Mohamed El-Alfy

Cell transplantation for myocardial repair: an experimental approach.

Myocardium lacks the ability to regenerate following injury. This is in contrast to skeletal muscle (SKM), in which capacity for tissue repair is attributed to the presence of satellite cells. It was hypothesized that SKM satellite cells multiplied in vitro could be used to repair injured heart muscle. Fourteen dogs underwent explantation of the anterior tibialis muscle. Satellite cells were multiplied in vitro and their nuclei were labelled with tritiated thymidine 24 h prior to implantation. The same dogs were then subjected successfully to a myocardial injury by the application of a cryoprobe. The cells were suspended in serum-free growth medium and autotransplanted within the damaged muscle. Medium without cells was injected into an adjacent site to serve as a control. Endpoints comprised histology using standard stains as well as Masson trichrome (specific for connective tissue), and radioautography. In five dogs, satellite cell isolation, culture, and implantation were technically satisfactory. In three implanted dogs, specimens were taken within 6-8 wk. There were persistence of the implantation channels in the experimental sites when compared to the controls. Macroscopically, muscle tissue completely surrounded by scar tissue could be seen. Masson trichrome staining showed homogeneous scar in the control site, but not in the test site where a patch of muscle fibres containing intercalated discs (characteristic of myocardial tissue) was observed. In two other dogs, specimens were taken at 14 wk postimplantation. Muscle tissue could not be found. These preliminary results could be consistent with the hypothesis that SKM satellite cells can form neo-myocardium within an appropriate environment. Our specimens failed to demonstrate the presence of myocyte nuclei. It is therefore further hypothesized that in the late postoperative period, the muscle regenerate failed to survive.

Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the eggs zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High‐salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose‐dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol‐anchored protein and that prostasomes may be implicated in this process. Mol. Reprod. Dev. 52:225–233, 1999.

Purification, cDNA Cloning, and Expression of a New Human Blood Plasma Glutamate Carboxypeptidase Homologous to N-Acetyl-aspartyl-α-glutamate Carboxypeptidase/Prostate-specific Membrane Antigen

We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a γ-interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed at 5′-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known asN-acetyl-aspartyl-α-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.

The RNA- and DNA-binding protein TB-RBP is spatially and developmentally regulated during spermatogenesis

Testis brain RNA‐binding protein (TB‐RBP) suppresses translation in vitro and attaches mRNAs to microtubules by binding to conserved elements in the 3 untranslated regions (UTRs) of specific testis and brain mRNAs. Purification of TB‐RBP from testicular and brain cytoplasmic extracts has revealed that mouse TB‐RBP is 99% identical to the human protein translin, a recombination “hot spot” binding protein associated with chromosomal translocations. Using a cDNA encoding TB‐RBP, the gene copy number and the developmental expression of TB‐RBP have been analyzed by Southern blotting, Northern blotting, and in situ hybridization. In the mouse, TB‐RBP is encoded by a single copy gene. In mouse testes, three TB‐RBP mRNAs of about 1.2, 1.7, and 3.0 kb are developmentally regulated with high levels of expression in meiotic and postmeiotic germ cells. A fourth TB‐RBP transcript of about 3.2 kb is seen in the brain. In situ hybridization confirms high levels of testicular TB‐RBP mRNAs in meiotic and postmeiotic cells, with the highest levels of TB‐RBP mRNAs in pachytene spermatocytes and round spermatids of the mouse and in round spermatids of the rat. RNase H digestion assays reveal that the three TB‐RBP mRNAs of mouse testes result from processing differences in their 3 untranslated regions. These data demonstrate that multiple TB‐RBP mRNAs are primarily expressed in meiotic and postmeiotic germ cells in the mammalian testis, and although the specific RNA‐binding ability of TB‐RBP appears limited to brain and testis, TB‐RBP mRNAs are widely expressed. Mol. Reprod. Dev. 49:219–228, 1998.

The eleven stages of the cell cycle, with emphasis on the changes in chromosomes and nucleoli during interphase and mitosis.

Since we had subdivided the cell cycle into 11 stages—four for mitosis and seven for the interphase—and since we had experience in detecting DNA in the electron microscope (EN) by the osmium‐amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041–3044), we combined the two approaches for the analysis of DNA‐containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde‐fixed mouse duodenum were stained by osmium‐amine for electron microscopic examination of the stages in the 12.3‐hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi‐thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA‐specific Hoechst 33258 dye and examined in the fluorescence microscope.

Expression of patched-1 and smoothened in testicular meiotic and post-meiotic cells

Desert hedgehog (Dhh) signaling plays an essential role in the normal development of the testis and in the process of spermatogenesis. Little is known about the involvement in spermatogenesis of the prototypic member of the family, Ptc1, which acts to suppress hedgehog signaling through Smoothened (Smo). Here, we have examined the expression of Ptc1, Smo, and Dhh in mouse and rat seminiferous epithelium. Our findings demonstrate that Ptc1 and Smo are expressed by primary spermatocytes and by round and condensing spermatids whereas Dhh is expressed by Sertoli cells. The findings suggest that Sertoli cells coordinate Dhh‐dependent spermatogenesis events via Ptc1 and Smo prior to the first meiotic division and in postmeiotic (haploid) cells, particularly during the first half of spermiogenesis. Microsc. Res. Tech. 2009.

Changes in the rate of RNA synthesis during the cell cycle

Although the rate of RNA synthesis is known to drop at mitosis, the recent identification of 11 stages in the cell cycle (El‐Alfy et al., 1994) makes it possible to measure the rate of this synthesis at each one of the stages and thus find out how it varies throughout the cell cycle.

Molecular cloning and developmental expression of a small ribonuclear protein in the mouse testis

U1 RNP C polypeptide is a ubiquitous and highly conserved protein that is found associated to the U1 small nuclear ribonuclear particle (U1 snRNP). The U1 snRNP is involved in pre‐mRNA splicing by defining introns and exons and by binding to consensus sequences within the pre‐mRNA. In the present study we immunoscreened a mouse testicular phagemid cDNA library with an anti‐Sm serum from patients with systemic lupus erythematosus. Sequence analysis of a positive clone containing a 0.75 kb cDNA insert revealed that it encodes the entire amino acid sequence of the U1 RNP C polypeptide. Northern blots of total RNA isolated from testes and various adult mouse tissues demonstrated that the 0.75 kb transcript is highly expressed in the testes and that it begins developmentally at day 18 postpartum, corresponding to the appearance of preleptotene spermatocytes. In situ hybridization confirmed the meiotic and post‐meiotic expression of this transcript. LM immunoperoxidase staining with the anti‐Sm serum localized spliceosome snRNPs predominantly in the nuclei of somatic and germinal testicular cells but not in elongated spermatids. EM immunogold labeling confirmed the LM observations but additionally showed that snRNP content peaked in the nuclei of pachytene spermatocytes and that 2 cytoplasmic components found exclusively in meiotic and post‐meiotic germ cells were intensively reactive. Immunoblots of testicular homogenates probed with the anti‐Sm serum revealed several reactive proteins, of which one, a 21 kDa polypeptide, could be the U1 RNP C based on its predicted molecular weight. In summary we report an isoform of U1 RNP C which is testis specific and which may play a role in mRNA splicing exclusively in meiotic and post‐meiotic germ cells during spermatogenesis. Mol. Reprod. Dev. 46: 459–470, 1997.