Biomedical subjects

R Kellner

Publications and source records attributed to R Kellner.

At least 19 recordsLinked to original sources

PubMed2001

[Screening for carrier state of Haemophilia B using indirect genomic detection].

The authors report the first data having applied the indirect genomic diagnosis in carrier screening in Hungary. 22 patients with haemophilia B and female family members of 14 out of them were examined by PCR based restriction fragment length polymorphism analysis. The combined use of 3 intra- and 1 extragenic polymorphisms have been examined at the same population. DNA fragments, containing the single nucleotide change polymorphic site (Xmnl, Hhal, Taql), or the 50 bp insertion/deletion element (Dde) were amplified. The products were digested by the appropriate restriction digestion enzyme and were detected on agarose gel following ethidium-bromide staining. 20 siblings were interested in the determination of their carrier-state. 15 (75%) of them could get definite diagnosis. The carrier-state was established in 7 cases, excluded in 8 subjects. For the remaining 5 participants studied, the absence of the parental DNA sample caused uncertainty, while in 2 cases (10%) none of the analyzed RFLP was informative. The heterozygosity rate, the gene and haplotype frequency were also recorded and compared with the international data. The indirect methods have proved to be sufficient and well suitable for routine carrier testing. The results provide the basis of the subsequent prenatal diagnosis.

Genetic Testing↗

PubMed2001

Mapping and expression pattern analysis of key components of the major histocompatibility complex class I antigen processing and presentation pathway in a representative human renal cell carcinoma cell line.

Renal cell carcinoma (RCC) represent approximately 5% of all cancer deaths. At the time of presentation, over 50% of the patients have already developed locally advanced or metastatic disease with five-year survival rates of less than 20%. Although relative resistant to conventional regimens, RCC are partially susceptible to T cell-based immunotherapy. To further develop this treatment modality, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied for both the mapping of the key components of the major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) and the characterization of the constitutive and cytokine-regulated protein expression profiles in a representative human RCC cell line. The latter aspect is based on the fact, that the expression level of some of the APM components can be altered in response to interferon (IFN)-gamma treatment. Total cell lysates from untreated and IFN-gamma-treated tumor cells were separated on 2-D PAGE gels using broad range immobilized pH gradient (IPG) strips. Serial Western blot analyses using sets of APM-specific antibodies were performed to target the relevant protein spots. Protein verification was mostly accomplished via peptide mass finger-printing using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). To date, the majority of the APM-related components have been identified and mapped. In addition, the different protein expression profiles of untreated and IFN-gamma-treated RCC cells are under investigation.

Antigen Presentation↗

PubMed2001

A comparative study on hypertrehalosaemic hormones in the Hymenoptera: sequence determination, physiological actions and biological significance.

A new hypertrehalosaemic peptide (Tea-HrTH; pQLNFSTGWGG-NH(2)) was isolated from the corpora cardiaca (CC) of the sawfly Tenthredo arcuata. The hypertrehalosaemic peptides found in the CC of five Bombus species and the paper wasp Polistes fuscata were identical to the adipokinetic hormone II of the desert locust, Schistocerca gregaria (Scg-AKH-II). The hypertrehalosaemic peptides found in the yellowjacket Vespula vulgaris and the hornet Vespa crabro were identical to the adipokinetic hormone of the cricket, Gryllus bimaculatus (Grb-AKH).All species examined had a large storage crop which, when filled with honey, held up to one-third of their total body weight. Overwintering queens of P. fuscata had large stores of carbohydrates and lipids in the abdomen, and were able to survive months of fasting. Workers of Bombus hortorum (bumble-bee), Apis mellifera (honey-bee) and V. vulgaris had little or no fat body. These species could fly as long as sugar was present in their crops, but they stopped flying as the carbohydrates in the crop disappeared. There was no significant increase in the haemolymph carbohydrate titres after injections of CC extracts or corresponding synthetic peptides into workers of B. hortorum or into males and females of T. arcuata. There was a moderate increase in haemolymph carbohydrate titres when these peptides were injected into overwintering queens of P. fuscata and into workers of V. crabro, both with significant amounts of fat body. However, well-fed V. vulgaris workers, with very little fat body, also responded to their own hypertrehalosaemic peptide.

Journal Article↗

PubMed2000

Glutathione peroxidase-1 but not -4 is involved in the regulation of cellular 5-lipoxygenase activity in monocytic cells.

In contrast to neutrophils or B-lymphocytes, cells of the monocytic lineage like rat macrophages, human peripheral blood monocytes and Mono Mac 6 cells contain a strong inhibitor of 5-lipoxygenase (5-LO) activity, which scavenges hydroperoxides and inhibits 5-LO activity in broken-cell preparations in the absence of exogenously added thiols. Chromatographic purification of the inhibitor from the human monocytic cell line Mono Mac 6 and amino acid sequence analysis revealed that the inhibitory factor is glutathione peroxidase-1 (GPx-1). In contrast to the peroxidase activity of GPx-1, 5-LO inhibition by GPx-1 was supported by beta-mercaptoethanol and there was no absolute requirement for millimolar concentrations of glutathione or dithiothreitol. These cofactor characteristics suggest that both activities address distinct catalytic properties of GPx-1. 5-LO inhibition by GPx-1 was not due to direct GPx-5-LO protein-protein interactions, since GPx-1 did not bind to immobilized 5-LO. Interestingly, 5-LO derived from granulocytes was significantly more resistant against GPx-1 inhibition than B-lymphocytic 5-LO, which correlates with the respective cellular 5-LO activities. In summary, the data suggest that, in addition to previously reported phospholipid hydroperoxide glutathione peroxidase (GPx-4), GPx-1 is an efficient inhibitor of 5-LO even at low thiol concentrations, and is involved in the regulation of cellular 5-LO activity in various cell types.

Animals↗

PubMed2000

A third active AKH is present in the pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans.

The corpora cardiaca of the African pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans contain three adipokinetic hormones (AKHs): besides two already known AKHs, Phm-AKH-I and Scg-AKH-II (Gäde et al., 1996 [Gäde, G., Kellner, R., Rinehart, K.L., 1996. Pyrgomorphid grasshoppers of the genus Phymateus contain species-specific decapeptides of the AKH/RPCH family regulating lipid-mobilisation during flight. Physiol. Entomol. 21, 193-202]), a new AKH-III, denoted Phm-AKH-III, pGlu-Ile-Asn-Phe-Thr-Pro-Trp-Trp-NH(2), has been characterised. This is only the second AKH-III identified so far, thus, only three insect species - all of them grasshoppers - contain three active AKHs. Phm-AKH-III differs from Lom-AKH-III from the migratory locust, Locusta migratoria, only in position 2: isoleucine is present instead of leucine. The structure of the Phm-AKH-III was confirmed by synthesis, subsequent mass determination and reversed-phase high-performance liquid chromatography. The synthetic peptide also induced hyperlipaemia in D. spumans and L. migratoria.

Animals↗

PubMed2000

A controlled study of imagery rehearsal for chronic nightmares in sexual assault survivors with PTSD: a preliminary report.

Imagery-rehearsal therapy for chronic nightmares was assessed in a randomized, controlled study of sexual assault survivors with posttraumatic stress disorder (PTSD). Nightmares, sleep quality, and PTSD were assessed at baseline for 169 women, who were randomized into two groups: treatment (n = 87) and wait-list control (n = 82). Treatment consisted of two 3-hr sessions and one 1-hr session conducted over 5 weeks. Of 169 participants, 91 women (Treatment, n = 43, Control, n = 48) completed a 3-month follow-up and 78 did not. At follow-up, nightmare frequency and PTSD severity decreased and sleep quality improved in the treatment group with small to minimal changes in the control group. Treatment effects were moderate to high (Cohen's d ranged from 0.57 to 1.26). Notwithstanding the large dropout rate, imagery-rehearsal therapy is an effective treatment for chronic nightmares in sexual assault survivors with PTSD and is associated with improvement in sleep quality and decreases in PTSD severity.

Adult↗

PubMed2000

Identification of novel periviscerokinins from single neurohaemal release sites in insects MS/MS fragmentation complemented by Edman degradation.

Three novel members of the periviscerokinin family could be identified directly from extracts of single abdominal perisympathetic organs of blaberoid cockroaches by means of electrospray ionization-quadrupole time of flight (ESI-QTOF) MS. Sequences of these periviscerokinins were confirmed by Edman degradation. Their primary structures are GSSGLIPFGRT-NH2 (Lem-PVK-1), GSSGLISMPRV-NH2 (Lem-PVK-2), and GSSGMIPFPRV-NH2 (Lem-PVK-3). Hitherto only known from the American cockroach, this neuropeptide family contains a highly conserved N-terminus whereas, at the C-terminus, only the penultimate amino-acid residue (Arg) has been found in all members of this peptide family. The identified periviscerokinins are the only abundant myoactive peptides in abdominal perisympathetic organs of blaberoid cockroches and they appear to be absent in the retrocerebral complex. Screening of extracts of single abdominal perisympathetic organs (70-90 microm in diameter), from five different species of the suborder Blaberoidea, revealed that they all contain the three neuropeptides which are described here for the first time.

Abdomen↗

PubMed2000

Structure elucidation and biological activity of an unusual adipokinetic hormone from corpora cardiaca of the butterfly, Vanessa cardui.

A structurally unusual member of the adipokinetic hormone/red pigment-concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano-electrospray-time-of-flight mass spectrometry as pQLTFTSSWGGK (Vac-AKH). Vac-AKH represents the first 11mer and the first nonamidated peptide in this family. The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac-AKH into 4-day-old decapitated males resulted in an approximately 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0. 1 pmol of Vac-AKH. During a 2-h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mM KCl, significant amounts of Vac-AKH were released from the glands.

Amino Acid Sequence↗

PubMed2000

Potencies of naturally-occurring AKH/RPCH peptides in Locusta migratoria in the acetate uptake assay in vitro and comparison with their potencies in the lipid mobilisation assay in vivo.

The biological potencies of a number of naturally-occurring octa- and decapeptides of the large AKH/RPCH family of peptides were determined in Locusta migratoria using the lipid-mobilising assay in vivo and the acetate uptake assay in vitro. The most potent of the newly-tested peptides in the in vitro assay, Phl-CC, differs from the endogenous major locust peptide, Lom-AKH-I, only by an exchange of serine versus threonine at position 10. However, the most active peptide in the in vitro assay remains Lom-AKH-III. At the other extreme is the peptide Mem-CC which contains a tyrosine residue at position 4 rather than the more typical phenylalanine. This peptide is over 20,000 times less potent than Lom-AKH-III in the in vitro assay, and also results in an unusual dose-response curve in the in vivo assay. Only a few peptides are approximately equipotent in both assays, but mostly the bioanalogues have a higher potency in vitro. The majority of them are 2- to 10-fold more potent in vitro, but Ani-AKH and Lom-AKH-III are 19- and 48-fold more potent. The results are discussed in relation to either the actions of proteases or of possible preferential binding of different receptors involved in the different assays.

Acetic Acid↗

PubMed1999

Increased stability upon heptamerization of the pore-forming toxin aerolysin.

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

Amino Acid Sequence↗

PubMed1999

Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors.

The assembly of signalling molecules into macromolecular complexes (transducisomes) provides specificity, sensitivity and speed in intracellular signalling pathways. Rod photoreceptors in the eye contain an unusual set of glutamic-acid-rich proteins (GARPs) of unknown function. GARPs exist as two soluble forms, GARP1 and GARP2, and as a large cytoplasmic domain (GARP' part) of the beta-subunit of the cyclic GMP-gated channel. Here we identify GARPs as multivalent proteins that interact with the key players of cGMP signalling, phosphodiesterase and guanylate cyclase, and with a retina-specific ATP-binding cassette transporter (ABCR), through four, short, repetitive sequences. In electron micrographs, GARPs are restricted to the rim region and incisures of discs in close proximity to the guanylate cyclase and ABCR, whereas the phosphodiesterase is randomly distributed. GARP2, the most abundant splice form, associates more strongly with light-activated than with inactive phosphodiesterase, and GARP2 potently inhibits phosphodiesterase activity. Thus, the GARPs organize a dynamic protein complex near the disc rim that may control cGMP turnover and possibly other light-dependent processes. Because there are no similar GARPs in cones, we propose that GARPs may prevent unnecessary cGMP turnover during daylight, when rods are held in saturation by the relatively high light levels.

ATP-Binding Cassette Transporters↗

PubMed1999

Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

The occurrence of allatostatins in retrocerebral complexes and antennal pulsatile organs of the American cockroach, Periplaneta americana, was investigated. Previously, molecular cloning of the P. americana allatostatin gene had predicted 14 peptides of this family [Ding et al., Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata. Eur J Biochem 1997;234:737-746], however, only two forms had been identified by peptide isolation procedures [Weaver et al., Identification of two allatostatins from the CNS of the cockroach Periplaneta americana: novel members of a family of neuropeptide inhibitors of insect juvenile hormone biosynthesis. Comp Biochem Physiol 1994;107(C):119-127]. Using an extract of only 200 corpora cardiaca/corpora allata, we have found that at least 11 allatostatins occur in the retrocerebral complex. These peptides were already separated from other substances of the crude extract in the first HPLC step with heptafluorobutyric acid as organic modifier, and subsequently identified by MALDI-TOF mass spectrometry. Moreover, we have demonstrated the occurrence of nearly all allatostatins, including the cleavage product of Pea-AST-2 (LPVYNFGL-NH2), in antennal pulsatile organs of males and females. Allatostatins are predominant neuropeptides in these organs. Additionally, only two other known peptides could be identified in these organs by mass screening: proctolin and leucomyosuppressin. The function of allatostatins in antennal pulsatile organs remains unclear. We assume a release into the hemolymph via the ampullac, which could act as neurohemal release sites. The method described for the identification of allatostatins is a very fast method for neuropeptide screening in neurohemal tissues.

Amino Acid Sequence↗

PubMed1999

Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.

Negative factor (Nef) is a regulatory myristoylated protein of human immunodeficiency virus (HIV) that has a two-domain structure consisting of an anchor domain and a core domain separated by a specific cleavage site of the HIV proteases. For structural analysis, the HIV-1 Nef anchor domain (residues 2-57) was synthesized with a myristoylated and non-myristoylated N terminus. The structures of the two peptides were studied by1H NMR spectroscopy and a structural model was obtained by restrained molecular dynamic simulations. The non-myristoylated peptide does not have a unique, compactly folded structure but occurs in a relatively extended conformation. The only rather well-defined canonical secondary structure element is a short two-turn alpha-helix (H2) between Arg35 and Gly41. A tendency for another helical secondary structure element (H1) can be observed for the arginine-rich region (Arg17 to Arg22). Myristoylation of the N-terminal glycine residue leads to stabilization of both helices, H1 and H2. The first helix in the arginine-rich region is stabilized by the myristoylation and now contains residues Pro14 to Arg22. The second helix appears to be better defined and to contain more residues (Ala33 to Gly41) than in the absence of myristoylation. In addition, the hydrophobic N-terminal myristic acid residue interacts closely with the side-chain of Trp5 and thereby forms a loop with Gly2, Gly3 and Lys4 in the kink region. This interaction could possibly be disturbed by phosphorylation of a nearby serine residue, and modifiy the characteristic membrane interactions of the HIV-1 Nef anchor domain.

Amino Acid Sequence↗

PubMed1999

Catalytic triad of microsomal epoxide hydrolase: replacement of Glu404 with Asp leads to a strongly increased turnover rate.

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431. Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae. For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction. The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp226. Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold). The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction. Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.

Amino Acid Sequence↗

PubMed1999

Hypertrehalosaemic peptides in the honeybee (Apis mellifera): purification, identification and function.

The corpora cardiaca (CC) of the Italian race (including also the africanised variety) of the honeybee (Apis mellifera ligustica) contain approximately 3 pmol of a hypertrehalosaemic peptide. This peptide is identical in structure to the adipokinetic hormone (AKH) found in Manduca sexta, Mas-AKH. The CC of the dark European race of the honeybee (Apis mellifera carnica) contain no detectable Mas-AKH or any other adipokinetic/hypertrehalosaemic peptide. This is the first report of the occurrence of this peptide in a non-lepidopteran insect and of an intraspecific variation with regards to the presence or absence of a hypertrehalosaemic peptide in the CC of an insect. Extracts of A. m. ligustica CC elicit a strong adipokinetic/hypertrehalosaemic response when injected into crickets and cockroaches but extracts of A. m. carnica CC elicit no such responses when injected into crickets, cockroaches and butterflies. A weak hypertrehalosaemic response to injected Mas-AKH was observed in winter bees of both races, but there was no response in spring/summer bees. However, if a seasonal difference exists, it is at best minimal. Honeybees always have access to a more than adequate supply of high energy food in the form of nectar or honey stored in the hive. Thus, though A. m. ligustica CC contain a hypertrehalosaemic peptide, there is neither a glycogen-mobilising function of this hormone nor an adequate glycogen store in their fat body for its effective utilisation.

Journal Article↗

PubMed1999

Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach.

Different pyrokinin isoforms were identified from major neurohemal organs of the American cockroach. During their isolation they were recognized by bioassay using a hyperneural muscle preparation that is sensitive to pyrokinins. All structures were elucidated by sequence analysis and mass spectrometry. The primary structures of the novel peptides isolated from the retrocerebral complex are LVPFRPRL-NH2 (designated Pea-PK-3) and DHLPHDVYSPRL-NH2 (designated Pea-PK-4). A pyrokinin, labeled Pea-PK-5, was isolated from abdominal perisympathetic organs. Structural analysis of this peptide yielded the sequence GGGGSGETSGMWFGPRL-NH2. The threshold concentrations of the identified pyrokinins for an eliciting effect on contractions of the hyperneural muscle preparations differed dramatically. This indicates that the different distribution of pyrokinin-isoform observed in neurohemal organs may be associated with different functions. This is the first report of a differential distribution of peptide-isoforms in the neurohemal organs of insects.

Animals↗

PubMed1999

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

Amino Acid Sequence↗

PubMed1998

Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2.

Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [35S]sulfate transfer from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS); and (ii) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. & Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896-2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an approximately 13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.

Adrenal Medulla↗