Biogenesis of the yeast vacuole (lysosome). Proteinase yscB contributes molecularly and kinetically to vacuolar hydrolase-precursor maturation.

The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro-CpY) to the mature wild-type form CpYb of 61 kDa. In PrB-deficient mutants, only the proteinase yscA processed form CpYa of 62 kDa is found [Mechler, B., Müller, H. & Wolf, D. H. (1987) EMBO J. 6, 2157-2163]. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB-deficient mutant cells, PrAa, migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild-type cells, had the known molecular mass of 42 kDa. In the PrB-deficient strain, pro-PrA and pro-CpY matured only to the higher-molecular-mass forms, PrAa and CpYa, and the maturation of both precursors was slower than in the isogenic wild-type strain. Pulse-labeling experiments indicated that the mature forms, PrAb or CpYb, are generated directly in the PrB-containing wild-type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42-kDa pro-PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.     

Clinic-based primary care of frail older patients in California.

We surveyed medical directors of primary care clinics in California to learn how those clinics cared for their frail older patients. Of 143 questionnaires sent, 127 (89%) were returned. A median of 30% of all patient encounters were with persons aged 65 or older, and a median of 20% of older patients were considered frail. A total of 20% of the clinics routinely provided house calls to homebound elderly patients. Of clinics involved in training medical students of physicians (teaching clinics), 70% had at least one physician with an interest in geriatrics, compared with 42% of nonteaching clinics (P less than .005). For frail patients, 40% of the clinics routinely performed functional assessment, while 20% routinely did an interdisciplinary evaluation. Continuing education in geriatrics emerged as a significant independent correlate of both functional assessment and interdisciplinary evaluation. Among the 94 clinics with a standard appointment length for the history and physical examination, only 11 (12%) allotted more than 60 minutes for frail patients. The data suggest that certain geriatric approaches are being incorporated into clinic-based primary care in California but do not provide insight into their content or clinical effects.     

Spatial and temporal distribution of pheromone biosynthesis-activating neuropeptide in Helicoverpa (Heliothis) armigera using RIA and in vitro bioassay.

A [3H]-PBAN (pheromone biosynthesis-activating neuropeptide) analog was synthesized, and binding of the radioligand to a specific PBAN-antiserum was achieved. The inhibition of binding of the radioligand by unlabeled PBAN, several PBAN analogs, and other competitors was studied and a specific radio-immunoassay was developed. Using this radioimmunoassay we found PBAN-like immunoreactivity in methanol extracts of hemolymph and neural tissues from females. Higher levels of PBAN-like immunoreactivity in extracts of brain-suboesophageal ganglion complexes, corpora cardiaca, thoracic ganglia, and abdominal ganglia were observed during the 4-5th h scotophase when compared to the PBAN-like immunoactivity levels during the 6-11th h photophase. On the other hand, the concentrations of PBAN-like immunoreactivity, in the terminal abdominal ganglion were higher during the photophase relative to minimal levels observed during the scotophase, indicating an accumulation before the onset of pheromone production. These differences in concentrations of PBAN were also reflected in the stimulation of in vitro pheromone glands, whereby significant stimulations were obtained by scotophase and photophase brain extracts, scotophase thoracic ganglia extracts, and photophase terminal abdominal ganglia extracts. No detectable levels of PBAN were found in hemolymph extracts during the sampling periods.     

Adenylate cyclase uncoupled beta-adrenergic receptors in salamander proximal tubules

The isolated perfused proximal tubule of the neotenic salamander Ambystoma tigrinum responds with either a hyperpolarization or depolarization of both the basolateral cell membrane and transepithelial potentials following the addition of 10(-5) M isoproterenol to the bath superfusate. Both responses were blocked by 10(-6) M propranolol but neither response was mimicked by 10(-4) M cAMP. beta-Adrenergic binding studies of individual microdissected proximal tubules using (-)-[3H]CGP-12177 as a hydrophyllic radioligand and (+/-)-timolol (0.1 mM) as the displacer drug revealed two distinct populations of proximal tubules possessing either low (KD = 153.8 nM; Bmax = 110.2 fM/mm) or high affinity (KD = 12.0 nM: Bmax = 3.9 fM/mm) binding characteristics. Competition studies indicated that the bound (-)-[3H]CGP-12177 behaved as a typical beta-adrenergic ligand, being displaced by (-)-isoproterenol but not by (+)-isoproterenol or (-)-phenylephrine. However, neither appeared to be coupled to the adenylate cyclase system. These data suggest the presence of functional beta-adrenergic receptors that do not appear to be coupled to the adenylate cyclase system.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.     

Discrimination and consistency of five myosin ATPase stains in human normal and duchenne dystrophic muscle

Summary Limitations in the ability of the human visual system to assess accurately the relative staining densities of individual fibers in muscle tissue stained for myosin ATPase can complicate the objective evaluation of fiber type populations. In this study a novel approach is employed which utilizes human visual capabilities to provide accurate fiber classification. Using this approach, the ability of five ATPase staining techniques to discriminate fiber type categories in single samples of human normal and Duchenne dystrophic skeletal muscle is evaluated, as is the consistency of the fiber type classifications between stains. While no major discrepancies in fiber typing were observed in the sample of normal muscle, significant differences in classification, along with a decrease in the ability to discriminate fiber types were noted in the sample of Duchenne muscular dystrophy. For the most part, these discrepancies were resolved by a re-interpretation of the staining characteristics of fibers in one stain.This work was supported in part by NIH grant NS 15584, and by a grant from the Muscular Dystrophy Association     

Host modification of Sindbis virus sialic acid content influences alternative complement pathway activation and virus clearance

Previous studies have shown that Sindbis virus, an enveloped alphavirus of the togavirus group, activates the alternative complement pathway in the absence of detectable antiviral immunoglobulin. The present studies examined the role of the host-determined sialic acid content of Sindbis virus on activation of the alternative complement pathway. Purified Sindbis virus grown in baby hamster kidney (BHK-SV) and in mosquito (MOSQ-SV) cells yielded virus with 10.2 and less than 2.0 nmol sialic acid/mg viral protein, respectively. Sindbis virus deficient in sialic acid (2.0 nmol sialic/mg) was also produced by treating the BHK-SV with neuraminidase (NANase-SV). When MOSQ-SV or NANase-SV was incubated in either C4DGPS or C2DHS, each consumed significantly more C3 than did BHK-SV, indicating that the ability of Sindbis virus to activate the alternative pathway is inversely related to its sialic acid content. Studies in vivo showed that virus deficient in sialic acid (MOSQ-SV) was cleared from the blood of mice much more efficiently than was virus rich in sialic acid (BHK-SV), after i.v. inoculation. Furthermore, when animals were depleted of C3 through C9 by cobra venom factor (CoVF) treatment, no differences in the clearance of high and low sialic acid-containing viruses were observed. Thus both the activation in vitro and complement-dependent clearance in vivo are significantly affected by the host-determined sialic acid content of Sindbis virus.     

Comparative studies on acetazolamide teratogenesis in pregnant rats, rabbits, and rhesus monkeys

Acetazolamide produces a characteristic forelimb reduction deformity when administered to pregnant rodents. Past studies indicated that non-rodent species (rabbit and monkey) are resistant to this effect. The present studies confirmed this fact and demonstrated that transport of acetazolamide into the rabbit embryo was similar to that in sensitive rat embryos. In monkeys, however, the concentrations of acetazolamide within maternal plasma and embryo were much lower than in rats. Carbonic anhydrase activity was also measured since inhibition of this enzyme is the primary pharmacologic effect of acetazolamide. Again the rabbit embryo had carbonic anhydrase specific activity levels similar to that of the rat. Monkey embryos, on the other hand, contained negligible levels of enzyme activity during the presumed sensitive period of development. Thus the resistance of monkey embryos to acetazolamide teratogenesis may be due to low carbonic anhydrase activity and/or the small amount of drug reaching the embryo. No basis for the resistance of rabbit embryos to acetazolamide teratogenesis was uncovered.     

Distribution of Polymorphus minutus among its intermediate hosts

Hirsch R. P. 1979. Distribution of Polymorphus minutus among its intermediate hosts. International journal for Parasitology10: 243–248. In 1971, Crofton investigated patterns of distribution of Polymorphus minutus in the intermediate host, Gammarus pulex. Among his conclusions were: (1) P. minutus populations occur in patterns similar to negative binomial distributions, and (2) parasite-induced host mortality results in patterns similar to truncated (high end) negative binomial distributions. Those conclusions, however, were not tested by statistical analyses. To test Crofton's observations, Chi-square goodness of fit tests were applied to data used by Crofton and an additional two stations sampled by Hynes & Nicholas in 1963. Analyses were expanded to include five theoretical distributions, four patterns of host mortality and various rates of host mortality. Truncated forms of negative binomial, positive binomial and Poisson distributions were also investigated where nontruncated distributions failed to fit observed distributions. It was found that negative binomial distributions most frequently describe patterns of P. minutus distribution with the exception of one population described by Poisson and another by positive binomial distributions. Crofton's assumption that truncated distributions result from parasite-induced host mortality seems unlikely in light of those analyses.     

An ultrastructural study of iron and manganese deposition associated with extracellular polymers of pedomicrobium-like budding bacteria

Morphological characteristics of two Pedomicrobium-like budding bacteria are described. A structured surface layer was regularly observed on strain 868. Ruthenium red- and Alcian blue-staining polymers were found on both strains.When either strain was grown in the presence of iron or manganese, the corresponding oxides accumulated on their surfaces. In thin sections iron oxides appeared as fine threads, arrays of particles or dense coatings, depending on the source of iron. Manganese oxides appeared as branching filaments or convoluted ribbons. Both metal oxides stained with ruthenium red. Extraction of the oxides followed by ruthenium red staining revealed that polyanionic polymers previously deposited on the cells were associated with the metals.Treatment of cultures with glutaraldehyde, HgCl₂, or heat, inhibited manganese but not iron deposition, suggesting that iron oxides accumulated by passive, non-biological processes. Manganese oxides apparently accumulated under control of a biological manganese-oxidizing factor. Incomplete inhibition of manganese deposition observed in cell suspensions suggested that, if the oxidizing factor was an enzyme, it was unusually stable.Based on these results, possible mechanisms of iron and manganese deposition in association with extracellular polymers are suggested.