Endogenous tissue engineering: PTH therapy for skeletal repair

Based on its proven anabolic effects on bone in osteoporosis patients, recombinant parathyroid hormone (PTH_1-34) has been evaluated as a potential therapy for skeletal repair. In animals, the effect of PTH_1-34 has been investigated in various skeletal repair models such as fractures, allografting, spinal arthrodesis and distraction osteogenesis. These studies have demonstrated that intermittent PTH_1-34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms, which include effects on mesenchymal stem cells, angiogenesis, chondrogenesis, bone formation and resorption. Furthermore, PTH_1-34 has been shown to enhance bone repair in challenged animal models of aging, inflammatory arthritis and glucocorticoid-induced bone loss. This pre-clinical success has led to off-label clinical use and a number of case reports documenting PTH_1-34 treatment of delayed-unions and non-unions have been published. Although a recently completed phase 2 clinical trial of PTH_1-34 treatment of patients with radius fracture has failed to achieve its primary outcome, largely because of effective healing in the placebo group, several secondary outcomes are statistically significant, highlighting important issues concerning the appropriate patient population for PTH_1-34 therapy in skeletal repair. Here, we review our current knowledge of the effects of PTH_1-34 therapy for bone healing, enumerate several critical unresolved issues (e.g., appropriate dosing regimen and indications) and discuss the long-term potential of this drug as an adjuvant for endogenous tissue engineering.     

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells

Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH₂-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH₂-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.     

Isolation and Characterization of a Photosystem II Core Complex Depleted in the 43 kDa-Chlorophyll-Binding Subunit

The photosystem II core complex purified from digitonin extractsof spinach chloroplasts was resolved into two chlorophyll-proteincomplexes by digitonin polyacrylamide gel electrophoresis aftertreatment with 1 M potassium thiocyanate. One of the chlorophyll-proteincomplexes resolved consisted of 47, 32, 30 and 9 kDa polypeptidesand the other was complementally composed of only the 43 kDapolypeptide. The former complex was highly active in the photoreductionof 2, 6-dichlorophenol indophenol by 1,5-diphenylcarbazide andretained all of the components responsible for the electrontransport from the secondary electron donor (Z) to the primaryelectron acceptor (Q_A). EPR signal II_fast and II_slow were alsopreserved in this complex although their hyperfine structureswere largely modified. The complex was estimated to contain1.8 molecules of plastoquinone A as well as 1.5, 3.7 and 3.9molecules of cytochrome b559, pheophytin and ß-carotene,respectively, per Q_A. These results indicate that potassiumthiocyanate specifically removes the 43 kDa polypeptide fromthe PS II core complex leaving the electron transport systemin an almost intact state. (Received June 17, 1987; Accepted October 23, 1987)     

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Cotyledons detached from light-grown radish (Raphanus sativusL. cv. Comet) seedlings were used as a model system to studythe changes in nuclear gene expression during dark-induced senescenceof green leaves. Polyadenylated RNA was prepared from the cotyledonsat different times and then translated in a wheat germ system.Approximately 1,000 different polypeptides of the translationproducts were separated from each other by two-dimensional gelelectrophoresis. As judged from the density of autoradiographicspots of the translation products, the induction of senescenceby dark treatment involved an increase in 26 species, a decreasein 11 species, and a temporary increase and subsequent decreasein 8 species of translatable mRNA. A similar pattern of changein protein synthesis was also observed in the dark-treated cotyledonswhen the cotyledons were pulse-labeled with ³⁵S-methionine andthe soluble proteins separated by two-dimensional gel electrophoresis,though the polypeptide pattern on the gel did not coincide exactlywith those of the cell-free translation products. These findingsstrongly suggest that the process of leaf senescence is notsimply a passive and gradual death of the tissue, but involvesa drastic and sequential response of the cells to environmentalstimuli with respect to the gene expression of the cells. (Received July 21, 1987; Accepted September 30, 1987)     

Statistical models of the overdispersed molecular clock

The most commonly used statistical model to describe the rate constancy of molecular evolution (molecular clock) is a simple Poisson process in which the variance of the number of amino acid or nucleotide substitutions in a particular gene should be equal to the mean and henceforth the dispersion index, the ratio of the variance to the mean, should be equal to one. Recent sequence data, however, have shown that the substitutional process in molecular evolution is often considerably overdispersed and have called into question the generality of using a simple Poisson process. Several efforts have been made to develop more realistic models of molecular evolution. In this paper, I will show that the spatial (site-specific) variation in the rate of molecular evolution is an improbable cause of the overdispersion and then review various statistical models which take the temporal variation into account. Although these models do not immediately specify what the mechanisms of molecular evolution might be, they do make qualitatively different predictions and give some insight into their inference. One way to distinguish them is suggested. In addition, effects of selected substitutions that presumably occur after a major change in a molecule are quasi-quantitatively examined. It is most likely that the overdispersion of molecular clock is due either to a major molecular reconfiguration (fluctuating neutral space) led by a series of subliminal neutral changes or to selected substitutions fine-tuning a molecule after a major molecular change. Although the latter possibility, of course, violates the simplest neutrality assumption, it would not impair the neutral theory as a whole.     

Gene Genealogy and Variance of Interpopulational Nucleotide Differences

A mathematical theory is developed for computing the probability that m genes sampled from one population (species) and n genes sampled from another are derived from l genes that existed at the time of population splitting. The expected time of divergence between the two most closely related genes sampled from two different populations and the time of divergence (coalescence) of all genes sampled are studied by using this theory. It is shown that the time of divergence between the two most closely related genes can be used as an approximate estimate of the time of population splitting (T) only when T identical to t/(2N) is small, where t and N are the number of generations and the effective population size, respectively. The variance of Nei and Li's estimate (d) of the number of net nucleotide differences between two populations is also studied. It is shown that the standard error (Sd) of d is larger than the mean when T is small (T much less than 1). In this case, Sd is reduced considerably by increasing sample size. When T is large (T greater than 1), however, a large proportion of the variance of d is caused by stochastic factors, and increase in the sample size does not help to reduce Sd. To reduce the stochastic variance of d, one must use data from many independent unlinked gene loci.     

Extranuclear differentiation and gene flow in the finite island model

Use of sequence information from extranuclear genomes to examine deme structure in natural populations has been hampered by lack of clear linkage between sequence relatedness and rates of mutation and migration among demes. Here, we approach this problem in two complementary ways. First, we develop a model of extranuclear genomes in a population divided into a finite number of demes. Sex-dependent migration, neutral mutation, unequal genetic contribution of separate sexes and random genetic drift in each deme are incorporated for generality. From this model, we derive the relationship between gene identity probabilities (between and within demes) and migration rate, mutation rate and effective deme size. Second, we show how within- and between-deme identity probabilities may be calculated from restriction maps of mitochondrial (mt) DNA. These results, when coupled with our results on gene flow and genetic differentiation, allow estimation of relative interdeme gene flow when deme sizes are constant and genetic variants are selectively neutral. We illustrate use of our results by reanalyzing published data on mtDNA in mouse populations from around the world and show that their geographic differentiation is consistent with an island model of deme structure.     

Gene identity and genetic differentiation of populations in the finite island model

A formula for the variance of gene identity (homozygosity) was derived for the case of neutral mutations using diffusion approximations for the changes of gene frequencies in a subdivided population. It is shown that when gene flow is extremely small, the variance of gene identity for the entire population at equilibrium is smaller than that of the panmictic population with the same mean gene identity. On the other hand, although a large amount of gene flow makes a subdivided population equivalent to a panmictic population, there is an intermediate range of gene flow in which population subdivision can increase the variance. This increase results from the increased variance between colonies. In such a case, each colony has a predominant allele, but the predominant type may differ from colony to colony. The formula for obtaining the variance allows us to study such statistics as the coefficient of gene differentiation and the correlation of heterozygosity. Computer simulations were conducted to study the distribution of gene identity as well as to check the validity of the analytical formulas. Effects of selection were also studied by simulations.