Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

Maturation of oral feeding skills in preterm infants.

AIM: Safe and successful oral feeding requires proper maturation of sucking, swallowing and respiration. We hypothesized that oral feeding difficulties result from different temporal development of the musculatures implicated in these functions. METHODS: Sixteen medically stable preterm infants (26 to 29 weeks gestation, GA) were recruited. Specific feeding skills were monitored as indirect markers for the maturational process of oral feeding musculatures: rate of milk intake (mL/min); percent milk leakage (lip seal); sucking stage, rate (#/s) and suction/expression ratio; suction amplitude (mmHg), rate and slope (mmHg/s); sucking/swallowing ratio; percent occurrence of swallows at specific phases of respiration. Coefficients of variation (COV) were used as indices of functional stability. Infants, born at 26/27- and 28/29-week GA, were at similar postmenstrual ages (PMA) when taking 1-2 and 6-8 oral feedings per day. RESULTS: Over time, feeding efficiency and several skills improved, some decreased and others remained unchanged. Differences in COVs between the two GA groups demonstrated that, despite similar oral feeding outcomes, maturation levels of certain skills differed. CONCLUSIONS: Components of sucking, swallowing, respiration and their coordinated activity matured at different times and rates. Differences in functional stability of particular outcomes confirm that maturation levels depend on infants' gestational rather than PMA.

THE ROLE OF MAP KINASES ON THE FUNCTIONAL HETEROGENEITY OF HUMAN CD8+ T CELL MATURATION SUBSETS

While prior studies have focused on naïve (CD45RA+CD27+) and early stage memory (CD45RA-CD27+) CD8+ T cells, late memory CD8+ T cells (CD45RA+CD27) have received less interest because this subset of T cells is generally recognized as effectors, which produce IFNγ (but no IL-2) and perforin. However, multiple studies suggest that late memory CD8+ T cells may provide inadequate protection in infectious diseases and cancer models. To better understand the unique function of late memory CD8+ T cells, I optimized multi-color flow cytometry techniques to assess the cytokine production of each human CD8+ T cell maturation subset. I demonstrated that late memory CD8+ T cells are the predominant producer of CC chemokines (e.g. MIP-1β), but rarely produce IL-2; therefore they do not co-produce IL-2/IFNγ (polyfunctionality), which has been shown to be critical for protective immunity against chronic viral infection. These data suggest that late memory CD8+ T cells are not just cytotoxic effectors, but may have unique functional properties. Determining the molecular signature of each CD8+ T cell maturation subset will help characterize the role of late memory CD8+ T cells. Prior studies suggest that ERK1 and ERK2 play a role in cytokine production including IL-2 in T cells. Therefore, I tested whether differential expression of ERK1 and ERK2 in CD8+ T cell maturation subsets contributes to their functional signature by a novel flow cytometry technique. I found that the expression of total ERK1, but not ERK2, is significantly diminished in late memory CD8+ T cells and that ERK1 expression is strongly associated with IL-2 production and CD28 expression. I also found that IL-2 production is increased in late memory CD8+ T cells by over-expressing ERK1. Collectively, these data suggest that ERK1 is required for IL-2 production in human CD8+ T cells. In summary, this dissertation demonstrated that ERK1 is down-regulated in human late memory CD8+ T cells, leading to decreased production of IL-2. The data in this dissertation also suggested that the functional heterogeneity in human CD8+ T cell maturation subsets results from their differential ERK1 expression.

Cryo-electron tomography of Kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the gamma-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain 3D reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of "B-capsid-like" particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detailed pathway of herpesvirus capsid assembly and maturation.

An examination of the characteristics, concentration and distribution of androgen receptor in rat testis during sexual maturation

Previously reported androgen receptor concentrations in rat testis and testicular cell types have varied widely. In the studies reported here a nuclear exchange assay was established in rat testis in which exchange after 86 hours at 4$\sp\circ$C was greater than 85% complete and receptor was stable. Receptor concentration per DNA measured by exchange declined between 15 and 25 days of age in the rat testis, then increased 4-fold during sexual maturation. Proliferation of germ cells which had low receptor concentration appeared to account for the early decline in testicular receptor concentration, whereas increase in receptor number per Sertoli cell between 25 and 35 days of age contributed to the later increase. Increase in Leydig cell number during maturation appeared to account for the remainder of the increase due to the high receptor concentration in these cells. Detailed studies showed that other possible explanations for changes in receptor number (e.g. shifts in receptor concentration between the cytosol and nuclear subcellular compartments or changes in the affinity of the receptor for its ligands) were not likely.^ Androgen receptor dynamics in testicular cells showed rapid, specific uptake of ($\sp3$H) -testosterone that was easily blocked by unlabeled testosterone (RA of 7 nM in both cell types), and medroxyprogesterone acetate (RA of 28 and 16 nM in Sertoli and peritubular cells, respectively), but not as well by the anti-androgens cyproterone acetate (RA of 116 and 68 nM) and hydroxyflutamide (RA of 300 and 180 nM). The affinity of the receptor for the ligand dimethylnortestosterone was similar in the two cell types (K$\rm\sb{d}$ values of 0.78 and 0.71 nM for Sertoli and peritubular cells) and was virtually identical with the affinity of the whole testis receptor (0.89 nM). Medroxyprogesterone acetate and testosterone significantly increased nuclear androgen receptor concentration relative to untreated controls in Sertoli and peritubular cells, whereas hydroxyflutamide and cyproterone acetate did not. Despite the different embryological origins of peritubular and Sertoli cells, their responses to both androgens and anti-androgens were similar. In addition, these studies suggest that peritubular cells are as likely as Sertoli cells to be primary androgen targets. ^

Molecular basis of maturation promoting factor

Maturation promoting factor (MPF), which is functionally defined by its ability to induce Xenopus oocyte maturation, is an M phase (meiosis and mitosis) specific activity that is present in all species tested. It was hypothesized that MPF is a universal trigger of the interphase to M phase transition during the cell cycle. The current model for the molecular basis of MPF is that MPF is a protein kinase having the cdc2 protein as its catalytic subunit and is identical to the M phase-specific histone H1 kinase. In the present study, I have shown that more than just cdc2 kinase contributes to MPF activity, and M phase-specific H1 kinase is composed of at least two entities, instead of just cdc2 kinase. Therefore, the simple model of MPF = cdc2 kinase = M phase-specific H1 kinase should be ruled out.^ My study began with the characterization of the mitosis-specific monoclonal antibody MPM-2. MPM-2 reacts specifically with M phase cells from different species by recognizing a discrete set of proteins once they are phosphorylated at the G$\sb2$/M transition. I found that phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity during oocyte maturation stimulated by progesterone. If MPM-2 was injected into oocytes before the stimulation, MPF activity failed to appear, and the oocytes could not mature. Furthermore, MPM-2 was able to deplete MPF activity from M phase extracts. These results identified MPM-2 as a probe that recognizes either MPF itself or a regulator of MPF.^ Since M phase-specific H1 kinase was believed to be identical to cdc2 kinase and MPF, I proceeded to determine whether MPM-2 recognized the M phase-specific H1 kinase. I found that MPM-2 did recognize an M phase-specific H1 kinase. However, this kinase was not cdc2 kinase. This kinase (MPM-2 kinase) is present in a latent form in immature oocytes and is activated in tandem with the activation of MPF during oocyte maturation. It appears to accelerate progesterone-induced oocyte maturation. Therefore, MPM-2 kinase may be a novel positive regulator of MPF activation.^ MPM-2 depletes MPF activity, but not cdc2 kinase activity. This discrepancy caused me to question the equivalency of MPF with cdc2 kinase. I found that when a high percentage of MPF activity was recovered from gel filtration of mature oocyte extract, the recovered MPF activity was due to two factors, cdc2 kinase and a factor recognized by MPM-2. This factor might activate and stabilize cdc2 kinase. Identification of this factor in the present study may contribute to the understanding of the autoactivation of MPF. ^

Xp95 is phosphorylated within the proline rich domain during Xenopus oocyte maturation

Xp95 is the Xenopus ortholog of a conserved family of scaffold proteins that have in common an N-terminal Bro1 domain and a C-terminal proline rich domain (PRD). The regulation of this protein family is poorly understood. We previously showed that Xp95 undergoes a phosphorylation-dependant gel mobility shift during meiotic maturation of Xenopus oocytes, the only natural biological system in which post-translational modifications of this family has been demonstrated. Here we characterized Xp95 phosphorylation via two approaches. First, we tested a series of Xp95 fragments for the ability to gel-shift during oocyte maturation, and found that a fragment containing amino acids 705-786 is sufficient to cause a gel-shift. This fragment is within the N-terminal region of Xp95's PRD (N-PRD). Second, we purified phosphorylated Xp95 and by mass spectrometry found that a 5080 Da peptide which maps to N-PRD (amino acids 706-756) contains two phosphorylation sites, one of which is T745, within the conserved CIN85 binding motif. By in vitro protein interaction assays, we that T745 is critical for CIN85/Xp95 interaction, and that Xp95 phosphorylation correlates with loss of binding to CIN85. We also show that an Alix fragment (amino acids 604-789) also undergoes a gel-shift during oocyte maturation and during colcemid-induced mitotic arrest of HeLa cells. These findings indicate that Xp95/Alix is phosphorylated on the PRD during M phase induction and that the PRD phosphorylation regulates partner protein interaction. ^

FEMALE SEXUAL MATURATION AND BLOOD PRESSURE (SECONDARY-SEX CHARACTERISTICS, GROWTH, DEVELOPMENT)

This study described the relationship of sexual maturation and blood pressure in a sample (n = 361) of white females, ages seven through 18, attending public schools in a defined area of Central Texas during October through December, 1984. Other correlates of blood pressure were also described for this sample.^ A survey was performed to obtain the data on height, weight, body mass, pulse rate, upper arm circumference and length, and blood pressure. Each subject self-assessed her secondary sex characteristics (breast and pubic hair) according to drawings of the Tanner stages of maturation. The subjects were interviewed to obtain data on personal health habits and menstrual status. Student age, ethnic group and place of residence were abstracted from school records. Parents or guardians of the subjects responded to a questionnaire pertaining to parental and subject health history and parents' occupation and educational attainment.^ In the simple linear regression analysis, sexual maturation and variables of body size were significantly (p < 0.001) and positively associated with systolic and fourth- and fifth-phase diastolic blood pressure. The demographic and socioeconomic variables were not sufficiently variant in this population to have differential effects on the relation between blood pressure and maturation. Stepwise multiple regression was used to assess the contribution of sexual maturation to the variance of blood pressure after accounting for the variables of body size. Sexual maturation (breast stage) along with weight, height and body mass remained in the multiple regression models for fourth- and fifth-phase diastolic blood pressure. Only height and body mass remained in the regression model for systolic blood pressure; sexual maturation did not contribute more to the explanation of the systolic blood pressure variance.^ The association of sexual maturation with blood pressure level was established in this sample of young white females. More research is needed first, to determine if this relationship prevails in other populations of young females, and second, to determine the relationship of sexual maturation sequence and change with the change of blood pressure during childhood and adolescence. ^

BLOOD PRESSURE CHANGES IN RELATION TO DEMOGRAPHIC, ANTHROPOMETRIC, AND SEXUAL MATURATION VARIABLES IN U.S. HEALTH EXAMINATION SURVEY OF CHILDREN AND YOUTHS (UNITED STATES)

The determinants of change in blood pressure during childhood and adolescence were studied in a cohort of U.S. national probability sample of 2146 children examined on two occasions during the Health Examination Survey. Significant negative correlations between the initial level and the subsequent changes in blood pressure were observed. The multiple regression analyses showed that the major determinants of systolic blood pressure (SBP) change were change in weight, baseline SBP, and baseline upper arm girth. Race, time interval between examinations, baseline age, and height change were also significant determinants in SBP change. For the change in diastolic blood pressure (DBP), baseline DBP, baseline weight, and weight change were the major determinants. Baseline SBP, time interval and race were also significant determinants. Sexual maturation variables were also considered in the subgroup analysis for girls. Weight change was the most important predictor of the change in SBP for the group of girls who were still in the pre-menarchal or pre-breast maturation status at the time of the follow-up examination, and who had started to menstruate or to develop breast maturation at sometime between the two examinations. Baseline triceps skinfold thickness or initial SBP were more important variables than weight change for the group of girls who had already experienced menarche or breast maturation at the time of the initial survey. For the total group, pubic hair maturation was found to be a significant predictor of SBP change at the 5% significance level. The importance of weight change and baseline weight for the changes in blood pressure warrants further study. ^

Longitudinal and logistic analysis of endocrine hormone profiles, physical maturation, and candidate genes related to pubertal events in children

Children who experience early pubertal development have an increased risk of developing cancer (breast, ovarian, and testicular), osteoporosis, insulin resistance, and obesity as adults. Early pubertal development has been associated with depression, aggressiveness, and increased sexual prowess. Possible explanations for the decline in age of pubertal onset include genetics, exposure to environmental toxins, better nutrition, and a reduction in childhood infections. In this study we (1) evaluated the association between 415 single nucleotide polymorphisms (SNPs) from hormonal pathways and early puberty, defined as menarche prior to age 12 in females and Tanner Stage 2 development prior to age 11 in males, and (2) measured endocrine hormone trajectories (estradiol, testosterone, and DHEAS) in relation to age, race, and Tanner Stage in a cohort of children from Project HeartBeat! At the end of the 4-year study, 193 females had onset of menarche and 121 males had pubertal staging at age 11. African American females had a younger mean age at menarche than Non-Hispanic White females. African American females and males had a lower mean age at each pubertal stage (1-5) than Non-Hispanic White females and males. African American females had higher mean BMI measures at each pubertal stage than Non-Hispanic White females. Of the 415 SNPs evaluated in females, 22 SNPs were associated with early menarche, when adjusted for race ( p<0.05), but none remained significant after adjusting for multiple testing by False Discovery Rate (p<0.00017). In males, 17 SNPs were associated with early pubertal development when adjusted for race (p<0.05), but none remained significant when adjusted for multiple testing (p<0.00017). ^ There were 4955 hormone measurements taken during the 4-year study period from 632 African American and Non-Hispanic White males and females. On average, African American females started and ended the pubertal process at a younger age than Non-Hispanic White females. The mean age of Tanner Stage 2 breast development in African American and Non-Hispanic White females was 9.7 (S.D.=0.8) and 10.2 (S.D.=1.1) years, respectively. There was a significant difference by race in mean age for each pubertal stage, except Tanner Stage 1 for pubic hair development. Both Estradiol and DHEAS levels in females varied significantly with age, but not by race. Estradiol and DHEAS levels increased from Tanner Stage 1 to Tanner Stage 5.^ African American males had a lower mean age at each Tanner Stage of development than Non-Hispanic White males. The mean age of Tanner Stage 2 genital development in African American and Non-Hispanic White males was 10.5 (S.D.=1.1) and 10.8 (S.D.=1.1) years, respectively, but this difference was not significant (p=0.11). Testosterone levels varied significantly with age and race. Non-Hispanic White males had higher levels of testosterone than African American males from Tanner Stage 1-4. Testosterone levels increased for both races from Tanner Stage 1 to Tanner Stage 5. Testosterone levels had the steepest increase from ages 11-15 for both races. DHEAS levels in males varied significantly with age, but not by race. DHEAS levels had the steepest increase from ages 14-17. ^ In conclusion, African American males and females experience pubertal onset at a younger age than Non-Hispanic White males and females, but in this study, we could not find a specific gene that explained the observed variation in age of pubertal onset. Future studies with larger study populations may provide a better understanding of the contribution of genes in early pubertal onset.^

Expression of TNF-$\alpha$ protein and C -FOS and TNF mRNA during human peripheral blood monocyte maturation and activation

Human peripheral blood monocytes (HPBM) were isolated by centrifugal elutriation from mononuclear cell enriched fractions after routine plateletapheresis and the relationship between maturation of HPBM to macrophage-like cells and activation for tumoricidal activity determined. HPBM were cultured for various times in RPMI 1640 supplemented with 5% pooled human AB serum and cytotoxicity to $\sp{125}$IUDR labeled A375M, a human melanoma cell line, and TNF-$\alpha$ release determined by cytolysis of actinomycin D treated L929 cells. Freshly isolated HPBM or those exposed to recombinant IFN-$\gamma$(1.0 U/ml) were not cytolytic and did not release TNF-$\alpha$ into culture supernatants. Exposure to bacterial lipopolysaccharide (LPS, 1.0 $\upsilon$g/ml) stimulated cytolytic activity and release of TNF-$\alpha$. Maximal release of TNF-$\alpha$ protein occurred at 8 hrs and returned to baseline by 72 hrs. Expression of TNF-$\alpha$ protein was determined by Western blotting. Neither freshly isolated nor IFN-$\gamma$ treated HPBM expressed TNF protein at any time during in vitro culture. LPS treated HPBM maximally expressed the 17KD TNF-$\alpha$ protein at 8 hrs, and protein was not detected after 36 hrs of in vitro culture. Expression of TNF-$\alpha$ mRNA was determined by Northern blotting. Freshly isolated HPBM express TNF-$\alpha$ mRNA which decays to basal levels by 6 hrs of in vitro culture. IFN-$\gamma$ treatment maintains TNF-$\alpha$ mRNA expression for up to 48 hrs of culture, after which it is undetectable. LPS induces TNF-$\alpha$ mRNA after 30 minutes of exposure with maximal accumulation occurring between 4 to 8 hrs. TNF mRNA was not detected in control HPBM at any time after 6 hrs or IFN-$\gamma$ treated HPBM after 48 hrs of in vitro culture. A pulse of LPS the last 24 hrs of in vitro culture induces the accumulation of TNF-$\alpha$ mRNA in HPBM cultured for 3, 5, and 7 days, with the magnitude of induction decreasing approximately 10 fold between 3 and 7 days. Induction of TNF-$\alpha$ mRNA occurred in the absence of detectable TNF-$\alpha$ protein or supernatant activity. Maturation of HPBM to macrophage-like cells controls competence for activation, magnitude and duration of the activation response. ^

Cloning and molecular analysis of paraxis: A {\it trans\/}-acting factor required for somite maturation

During vertebrate embryogenesis, cells from the paraxial mesoderm coalesce in a rostral-to-caudal progression to form the somites. Subsequent compartmentalization of the somites yields the sclerotome, myotome and dermatome, which give rise to the axial skeleton, axial musculature, and dermis, respectively. Recently, we cloned a novel basic-Helix-Loop-Helix (bHLH) protein, called scleraxis, which is expressed in the sclerotome, in mesenchymal precursors of bone and cartilage, and in connective tissues. This dissertation focuses on the cloning, expression and functional analysis of a bHLH protein termed paraxis, which is nearly identical to scleraxis within the bHLH region but diverges in both its amino and carboxyl termini. During the process of mouse embryogenesis, paraxis transcripts are first detected at about day 7.5 post coitum within the primitive mesoderm lying posterior to the head and heart primordia. Subsequently, paraxis expression progresses caudally through the paraxial mesoderm, immediately preceding somite formation. Paraxis is expressed at high levels in newly formed somites before the first detectable expression of the myogenic bHLH genes, and as the somite becomes compartmentalized, paraxis becomes downregulated within the myotome.^ To determine the function of paraxis during mammalian embryogenesis, mice were generated with a null mutation in the paraxis locus. Paraxis null mice survived until birth, but exhibited severe foreshortening along the anteroposterior axis due to the absence of vertebrae caudal to the midthoracic region. The phenotype also included axial skeletal defects, particularly shortened bifurcated ribs which were detached from the vertebral column, fused vertebrae and extensive truncation and disorganization caudal to the hindlimbs. Mutant neonates also lacked normal levels of trunk muscle and exhibited defects in the dermis as well as the stratification of the epidermis. Analysis of paraxis -/- mutant embryos has revealed a failure of the somites to both properly epithelialize and compartmentalize, resulting in defects in somite-derived cell lineages. These results suggest that paraxis is an essential component of the genetic pathway regulating somitogenesis. ^