The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender.

Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 µg mL⁻¹) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48h and this experiment was repeated using 25 × 106 and 800 × 106 spermatozoa mL⁻¹. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.

CVB translation: lessons from the polioviruses.

Our understanding of coxsackie B virus translation and replication has benefited greatly from half a century of research on the closely related polioviruses. Like poliovirus, coxsackievirus gene expression is controlled largely at the translation level and coxsackievirus infection results in profound changes in the profile of mRNAs with access to the protein synthesis machinery of the host cell. This review chronicles the advances in understanding translational control by the enteroviruses, primarily in poliovirus and clarified by related viruses, and highlights areas where coxsackievirus conforms to or differs from the aggregate model. Basic IRES structure and function, proteins involved in cap-dependent and viral translation, viral modification of translation factors to achieve host translation shutoff and promotion of viral translation are discussed. The translational bases for neurovirulent phenotypes and tissue specificity are also addressed.

Cell-to-cell transfer of interferon-induced antiproliferative activity.

Interferon-treated cells rapidly and efficiently transferred the antiproliferative activity of interferon to untreated cells. This phenomenon was not due to the carry-over of interferon by the interferon-treated cells. Thus, to evoke an antiproliferative state, interferon did not directly contact each cell in a population. The results suggest a novel mechanism by which interferon may indirectly regulate cell growth, and suggests that cells other than those of the immune system may play a role in controlling tumor growth in tissue where cell-to-cell contact occurs.

Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro.

Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39 degrees C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.

Artificial insemination for the propagation of CANDES: the reality!

Conservation is about protecting and nurturing species so that they can survive, not only now, but also into the future. Ideally this means protecting genetically diverse populations and not simply breeding a few individuals. Unfortunately, this point is often overlooked by reproductive technologists, especially if they are more accustomed to working with humans, companion animals or agricultural species, where the goals are more usually directed towards obtaining offspring from particular individuals. This approach has tended to antagonise the conservation community, who are quick to develop an unreasonable suspicion of technological solutions, partly because they are unfamiliar with the scientific principles that underpin the reproductive technology. Unfortunately, this mutual failure to recognise that all parties are actually well meaning, has led to separate cultures that barely communicate with each other and thus fail to capitalise on the potential benefits that would come from a good working relationship. Notable successes with reproductive technology have only emerged where such relationships have been forged. In this review, we highlight, mainly for the benefit of the technologist community, the need to foster good working relationships with conservation managers and to recognise that the latest hi-tech approach to animal breeding is more likely to engender suspicion than enthusiasm.

Temporal dynamics of ram sperm binding and survival during 48-h coculture with oviducal epithelial cells.

Following insemination, ram spermatozoa bind to oviducal epithelial cells (OEC) in vivo and remain viable for several hours before fertilisation. In the present study, we investigated whether OEC monolayers reproduce this effect in vitro, performing an analysis of ram sperm binding and survival over an extended (48 h) period at 39 degrees C. We wanted to determine whether the reproductive cycle phase and/or oviducal region would influence ram sperm binding and survival in coculture with OEC and whether reproductive and non-reproductive epithelial cells bound and maintained the viability of ram spermatozoa equivalently. Oviducts were separated into groups based on their ovarian state (follicular or luteal) and then divided into two parts (isthmus and ampulla) for OEC isolation. Sheep kidney epithelial cells (Madin-Darby ovine kidney; MDOK) were purchased commercially. Reproductive cycle phase, but not oviducal region, affected sperm binding to OEC. Although more spermatozoa bound to luteal OEC than to follicular OEC at 1 h, at 24 h follicular OEC had bound more spermatozoa than luteal OEC. Generally, spermatozoa that were bound to OEC and MDOK had enhanced viability at each of the time points investigated (1, 6, 24 and 48 h), but the viability of the OEC-bound spermatozoa was greater than that of the MDOK-bound spermatozoa at 48 h. In conclusion, ram sperm-epithelial cell interactions are temporal, dynamic and depend on the origin of the epithelial cells.

Eukaryotic translation initiation factor 4G is targeted for proteolytic cleavage by caspase 3 during inhibition of translation in apoptotic cells.

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.

Cleavage of eukaryotic translation initiation factor 4GII correlates with translation inhibition during apoptosis.

Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage.

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.

Haematoma of anterior ethmoidal artery after reduction of fracture of zygomatic complex.

Extraconal intraorbital haemorrhage is a rare complication after reduction of a fracture of the zygomatic complex. We present a case of postoperative intraorbital haematoma that arose from the anterior ethmoidal artery. We stress the advantages of imaging before decompression and of the medial approach.

One-day admission for lung lobectomy: an incidental result of a clinical pathway.

BACKGROUND: Most complications after lung lobectomy are related to pain, narcotic analgesia, and inactivity. When the operation is performed with the goal of minimizing postoperative pain, and when rapid restoration of activity and patient independence can be achieved, most postoperative complications can be obviated and early discharge can be attained. METHODS: Since March 1996, we have performed 10 consecutive elective major lung resections (8 lobectomies and 2 bilobectomies) for neoplastic (n = 8) and benign inflammatory (n = 2) lesions. Of the 10 patients, 4 were men and 6 were women ranging in age from 58 to 77 years (mean age, 66 years). Extensive preoperative patient and family education was provided in the surgeon's office. Same-day admission was followed by an oblique muscle-sparing minithoracotomy to access the chest cavity. A meticulous operation, with special attention to minimizing air leak and postoperative discomfort, was performed. Intercostal nerve cryolysis was used as the main method of analgesia. RESULTS: All patients underwent the planned operation through a minithoracotomy and were extubated in the operating room. All patients exhibited normal ipsilateral shoulder girdle mobility in the recovery room and none required intravenous narcotics after leaving this unit. All patients were out of bed the day of the operation. The chest tube was removed the night of the operation in 2 patients, the morning after the operation in 6 patients, and on the second postoperative day in 1 patient. One patient who was discharged with a Heimlich valve had this device removed in the office 4 days after the operation. After the chest tubes were removed, there were no instances of pneumothorax. All 10 patients were able to ambulate independently on the first postoperative day. Eight patients were discharged home the morning after the operation and 2 on the second postoperative day. None of the patients have required readmission related to their operation or have exhibited evidence of postthoracotomy pain syndrome. CONCLUSIONS: We have developed a clinical pathway based on patient education, meticulous minimally invasive operation, cryoanalgesia, and quick resumption of physical activity. Our preliminary experience with this approach has shown minimal morbidity, rapid restoration to preoperative status, and, for most patients, a 1-day hospital stay after major lung resection.

Growing Escherichia coli mutants deficient in riboflavin biosynthesis with non-limiting riboflavin results in sensitization to inactivation by broad-spectrum near-ultraviolet light (320-400 nm).

Two mutants of Escherichia coli unable to synthesize riboflavin were grown with limiting (2 micrograms ml-1) and non-limiting (10 micrograms ml-1) concentrations of riboflavin. These riboflavin auxotrophs when grown to exponential phase with non-limiting riboflavin are more sensitive to broad spectrum near-ultraviolet light (NUV, 320-400 nm) inactivation than when they are grown with limiting riboflavin. Exponential phase cells of the riboflavin auxotrophs grown with limiting riboflavin are sensitized when irradiated in saline supplemented with riboflavin. This suggests that extracellular riboflavin is important as a NUV sensitizer when intracellular levels of riboflavin are reduced. The concentration of riboflavin in crude extracts from exponentially growing cells correlates well with the sensitivity of these mutants to NUV inactivation. The level of riboflavin supplementation has little effect on the NUV sensitivity of the parental strain.

Cu(II) sensitizes pBR322 plasmid DNA to inactivation by UV-B (280-315 nm).

Copper(II), in the presence of UV-B radiation (280-315 nm), can generate single-strand breaks in the sugar-phosphate backbone of pBR322 plasmid DNA. A low level of single-strand backbone breaks occurs in the presence of Cu(II) alone, but UV-B irradiation increases the rate by the more than 100-fold. Concomitant with the damage to the DNA backbone is a loss of transforming activity. Oxygen is required for generation of the single-strand breaks but not for the loss of transforming activity. A DNA glycosylase (Fpg), which participates in the repair of certain DNA nitrogenous base damage, does not repair plasmid DNA damaged by Cu(II). The hydroxyl radical scavenging compound DMSO is only somewhat effective at protecting the physical and biological properties of the DNA. These results with Cu(II) are compared to those obtained previously with pBR322 plasmid DNA in the presence of Fe(III) and UV-A.

Ferric-ion-photosensitized damage to DNA by hydroxyl and non-hydroxyl radical mechanisms.

Iron(III) and UVA (320-400 nm) light strongly diminished the transforming activity of Haemophilus influenzae DNA in the presence of oxygen. Iron(III) alone in the absence of light had no measurable effect on the transforming activity. The chelating agent ethylenediaminetetraacetic acid (EDTA) conferred virtually complete protection, but hydroxyl radical scavengers (mannitol, methanol, ethanol, isopropanol and dimethyl sulfoxide) inhibited only a small fraction of the inactivation. Treatment of plasmid DNA (pBR322) with iron(III) results in the conversion of the covalently closed circular form of the plasmid to open circles and ultimately to the linear form. Concomitant with the alteration in the conformation of the plasmid, the ability to transform Escherichia coli was reduced. In model systems, iron(III) photoreacted with the DNA backbone causing nicking and double-strand breakage. The results are consistent with a mechanism involving a preliminary complexation of iron(III) by DNA followed by the generation of reactive free radicals other than .OH. We suggest that bound iron, or other UV-absorbing transition metal complexes, may be chromophores capable of causing DNA damage in the long-wave near-UV region.

Muscle-sparing minithoracotomy with intercostal nerve cryoanalgesia: an improved method for major lung resections.

To decrease incisional pain, morbidity, and length of hospital stay (LOS) and, hopefully, to reduce costs, most surgical specialties have turned to minimally invasive procedures to access the body cavities during commonly performed operations. Video-assisted thoracic surgery (VATS) has emerged as the standard approach for a number of diagnostic and therapeutic procedures in thoracic surgery. Major lung resections (lobectomy, bilobectomy, and pneumonectomy), however, can be performed through an incision similar in size to the utility or access thoracotomy used in VATS to remove the specimen. The purpose of this study was to compare an oblique muscle-sparing minithoracotomy with intercostal nerve cryoanalgesia with the standard posterolateral thoracotomy incision and VATS to perform major lung resections. Forty consecutive patients with bronchogenic carcinoma, operated on by a single surgeon, were chronologically divided into two groups, each with equivalent age, sex distribution, physiologic parameters, tumor size, and clinical stage. In addition, data were collected from a MEDLINE search of all published studies in which major lung resections were performed via VATS. The first group (group A, n = 20) underwent posterolateral thoracotomy to access the chest cavity, whereas the patients in the second group (group B, n = 20) underwent oblique minithoracotomy with intercostal nerve cryoanalgesia. Group B compared favorably with group A in LOS (P = 0.002), narcotic requirements (P = 0.001), morbidity (P = 0.042), and cost (P = 0.058). Group B also compared favorably with VATS major lung resection published data regarding LOS and morbidity.

Necrotising fasciitis of the orbit.

A rare case of necrotising fasciitis affecting the face is described which, following prompt treatment and reconstruction, resulted in a successful outcome.

Lymphoepithelial cyst of the parotid gland.

A case of a lymphoepithelial cyst presenting as a parotid tumour is described. Aspects of diagnosis and management are discussed, as well as current theories on the origin and development of lymphoepithelial (branchial) cysts.

Int6 regulates both proteasomal degradation and translation initiation and is critical for proper formation of acini by human mammary epithelium.

INT6/EIF3E has been implicated in breast tumorigenesis, but its functional activities remain poorly defined. We found that, repressing INT6 expression induced transformed properties in normal human mammary epithelium (MCF10A); in contrast, Int6 silencing induced apoptosis in HeLa cells. As in fission yeast, Int6 in human cells was required for assembly of active proteasomes. A reverse-phase protein array screen identified SRC3/AIB1 as one oncoprotein the level and stability of which increased when Int6 was silenced in MCF10A cells. Our data further show that Int6 binds SRC3 and its ubiquitin ligase Fbw7, thus perhaps mediating the interaction between SRC3-Fbw7 and proteasomes. Consistent with this, Int6 silencing did not increase SRC3 levels in HeLa cells, which have low Fbw7 levels. It is surprising that, however, polyubiquitylated proteins do not accumulate or may even decrease in Int6-silenced cells that contain defective proteasomes. Considering that decreased ubiquitin might explain this observation and that Int6 might control ubiquitin levels in its role as a subunit of eIF3 (eukaryote translation initiation factor 3), we found that silencing Int6 reduced monoubiquitin protein levels, which correlated with a shift of ubiquitin mRNAs from larger polysomes to non-translating ribosomes. In contrast, levels of many housekeeping proteins did not change. This apparent reduction in the translation of ubiquitin genes correlated with a modest reduction in protein synthesis rate and formation of large polysomes. To further determine whether Int6 can selectively control translation, we analyzed translation of different 5'-untranslated region reporters and found that indeed, loss of Int6 had differential effects on these reporters. Together the data suggest that Int6 depletion blocks ubiquitin-dependent proteolysis by decreasing both ubiquitin levels and the assembly of functional proteasome machinery, leading to accumulation of oncoproteins, such as SRC3 that can transform mammary epithelium. Our data also raise the possibility that Int6 can further fine-tune protein levels by selectively controlling translation of specific mRNAs.

Sperm storage in the vertebrate female reproductive tract: how does it work so well?

The capacity for sperm storage within the female reproductive tract occurs widely across all groups of vertebrate species and is exceptionally well developed in some reptiles (maximum duration, 7 yr) and fish (maximum duration, >1 yr). Amphibians (most salamanders and one species of frog; duration approximately 5 mo), all birds examined to date and some bats, have also evolved the ability to store spermatozoa in the female reproductive tract. Although there are many reports on both the occurrence of female sperm storage and its adaptive benefits, few studies have been directed toward explaining the mechanisms involved. Phylogenetic evidence suggests that the capacity for sperm storage has evolved independently within different taxonomic groups, and it is by no means clear whether these groups have established similar or different mechanisms or whether simple and common principles have been exploited during evolution. If the process has indeed developed by the invention of numerous different and species-specific mechanisms, it is surprising that none have yet been elucidated by technologists wishing to improve the long-term storage of fresh semen. On the other hand, if there is a simple and common solution to the problem, readily accessed by diverse groups of species, it is equally logical to suppose that the mechanism should be easily discovered in the laboratory. While recognizing that studies on wild species are usually neither practically or ethically easy to undertake, it is clear that there is a huge and largely unexplored field to be investigated.