Fine structure of the spermatozoa of Crassostrea gigas (Mollusca,Bivalvia)

We describe sperm ultrastructure and acrosome differentiation during spermiogenesis in Crassostrea gigas (Mollusca Bivalvia). The sperm cell is a uniflagellated cell of the primitive type. The head region contains a rounded or conical nucleus surmounted by small acrosome. This organelle consists of a membrane-bound acrosomal granule, the contents of which have a homogeneous density, except in the anterior region, which is positive for PTA. The acrosome also surrounds the perforatorium, which includes oriented fibrillar elements: this is the axial body. The middle piece contains four mitochondria encircling two perpendicular centrioles. The distal centriole is provided with a system of mechanical fixation to the plasma membrane, consisting of nine fibers in radial arrangement. The tail flagellum, about 50 m?m long, contains the usual microtubular axoneme. © 1993 Wiley-Liss, Inc.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

The spermatogenesis of crustaceans. VII. Review of spermatozoon of the crayfish Astacus astacus (Malacostraca,Decapoda, Macrura,Reptantia)

The star-like spermatozoon of Astacus astacus consists of a spheroidal central body around which various prolongations of same, denominated spines, are arranged. In the interior of the gamete the following parts may be distinguished: (1) The acrosomic region, formed by a complex vesicle, or thick-walled, helmetshaped body, whose opening is orientated towards the nuclear region. In the interior of the vesicle different structures can be appreciated. (2) The nuclear region, which is formed by a large cupuliform nucleus limited by a double membrane. In the nucleoplasm numerous bundles of microtubules, mixed with noncondensated chromatin fibers, are found. (3) The laminar region, present in other Decapoda, is practically nonexistent. Within the spines of these spermatozoa, only microtubules can be observed. The morphology of this crayfish is similar to that presented by Brachiura, another group of Reptantia.     

Comparison of the effects of human erythrocyte ghosts and intact erythrocytes on platelet interactions with subendothelium in flowing blood

We investigated whether ghosts behaved similarly to intact erythrocytes to maintain regular primary hemostasis under flow conditions. To this end we performed perfusion experiments with whole blood in which erythrocytes were replaced by pink ghosts, and platelet interaction with the subendothelial surface of a damaged vessel was morphometrically evaluated. The same objective was sought by means of studies with a platelet function analyzer (PFA-100(TM) instrument). Perfusions performed with control blood reconstituted with intact erythrocytes gave rise to 0.4+/-0.2% contact but not spread platelets, 10.8+/-3.4% adhering and spread platelets, 16.3+/-4.6% platelets in thrombi, with 27.5+/-7.4% of the surface covered. Even though the average diameter of the ghosts was smaller than that of intact erythrocytes (5.3 microm vs. 7.7 microm), the values obtained in perfusions performed with ghosts were similar to those of the erythrocyte controls. Studies performed with the PFA-100(TM) analyzer were consistent with those observed in perfusion studies. The viscosity of control blood was compared with that of blood reconstituted with ghosts. At shear rates lower than 450 s(-1), the viscosity of the ghost samples was higher than that of the controls, but the difference progressively decreased as shear rate increased up to 750 s(-1) (3.61+/-0.15 and 3.71+/-0.17 cP, respectively). In conclusion, the results of our study showed that ghosts behaved similarly to intact erythrocytes in maintaining a normal platelet interaction with digested subendothelium, under conditions of moderate shear rate and constant hematocrit (40%). The rheological activity of ghosts, bodies that are metabolically less active, was sufficient for them to satisfactorily act as substitutes for intact erythrocytes in our system.     

Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus

In striated muscles myosin light chain (MLC)2 phosphorylation regulates calcium sensitivity and mediates sarcomere organization. Little is known about the changes in MLC2 phosphorylation in relation to skeletal muscle plasticity. We studied changes in MLC2 phosphorylation in rats receiving three treatment conditions causing slow-to-fast transitions: 1) atrophy induced by 14 days of hindlimb suspension (HS), 2) hypertrophy induced by 14 days of clenbuterol administration (CB), and 3) 14 days of combined treatment (CB-HS). Three variants of the slow (MLC2s) and two variants of the fast MLC2 (MLC2f) isoform were separated with two-dimensional electrophoresis and identified with monoclonal and polyclonal antibodies specific for MLC2; their relative proportions were densitometrically quantified. In control soleus muscle MLC2s predominated over MLC2f (91.4 ± 3.9% vs. 8.5 ± 3.9%) and was separated into two spots, the less acidic spot being 73.5 ± 4.3% of the total. All treatments caused a decrease of the less acidic unphosphorylated spot of MLC2s (CB: 64.1 ± 5.6%, HS: 62.4 ± 6.8%, CB-HS: 56.4 ± 4.4%), the appearance of a third more acidic variant of MLC2s (representing 3.9–5.9% of total MLC2s), an increase of MLC2f (CB: 30.9 ± 3.1%, HS: 23.9 ± 3.3%, CB-HS: 25.3 ± 3.9%), and the phosphorylation of a large fraction of MLC2f (CB: 30.4 ± 6.7%, HS: 28.7 ± 6.5%, CB-HS: 21.8 ± 2.1%). Treatment with alkaline phosphatase or with protein phosphatase 1 (PP1) removed the most acidic spots of both MLC2f and MLC2s. We conclude that in rat skeletal muscles an increase of MLC2 phosphorylation is associated with the slow-to-fast transition regardless of whether hypertrophy or atrophy develops. muscle atrophy; muscle hypertrophy; clenbuterol; hindlimb suspension     

Measurement of chemopreventive efficacy in skin biopsies

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.     

啤酒酵母菌的细胞融合

啤酒多倍体酵母菌原生质体已成功地与单倍体原生质体进行融合。经细胞壁再生后,稳定的融合重组体被分离出来。这些融合体的基因分析表明,融合体中含有双亲的基因型。孢子形成良好,且每个子囊中含有四个孢子,每个孢子确实是二倍体。这样原生质体融合就提供了一个对啤酒酿造酵母进行遗传分析的方法。但是如果没有一个方便的杂交技术,这个方法将是很困难的。     

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.