high-definition microscope. These studies have demonstrated that neural tube closure in the trunk region differs from that observed in the head. The cephalic neural folds elevate slowly, then make contact rapidly. Following this initial apposition, they gradually "zip-up" in the rostrad and caudad direction. In the trunk region where the neuroepithelium bulges adjacent to the somites, the edges of the folds pulsate and forcefully touch-retract-touch in these bulging regions; the intersomitic epithelia retract, remain open even after more posterior somitic regions have apposed, and then close slowly. Epithelial blebs and N-CAM antibody were observed at the leading edges of the neuroepithelia. Between the open folds only a few bridging cells were seen; they probably represent the sites of initial cell adhesion following epithelial retraction. Focusing into the developing embryo shows that neuroepithelial fusion occurs prior to surface epithelial fusion. A meshwork of synchronously pulsating neural crest cells was identified below the surface epithelium and a preliminary investigation of their initial migration was conducted.

The effects of chorion type on normal and abnormal developmental variation in monozygous twins.

To determine the effects, if any, of chorion type on normal and abnormal developmental variation in monozygous (MZ) twins, we tested the hypothesis that disparate environments that are related to chorion type have no effect on this variation. The parameters studied included congenital anomalies and dermatoglyphics (total ridge count and right-left asymmetry). With the exception of total ridge count, analyses of these data failed to reject the null hypothesis. Dichorionic MZ twins had a significantly greater within-pair variation than monochorionic MZ twins for total ridge count. In summary, then, these data could offer little support to prior speculation that monochorial placenta may present less favorable environments for feta development.