An analysis of white-handed gibbon male song reveals speech-like phrases.

OBJECTIVES: Our goal was to document song phrases of the white-handed gibbon (Hylobates lar), an Asian ape that produces elaborate songs, often in well-coordinated male/female duets. We focused on the male coda, which is produced during vocal turn-taking with one's mate, and particularly its phrases containing rapid spectral and temporal variation, to investigate if modulation rates resemble those of lip-smacking in other nonhuman primates and human speech rhythm. MATERIALS AND METHODS: We produced recordings from a large population of wild gibbons. Using terminology consistent with that used to describe vocalizations in other singing species, we analyzed coda phrases, overall coda properties, coda distinctiveness across individuals, and flexibility of phrase production within song bouts. RESULTS: Our song phrase-level analysis showed that male codas differed between individuals and increase in complexity within song bouts by the addition of the only two male-specific phrases of the species' repertoire. These phrases differ from all others of the species and from vocalizations typical of the larger, nonhuman great apes, in that they contain rapid within-phrase modulation. Their modulation rates (6.82 and 7.34 Hz) are similar to that of lip-smacking in other nonhuman primates and speech in humans and, like human speech, are produced exclusively during exhalation. One phrase type (trills) contains multiple notes per exhalation, another characteristic similar to speech but not most primate vocalizations. DISCUSSION: Our data highlight the complexity and flexibility of gibbon song, and show that particular phrase features likely arose from sexual selection pressures and possess similarities to human speech rhythm.

Age related decline in female lar gibbon great call performance suggests that call features correlate with physical condition.

BACKGROUND: White-handed gibbons (Hylobates lar) are small Asian apes known for living in stable territories and producing loud, elaborate vocalizations (songs), often in well-coordinated male/female duets. The female great call, the most conspicuous phrase of the repertoire, has been hypothesized to function in intra-sexual territorial defense. We therefore predicted that characteristics of the great call would correlate with a caller's physical condition, and thus might honestly reflect resource holding potential (RHP). Because measurement of RHP is virtually impossible for wild animals, we used age as a proxy, hypothesizing that great call climaxes are difficult to produce and maintain over time, and that older adults will therefore perform lower quality great calls than young adults. To test this we analyzed the great call climaxes of 15 wild lar gibbon females at Khao Yai National Park, Thailand and 2 captive females at Leo Conservation Center, Greenwich, CT. RESULTS: Findings show that call climaxes correlate with female age, as young animals (n = 8, mean age: 12.9 years) produced climaxes with a higher frequency range (delta F0), maximum F0 frequency and duty cycle than old animals (n = 9, mean age: 29.6 years). A permuted discriminant function analysis also correctly classified calls by age group. During long song bouts the maximum F0 frequency of great call climaxes' also decreased. Additional data support the hypothesis that short high notes, associated with rapid inhalation as an individual catches its breath, reflect increased caller effort. Older females produced more high notes than younger females, but the difference only approached statistical significance, suggesting that calling effort may be similar across different ages. Finally, for the first time in this species, we measured peak intensity of calls in captive females. They were capable of producing climaxes in excess of 100 dB at close range (2.7 m). CONCLUSIONS: Age and within-bout differences in the lar gibbon great call climax suggest that call features correlate with physical condition and thus the call may have evolved as an honest signal in the context of intra-sexual territorial defense and possibly also in male mate choice via sexual selection, although further testing of these hypotheses is necessary.

Lar gibbon (Hylobates lar) great call reveals individual caller identity.

Gibbons (family Hylobatidae) produce loud, elaborate vocalizations (songs), often in well-coordinated male/female duets. The female's great call, the most conspicuous phrase of the gibbon vocal repertoire, functions primarily to mediate territorial defense. Despite the fact that lar gibbons (Hylobates lar) are the most widely distributed and well researched hylobatid species and produce a rich vocal repertoire, the individual-specificity of their great calls has not previously been quantified. In addition, spectral and temporal features of notes occurring at specific locations within the lar great call have not been described. Here we provide such a description, and test the hypothesis that great calls are statistically discriminable between a large sample of individual callers. We compared recordings of great calls from 14 wild lar females in Khao Yai National Park, Thailand. Our analyses of principal components derived from spectral and temporal measures, as well as spectrograms from the entire great call, indicate that acoustic variation is sufficient to allow identification of individual callers (83.5% discriminability based on principal components, and inter-individual call variation exceeding intra-individual variation in overall spectrogram). These vocalizations potentially allow individual recognition of animals.

[Incidence of Candidaemia Following Abdominal Surgery in German Hospitals]. / Inzidenz der Candidämie nach gastrointestinal-chirurgischen Eingriffen in deutschen Akutkrankenhäusern.

BACKGROUND: Invasive Candida infections following abdominal surgery represent a significant medical problem. This study initiates a benchmarking project to pinpoint the current role and epidemiology of candidemia in this patient group in German hospitals. MATERIAL AND METHODS: During the year 2010 data derived from 47 704 abdominal surgery cases in hospitals from Germany were analysed in order to determine benchmarking incidences for candidemia. RESULTS AND CONCLUSION: In 20.3 % of all recognised bloodstream infections Candida spp. were identified as the responsible organisms. If related to all abdominal surgery cases analysed in this study, a candidemia-benchmarking incidence of 0.15 % (95 % CI: 0.10-0.21 %) was determined. In patients who required intensive care after surgery the incidence of candidemia was found to be 0.89 % (95 % CI: 0.57-1.38 %). The incidence increased to 3.13 % (95 % CI: 2.09-4.66 %) in patients who received blood culture diagnosis. The German National Reference Centre of Systemic Mycosis provides hospital specific data for participants of this study to enable benchmarking and infection control (www.nrz-mykosen.de/gastrointestinalchirurgie).

[Successful management of malaria tropica with 50% parasitaemia]. / Erfolgreiche Therapie einer Malaria tropica mit 50%-iger Parasitämie.

HISTORY: A 52-year-old woman was hospitalized with fever after a 3-week stay in tropical Kenya. Prophylaxis against malaria had been carried out with chloroquine. DIAGNOSIS: Falciparum malaria with 28% parasitaemia at first examination, rising to 50% after 3 hours. TREATMENT AND COURSE: Treatment with quinine dihydrochloride i.v. was initiated immediately after diagnosis. In addition, in view of increasing parasitemia of up to 50%, a partial exchange blood transfusion was carried out. No clinical signs of organ damage caused by malaria were observed. Because of a drop in blood pressure the patient needed catecholamine treatment for a short time. After decrease of the parasitemia the patient rapidly recovered and complete cure was achieved. CONCLUSION: Despite extremely high parasitemia the clinical signs were unusually mild. Standard treatment for severe malaria is intravenous administration of quinine. However, this drug is no longer sold in Germany, so that difficulty in obtaining it must be expected. A stockpiling of quinine is recommended for hospitals treating patients with malaria. Transfusion may improve outcome and must be considered if parasite counts are high or if there are clinical signs of malaria complications.

Molecular cloning of an extracellular aspartic proteinase from Rhizopus microsporus and evidence for its expression during infection.

An extracellular aspartic proteinase (Rmap) from Rhizopus microsporus var. rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA). The proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose. Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids. The preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids. The deduced amino-acid sequence of the preproenzyme was 82% homologous to an extracellular aspartic proteinase of Rhizopus niveus. Low stringency Southern blot analysis of R. microsporus DNA suggested the presence of other homologous genes. Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant. Both enzyme preparations exhibited a similar optimum of activity in the pH 2.5 region. Furthermore, Rmap was shown to activate bovine blood coagulation factor X at slightly acidic pH in vitro. Expression of the proteinase during mycosis was proven by a specific immune response of patient HA.

Secreted aspartic proteinase family of Candida tropicalis.

Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis lambdaEMBL3 genomic library. All bands identified by Southern blotting of EcoRI-digested C. tropicalis genomic DNA with SAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicalis cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other SAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.

Molecular characterization and influence on fungal development of ALP2, a novel serine proteinase from Aspergillus fumigatus.

A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, Int. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries. ALP2 shares a 49% identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae. In addition there is a 78% identity with PEPC, a serine proteinase which has been described in Aspergillus niger. Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80% reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50% reduction of the median diameter of conidiophore vesicles. The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A. fumigatus.

Intra- and intermolecular events direct the propeptide-mediated maturation of the Candida albicans secreted aspartic proteinase Sap1p.

Pathogenic yeasts of the genus Candida secrete aspartic proteinases (Sap) which are synthesized as preproenzymes. Expression of the C. albicans SAP1 gene lacking the propeptide-coding region in the methylotrophic yeast Pichia pastoris does not lead to the secretion of the enzyme into the culture supernatant, but results in an accumulation of recombinant protein in the cell. Co-expression in this system of the unattached propeptide from Sap1p, as well as from other Saps, restored Sap1p secretion. A deletion analysis revealed that only a 12 aa sequence in the propeptide, corresponding to a highly conserved region in all Sap propeptides, was necessary and sufficient to produce a large amount of Sap1p in culture supernatant. No Sap1p was secreted when Sap1p was produced with a propeptide carrying an F to D mutation in the identified 12 aa sequence. However, the simultaneous production of equivalent amounts of Sap1p and His-tagged Sap1p (H(6)-Sap1p) with a mutated and a non-mutated propeptide, respectively, led to the secretion of both proteins in a ratio of approximately 1:2. The restoration of Sap1p secretion occurred at the expense of secretion of H(6)-Sap1p since the total activity was comparable to that of strains producing only H(6)-Sap1p with a non-mutated propeptide. In contrast, the proteolytic activity of strains secreting Sap1p and H(6)-Sap1p both with a functional propeptide was twice that of strains producing either Sap1p or H(6)-Sap1p alone, and the two enzymes were found in an equivalent amount in the culture supernatant. Altogether, these results show that the propeptide can only function once and that the maturation of recombinant C. albicans secreted aspartic proteinase Sap1p is directed through a combination of intra- and inter-molecular pathways.

Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus.

An aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes. PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis.

Disruption of the gene which encodes a serodiagnostic antigen and chitinase of the human fungal pathogen Coccidioides immitis.

Disruption of genes in medically important fungi has proved to be a powerful tool for evaluation of putative virulence factors and identification of potential protein targets for novel antifungal drugs. Chitinase has been suggested to play a pivotal role in autolysis of the parasitic cell wall of Coccidioides immitis during the asexual reproductive cycle (endosporulation) of this systemic pathogen. Two chitinase genes (CTS1 and CTS2) of C. immitis have been cloned. Preliminary evidence has suggested that expression of CTS1 is markedly increased during endospore formation. The secreted CTS1 chitinase has also been shown to react with patient anti-Coccidioides complement-fixing (CF) antibody and is a valuable aid in the serodiagnosis of coccidioidomycosis. To examine the role of CTS1 in the morphogenesis of parasitic cells, the CTS1 gene was disrupted by a single, locus-specific crossover event. This resulted in homologous integration of a pAN7.1 plasmid construct that contained a 1.1-kb fragment of the chitinase gene into the chromosomal DNA of C. immitis. Results of Southern hybridizations, immunoblot analyses of culture filtrates using both CTS1-specific murine antiserum and serum from a patient with confirmed coccidioidal infection, an immunodiffusion test for CF antigenicity, and substrate gel electrophoresis assays of chitinase activity confirmed that the CTS1 gene was disrupted and nonfunctional. This is the first report of a successful targeted gene disruption in C. immitis. However, loss of CTS1 function had no effect on virulence or endosporulation. Comparative assays of chitinase activity in the parental and Deltacts1 strains suggested that the absence of a functional CTS1 gene can be compensated for by elevated expression of the CTS2 gene. Current investigations are focused on disruption of CTS2 in the Deltacts1 host to further evaluate the significance of chitinase activity in the parasitic cycle of C. immitis.

Effects on the mitosis of normal and tumor cells induced by light treatment of different wavelengths.

OBJECTIVE: Although the background of laser therapy by means of low level energy and power is still only partially understood, there are nevertheless promising reports from clinical studies concerning pain treatment, the acceleration of wound healing, and the modulation of cell functions. In order to contribute to the understanding of such a phototherapeutic procedure cell experiments were performed. MATERIALS AND METHODS: The influence of light (lambda = 410, 488, 630, 635, 640, 805, and 1,064 nm and broad band white light) on the proliferation of cells was investigated on skeletal myotubes (C2), normal urothelial cells (HCV29), human squamous carcinoma cells of the gingival mucosa (ZMK1), urothelial carcinoma cells (J82), glioblastoma cells (U373MG), and mamma adenocarcinoma cells (MCF7) in a computer-controlled light treatment chamber. The cellular response was tested by way of the following methods: The rate of mitosis was determined by counting the single cells after Orcein-staining. The proliferation index measurements were based on the BrdU incorporation during the DNA synthesis. Statistics were performed using unpaired Student's t-test procedures, stating P < 0. 05 to be significant and P>0.05 not to be significant. RESULTS: Twenty-four hours after light treatment, a significant increase in the mitotic rate of J82 and HCV29 cells was determined when illuminated with lambda = 410 nm, lambda = 635 nm and lambda = 805 nm, respectively. C2 cells showed an increase only after lambda = 635 nm illumination. In all three cell lines, a maximum mitotic rate was determined after an irradiation between 4 and 8 J/cm(2), while a reduced mitotic rate was measured at 20 J/cm(2). MCF7, U373MG, and ZMK1 cells showed a slight decrease in the mitotic rate with increasing irradiation independent of the wavelength used. When an irradiation of 20 J/cm(2) was applied, all cell lines showed a slight decrease compared to the controls independent to the wavelength used. White light as well as lambda = 1,064 nm does not affect the mitotic rate in this irradiation range. No significant differences in the effects could be determined when the irradiance changed between 10 and 150 mW/cm(2) at certain irradiation values. The BrdU test did not show any significant alterations with respect to possible light induced processes compared to the controls. CONCLUSIONS: Dependent upon the irradiation parameter, light of a defined wavelength does affect the mitotic rate of both normal as well as tumor cells. It could be hypothesized that the action spectra of the cellular response indicate the participation of endogenous porphyrins and cytochromes as primary photoreceptors. Taking into account all light induced processes, the term biomodulation should preferably be used.

Sequences from the aspergillopepsin PEP gene of Aspergillus fumigatus: evidence on their use in selective PCR identification of Aspergillus species in infected clinical samples.

In immunodeficient patients, Aspergillus species emerge as circumstantial pathogens. Aspergillus fumigatus is a distant first among the pathogenic aspergilli, which cause deep-seated mycoses. Sequences of the pep gene of A. fumigatus as potential PCR primers, which have not been tested before, were used to identify this species and if possible, differentiate it from other, co-identified, clinically important species of the genus. We present results of the three most promising primer pairs, pep-1/pep-22, pep-15/pep-22 and pep-21/pep32. The second pair was of better specificity when tested with DNA extracted from pure cultures of a multitude of aspergilli, whereas the first co-amplified four clinically significant Aspergillus species. The compatibility of the PCR method with the CTAB DNA extraction protocol varied according to the biological fluid tested and the primer pair used. The first two pairs showed moderate adaptability to the different commercial DNA extraction kits, which were tested in whole blood, spiked with Aspergillus fumigatus hyphae and conidia - as were all the biological fluids used. Restriction of the amplification products with MspI produced distinct patterns for different Aspergillus spp. This approach, as a potential diagnostic tool, seems reliable and sensitive due to its flexibility, speed, low cost, ease of application and selectable breadth of detection.

Sleeping sites, sleeping places, and presleep behavior of gibbons (Hylobates lar).

The sleeping habits of wild white-handed gibbons (Hylobates lar) were investigated to assess the risk of predation and predation-avoidance behavior. Sleeping sites were distributed throughout home ranges, including areas where they overlapped with neighbors, and appeared to be selected independently of habitat characteristics. Individuals did not build night nests or otherwise manipulate the vegetation around the sleeping place but slept on open branches. Group members usually slept in separate trees, and, except for females with infants, they never shared a sleeping place. Sleeping trees were entered several hours before dusk and were used for about 14-17 h. The majority of sleeping trees were used only once, and fewer were selected repeatedly by the same or other group members. Usually females with infants went into a sleeping tree first, then juveniles, and last were mostly subadult and adult males. Intragroup competition over access to a sleeping place was observed once. Average time difference between the first and last group member to enter a sleeping tree was 13 min. The sequence of departure from sleeping trees was more variable. Gibbon sleeping habits seem to primarily reflect adaptations to minimize predation risk. The predation-risk hypothesis was indirectly supported by observations of mobbing pythons, alarm calls given in response to birdes of prey flying low over the canopy, and more importantly by 1) the predominant use of large sleeping trees, which were among the tallest trees available, particularly by adult females with small infants and juveniles, 2) an unpredictable long-term pattern of reuse of sleeping places, and 3) inconspicuous presleep behavior.

[The significance of secretory and structure-associated proteases of Aspergillus fumigatus for the pathogenesis of invasive aspergillosis]. / Zur Bedeutung von sekretorischen und stukturassoziierten Proteasen von Aspergillus fumigatus für die Pathogenese der invasiven Aspergillose.

In the course of invasive aspergillosis, Aspergillus fumigatus is capable of penetrating any tissue of the host. Secretory proteinases of the fungus might facilitate the hyphae to grow through fibrillar proteins like elastin and collagen. However, using systemic infection models, no significantly reduced virulence could be shown with fungal mutants deficient for all known secretory proteinases. Thus, secretory proteinases might be of minor relevance for the pathogenesis of invasive aspergillosis. In addition, microscopic examination of aspergilli penetrating vessel walls did not reveal obvious lysis of wall proteins, thus emphasizing a mechanical disruption of fibrillar proteins by the growing hyphae. However, a strictly localized proteolysis at the tips of growing hyphae caused by wall associated proteinases might be involved. Candidates for such a mechanism are the activities of aspartic and serine proteinases which we have discovered in the cell wall fraction of A. fumigatus.

Virulence of an aspergillopepsin-deficient mutant of Aspergillus fumigatus and evidence for another aspartic proteinase linked to the fungal cell wall.

A gene replacement was performed to produce mutants of Aspergillus fumigatus deficient in the aspergillopepsin PEP (E.C. 3.4.23.18). The correct replacement of the PEP gene was confirmed by PCR and Southern hybridization experiments, whereas the absence of PEP production was demonstrated by Western blots. The culture supernatant of the transformants showed no detectable acid proteinase activity, suggesting that there is only one acid proteinase secreted by the fungus. The wild-type strain and the PEP-deficient mutants invaded tissues to a similar extent and produced comparable mortality in guinea pigs. As PEP represents a third secretory proteinase of A. fumigatus and the other two proteinases also did not show significant influence on fungal invasiveness, it is probable that secreted proteinases do not contribute decisively to tissue invasion in the pathogenesis of systemic aspergillosis. However, immunofluorescence on A. fumigatus colonies using polyclonal antibodies to PEP showed a similar pattern for the wild-type and for the mutants, with a bright fluorescence on young conidiophores, on submerged mycelium and on the tips of growing aerial mycelium. Conidia and mature aerial hyphae were not fluorescent. This pattern could reflect the existence of another crossreacting aspartic proteinase (PEP2) which was found to be sensitive to pepstatin but tightly linked to the fungal cell wall.