Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway.

The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.

In vivo processing of the precursor of the major exoglucanase by KEX2 endoprotease in the Saccharomyces cerevisiae secretory pathway.

We have established the main post-translational modification of the major exoglucanase of Saccharomyces cerevisiae as the enzyme progresses through the secretory pathway. The protein portion of the enzyme accumulated by sec18 cells was about 2 kDa larger than that of the secreted enzyme. This precursor (form A) was stable when maintained in the endoplasmic reticulum but was processed to the mature form (form B) before the block imposed by the sec7 mutation. Sec7 cells, when incubated at 37 degrees C, accumulated form B first, but upon prolonged incubation, form A was preferentially accumulated. When the supply of newly synthesized exoglucanase was prevented by the addition of cycloheximide, the accumulated A was transformed into B in the presence of altered Sec7p that still prevented secretion. Conversion of A into B was prevented in the double mutant sec7 kex2-1, indicating that Kex2p is central to the in vivo processing. Consistent with this, a KEX2 deletion mutant secreted form A exclusively. Conversion of A into B was also prevented in sec7 cells by the presence of dinitrophenol, a poison that depletes ATP levels, indicating that processing is dependent upon intracellular transport which involves ER --> Golgi and/or, at least, one intra-Golgi step(s). It follows that this transport step(s) is independent of functional Sec7p.

Preferential transfer to truncated oligosaccharides to the first sequon of yeast exoglucanase in Saccharomyces cerevisiae alg3 cells.

In addition to the exoglucanases (Exg) secreted into the culture medium by wild type cells, ExgIa and ExgIb, which have oligosaccharides attached to both potential N-glycosylation sites, Saccharomyces cerevisiae alg3 mutant secreted substantial amounts (35--44%) of underglycosylated and unglycosylated forms. Quantification of these forms indicated that no more than 78% of the available N-sites were occupied. About 50% of the transferred oligosaccharides were endo H sensitive, indicating that the lipid-linked precursor had completed its synthesis to Glc3-Man9-GlcNAc2. The other 50% remained endo H-resistant and, accordingly, it should be derived from the precursor oligosaccharide Man5-GlcNAc2 synthesized by this mutant. A closer analysis of forms that have received two oligosaccharides (ExgIb) showed that the first sequon was enriched in truncated residues, whereas the second one was enriched in regular counterparts. Similarly, analysis of the individual underglycosylated glycoforms indicated that 38% of the oligosaccharides attached to the second site were regular. This percentage dropped to 20% for glycoforms carrying the oligosaccharide in the first sequon. The preferential transfer of truncated oligosaccharides to the first glycosylation site seems to be a consequence of (1) the low percentage of truncated lipid linked oligosaccharides that receives the glucotriose unit, and (2) the effect of the glucotriose unit on the selection of N-sites to be glycosylated.

Transcriptional regulation of the yeast vacuolar aminopeptidase yscI encoding gene (APE1) by carbon sources.

Transcription of the vacuolar aminopeptidase yscI-encoding gene (APE1) is regulated by the carbon source used for yeast growth, responding to carbon catabolite repression. By Northern blot analyses, we determined the kinetics of glucose repression in growth-shift experiments. When added to induced cells, glucose leads to the disappearance of hybridizable aminopeptidase yscI RNA sequences within 30 min. However, the amount of inmunoreactive protein, once induced, is not affected by the addition of glucose. By deletion analysis of the fusion gene APE1-lacZ we have identified a number of strong regulatory regions in the APE1 promoter. Consensus sequences for the binding of yAP1 and the HAP2/HAP3/HAP4 complex are contained in those regions. Control of the APE1 gene expression is not mediated by the HXK2 regulatory gene, but a strain bearing a deletion in the CAT1 gene can not derepress APE1 transcription to wild-type levels.

Yeast vacuolar aminopeptidase yscI. Isolation and regulation of the APE1 (LAP4) structural gene.

The structural gene, APE1, (LAP4), for the vacuolar aminopeptidase I of Saccharomyces cerevisiae was cloned with the aid of a staining technique which permitted monitoring of aminopeptidase activity in yeast colonies. Genetic linkage data demonstrate that integrated copies of the cloned gene map to the APE1 locus. The nucleotide sequence of the cloned gene was determined. The open reading frame of APE1 consists of 514 codons and, therefore, encodes a larger protein (MW 57,003) than the reported mature aminopeptidase yscI (MW 44,800), suggesting that proteolytic processing must occur. A 1.75-kb mRNA, which is made in substantial amounts only when yeast cells have exhausted the glucose supply, was identified.

The major exoglucanase secreted by Saccharomyces cerevisiae as a model to study protein glycosylation.

The major yeast exoglucanase (ExgIb) consists of a 408 amino acid polypeptide carrying two short N-linked oligosaccharides attached to asparagines 165 (Asn(165)) and 325 (Asn(325)). These oligosaccharides are very similar, in both length and composition, to those present in the vacuolar protease carboxypeptidase Y. Minor glycoforms of exoglucanase arise by underglycosylation of the protein precursor (Exg(165) and Exg(325)) or by elongation of the second oligosaccharide (ExgIa). The fact that these glycoforms can be readily separated and identified by HPLC and/or Western blots converts ExgI in an excellent model to study the role of the several components or branches of the precursor oligosaccharide in the efficiency and selectivity of the oligosaccharidyl transferase in vivo. We have found that the presence of a single glucose attached to Dol-PP-GlcNAc(2)-Man(9) increases the efficiency of transfer of that oligosaccharide to the protein acceptor. Also, the glucotriose unit appears to be involved in the selection of the sequons to be occupied, in such a way that its absence results in a bias towards the glycosylation of a particular sequon. Finally, we have shown the transfer of GlcNAc(2) from Dol-PP-GlcNAc(2) to exoglucanase, an indication that this intermediate is able to translocate from the cytoplasmic to the lumenal face of the endoplasmic reticulum membrane.

Biochemical and genetic analysis of an alpha-mannosidase mutant from Saccharomyces cerevisiae.

A yeast mutant lacking non-specific alpha-mannosidase activity was found as a background marker during our search for dap2 mutants (Suárez-Rendueles, P. and Wolf, D. H. (1987) J. Bacteriol. 169, 4041-4048). The mutant (DPS-15) is characterized in detail. The mutation called amd1 segregates 2:2 in meiotic tetrads, indicating a single chromosomal gene mutation which is recessive. Diploids heterozygous for amd1 show gene dosage. Thus, it appears that AMD1 might be the structural gene for alpha-mannosidase. Results obtained with this mutant show that alpha-mannosidase is not a vital component of the vegetative cell cycle. The differentiation process of sporulation is not disturbed in homozygous mutant diploids. Mannose turnover does not seem to be altered in mutant cells.

Molecular biology of yeast exoglucanases.

Three exoglucanase (Exg) genes have been reported in Saccharomyces cerevisiae. Gene EXG1 encodes the major isoenzyme (ExgI). Differential glycosylation of the primary translation product throughout the secretory pathway results in the secretion of several glycoforms. The major glycoform (ExgIb) contains two short carboxypeptidase Y-like oligosaccharides attached to both potential glycosylation sites present in the molecule. A minor glycoform (ExgIa) arises from the former by elongation of the second oligosaccharide. The protein portion is processed in the secretory pathway by the Kex2 protease. Gene EXG2 encodes a 63 kDa polypeptide with 12 potential glycosylation sites. The predicted protein, ExgII, carries a signal peptide at the amino terminus and a glycosyl-phosphatidyl inositol anchoring motif at the carboxyl end. The latter appears responsible for the particulate nature of this isoenzyme, since its elimination results in the secretion of this activity into the culture medium. Gene SSG1 encodes a 52 kDa polypeptide which is specifically synthesized during sporulation of diploids. SSG1 expression is under control of both sexual (a1-alpha 2 element) and nutritional control. Although homozygous ssg1/ssg1 diploid strains are still able to complete sporulation, they exhibited a delay in the appearance of mature asci. Single or double disruption of EXG1 and EXG2 did not result in any relevant phenotype and the triple mutant behaved as ssg1/ssg1. A ExgI-related enzyme is secreted by Candida albicans. All these four enzymes share 8 highly conserved regions in the same relative positions, indicating that they derived from a common ancestor. However, no clear function has so far been demonstrated for them.

Treatment of an acoustic neuroma in an only-hearing ear: case reports and considerations for the future.

Current options regarding the treatment of acoustic neuroma in an only-hearing ear include: observation, attempted hearing preservation surgery, and stereotactically guided radiation therapy. A patient who had a left labyrinthectomy for Menière's disease presented 15 years later with a large right acoustic neuroma. Due to anticipated profound deafness, he fit the criteria for cochlear implantation. Promontory stimulation of the left ear was positive. He underwent successful left cochlear implantation with the Nucleus 22-channel device and was successfully rehabilitated. He then underwent translabyrinthine removal of his right-sided 2.5-cm acoustic neuroma. This case is used to illustrate a new option available to those faced with treating a patient with acoustic neuroma in an only-hearing ear. How this approach may fit in with other available options will be discussed.

Conservation surgery for glomus jugulare tumors: the value of early diagnosis.

The results of lateral cranial base surgery for glomus jugulare tumors are gratifying when normal anatomy and function can be preserved. The goal of conservation surgery is to preserve normal ear anatomy and cranial nerve function. In general, conservation surgery is tumor-size dependent. Thus, excellent states of functional recovery depend upon accurate early diagnosis. This paper reviews the technical aspects of transtemporal conservation skull base tumor surgery while also reviewing our experience with nearly 100 glomus jugulare patients. Adjuvants to early diagnosis will be highlighted from a review of presenting symptoms, clinical signs, and related diagnosis. Our objective is to provoke a high index of suspicion in physicians charged with the responsibility of diagnosing these tumors. Diagnostic guidelines are proposed.

Postoperative radiographic evaluation after acoustic neuroma and glomus jugulare tumor removal.

The role of postoperative radiographic follow-up of patients after acoustic neuroma or glomus jugulare tumor removal is unclear and not standardized. The incidence of both lesions is rare and recurrence thought to be unusual. However, a patient with an acoustic neuroma arising in the same ear afflicted with a glomus jugulare tumor removed 5 years earlier prompted a retrospective review of 999 acoustic neuroma and 98 glomus jugulare tumor patients. This review helps to determine the role and frequency of postoperative follow-up, not only to assess recurrence of these lesions but to monitor for the possible development of potential clinically silent new lesions.

Electrical promontory stimulation in patients with intact cochlear nerve and anacusis following acoustic neuroma surgery.

Anacusis following hearing preservation surgery for acoustic neuroma removal in which the cochlear nerve was preserved has been explained on the basis of neural or vascular compromise. In the absence of pathologic evidence for either theory, a physiologic model was chosen. Electrical promontory stimulation with monitoring of subjective and electrically evoked auditory brainstem responses was undertaken. A positive response to stimulation suggests a vascular impairment of the cochlea sparing the cochlear nerve and spiral ganglion. The absence of response suggests loss of neural integrity at the level of the spiral ganglion or cochlear nerve. Six patients who suffered anacusis following hearing preservation surgery for acoustic neuroma were studied. Data regarding electrical promontory stimulation, auditory brainstem responses, and implications of the possible role of cochlear implantation are discussed.

Pasteurella infections of the head and neck.

Infections caused by Pasteurella occur most frequently after domestic animal bites or scratches and in individuals with agricultural or veterinary contact with animals. A serious Pasteurella infection developed in an agricultural worker following tumor extirpation of a head and neck neoplasm. Review of Pasteurella infections in humans disclosed that 31 of 446 reported infections involved head and neck structures. The most serious of these involved the adjacent central nervous system. Surgical drainage combined with parenteral penicillin remains the treatment of choice in these infections. Aminoglycosides are not effective in treating this organism.

An analysis of the retrolabyrinthine vs. the retrosigmoid vestibular nerve section.

Vestibular neurectomy is gaining widespread acceptance as a primary means of controlling medically refractory vertigo. However, debate continues over the adequacy of vestibular neurectomy within the cerebellopontine angle, long-term control, and the most appropriate surgical approach. To address these issues, we retrospectively reviewed 118 patients who underwent vestibular neurectomy between October 1984 and January 1988. Forty-two patients who underwent a retrolabyrinthine approach and 44 patients who underwent a retrosigmoid approach completed a written questionnaire and provided a recent audiogram. According to American Academy of Otolaryngology-Head and Neck Surgery guidelines, complete or substantial vertigo control was achieved and maintained in 95% of patients in both surgical groups. Hearing, tinnitus, and fullness results over the long term are variable. The advantages and disadvantages of the various vestibular neurectomy approaches will be detailed. On review of our results and surgical experience, we now prefer the retrosigmoid approach.

Cranial nerve preservation in lesions of the jugular fossa.

The most significant objection to the proposal of lateral transtemporal cranial base surgery for the treatment of jugular foramen tumors is the perceived lasting morbidity attendant to aggregate cranial nerve loss. As techniques become more standardized and earlier diagnosis generates smaller tumors for treatment, outcome has become more predictable. Surgery has become the recognized management preference for these cranial base lesions. The purpose of this article is to assess the role of surgery in the treatment of jugular foramen lesions, as well as to review some of the technical highlights of conservation surgery, its clinical prerequisites, and reasonable expectations. We review 100 lateral skull base surgical cases of lesions involving the jugular foramen. The majority of these lesions--77 of 100--were paragangliomas. For these glomus tumors, cranial nerve preservation correlated well to tumor size and location. The diversity of the remaining 23 cases prevented any substantive conclusions.

Leiomyoma of the nasal septum.

Leiomyoma is a benign myogenic tumor that may develop wherever smooth muscle is present. It occurs commonly in the uterus, skin, and gastrointestinal tract and is rare within the nasal cavity. Only three of twenty-four reported cases of sinonasal leiomyoma have been found to originate from the nasal septum. Treatment of choice for these neoplasms is surgical excision. We present two cases of nasal septal leiomyoma. Unique features discussed include recurrence of one neoplasm and the technique used to endoscopically repair a cerebrospinal fluid leak resulting from resection of the neoplasm.

Mejorando la respuesta patológica completa con tres esquemas diferentes de antraciclinas en densidad de dosis como primera línea en el tratamiento preoperatorio del cáncer de mama localmente avanzado / Improving the complete pathological response with three different schemes anthracyclines dose density as the first line in the preoperative treatment locally advanced breast cancer

Es un estudio prospectivo multiinstitucional que conlleva, a su vez, tres subestudios y luego se hace el metaanalisis de estos estudios piloto en pacientes con cáncer de mama localmente avanzado que reciben quimioterapia preoperatoria con antraciclinas en densidad de dosis seguido de tres esquemas diferentes, teniendo como objetivo llegar a la respuesta patológica completa (pCR). MATERIAL Y METODOS. Participaron 150 pacientes, 28 pacientes en el primer grupo (4AC+4AT), 57 pacientes en el segundo grupo (4AC+4CptT) y 65 pacientes en el tercer grupo (4AC+ 12 TXe), todos de inicio cánceres inoperables no metastásicos. RESULTADOS. En el primer grupo la RPC fue de 28 %, en el segundo grupo 20 % y en el tercer grupo 24 %, que se incrementó a 35 %, 19 %y 30 %, respectivamente, cuando solo se tabulo los datos de las pacientes que culminaron todo el tratamiento y que no presentaron progresión de enfermedad. CONCLUSIONES. En pacientes con tumores gigantes y en mds de 90 % EC III, las respuestas obtenidas son muy significativas solo con uso de quimioterapia, además de un ahorro económico importante al no usar biológicos. Con esto no se pretende ignorar la gran ayuda de los biológicos, simplemente que, para la realidad peruana, se proponen nuevas alternativas...

Is a prospective multi-institutional study involved three substudies in turn and then the meta-analysis of these pilot studies in patients with breast cancer with locally advanced receiving preoperative chemotherapy with anthracycline dose density followed by 3 different schemes, taking aim to reach the pCR. MATERIAL AND PATIENTS METHODS. 150 patients, 28 patients in the first group (4AC + 4AT), 57 patients in the second group (4CptT 4AC +) and 65 patients in the third group (4AC + I2TXe), all of them with inoperable cancers with nonmetastatic disease. RESULTS. The pCR In the first group was 28 %, in the second group 20% and in the third group 24 %, which increased to 35 %, 19 % and 30 % respectively when only the data of the patients culminating all treatment and no progression of disease was tabulated. CONCLUSIONS. Whereas these patients with giant tumors and in 90 % EC III responses obtained are significant only with use of chemotherapy, in addition to significant cost savings by not using biological agents. We not pretended ignore the evidence that the biological products help in excellent manner, but for our country this is an alternative good way...