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This handbook for secondary teachers contains activities, exercises, and strategies designed to make more obvious and concrete the opportunities local communities and states provide for students to learn about and to participate in global affairs. A major objective is to help high school students develop the competencies needed to identify and to assess the significance of the contacts and connections between life in their states and hometowns and life in cities and villages around the world. Each of the 33 activities included in the handbook is self-contained with objectives, materials needed, and suggested procedures for classroom use. Many different kinds of activities are included. For example, in one activity students use a questionnaire to survey local civic, service, or religious organizations to determine ways they are linked to the world. Students organize the data from the questionnaires into charts and tables which they then analyze. In the "Take a Trip Abroad" activity students fill out a passport application, make plans for an imaginary trip, keep a record of their trip, and analyze passport information for their state. Other activities help students assess the impact of foreign students, recent immigrants, and ethnic restaurants on Indiana and its communities. In addition, ethnic ties and language influences are examined. Although the handbook was written for Indiana teachers, teachers from other states can easily use or adapt the activities. (Author/RM)

The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself - and motor organization on the cargo-affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their 'on' rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.

Textbook chapter outlining the molecular pathways of sex determination

Textbook chapter covering the molecular mechanisms of sex determination and differentiation

Niemann Pick Type C (NPC) disease is a rare genetic disease which is most often diagnosed in children, causes tragic irreversible neurologic deterioration, and is universally fatal. Many therapies and treatments are in development and would benefit from improved methods of assessing disease progression and treatment response. A large amount of NPC research is carried out in animal models such as the Npc1^(-/-) mouse model of the most common type of NPC disease, NPC1. This dissertation investigates three methods of noninvasive assessments of disease state in the Npc1^(-/-) mouse model with the use of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS).MRI and MRS provide safe and widely available methods of measuring and visualizing internal tissue characteristics, suitable for longitudinal studies of disease progression and response to therapy. In this work, disease-associated dysmyelination of white matter tracts in the brain of Npc1^(-/-) mice was quantitatively measured at multiple time points with MRI methods of diffusion tensor imaging (DTI) and T2-mapping. These quantitative in vivo measures of disease status show promise as biomarkers for use in future studies of disease progression and treatment response in NPC disease models. High resolution MRI data was also collected and analyzed at multiple time points to quantify differences in both global and regional brain volumes in the Npc1^(-/-) mice as brain atrophy develops with disease progression. MRS was utilized to quantitatively examine changes in brain metabolite levels previously reported in clinical NPC disease studies. The results of the MRI and MRS studies in the Npc1^(-/-) mouse model demonstrate the ability to quantify changes in the brain due to neurodegeneration at multiple time points along the progression of neurological Npc1^(-/-) disease. MRI methods of quantifying white matter pathology with currently available DTI and T2-mapping techniques appear to be promising in vivo

Despite recent advances in lymphology, the molecular mechanisms regulating the development of the lymphatic system have not been fully delineated. Here I show that the growth factors Angiopoietin-2 (Ang2) and Vascular endothelial growth factor-c (Vegfc) serve distinct roles in the development of the lymphatic system.Adult Ang2-/- mice exhibit dermal lymphatic hypoplasia, abnormal smooth muscle cell (SMC) coverage of initial lymphatic vessels, and a severe deficiency of collecting lymphatic vessels. To determine whether these abnormalities were due to defects in the remodeling of the lymphatic vasculature, I characterized this process in Ang2-/- mice. Indeed, lymphatic vessels in the skin of Ang2-/- pups prematurely recruited SMCs and did not mature into collecting vessels during the remodeling stage of lymphatic development. In contrast, Ang2 knockout Ang1 knock-in mice did develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. To further delineate the molecular mechanisms regulating lymphatic development, I also characterized Chylous ascites-3 (Chy-3) mice, a strain missing cytobands 8A4 to 8B3 from one copy of chromosome 8. Real-time PCR using genomic DNA demonstrated that the lymphangiogenesis gene, Vegfc, was included in the deleted region. All of the key components of a normal lymphatic network had developed in Chy-3 mice; however, the number of lymphatic vessels was reduced. Although the patterning of lymphatic vasculature in Chy-3 mice was altered, the architecture of the blood vasculature appeared normal. Ang2-/- and Chy-3 mice are grossly indistinguishable from one another; however, their underlying lymphatic defects are dramatically different. Ang2-/- mice exhibit defects in lymphangiogenic remodeling and lymphangiogenesis, whereas Chy-3 mice show a reduced capacity for lymphangiogenesis. I propose that Ang2 maintains lymphatic vessel plasticity by preventing SMC-lymphatic vessel associ

Five patients were previously described with the Opitz (GBBB) syndrome (OMIM 145410) phenotype and 22q11.2 deletion determined by FISH but the precise limits of their deletions have not been determined. Since one locus for Opitz syndrome maps to 22q11.2 and chromosomal arrangements are frequently complex and could inactivate such a locus, we performed high-resolution array-based comparative genomic hybridization (CGH) on a new Opitz syndrome-like phenotype patient with a 22q11.2 deletion. He shares the same deletion as patients with velocardiofacial and DiGeorge syndrome.

The possible role of GATA/GACA repeated sequences in mammalian sex determination was investigated using Northern analyses of mouse and human RNA. Brain, liver, and gonadal RNA from three developmental stages of mice of both sexes and also human fetal RNA from various tissues were hybridized to both sense and antisense Bkm riboprobes as well as to the synthetic oligonucleotide (GATA) 5 . At low levels of stringency, putative transcripts of various sizes were observed in all tissue samples with all probes. At high stringency, only a putative transcript of approximately 12 kb was observed, but this was later shown to consist of contaminating DNA. No sex-specific differences were observed in any tissue or developmental stage. Thus, we find no evidence that the GATA/GACA repeated sequences are specifically expressed in quantities detectable by Northern analyses in a manner important to mammalian sex determination.

The DNA probe Y97 was derived from a repeat sequence in the human Y centromere, a region which must be present in a mitotically functional Y chromosome. We have demonstrated that Y97, which detects a Y-specific 5.5-kb Eco RI fragment by Southern analysis, is very useful for the molecular detection of small amounts of Y-derived material and represents a significant improvement over previous tests for molecular diagnosis of sex. The male-female difference in hybridization was unequivocal even when only 25 ng of total DNA was used per lane. Furthermore, in mixing experiments the 5.5-kb Eco RI fragment was detectable even when only 5% of the total DNA was male. By increasing hybridization stringency, we have developed a rapid, sensitive, and accurate method to detect Y chromosomal DNA in unrestricted samples.

In a search for genetic differences in susceptibility to cleft palate, congenic and recombinant inbred strains of mice were treated with 6-aminonicotinamide or control injections. Of six loci tested, only the chromosome segment marked by N-acetyl transferase was found to affect susceptibility to 6-aminonicotinamide-induced cleft palate. This chromosome segment is known to affect glucocorticoid-induced cleft palate and phenytoin-induced cleft lip with or without cleft palate in these strains of mice.

In order to obtain a genomic clone of Zfy-1 from a Y chromosome of (YDOM) origin, we cloned size-fractionated SJL/J DNA in EMBL-4 and selected colonies which hybridized to pDP1007, a human zinc finger Y clone. The specificity of the clone in hybridizations to mouse and human DNA and partial sequencing confirmed that the clone (subcloned as pGZfy1D) was of Zfy-1 origin. Studies on the expression during testicular development of mRNAs hybridizing to the clone suggested that the gene is expressed post-meiotically.

Several recent studies indicate that the von Recklinghausen neurofibromatosis (NF1) gene is located near the centromere of chromosome 17 in some families. However, variable expressivity and a very high mutation rate suggest that defects at several different loci could result in phenotypes categorized as NF1. In order to assess this possibility and to map the NF1 gene more precisely, we have used two polymorphic DNA markers from chromosome 17 to screen several pedigrees for linkage to NF1. We ascertained a large Caucasian pedigree (33 individuals sampled, 17 NF1 affected) as well as eight smaller pedigrees and nuclear families (50 individuals sampled, 30 NF1 affected). Here, we report strong evidence of linkage of NF1 to the centromeric marker D17Z1 (maximum LOD = 4.42) and a weaker suggestion of linkage to the ERBA1 oncogene (maximum LOD = 0.57), both at a recombination fraction of zero. Since obligate crossovers with NF1 were not observed for either marker in any of the informative families tested, the possibility of NF1 locus heterogeneity is not supported.

The effects of the mouse major histocompatibility complex, H-2, on phospholipid methyltransferase I and II activities were investigated on hepatocyte membranes from inbred, congenic and recombinant strains. Each methyltransferase was assayed individually by measuring the incorporation of radiolabel from into endogenous phospholipids. Our results indicate that H-2 exerted a significant effect on methyltransferase I but not on methyltransferase II activity. Thus, as in lower eukaryotes, two distinct enzymes were involved in methylation of phosphotidylethanolamine (PE) to phosphatidylcholine (PC). In addition, this effect was localized to the K end of the major histocompatibility complex by the use of recombinant hepalotypes.

The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.

We have examined effects of an autosomal gene on the expression of serologically detectable H-Y, a male-specific minor histocompatibility antigen for which a role in primary sex determination has been postulated. Quantitative immunoabsorption studies using three different male specific antisera revealed variation in the level of H-Y among inbred mouse strains and mice bearing variants of the T/t complex. Two patterns of expression in inbred strains were observed; high, C57BL/6J (B6) and low, which included A/J (A), C3H/DiSn and DBA/2J. In F1 hybrids between A (female parent) and B6 (male parent), the F1 expressed H-Y levels indistinguishable from A, the low parent. Among T/t complex variants, mice bearing T or t0 showed high expression whereas those of the t12 haplotype showed low expression. The co-segregation of H-Y expression types with these T/t complex variants provided further evidence for the location of a gene determining the level of H-Y antigen on the proximal portion of chromosome 17.

It has been demonstrated that susceptibility to glucocorticoid-induced formation of cleft palate is regulated by the mouse histocompatibility complex (H-2). This has encouraged us to examine H-2 effects on glucocorticoid binding in tissues of adult animals which would provide sufficient material with which to study the biochemical mechanism of the H-2 effect. Although it has been reported that cytosol prepared from lungs of adult mice with a high susceptibility to steroid-induced cleft palate formation have a higher level of glucocorticoid binding than lung cytosol prepared from a low-susceptibility strain, we are unable to demonstrate any influence of H-2 on binding capacity in this tissue from adult animals when glucocorticoid receptors are assayed in the presence of receptor reducing and stabilizing agents that maximize binding capacity. Cytosol prepared from rat liver contains an endogenous receptor-reducing system composed of NADPH and thioredoxin. It has also been reported that the murine H-2 complex contains a gene(s) that regulates the level of a modifier(s) in fetal hepatic cytosol that affects the binding of glucocorticoids to the receptor. Of two known low molecular weight modifiers that could account for this effect, we have previously established that the heat-stable, steroid receptor "modulator" is not regulated by the H-2 complex. In the present work we have assayed thioredoxin, a second potential modifier, in liver cytosols prepared from adults of two pairs of two H-2 congenic mouse strains. Our results show that the amount of thioredoxin is the same in all four mouse strains and that it is not regulated by the H-2 locus. At this time, we are unable to identify a system in adult mice in which the widely reported regulation of glucocorticoid binding by the mouse histocompatibility locus can be submitted to definitive biochemical study.

The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [3H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.

The development of a spermatozoon (sperm) from a spermatid involves a complex process of differentiation during which a variety of new gene products appear. It has been generally assumed that no genetic transcription occurs after meiosis and, if this were so, that all the new sperm proteins would have to be transcribed from stored messenger RNA. However, the biochemical evidence suggests that there is no abrupt change in the rate of RNA synthesis during meiosis and that qualitative changes in RNA synthesis, to the extent that they are known, favor the likelihood of continuing messenger RNA synthesis. Experimental analyses of distorted transmission ratios of t-alleles and unbalanced chromosomal states in males also suggest that genes are expressed in haploid nuclei after meiosis. It is probable that spermatozoa are functionally equivalent in most respects because of intercellular bridges that create a continuous cytoplasm between developing spermatozoa, facilitating an exchange of most post-meiotic gene products. Plasma membrane proteins which are potential antigens might not be shared across the intercellular bridges but the evidence to date for haploid expression of sperm antigens is poor.

It has recently been suggested that the level of the endogenous glucocorticoid receptor stabilizing factor in mouse liver is regulated by the major hisiocompatibility, H-2. complex (Katsumata et al. [1]). We have developed an asssay for the activity of the endogenous heat-stable factor in mouse liver and have assayed this factor in liver cytosols prepared from two pairs of two H-2 congenic mouse strains. Our results show that the amount of the endogenous factor is the same in all four mouse strains and that it is not regulated by the H-2 locus.

In order to define the nature of the genetic lesion which gave rise to Sxr, the sex-limited autosomal dominant sex reversal condition in the mouse

We have investigated the changes occurring in the pattern of translatable mRNA species in germ cells during spermatogenesis of mice. Highly homogeneous cell populations of spermatocytes or spermatids were purified. Poly(A)+ mRNA was isolated from each cell population by oligo(dT)-cellulose chromatography. A comparison of meiotic and post-meiotic mRNAs was made by two-dimensional gel analyses of their in vitro synthesized translation products. Among spots identified in the fluorograms of two-dimensional gels, a number of qualitatively new proteins appeared after meiosis. The results suggest that some of poly(A)+ mRNAs are transcribed post-meiotically in haploid germ cells.

The present dissertation investigated the possible role of proteoglycans (PGs) in acetycholine receptor (AChR) clustering on cultured C2 myotubes. Analysis of variant muscle cell lines and their hybrid products supported the hypothesis that PGs are required for the clustering of AChRs. Three PG-defective genetic variants derived from the C2 cell line form myotubes but fail to spontaneously cluster AChRs. The three variants show different broad-spectrum defects in glycosaminoglycan (GAG) biosynthesis and are especially deficient in the synthesis of chondroitin sulfate (CS) chain. Formation of heterokaryon myotubes containing nuclei from two different variants spontaneously clustered AChRs and recovered synthesis of GAGs, especially of CS. It strongly suggests that there is a requirement for proper GAG biosynthesis in AChR clustering. Chlorate was found to inhibit both GAG synthesis and the clustering of AChRs in a dose-dependent manner. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild type C2 myotubes, both the frequency of spontaneous AChR clusters and the level of CS were increased. Culture of wild type C2 myotubes in the presence of chondroitinase ABC eliminated CS and prevented the formation of AChR clusters. Treatment with chondroitinase ABC only prevented AChR clustering if begun prior to the formation of spontaneous clusters. This suggests that CS is required in the initiation but not the maintenance of AChR clusters. Agrin-induced AChR clustering was dramatically reduced by digestion of CS. Unlike calcium, however, agrin action on AChR clustering did not affect levels of CS. Also, agrin-induced AChR clustering was restored on S27 myotubes by adding calcium-treated C2 conditioned medium. The IIH6 monoclonal antibody against α-dystroglycan, laminin, and agrin all bound to a specific fraction of C2 cell extracts separated on ion exchange chromatography. Also IIH6, laminin, and agrin affinity-precipitations showed a smeared sulfate lab

Various aspects of the neural bases of taste were studied in rats. The main data were of the activity of single neurons in the peripheral nerve (chorda tympani) and medulla (nucleus of the solitary tract), although some studies were made of central taste projections with the 2-DG method, and data were obtained of the electrical-responses in the cerebellum to simulation of oral structures. The data were treated mathematically to determine various aspects of their organization; previous to this, such treatments were largely lacking, leading to conclusions contested in the present research. The present findings were that the previous conclusions that taste is organized in terms of 'four primary tastes' is an artifact of the methods used in characterizing the data; instead, the underlying organization is more continuous, as in the frequency dimension in audition, or wavelength in vision. This conclusion holds for both the stimulus and neural domains. (Author)