Chromosome analysis of BALB/c mouse spermatozoa with normal and abnormal head morphology.

To assess the relationship between mouse sperm head morphology and karyotype, sperm heads with either a normal or an abnormal morphology were injected individually into enucleated mouse oocytes that were karyotyped at the metaphase of the first cleavage. BALB/c male mice that produce an unusually high proportion of morphologically abnormal spermatozoa were used as sperm donors. Abnormal karyotypes were found in a significantly higher proportion of eggs injected with severely misshapen sperm heads (36-38%) as compared to those injected with normal and quasi-normal heads (15-21%) (p < 0.01). Most karyotype abnormalities were structural rather than numerical, the most common being breaks and exchanges of chromosome type in both normal and abnormal spermatozoa.     

Maturation and pro-peptide cleavage of beta-secretase

Amyloid beta-peptide is generated by two sequential proteolytic cleavages mediated by beta-secretase (BACE) and gamma-secretase. BACE was recently identified as a membrane-associated aspartyl protease. We have now analyzed the maturation and pro-peptide cleavage of BACE. Pulse-chase experiments revealed that BACE is post-translationally modified during transport to the cell surface, which can be monitored by a significant increase in the molecular mass. The increase in molecular mass is caused by complex N-glycosylation. Treatment with tunicamycin and N-glycosidase F led to a BACE derivative with a molecular weight corresponding to an unmodified version. In contrast, the mature form of BACE was resistant to endoglycosidase H treatment. The cytoplasmic tail of BACE was required for efficient maturation and trafficking through the Golgi; a BACE variant lacking the cytoplasmic tail undergoes inefficient maturation. In contrast a soluble BACE variant that does not contain a membrane anchor matured more rapidly than full-length BACE. Pro-BACE was predominantly located within the endoplasmic reticulum. Pro-peptide cleavage occurred immediately before full maturation and trafficking through the Golgi.     

Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer.

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.     

Coexistence of inverted Y, chromosome 15p+ and abnormal phenotype.

In this study, we report conventional and molecular cytogenetic studies in a patient with multiple anomalies who is a carrier of a pericentric inversion on chromosome Y and a chromosome 15p+. His parents were phenotypically normal. The father is a carrier of a pericentric inversion of chromosome Y, and the mother carries a large chromosome 15p+ variant. The inverted Y chromosome was demonstrated by GTG- and CBG-banding, and DAPI-staining. The presence of extra chromosomal material on the chromosome 15p, that was C-band and DAPI positive, was demonstrated by trypsin G-banding. This suggests that the extra chromosomal material contained repetitive DNA sequences. NOR-staining indicated the presence a nuclear organizer region at the junction of the chromosome 15p+ material. Fluorescence in situ hybridization (FISH), with chromosome X and Y painting probes, alpha- and classic-satellite probes specific for chromosome Y, alpha- and beta-satellite III probes for chromosome 15 were used to elucidate the nature of both the inverted Y chromosome and chromosome 15p+. The result with chromosome X and Y painting probes, alpha-satellite, classic-satellite, and DYS59 probes specific for chromosome Y revealed the rearrangement of the Y chromosome was an inv(Y)(p11.2q11.22 or q11.23). FISH with alpha-satellite and beta-satellite III probes for chromosome 15 demonstrated that the extra chromosomal material on the chromosome 15 probably represents beta-satellite III sequences. The possible roles of the simultaneous occurrence of an inverted Y and the amplified DNA sequence on chromosome 15p in the abnormal phenotype of the proband are discussed.     

The transport of morphologically abnormal sperm in the female reproductive tract of mice

Mice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.     

Molecular characterization of an aggregation-prone variant of alpha-synuclein used to model synucleinopathies

The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells.     

Spermatozoa of chromosomally heterozygous mice and their fate in male and female genital tracts

The fate of morphologically normal but chromosomally abnormal spermatozoa derived from mice with variable degrees and complexity of Robertsonian heterozygosity was studied at different sites along the male and female genital tract by Feulgen-DNA measurements. In addition, the percentage frequencies of morphologically abnormal spermatozoa in transit along the male and female genital tracts were studied. It was found that during transit from the epididymis to the vas deferens the distribution of the Feulgen-DNA contents of morphologically normal spermatozoa changed: Spermatozoa with chromatin with the extremely low or high Feulgen staining intensity disappeared. The percentages of morphologically abnormal sperm cells did not change at these levels. In the female genital tracts, the distribution of Feulgen-DNA content of morphologically normal spermatozoa did not show significant changes. This indicates that spermatozoa are able to reach the fallopian tube in spite of gross genome unbalance. There is evidence that unbalanced spermatozoa take part in the fertilization process, producing abnormal zygotes subject to postzygotic loss. Conversely morphologically abnormal spermatozoa were preferentially lost before they reached the fallopian tube, suggesting they had been eliminated prezygotically.     

Preliminary evaluation of a single-day tubeless test of pancreatic function.

The test for pancreatic exocrine function using N-benzoyl-L-tyrosyl-p-aminobenzoic acid (BTP test) does not require duodenal intubation, but misleadingly abnormal results often occur in patients with liver or bowel disease because the p-aminobenzoic acid (PABA) released by chymotrypsin hydrolysis of the peptide either is not conjugated or is malabsorbed. This study evaluated a modified BTP test, using a tracer dose of 14C-PABA to eliminate misleading results, to assess exocrine function from a single six-hour collection of urine. The test clearly distinguished all patients with pancreatic steatorrhoea from normal subjects and identified patients with less severe pancreatitis as often as did the Lundh test. Furthermore, in patients with bowel or liver disease the misleadingly abnormal results of the unmodified BTP test were eliminated by the modified test in all but one case. These findings suggest that the modified BTP test provides a practical alternative to conventional tests of pancreatic function that entail duodenal intubation.     

Isolation and characterization of temperature-sensitivelethal(2)giant larva alleles

Summary In this paper we present an analysis of the behavior ofl(2)gl ^tsimaginal wing discs during culture in adult hosts. Thel(2)gl ^tslarvae reared at 29° C contain two types of wing discs, those that are morphologically normal and those that are abnormal. When discs of both types are cultured in adult hosts at 29° C, the restrictive temperature, they give rise to transplantable neoplastic tissue. However, when the 29° C reared discs are cultured at 15° C, the permissive temperature, the morphologically normal discs maintain their morphology, but the morphologically abnormal discs give rise to neoplasms. Thel(2)gl ^tslarvae reared at 15° C contain only morphologically normal discs. When these discs are cultured in adult hosts at 29° C they give rise to neoplasms, however if the discs are cultured at 15° C they maintain their normal morphology. These results demonstrate: (1) that all wing imaginal discs obtained from 29° C rearedl(2)gl ^tslarvae are competent to undergo neoplastic development, (2) the morphologically abnormal discs obtained from the 29° C rearedl(2)gl ^tslarvae are committed to neoplastic development, (3) the neoplastic development of the morphologically normal discs is temperature dependent, (4) once the neoplastic development of thel(2)gl ^tsdiscs has been initiated the process is not readily reversible. In addition, the ability ofl(2)gl ^tswing discs to perform epimorphic regulation was tested by amputating morphologically normal permissively rearedl(2)gl ^tswing discs and culturing both fragiments at the permissive temperature. Fragments of control wild-type discs maintained their morphology during culture at the permissive temperature. However, both fragments of txel(2)gl ^tsdiscs became neoplastic. This result is discussed with respect to a possible role for thel(2)gl ⁺function in epimorphic regulation and with respect to the phenomena of tumor promotion in vertebrates.     

A prevalent Y;15 translocation in the Ethiopian Beta Israel community in Israel

We describe 7 cases of abnormal karyotypes involving chromosomes Y and 15 in Ethiopian Beta Israel patients: 46,XX, der(15)t(Y;15)(q12;p12) and 46,XY,der(15)t(Y;15)(q12;p12). Six cases were incidentally found in amniocentesis performed for various indications; the indication for karyotyping in 1 case was recurrent abortions. To the best of our knowledge, this is the first report of this translocation in a specific ethnic group. We conclude that the derivative chromosome 15 with chromosome Y is probably a normal variant in Ethiopian Beta Israel occurring at an estimated frequency of 4/74 (5.4%). The prenatal diagnosis of this translocation in this population probably does not require further parental testing.     

POLYMORPHIC POSTTRANSLATIONAL MODIFICATION OF ALKALINE PHOSPHATASE IN ESCHERICHIA COLI

Natural isolates of Escherichia coli have been examined by polyacrylamide gel electrophoresis for variant isozyme patterns of alkaline phosphatase. The polypeptide chains of this enzyme normally exist in two forms—an unmodified polypeptide product of the phoA gene and a posttranslationally modified version of the same polypeptide that has had its N-terminal arginine removed through the action of the iap gene product. These two forms of the polypeptide aggregate as dimers and thus normally form three electrophoretically distinguishable alkaline phosphatase isozymes. Among 104 strains screened, three had variant isozyme patterns. Two of these were deficient in the posttranslationally modified polypeptide, and cotransduction with cysI indicates that they carry mutant iap genes. The third variant is deficient in the unmodified form of the polypeptide. These results provide an unambiguous case of polymorphic posttranslational modification in E. coli.     

Assembly properties of tubulin after carboxyl group modification

By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.     

Human spleen histone H1. Isolation and amino acid sequences of three minor variants, H1a, H1c, and H1d

Following the previous determination of the main variant H1b of human spleen histone H1, we have determined the complete amino acid sequence of another variant, H1d. Limited chymotryptic digestion of H1d produced four fragments, I to IV, and one partial fragment I-II, as in the case of H1b. These fragments were aligned with two overlapping peptides, produced by another enzyme from the intact H1d. We also confirmed the C-terminal sequence of H1d by carboxypeptidase digestion. This H1d has an acetylated N-terminal serine, equimolar alanine or valine residue at 17, and is composed of 212 residues. The molecular weight was 21,233 for the alanine variant and 21,261 for the valine variant in the unmodified form. We also deduced the total sequences of H1a and H1c in a similar way, considering the maximum homology with H1b and H1d. Each N-terminal serine residue is acetylated, too. H1a consists of 222 amino acid residues and has a molecular weight of 22,178 in its unmodified form; the H1c consists of 220 residues and has a molecular weight of 22,218 in that form. The human spleen H1 sequences varied to about the same extent in the N-terminal 40 and C-terminal 110 residues. However, the sequences of the about 70 internal residues are well conserved between the variants. The extent of differences among the human H1 variants is similar to, or rather smaller than, those among the mammalian somatic H1 species. The implications of these differences in the sequence for H1 function are discussed from the evolutionary viewpoint.     

Identification of a Novel MYO15A Mutation in a Chinese Family with Autosomal Recessive Nonsyndromic Hearing Loss

Autosomal recessive nonsyndromic hearing loss (ARNSHL) is a genetically heterogeneous sensorineural disorder, generally manifested with prelingual hearing loss and absence of other clinical manifestations. The aim of this study is to identify the pathogenic gene in a four-generation consanguineous Chinese family with ARNSHL. A novel homozygous variant, c.9316dupC (p.H3106Pfs*2), in the myoxin XVa gene (MYO15A) was identified by exome sequencing and Sanger sequencing. The homozygous MYO15A c.9316dupC variant co-segregated with the phenotypes in the ARNSHL family and was absent in two hundred normal controls. The variant was predicted to interfere with the formation of the Myosin XVa-whirlin-Eps8 complex at the tip of stereocilia, which is indispensable for stereocilia elongation. Our data suggest that the homozygous MYO15A c.9316dupC variant might be the pathogenic mutation, and exome sequencing is a powerful molecular diagnostic strategy for ARNSHL, an extremely heterogeneous disorder. Our findings extend the mutation spectrum of the MYO15A gene and have important implications for genetic counseling for the family.     

Influence of diandric and digynic triploid genotypes on early mouse embryogenesis

Standard micromanipulatory techniques were used to produce tripronucleate diandric and digynic triploid mouse conceptuses. When these were transferred to suitable recipients, most implanted. A wide range of embryonic stages from the primitive streak to the 15- to 25-somite stage were isolated in both triploid series in otherwise identical recipients. In the diandric triploid series, all of the embryos recovered appeared to be morphologically normal, but considerably smaller than fertilized embryos analysed at similar stages of development. This contrasts with the digynic triploid conceptuses which, though also ranging from the primitive-streak stage to about the 10- to 15-somite stage at the time of their isolation, generally showed poorer embryonic development than the diandric triploids, and were invariably morphologically abnormal. Unlike the situation observed in man, where the placentas of diandric triploid conceptuses commonly display widespread trophoblastic hyperplasia and form the characteristic 'partial' or 'incomplete' type of hydatidiform moles, the extraembryonic membranes of the diandric triploid mouse conceptuses (as well as the digynic triploids) did not appear to be grossly abnormal).     

修饰SOD及未修饰SOD的稳定性比较研究

测定了分子修饰ＳＯＤ的酶活力及其在不同温度下的稳定性,与未修饰的ＳＯＤ进行了比较,对在不同温度下测定的结果进行数据处理,经推算得出分子修饰ＳＯＤ在２５℃条件下,保存９５％的酶活力达３．５年,保存９０％的酶活力达７．２年,未修饰ＳＯＤ在２５℃条件下,保存９５％的酶活力为１０３ｄ,保存９０％的酶活力为２１３ｄ。     

Molecular characterization of a Y;15 translocation segregating in a family

Summary We have used Y-specific and Y-derived DNA probes for in situ hybridization and Southern blotting analysis to characterize a Y;15 translocation showing normal Mendelian inheritance in a family. Cytogenetically there appeared to be an unbalanced translocation of Yqh to 15p; this translocation may be considered as a prototype of those translocations between Yq and the short arm of an acrocentric chromosome which have a population incidence of approximately 1 in 2,000. Our molecular studies showed that, in all probability, the breakpoints were near the border between Yq11.23 and Yq12, and in 15p11, respectively; the translocation is abbreviated t(Y;15)(q12;p11). Using the Y-specific probe pY431 in a quantitative Southern hybridization assay, normal females had no hybridization, female carriers and normal men had the same amount, and male carriers had twice that amount. Cytogenetic analysis and quantitative in situ hybridization using probes pY431 and pY3.4 were consistent with the hypothesis that the portion of Yq translocated to 15p comprised all of Yq12 and none of Yq11. The absence of Southern hybridization with probes specific for Yp and Yq11 confirmed this observation. Even though the family was ascertained through two brothers who both had schizophrenia and were carriers of the translocation, the clinical evaluation of a total of nine individuals with the translocation and five without it did not suggest its association with an abnormal phenotype.     

表面改性SBA-15材料固载猪胰脂肪酶

采用试剂y-氯丙基三乙氧基硅烷（cvrEs）对介孔硅材料SBA-15进行表面改性,并通过红外图谱（FT-IR）和N2吸附脱附等温图（BET）对其进行表征。结果表明：改性前原材料的比表面积为460．9m2／g,改性后材料比表面积提高到512．0m2／g。利用改性前和改性后的SBA-15对猪胰脂肪酶进行固载实验,并对实验结果进行比较,发现改性后的SBA-15在脂肪酶活性、pH环境适应性、热耐受性和可操作性都优于改性前的SBA-15,在最优条件下的酶活力提高超过60％。     

Isolation and partial characterization of a human prothrombin variant: prothrombin Barcelona

An abnormal prothrombin variant, Prothrombin Barcelona, has been isolated by chromatography on DEAE Sephadex, from several members of the same family. In the absence of any normal component, it was eluted in two unequal peaks. The second peak was homogeneous. This component had the same molecular weight as normal prothrombin but migrated slightly faster on disc gel electrophoresis. The first peak, the smaller one, was heterogeneous: in addition to a minor band similar to that of the second peak, a major one with less anodic mobility and with a molecular weight of 32,000 was found. A possible chromatographic artefact has been eliminated. The family study gave good arguments for an heterozygote state of both parents, the siblings being homozygote.