A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function.

Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.

Polyploids require Bik1 for kinetochore-microtubule attachment.

The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle.

The molecular mechanisms that link cell-cycle controls to the mitotic apparatus are poorly understood. A component of the Saccharomyces cerevisiae spindle, Ase1, was observed to undergo cell cycle-specific degradation mediated by the cyclosome, or anaphase promoting complex (APC). Ase1 was degraded when cells exited from mitosis and entered G1. Inappropriate expression of stable Ase1 during G1 produced a spindle defect that is sensed by the spindle assembly checkpoint. In addition, loss of ASE1 function destabilized telophase spindles, and expression of a nondegradable Ase1 mutant delayed spindle disassembly. APC-mediated proteolysis therefore appears to regulate both spindle assembly and disassembly.

The role of cytoplasmic dynein in the human brain developmental disease lissencephaly.

Lissencephaly is a brain developmental disorder characterized by disorganization of the cortical regions resulting from defects in neuronal migration. Recent evidence has implicated the human LIS-1 gene in Miller-Dieker lissencephaly and isolated lissencephaly sequence. LIS-1 is homologous to the fungal genes NudF and PAC1, which are involved in cytoplasmic dynein mediated nuclear transport, but it is also almost identical to a subunit of PAF acetylhydrolase, an enzyme which inactivates the lipid mediator platelet activating factor. Recent evidence from our laboratory has revealed that cytoplasmic dynein coimmunoprecipitates with LIS-1 in bovine brain cytosol, supporting a role in the dynein pathway in vertebrates. Overexpression of LIS-1 interferes with cell division, with noteworthy effects on chromosome attachment to the mitotic spindle and on the interaction of astral microtubules with the cell cortex. Other aspects of dynein function, such as the organization of the Golgi apparatus, are not affected. Together, these results suggest a role for LIS-1 in cytoplasmic dynein functions involving microtubule plus-ends. Furthermore, they suggest that mutations in LIS-1 may produce a lissencephalic phenotype either by interfering with the movement of neuronal nuclei within extending processes, or by interference with the division cycle of neuronal progenitor cells in the ventricular and subventricular zones of the developing nervous system.

The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo.

Oseltamivir carboxylate is a potent and specific inhibitor of influenza A and B neuraminidase (NA). Oseltamivir phosphate, the ethyl ester prodrug of oseltamivir carboxylate, is the first orally active NA inhibitor available for the prophylaxis and treatment of influenza A and B. It offers an improvement over amantadine and rimantadine which are active only against influenza A and rapidly generate resistant virus. The emergence of virus resistant to oseltamivir carboxylate in the treatment of naturally acquired influenza infection is low (about 1%). The types of NA mutation to arise are sub-type specific and largely predicted from in vitro drug selection studies. A substitution of the conserved histidine at position 274 for tyrosine in the NA active site has been selected via site directed mutagenesis, serial passage in culture under drug pressure in H1N1 and during the treatment of experimental H1N1 infection in man. Virus carrying H274Y NA enzyme selected in vivo has reduced sensitivity to oseltamivir carboxylate. The replicative ability in cell culture was reduced up to 3 logs, as was infectivity in animal models of influenza virus infection. Additionally, pathogenicity of the mutant virus is significantly compromised in ferret, compared to the corresponding wild type virus. Virus carrying a H274Y mutation is unlikely to be of clinical consequence in man.

Nucleotide sequence of the extracellular alpha-amylase gene in the yeast Schwanniomyces occidentalis ATCC 26077.

The Schwanniomyces occidentalis (formerly castellii) ATCC 26077 (CBS 2863) alpha-amylase (AMY 26077) gene was cloned in Saccharomyces cerevisiae and sequenced. An open-reading frame encoding the AMY consists of 1536 base pairs and contains 512 amino-acid residues, which is almost the same in size as the AMY of Sch. occidentalis ATCC 26076 and CCRC 21164. The amino-acid sequence of AMY 26077 differed from that of ATCC 26076 alpha-amylase (AMY 26076) at two residues and from that of CCRC 21164 alpha-amylase (AMY 21164) at three residues. Comparison of the AMY 26077 gene with its homologues from two other strains (Sch. alluvius CBS 1153 and Sch. persoonii CBS 2169) using several restriction enzymes revealed that the AMY 26077 was very similar to AMY CBS 1153 but different from that of CBS 2169.

Calcium influxes into brain and cerebrospinal fluid are linearly related to plasma ionized calcium concentration.

Unidirectional Ca influxes into brain and cerebrospinal fluid (CSF) were measured at different plasma concentrations of ionized Ca ([Ca]i) in pentobarbital-anesthetized rats. Plasma [Ca]i was varied acutely from 0.6 to 3.0 mumol/ml by intravenous infusion of EGTA, NaCl or CaCl2 or by thyroparathyroidectomy. Ca influx was determined from the 15-min uptake of 45Ca after intravenous injection. There were significant regional differences in 45Ca uptake into the CNS, with a approximately 20-fold greater rate into ventricular CSF than into frontal cortex. Autoradiographs of 45Ca uptake demonstrated that uptake into frontal cortex reflects primarily transport across the cerebral capillaries, whereas uptake into ventricular CSF reflects transport across the choroid plexuses. At both sites, Ca influx was a linear function of plasma [Ca]i and extrapolated to zero at [Ca]i = 0. Infusion of EGTA or CaCl2 did not alter the integrity of the blood-brain barrier, as determined by the permeability to [14C]sucrose. These results indicate that Ca influx into the CNS is not regulated by a saturable mechanism that is sensitive to acute changes in plasma [Ca]i. The proportionality between influx and concentration is suggestive of passive diffusional transport. The brain is protected from acute changes in plasma [Ca]i by the low cerebrovascular permeability to Ca, approximately 5 X 10(-8) cm/s.

Improved liquid chromatographic method for determination of carotenoids in Taiwanese mango (Mangifera indica L.).

An HPLC method was developed to determine the various carotenoids in Taiwanese mango (Mangifera indica L.). Initially, the peel and seed of mangoes were removed, the pulps were cut into pieces, freeze-dried, ground into powder, extracted and subjected to HPLC analysis. A mobile phase of methanol-isopropanol (99:1, v/v) (A) and methylene chloride (100%) (B) with the following gradient elution was developed: 100% A and 0% B in the beginning, maintained for 15 min, decreased to 70% A in 45 min, maintained for 15 min and returned to 100% A in 65 min. A total of 25 carotenoids were resolved within 53 min by using a C-30 column with flow rate at 1 mL/min and detection at 450 nm. alpha-Carotene was used as an internal standard to quantify all the carotenoids. All-trans-beta-carotene was present in largest amount (29.34 microg/g), followed by cis isomers of beta-carotene (9.86 microg/g), violaxanthin and its cis isomers (6.40 microg/g), neochrome (5.03 microg/g), luteoxanthin (3.6 microg/g), neoxanthin and its cis isomers (1.88 microg/g), zeaxanthin (1.16 microg/g) and 9- or 9'-cis-lutein (0.78 microg/g).

Analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by various treatments.

The analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by soaking and drying treatments were studied. The various carotenoids in the flowers of daylily were analyzed using a reversed-phase C(30) HPLC column and a mobile phase of methanol/methylene chloride/2-propanol (89:1:10, v/v/v) with methanol/methylene chloride (45:55, v/v) as sample solvent. Twenty-one pigments were resolved, of which 14 carotenoids were identified, including neoxanthin, violaxanthin, violeoxanthin, lutein-5,6-epoxide, lutein, zeaxanthin, beta-cryptoxanthin, all-trans-beta-carotene, and their cis isomers, based on spectral characteristics and Q ratios. Prior to hot-air-drying (50 degrees C) or freeze-drying, some of the daylily flowers were subjected to soaking in a sodium sulfite solution (1%) for 4 h. Under either the hot-air- or the freeze-drying treatment, the amounts of most carotenoids were higher in the soaked daylily flowers than in those that were not soaked. With hot-air-drying, the amount of cis carotenoids showed a higher yield in soaked samples than in nonsoaked samples. However, with freeze-drying, only a minor change of each carotenoid was observed for both soaked and nonsoaked samples. Also, air-drying resulted in a higher loss of carotenoids than freeze-drying.

Formation of heterocyclic amines in fried fish fiber during processing and storage.

The formation of heterocyclic amines (HAs) in fried fish fiber during processing and storage was studied. Fried fish fiber was prepared by boiling of raw fish, followed by eviscerating, pressing, chopping, and then the fish meat was subjected to frying, during which the various additives such as sugar, soybean sauce, and edible oil were added. The various HAs in fried fish fiber were analyzed by high-performance liquid chromatography with photodiode-array detection. Only four HAs, Norharman, Harman, 2-amino-9H-pyrido[2,3-b]indole, and 2-amino-3-methyl-9H-pyrido[2,3-b]indole were detected in fried fish fiber. The amount of HAs increased with increasing frying temperature. Amino acids might play a more important role for HA formation than reducing sugar during processing of fried fish fiber. During storage, the HAs increased with increasing storage temperature when the fried fish fiber was packed in an aluminum foil bag. However, the relationship between storage temperature and HAs formation was not consistent when the fried fish fiber was packed in a plastic bag.

Pharmacology of procaine in the horse: a preliminary report.

Rapid intravenous injection of 1 g of procaine hydrochloride in Thoroughbred mares produced variable signs of central nervous system excitation for as long as 4 minutes. Plasma concentrations of procaine were similarly variable and transient, decreasing with a half-life of approximately 25 minutes. In vitro, plasma from freshly collected equine blood hydrolyzed procaine with a half-life of approximately 7.5 minutes. This hydrolysis was apparently due to plasma esterases. Penicillin, when added free or complexed as procaine-penicillin, did not protect procaine against hydrolysis by these plasma esterases at pH 7.4.

Pharmacology of procaine in the horse: evidence against the existence of a "procaine - penicillin" complex.

It has recently been suggested that procaine penicillin existed in solution in vitro and in vivo as a "procaine - penicillin" complex rather than as dissociated ions. In vivo, this complexed procaine was considered unavailable for hydrolysis by plasma esterases or for interaction with pharmacologic receptors for procaine. When procaine penicillin was intramuscularly given to horses, about 90% of the procaine in blood drawn from these horses was split at the same rate as authentic procaine or procaine penicillin added to equine blood in vitro. In vitro, procaine and procaine penicillin partitioned similarly from aqueous medium at physiologic pH into several organic solvents and were split at the same rate by blood or plasma esterases. Experiments on the time course of the partitioning of procaine from procaine penicillin into benzene showed no evidence for the existence of a "procaine - penicillin" complex within seconds after procaine penicillin was added to aqueous medium. Thin layer chromatography in 2 dimensions also yielded no evidence for the existence of this postulated complex. These results show no evidence in support of the "procaine - penicillin" hypothesis and argue against the physical and pharmacologic and forensic implications of this hypothesis.

Transport of weak bases across rat gastric mucosa in vivo and in vitro.

The transport of weak bases (pKa values 1.4 through 10.3) across in vitro and in vivo preparations of rat gastric mucosa have been compared. Asymmetric movements were observed in all cases, and the in vitro and in vivo data showed similar profiles of relation between the degrees of asymmetry and the pKa values of transported compounds. These profiles could be described by an equation based on a three-compartment model system, but it was necessary to postulate two such systems in parallel to give a satisfactory fit to the full range of pKa values tested. One system (I) is analogous to that described previously in small intestine. The properties of this system [(Pi/Pni)a = 2.5 x 10(-3), (Pi/Pni)b = 7.0 x 10(-1)] suggested that the intermediate compartment is a subepithelial extracellular compartment in which an alkaline pH is maintained [pHx = 8.5]. The second system (II) is the opposite polarity [(Pi/Pni)a = 1 x 10(0), (Pi/Pni)b = 3 x 10(-1)], and this system may be represented by the acidic lumen of the gastric tube [pHx = 2.0]. The principal differences between the in vivo and in vitro data could be ascribed to poor stirring of the luminal bulk phase in the in vivo situation. It was concluded that the determinants of weak base transport identified in studies with in vitro preparations are pertinent to the absorptive and secretory processes that occur in the intact stomach.

Weak-acid transport in the small intestine: discrimination in the lamina propria.

Studies on the intestinal transport of weak acids suggest that the subepithelial tissues exhibit a modest, but significant, ability to discriminate between the ionized and nonionized forms. This suggestion has been tested directly in experiments using an in vitro preparation of rat small intestine from which the epithelial cells were removed, but in which the structural and functional integrity of the subepithelial tissues was maintained. Studies on the effects of potential difference on the fluxes of weak acids in this preparation showed that the ratio of permeabilities for the ionized and nonionized species (Pi/Pni) was indeed less than one, and of a magnitude comparable to the value suggested by analysis of transport in the intact tissue. (Pi/Pni) for the subepithelial tissue decreased as pH was increased, and the discriminatory properties of the tissue were abolished [(Pi/Pni)=1] on treatment with the cationic macromolecule polyethyleneimine (PEI). These observations suggested that the discriminatory properties of the subepithelial tissues were determined by fixed anionic sites, and morphological studies with PEI indicated that such sites were concentrated in the region of the basement membrane.

The conventional short-circuiting technique under-short-circuits most epithelia.

The relationships among ion current, membrane potential difference, and resistance of an epithelium are studied. The short-circuit technique introduced by Ussing and Zerahn does not completely short circuit the epithelium if the series resistance parallel to the cell layer between the voltage electrodes is not properly compensated. The residual potential difference across the epithelial cell layer in the "short-circuit state" is proportional to both the measured short-circuited small intestinal mucosa the villus and crypt areas are hypo-polarized to different degrees rather than simultaneously hyper- and hypo-polarized. Short-circuiting the whole tissue reduces but does not abolish the passive net ion movement across the tissue. Measurements of the electrical properties of the whole and denuded rat distal small intestine in HCO3-Ringer solution containing 10 mM glucose reveal that the measured short-circuit current has under-estimated approximately 33% of the true short-circuit current and that the passive net Na flux from serosa to mucosa and Cl flux from mucosa to serosa are not negligible in the "short circuit state."

Plasma membrane intrinsic proteins of Beta vulgaris L.

The plasma membrane (PM) of higher plants contains numerous proteins; however, due to their low abundance, only a few have been identified and characterized by direct biochemical approaches. The major intrinsic protein (MIP) family is a class of highly hydrophobic integral membrane proteins thought to function as channels that facilitate the passage of water, small solutes, and possibly other moieties through the membrane. A family of PM intrinsic proteins was purified and characterized from PM vesicles derived from storage tissue of Beta vulgaris L. using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate. This PM intrinsic protein-enriched fraction also contains high levels of UDP-glucose:(1,3)-beta-glucan (callose) synthase activity. Dithiothreitol is required to visualize the monomeric species of these highly hydrophobic integral membrane proteins. Sequence analysis of tryptic fragments derived from polypeptides of 31 and 27 kD revealed significant homologies to plant MIPs identified from cloned sequences. These MIPs include clone 7a from pea and RD28 from Arabidopsis, both of which are water-stress proteins, a tomato ripening-associated membrane protein, and PIP 2b, a PM-bound water channel protein from Arabidopsis. MIPs, therefore, represent abundantly occurring components of PMs derived from beet storage tissue.

Weak electrolyte permeation in alimentary epithelia.

The potential significance of ionized species in weak electrolyte absorption or secretion has been reexamined using a mathematical model that represents the epithelium as a system of parallel ion-permeable and ion-impermeable channels. An important determinant of weak electrolyte movement in this system is the ratio of ionized and nonionized permeabilities (Pi/Pni). This variable, which has been termed the discrimination coefficient, interacts with the degree of ionization in determining the contributions of ionized and nonionized species to the transepithelial movement of a weak electrolyte. Calculations based on the model suggest that ionized species may contribute significantly to the absorption or secretion of many common weak electrolytes. It is concluded that the frequently made assumption that ionized species do not contribute significantly to transepithelial movements of weak electrolytes in the alimentary tract is not generally valid. Further work is required to delineate the quantitative determinants of discrimination in alimentary epithelia, and two methods for evaluation of epithelial discrimination coefficients are described.

Recovery of procaine from biological fluids.

A published method for the recovery of procaine from human plasma using 5M NaOH gave very poor recoveries. Investigation showed that under the recommended extraction conditions procaine was rapidly hydrolysed. Extraction into benzene of samples buffered to pH 9.0 with borate buffer allowed essentially 100% recovery of procaine from equine plasma and urine.

Arabidopsis mutants with increased sensitivity to aluminum.

Al-sensitive (als) mutants of Arabidopsis were isolated and characterized with the aim of defining mechanisms of Al toxicity and resistance. Most als mutants selected on the basis of root growth sensitivity to Al were recessive, and together the mutants constituted eight complementation groups. Also, in most als mutants, Al sensitivity appeared to be specific for Al relative to La (another trivalent cation), except als2, which was more sensitive to La than wild type. The tendency of roots on mutant seedlings to accumulate Al was examined by staining with morin and hematoxylin, dyes used to indicate the presence of Al. A significant increase in morin staining was observed in als5, consistent with its increased sensitivity to Al. Unexpectedly, als7 and als4 showed less morin staining, suggesting that the roots on these mutants accumulate less Al than wild type seedlings after exposure to Al-containing solutions. Roots of wild-type seedlings produce callose in response to AlCl3 concentrations that inhibit root growth. Only als5 accumulated more callose than wild type in response to low levels (25 mu M) of AICI3 However, als4 and als7 did not accumulate callose at this AlCl3 concentration even though root growth was significantly inhibited. The lack of callose accumulation in als4 and als7 suggests that there is not an obligatory relationship between callose deposition and Al-induced inhibition of root growth.