The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

STUDY QUESTION: Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? SUMMARY ANSWER: In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. WHAT IS KNOWN ALREADY: In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. STUDY DESIGN, SIZE, AND DURATION: We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. PARTICIPANTS/MATERIALS SETTING METHODS: Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. MAIN RESULTS AND THE ROLE OF CHANCE: Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. WIDER IMPLICATIONS OF THE FINDINGS: We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests.

BRCA1 mRNA is expressed highly during meiosis and spermiogenesis but not during mitosis of male germ cells.

The 17q-linked breast and ovarian cancer susceptibility gene (BRCA1) is believed to function as a tumor suppressor gene (Miki et al., 1994). In this report BRCA1 RNA expression has been analysed in adult mouse tissues with detailed attention to its expression in prepuberal and adult testis. Measurements of BRCA1 mRNA levels in highly purified somatic cells of the testis and in staged germ cells showed that high level BRCA1 mRNA expression is limited to the germ cells. Within the germ cell lineage, the high level expression was detected in meiotic cells, specifically pachytene spermatocytes and in post-meiotic round spermatids. This is in contrast to premeiotic germ cells which were found to express little or no BRCA1 mRNA. These observations, considered together with recent data on the expression of BRCA1 in breast epithelium, argues against a function for BRACA1 in early progenitor cells in both tissues and cells attention instead to roles intimately associated with terminal differentiation or with final rounds of cell division.

Nitric oxide diffusion coefficients in solutions, proteins and membranes determined by phosphorescence.

The reactivity of nitric oxide under a given condition is a complex function of its diffusivity and the concentration of reacting partners. Quenching by NO of luminescence from Ru and Pd chelates of mesoporphyrin IX, two molecules which exhibit phosphorescence at room temperature, was utilized to evaluate the gas concentration and apparent diffusion coefficients. The properties of Ru-mesoporphyrin, a dye not previously employed as a probe for O2 or NO, were determined and the assay was verified and used to quantify NO produced by decomposition of nitrosocysteine. The pseudo-second order quenching constants were obtained from Stern-Volmer plots measured under various conditions and used to calculate diffusion coefficients for nitric oxide in solutions, proteins and membranes. The diffusion coefficients were greater at 37 than at 25 degrees C and, at a given temperature, smaller in proteins and membranes than in water. The conclusion is that NO and O2 closely resemble each other in diffusivity but that NO is slightly less lipophilic, resulting in somewhat faster apparent diffusion in protein and slower diffusivity in lipid, relative to O2. Taking a mean diffusion coefficient for NO of 10(-7) cm2s-1, then within 10 s the mean path is 10(-3) cm, or less than the diameter of a single cell. However, at low NO and O2 concentrations, the halflife of NO will be considerably longer than 10 s, and consequently the path of NO diffusion much greater.

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.

Horseradish peroxidase monitored by infrared spectroscopy: effect of temperature, substrate and calcium.

Horseradish peroxidase was examined as a function of Ca and substrate binding using infrared spectroscopy in the temperature range of 10-300 K. The Ca complex could be identified by the carboxylate stretches. The amide peak positions indicate that the protein remains stable from room temperature to 10 K. Shifts in these peaks are consistent with increased hydrogen bonding as temperature decreases, but the protein conformation is maintained at cryogenic temperatures. The substrate, benzohydroxamic acid, produced no detectable change in the infrared spectrum, consistent with X-ray crystallography results. With removal of Ca, the protein maintained its overall helicity.

Cyclic protein-2, a secretory product of rat Sertoli cells, is the proenzyme form of cathepsin L.

Previous studies demonstrated that secretion of Cyclic Protein-2 (CP-2) by mature rat Sertoli cells increased 30-fold from stage II to stages VI-VII of the cycle of the seminiferous epithelium and suggested that this protein was concentrated around compacted spermatids. Analysis of other organs revealed that CP-2 was also detectable in the epithelium of the proximal kidney tubule and in neurons originating from the supraoptic and paraventricular nuclei of the hypothalamus. We now have isolated a partial 1.8-kilobase (kb) cDNA for CP-2 mRNA, and sequence analysis revealed that CP-2 was the proenzyme form of the cysteine protease cathepsin L; this was corroborated by immunoprecipitation of CP-2 by anticathepsin L immunoglobulin G and by enzymatic analysis of purified CP-2. Northern analysis of testis mRNA revealed major (1.7 kb) and minor (2.2 kb) transcripts which differed in the length of their 3'-untranslated sequences. Low levels of CP-2/cathepsin L transcripts were detected in many organs, while high levels were only detected in testis, kidney, and liver. In seminiferous tubules, CP-2/cathepsin L mRNA was undetectable at stage II, increased to maximal levels at stages VI and VIIa,b, and was again undetectable at stage XII. At stages VI-VII, CP-2/cathepsin L mRNA was present in Sertoli but not germ cells. Taken together, these data suggest that CP-2/cathepsin L gene expression is regulated in a cell-specific manner and that in Sertoli cells this expression is influenced by germ cells at specific steps of development. We propose that at stages V-VII, secreted CP-2/cathepsin L degrades adhesion molecules which bind compacted spermatids to Sertoli cells, thereby facilitating movement of these spermatids toward the lumen of the tubule.

Characterization of the androgen receptor in nuclei of seminiferous tubules of the mature male rat based upon radioimmunoassay of endogenous levels of bound testosterone.

A macromolecular testosterone complex which was salt-extracted from nuclei isolated from seminiferous tubules of the adult male rat was investigated by quantifying the testosterone directly by RIA. The steroid-binding capacity of the complex was destroyed by heating at both 12 and 50 C, suggesting the presence of an extremely thermolabile complex. This tubular complex was shown to be similar to the radioactively labeled androgen receptor isolated from ventral prostatic nuclei by their coelution during adsorption chromatography, by their coleution during ion exchange chromatography, their similar migration rate (4-4.46S) on both sucrose and glycerol density gradients, and their similar slow dissociation at 0 C. These observations indicate that the tubular complex contains a high affinity androgen receptor and suggest that the direct measurement of testosterone in this complex by RIA may be useful in identifying its cellular distribution.

Male germ cells regulate transcription of the cathepsin l gene by rat Sertoli cells.

It is well known that male germ cells regulate the steady state levels of numerous transcripts expressed by Sertoli cells. To date, however, there has been no direct test of whether this regulation reflects changes in gene transcription and/or transcript stability. This study used two experimental approaches to test the hypothesis that germ cells regulate transcription of the cathepsin L gene by rat Sertoli cells. We examined this gene because, in vivo, steady state levels of cath L messenger RNA in Sertoli cells change in a stage-specific manner as the surrounding germ cells progress through the 14 stages of the cycle of the seminiferous epithelium. In the first experimental approach, seminiferous tubules at stages VI-VII and stages IX-XII were incubated for 1 h in 4-thiouridine, and the amount of metabolically labeled cath L messenger RNA was quantified. The results demonstrate that transcription of the cath L gene by Sertoli cells is 7-fold higher at stages VI-VII than at stages IX-XII. The second experimental approach examined the ability of germ cells to regulate the activity of cath L reporter constructs in mature Sertoli cells. Before these studies, we isolated a cath L genomic clone and demonstrated that this clone contains the transcription start site of the cath L gene expressed by Sertoli cells. Transient transfection analysis then demonstrated that two reporter constructs, containing 244 and about 2.1 kb of sequence upstream from the transcription start site, had similar activities in mature Sertoli cells. However, germ cells only affected the activity of the larger construct in Sertoli cells, which was reduced by 30%. We conclude that germ cells regulate transcription of the cath L gene by Sertoli cells and that repressive effects of germ cells are mediated by elements upstream from nucleotide -244 of this gene.

Evidence that rodent epididymal sperm contain the Mr approximately 94,000 glucocorticoid receptor but lack the Mr approximately 90,000 heat shock protein.

Monoclonal antibodies directed against four different polypeptide epitopes on the Mr approximately 94,000 steroid-binding subunit of the rat liver cytosolic glucocorticoid receptor (GcR) were used to probe Western blots of epididymal spermatozoa from rats and mice. Two sperm polypeptides with apparent molecular weights of 94,000 (indistinguishable in size from the liver GcR subunit) and 150,000 reacted with these antibodies. Other polypeptides that are present in a wide variety of somatic cells [lamin-A, -B, and -C; topoisomerase-I; poly(ADP-ribose) polymerase; the 62-kilodalton internal nuclear matrix protein; the nucleolar protein B23; and histone H1] could not be detected in these preparations of spermatozoa, thus appearing to rule out contamination by somatic cells. Rat and mouse pachytene spermatocytes and round spermatids contained much lower amounts of the Mr approximately 94,000 and 150,000 polypeptides. These results suggested that the steroid-binding subunit of the GcR might be accumulated late in spermatogenesis. Consistent with this view, a 6-kilobase mRNA (identical in size to a mRNA detected in mouse somatic cell lines) was detected when Northern blots of mouse round spermatid RNA were probed with a cDNA to the steroid-binding GcR subunit. Although the results described above suggest the presence of GcR in rodent sperm, high affinity binding of glucocorticoids to epididymal sperm could not be detected in a whole cell binding assay. Further analysis revealed that the Mr approximately 90,000 heat shock protein (hsp90), a component reportedly required for high affinity ligand binding to the GcR, was present in early germ cells, but absent from rodent epididymal sperm. These results suggest that the Mr approximately 94,000 steroid-binding subunit of the GcR and an immunologically related Mr approximately 150,000 polypeptide are specifically accumulated during the later stages of rodent spermatogenesis, but are not assembled into receptor complexes capable of binding steroid. In addition, these results support the view that hsp90 is required for high affinity binding of glucocorticoids to the Mr approximately 94,000 GcR subunit in intact cells.

Spermatogenesis in vitro: completion of meiosis and early spermiogenesis.

In vitro formation of haploid spermatids has not been convincingly demonstrated in mammals. To investigate this problem we selected defined segments of rat seminiferous tubules containing late pachytene and diakinetic primary spermatocytes (Stages XII and XIII of the cycle) for culture in a chemically defined medium. After 2 days, most spermatocytes completed both meiotic divisions, and by 6 days the tubular epithelium developed morphologic characteristics of Stage V in which the newly formed spermatids had acrosomic systems characteristic of step 5 spermiogenesis. The seminiferous tubules also differentiated biochemically as evidenced by increased production of proteins characteristically secreted by Stage V. Since this in vitro differentiation of the germinal epithelium occurred in the absence of testosterone and FSH, we conclude that late pachytene spermatocytes and their associated Sertoli cells have all the information required for both meiotic divisions and early spermiogenesis.

The hemicastrated rat: definition of a model for the study of the regulation of testicular steroidogenesis.

A comprehensive study has been made of the hemicastrated rat from 2 to 12 months of age in order to define what might represent an ideal model in which to study testicular regulation. Although there was no compensatory hypertrophy in the remaining testis of the mature hemicastrated rat, levels of plasma testosterone fell significantly within 4 h after surgery in all age groups older than 3 months, and were restored to normal levels almost immediately, usually within 8 h. There were no significant changes in LH and prolactin, and the significant rise in FSH was sufficiently delayed (2 days or more) to suggest that none of these three hormones was implicated in any obvious way in the compensatory restoration of plasma testosterone levels. Although a single testis was capable of maintaining normal plasma testosterone concentrations, its response to human chorionic gonadotrophin at 24 h after hemicastration was significantly less than that of intact animals, suggesting that the single testis was functioning at near-maximal capacity. The hormonal responses to repetitive blood sampling and to sham-surgery simulated the response to hemicastration remarkably. However, these responses were never statistically significant in within-group analysis, and therefore did not obscure the significant fall of plasma testosterone levels in response to hemicastration. The basic mechanism by which plasma testosterone is restored in the hemicastrated rat is still unknown, but the options have been narrowed.

Follicle-stimulating hormone regulates the expression of cyclic protein-2/cathepsin L messenger ribonucleic acid in rat Sertoli cells in a stage-specific manner.

Cyclic protein-2/cathepsin L (CP-2) is secreted by Sertoli cells in a highly stage-specific manner, maximally during stages VI-VII of the rat seminiferous epithelial cycle. We investigated FSH regulation of CP-2 mRNA expression of its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA expression and its cellular localization in isolated staged seminiferous tubular segments. FSH induced a significant increase of CP-2 mRNA levels in stages IX-I, whereas in stages II-VIII, the levels of CP-2 mRNA were reduced. A similar effect was produced by two cAMP analogs, dbcAMP (0.2 mM) and Sp cAMP (20 microM). FSH and cAMP did not affect on the levels of SGP-2 mRNA during the seminiferous epithelial cycle. The magnitude of the response was time- and dose-dependent; the maximum was obtained with 100 ng/ml of FSH. It is likely that FSH regulates Cp-2 gene transcription, since de novo RNA synthesis was required for the stimulatory FSH effect on CP-2 mRNA levels, while ongoing protein synthesis was not. In conclusion, the data suggest that FSH, via cAMP-mediated pathway, regulates CP-2/cathepsin L gene transcription in rat Sertoli cells and modulated the stage-specific expression pattern.

Visually evoked potentials to pseudorandom binary sequence stimulation. Preliminary clinical trails.

We report results using a new stimulus for clinical testing of visually evoked potentials (VEPs). The stimulus was the modulation of the luminance of a large unpatterned field by a temporal pseudorandom binary sequence. The stimulus was similar to Gaussian white noise in that a large number of sinusoidal harmonics were presented concurrently. It has the important advantage that conventional signal averaging techniques can be used to analyze the VEP. Abnormal VEP responses were obtained when the stimulus was applied to patients with minimal macular and optic nerve disease.

Grating acuity development after early surgery for congenital unilateral cataract.

Longitudinal measures of grating acuity were obtained from 16 children after early surgery, optical correction, and occlusion therapy for congenital unilateral cataract. Compliance with contact lens wear and occlusion therapy was good in this population. Monocular grating acuities were obtained by preferential looking during months 1 through 15 and by an operant procedure during months 16 through 53. Grating acuities of normal eyes did not differ from those obtained in an age-matched normal population and showed no evidence of occlusion amblyopia. During the first year of life, grating acuities of aphakic eyes were typically within the normal range, but lagged behind normal development during years 2 through 4. These results suggest that early surgery is associated with favorable long-term visual results but does not, even with good compliance, completely eliminate deprivation amblyopia.

Germ cell-Sertoli cell interactions. Studies of cyclic protein-2 in the seminiferous tubule.

This review briefly describes the discovery and isolation of a novel Sertoli cell product, cyclic protein-2, (CP-2) and the generation of an antiserum against this protein. Using this antiserum, we demonstrated a stage-specific change in the synthesis of CP-2 by Sertoli cells within intact seminiferous tubules; synthesis is maximal at stages VI and VIIa,b of the cycle and minimal at stage XII. That CP-2 is a product of Sertoli cells was confirmed by immunohistochemical analysis. Comparison of CP-2 and transferrin synthesis by immature (17-day) and mature (75-day) Sertoli cells within intact seminiferous tubules has documented a significant increase in the synthesis of both proteins during testicular maturation. It was noteworthy, however, that the increase in CP-2 synthesis was much greater than the increase in transferrin synthesis. These data in conjunction with previous comparisons of the stage-specific changes in CP-2 and transferrin synthesis and secretion led to the hypothesis that the synthesis of these two proteins is regulated by different cellular interactions. Examination of cultured Sertoli cells obtained from mature rats demonstrated that transferrin synthesis and secretion were stimulated by hormones and vitamins, whereas CP-2 synthesis and secretion were not significantly affected by the same factors. Therefore, these data demonstrate that hormonal regulation of transferrin synthesis by Sertoli cells differs from hormonal regulation of CP-2 synthesis. Indeed, our data suggest that CP-2 synthesis is not directly regulated by hormones and vitamins. Finally, we demonstrated that when Sertoli cells are separated from germ cells and the Sertoli cells placed in culture, the age-dependent increase in CP-2 synthesis, noted with cultured tubules, is lost. In contrast, significantly more transferrin is synthesized by primary cultures of Sertoli cells obtained from old animals than from young animals. Taken together, all of these data indicate that the regulation of CP-2 synthesis and secretion by the Sertoli cell is unique and is primarily stimulated by paracrine signals or direct cell contact with the germ cells. Which of these mechanisms of cell-cell communication in the testis is important to regulation of CP-2 synthesis by Sertoli cells is unknown. Neither do we know which spermatogenic cell type provides this stimulus. These issues can now be addressed, however, because we have developed the protocols for isolating and culturing Sertoli cells from mature rat testes.

The effect of aging on the seminiferous epithelium of the brown Norway rat.

Aging of the mammalian testis is often accompanied by loss of germ cells and, as a result, decreased daily sperm production. It is currently unknown whether cell loss is the result of aging-related changes in germ cells or whether there are also aging-related changes in the Sertoli cells that normally support germ development and differentiation. To begin to compare the effects of age on germ cells and on Sertoli cells, we examined brown Norway rats of 6, 12, 18, 21, and 24 months of age for the frequency of seminiferous tubule regression and total testis contents of transcripts for three Sertoli cell products: SGP-2, transferrin, and cyclic protein-2 (CP-2)/cathepsin L. Histological analysis revealed no changes in the seminiferous epithelium from 6 to 12 months of age. However, from 12 to 24 months of age, the percentage of normal tubules gradually decreased from 95% to 15% of the total while the percentage of fully regressed tubules (containing no germ cells per tubule cross section) increased from 0% to 78%. In our analysis of specific Sertoli cell transcripts, we noted no change in total testis content of SGP-2 mRNA from 6 to 24 months. However, total testis content of transferrin mRNA was unchanged from 6 to 18 months, but increased by 24 months to 368% of the content of a 6-month-old testis. In contrast, total testis content of CP-2/cathepsin L mRNA was unchanged from 6 to 12 months, but decreased by 24 months to 58% of the content of a 6-month-old testis.(ABSTRACT TRUNCATED AT 250 WORDS)

Testicular steroidogenesis in the aging brown Norway rat.

In seeking an animal model of age-associated changes in the male reproductive tract, we examined the effects of age on the health and testicular steroidogenic activity of the Brown Norway rat, with comparisons made to the Sprague-Dawley rat. When perfused in vitro under conditions of maximally stimulating luteinizing hormone significant age-associated reductions were seen in testosterone production by testes of Sprague-Dawley rats of 21-24 months of age and by testes of Brown Norway rats of 18-30 months of age. Decreases in the capacity of the testes to produce testosterone were reflected in age-associated decreases in both serum testosterone and in testosterone concentration within the seminiferous tubule fluid. In contrast to the Sprague-Dawley rat, changes in steroidogenic activity in the Brown Norway rat were not accompanied by the occurrence of pituitary adenomas, obesity, or testicular tumors. This along with its longevity, make the Brown Norway strain a highly promising model for testicular aging.

Solvent dependent and independent motions of CO-horseradish peroxidase examined by infrared spectroscopy and molecular dynamics calculations.

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe[bond]CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.

Characterization and physiological validation of a radioimmunoassay for plasma testosterone in the male rat.

A radioimmunoassay (RIA) to measure testosterone concentration in the plasma of male rats was formalized, tested and physiologically validated. The procedure measured testosterone equally well, whether or not estimation of recovery and chromatographic purification preceded the RIA. The results were equivalent to those achieved by the method of competitive protein binding. No 17beta-hydroxy-5alpha-androstan-3-one was found in the plasma of male rats. Adrenalectomy did not significantly decrease plasma testosterone in either male or female rats. The simplified protocol met all requirements of precision, sensitivity, accuracy and specificity. A single investigator can analyze 1,000 plasma samples for testosterone in one week if necessary.

Clinical evaluation of a posterior composite resin containing a semiporous filler particle.

Bowen recently developed a new filler particle for composite resin restorations. The surface particles of a diphasic strontium glass are superficially etched and after silanating the surface, the microscopic defects are filled with prepolymerized resin. The filler particles are then bonded chemically and mechanically to the resin matrix. This paper discusses the clinical performance of this material as a posterior composite resin over a two year period.